Use of a compound and derivatives thereof in the manufacture of a medicament for the treatment of cancer

Abstract

The application provides application of a compound and derivatives thereof in preparation of an anticancer agent resistance medicine, relates to a heterocyclic compound lanostane type triterpene lactone glycoside compound and a medical technology field. In the application, it is first disclosed that three lanostane type triterpene lactone glycoside compounds have cytotoxic activity on tumor cells and tumor stem cells, can be used as a new anticancer agent, provide a new functional medicine for cancer treatment, can also be applied to a cancer treatment process of targeting tumor stem cells, and effectively prevent tumor recurrence and metastasis. In the application, it is also found that desulfated holothurin A has good killing power on drug-resistant tumor cells, and tumor cells still have sensitivity to desulfated holothurin A after drug resistance to a chemotherapeutic medicine is generated, so that desulfated holothurin A can avoid the drug resistance mechanism, effectively kill drug-resistant tumor cells, can be used as a potential candidate medicine for drug-resistant tumor treatment, and is expected to solve the clinical difficulties that drug-resistant tumor patients have no available medicine.

Description

Technical Field

[0001] This application relates to the field of heterocyclic compounds, lanostane-type triterpenoid lactone glycosides, and pharmaceutical technology, and particularly to the application of a compound and its derivatives in the preparation of anticancer resistant drugs. Background Technology

[0002] Currently, the incidence and mortality rates of malignant tumors are rising year by year, seriously threatening human health. In clinical treatment, the high invasiveness and multidrug resistance (MDR) of tumor cells have become the main reasons for the failure of traditional chemotherapy, leading to poor patient prognosis and disease recurrence.

[0003] Cancer stem cells (CSCs) are key mediators of drug resistance, recurrence, and metastasis. These cells account for 3%-5% of all tumor cells, possess self-renewal and multi-lineage differentiation capabilities, are often in a quiescent state, have strong DNA repair capabilities, and highly express drug efflux systems, enabling them to evade conventional radiotherapy and chemotherapy, thus becoming "seed cells" for tumor recurrence. Therefore, drugs capable of eliminating CSCs are considered crucial for a complete cure of cancer.

[0004] Therefore, the search for novel anti-tumor drug candidates that are highly effective, low in toxicity, and can overcome drug resistance, as well as drugs that target tumor stem cells, has become a research hotspot in the pharmaceutical field. Summary of the Invention

[0005] This invention relates to three sea cucumber saponins (i.e., lanostane-type triterpenoid lactone glycosides) isolated from sea cucumbers. Experiments have shown that they have cytotoxic activity against tumor cells and significant cytotoxic activity against various drug-resistant malignant tumor cells (cancer cells), laying the foundation for the development of novel anti-drug-resistant tumor drugs.

[0006] On the one hand, this application provides the use of lanostane-type triterpenoid lactone glycosides and their derivatives in the preparation of therapeutic and / or preventive cancer drugs, wherein the lanostane-type triterpenoid lactone glycosides and their derivatives include a sea cucumber-type aglycone core having a 3,12,17-trihydroxy-22,25-epoxylanostane-9(11)-ene-18-acid-18,20-lactone structure. The sea cucumber alkyl aglycone core contains a double bond at position C-9(11) and hydroxyl groups at positions C-12 and C-17. The structure of the sea cucumber alkyl aglycone core is shown in the following chemical structural formula I: ...Formula I; Where R is a sugar chain containing 2-4 monosaccharide units.

[0007] Preferably, R is one or more of β-D-pyrano-xylose and its sulfate ester or sodium sulfate salt, β-D-pyrano-quinoose, β-D-pyrano-glucose, 3-O-methyl-β-D-pyrano-xylose, and 3-O-methyl-βD-pyrano-glucose.

[0008] More preferably, the β-D-pyran-xylose and its sulfate ester or sodium sulfate salt form have the following chemical structural formula II: ...Formula II, where R can be one of H, SO3Na, or SO3H; More preferably, the β-D-pyranoquinoose structure is shown in the following chemical structural formula III: ...Formula III; More preferably, the β-D-pyranose-glucose structure is shown in the following chemical structural formula IV: ...Formula IV; More preferably, the 3-O-methyl-β-D-pyran-xylose structure is shown in the following chemical structural formula V: ...Form V; More preferably, the 3-O-methyl-βD-pyran-glucose structure is shown in the following chemical structural formula VI: ...Form VI.

[0009] Preferably, the length of the R sugar chain is greater than 2.

[0010] More preferably, the length of the R sugar chain is greater than or equal to 4.

[0011] Preferably, the lanostane-type triterpenoid lactone glycoside compound includes one or more of desulfated holothurin A, holothurin B, and desodium holothurin B.

[0012] This application discloses for the first time that desulfated holothurin A, holothurin B, and desodium holothurin B can be used as anticancer agents to treat and / or prevent cancer. Furthermore, through structural studies of lanostane-type triterpenoid lactone glycosides, it was found that the antitumor activity of lanostane-type triterpenoid lactone glycosides is positively correlated with the complexity of the sugar chain at the C-3 position of its aglycone, and the activity of tetrasaccharide chain structures (such as desulfated holothurin A) is significantly better than that of disaccharide chain structures.

[0013] Preferably, the lanostane-type triterpenoid lactone glycoside compound includes desulfated holothurin A.

[0014] On the other hand, this application also provides the application of lanostane-type triterpenoid lactone glycosides and their derivatives in the preparation of drugs to reduce cancer recurrence, wherein the lanostane-type triterpenoid lactone glycosides and their derivatives include a sea cucumber-type aglycone core having a 3,12,17-trihydroxy-22,25-epoxylanostane-9(11)-ene-18-acid-18,20-lactone structure. The sea cucumber alkyl aglycone core contains a double bond at position C-9(11) and hydroxyl groups at positions C-12 and C-17. The structure of the sea cucumber alkyl aglycone core is shown in the following chemical structural formula I: ...Formula I; Where R is a sugar chain containing 2-4 monosaccharide units.

[0015] Preferably, the lanostane-type triterpenoid lactone glycoside compound includes one or more of desulfated holothurin A, holothurin B, and desodium holothurin B.

[0016] Preferably, the reduction in cancer recurrence is achieved by inhibiting the activity of tumor stem cells.

[0017] On the other hand, this application also provides the application of lanostane-type triterpenoid lactone glycosides and their derivatives in the preparation of drugs for inhibiting cancer metastasis, wherein the lanostane-type triterpenoid lactone glycosides and their derivatives include a sea cucumber-type aglycone core having a 3,12,17-trihydroxy-22,25-epoxylanostane-9(11)-ene-18-acid-18,20-lactone structure. The sea cucumber alkyl aglycone core contains a double bond at position C-9(11) and hydroxyl groups at positions C-12 and C-17. The structure of the sea cucumber alkyl aglycone core is shown in the following chemical structural formula I: ...Formula I; Where R is a sugar chain containing 2-4 monosaccharide units.

[0018] Preferably, the lanostane-type triterpenoid lactone glycoside compound includes one or more of desulfated holothurin A, holothurin B, and desodium holothurin B.

[0019] Preferably, the inhibition of cancer metastasis is achieved by inhibiting the activity of tumor stem cells.

[0020] Furthermore, the lanostane-type triterpenoid lactone glycosides and their derivatives include any one or more of the following chemical structural formulas VII-IX: ... (VII); ... (VIII); ... (IX).

[0021] Furthermore, the cancer includes one or more of the following: brain cancer, breast cancer, lung cancer, and liver cancer.

[0022] Furthermore, the lung cancer includes non-small cell lung cancer.

[0023] Furthermore, the brain cancer includes glioblastoma.

[0024] Preferably, the cancer is a malignant cancer.

[0025] On the other hand, this application also provides the use of lanostane-type triterpenoid lactone glycosides and their derivatives in the preparation of therapeutic and / or preventative anticancer drugs, wherein the lanostane-type triterpenoid lactone glycosides and their derivatives include a lanostane-type aglycone core having a 3,12,17-trihydroxy-22,25-epoxylanostane-9(11)-ene-18-acid-18,20-lactone structure. The sea cucumber alkyl aglycone core contains a double bond at position C-9(11) and hydroxyl groups at positions C-12 and C-17. The structure of the sea cucumber alkyl aglycone core is shown in the following chemical structural formula I: ...Formula I; Where R is a sugar chain containing 2-4 monosaccharide units.

[0026] Furthermore, the treatment and / or prevention of anticancer drug-resistant cancers include prolonging the survival of cancer patients, inhibiting tumor growth, inhibiting tumor metastasis, and / or inhibiting tumor recurrence.

[0027] Furthermore, the anticancer agents include platinum-based drugs, anthracycline topoisomerase inhibitors, and / or alkylating agents.

[0028] Preferably, the platinum-based drug includes cisplatin.

[0029] Preferably, the alkylating agent includes temozolomide.

[0030] Preferably, the anthracycline topoisomerase inhibitor includes doxorubicin.

[0031] Furthermore, the cancer is a drug-resistant (anti-cancer agent resistant) cancer.

[0032] Furthermore, the drug-resistant (anticancer agent resistant) cancers include platinum-resistant cancers, anthracycline topoisomerase inhibitor-resistant cancers, and alkylating agent-resistant cancers.

[0033] Preferably, the drug-resistant cancers include cisplatin-resistant cancers, doxorubicin-resistant cancers, and temozolomide-resistant cancers.

[0034] More preferably, the drug-resistant (anticancer agent resistant) cancers include cisplatin-resistant breast cancer, doxorubicin-resistant liver cancer, temozolomide-resistant glioblastoma (brain cancer) and / or cisplatin-resistant non-small cell lung cancer.

[0035] Furthermore, treating and / or preventing cancer includes killing tumor cells and / or inhibiting the activity of tumor stem cells.

[0036] Preferably, the tumor cells include one or more of human glioblastoma cells LN229, human liver cancer cells HepG2, human non-small cell lung cancer cells A549, and human triple-negative breast cancer cells MDA-MB-231.

[0037] Preferably, the tumor stem cells are glioblastoma stem cells, specifically one or more of glioblastoma stem cells GSC0617 and glioblastoma stem cells GSC0722.

[0038] In a preferred embodiment, IC50 of desulfated holothurin A against human triple-negative breast cancer cells MDA-MB-231 50 The IC50 value for human glioblastoma cells LN229 was 0.6438 μM. 50 The IC50 value for human non-small cell lung cancer cells A549 was 0.2261 μM. 50 The IC50 value for glioblastoma stem cell line GSC0722 was 1.607 μM. 50 The IC50 value for glioblastoma stem cell GSC0617 was 0.9877 μM. 50 The value is 1.080 μM.

[0039] Holothurin B's IC50 effect on human triple-negative breast cancer cells MDA-MB-231 50 The IC50 concentration was 3.124 μM, and the IC50 against human glioblastoma cells LN229 was [value missing]. 50 The IC50 concentration was 1.567 μM, and the IC50 value against human non-small cell lung cancer cells A549 was [missing value]. 50 The IC50 value for glioblastoma stem cell GSC0722 was 4.504 μM. 50 The IC50 value for glioblastoma stem cell GSC0617 was 3.055 μM. 50 It is 2.614 μM.

[0040] Desodium-desodium holothurin B's IC50 on human triple-negative breast cancer cells MDA-MB-231 50 The IC50 concentration was 2.539 μM, and the IC50 against human glioblastoma cells LN229 was [missing value]. 50 The IC50 concentration was 7.292 μM, and the IC50 against human non-small cell lung cancer cells A549 was [missing value]. 50The IC50 value for glioblastoma stem cell GSC0722 was 4.992 μM. 50 The IC50 value for glioblastoma stem cell GSC0617 was 3.100 μM. 50 The value is 1.563 μM.

[0041] Preferably, the lanostane-type triterpenoid lactone glycoside compound treats and / or prevents cancer by killing tumor cells and / or inhibiting the activity of tumor stem cells in a dose-dependent manner, with the killing effect increasing with increasing administered concentration.

[0042] Preferably, the lanostane-type triterpenoid lactone glycosides are used to treat and / or prevent cancer by killing tumor cells and / or inhibiting the activity of tumor stem cells through inducing cell membrane damage.

[0043] Preferably, the lanostane-type triterpenoid lactone glycosides are used to treat and / or prevent cancer by killing tumor cells and / or inhibiting the activity of tumor stem cells through inducing apoptosis.

[0044] Furthermore, the dosage form of the drug may be one or more of the following: injection, tablet, capsule, powder, patch, and sustained-release formulation.

[0045] The drug of this application may also contain physiologically acceptable excipients, which may include appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0046] The drug of this application can be prepared by a general method, wherein one or more diluents or carriers can be added to meet the requirements of use and become a specific dosage form.

[0047] Those skilled in the art can adjust the concentration used as needed, and no specific limitation is made here. The concentration used can be lanostane-type triterpenoid lactone glycosides and their derivatives at IC50. 50 0.1-100 times.

[0048] The concentration of lanostane-type triterpenoid lactone glycosides and their derivatives can range from 0.01% to 99%.

[0049] The upper or lower limits of the concentration and range of lanostane-type triterpenoid lactone glycosides and their derivatives may be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 99%, 94%, 95%, 96%, 97%, 98%, 99%, and any value thereof.

[0050] On the other hand, this application provides a method for preparing lanosterane-type triterpenoid lactone glycosides and their derivatives, the method comprising: Step 1: Take sea cucumber, crush it, extract it with ethanol, concentrate it under reduced pressure, extract it with butanone, and recover the butanone extract to obtain crude extract of lanostane-type triterpenoid lactone glycosides. Step 2: The crude extract of lanostane-type triterpenoid lactone glycosides was separated by chromatography, and gradient elution was used to obtain different preliminary separated fractions of lanostane-type triterpenoid lactone glycosides. Step 3: Based on the different polarities of the preliminarily separated components of lanostane-type triterpenoid lactone glycosides, purify them to obtain lanostane-type triterpenoid lactone glycosides and their derivatives.

[0051] Preferably, the sea cucumber includes the jade-foot sea cucumber (Sea Cucumber spp.) Holothuria leucospilota ).

[0052] Preferably, the pulverization method includes wet pulverization.

[0053] Preferably, the ethanol concentration in step one is 70%-90%.

[0054] Preferably, the ethanol extraction includes ultrasonic extraction 1-10 times, each time for 10-30 minutes.

[0055] Preferably, the reduced pressure concentration conditions include a pressure of 100-130 bar and a temperature of 35°C-40°C.

[0056] Preferably, the chromatographic separation in step two includes gradient elution using silica gel column chromatography (100-200 mesh) with a dichloromethane:methanol system, grouping the lanostane-type triterpenoid lactone glycosides according to their polarity.

[0057] Preferably, the purification in step three includes: component separation by gel chromatography, silica gel column chromatography and / or medium-pressure reversed-phase chromatography.

[0058] More preferably, silica gel column chromatography is used for separation, with dichloromethane:methanol gradient elution.

[0059] More preferably, the medium-pressure reversed-phase chromatographic packing material includes C18, eluted with a methanol:water gradient.

[0060] More preferably, the Sephadex LH-20 gel chromatography column is eluted with methanol.

[0061] Preferably, step three includes purifying lanostane-type triterpenoid lactone glycosides and their derivatives by combining components with similar polarity.

[0062] In a preferred embodiment, a method for preparing a lanostane-type triterpenoid lactone glycoside compound and its derivatives includes: Step 1: Take the jade-foot sea cucumber ( Holothuria leucospilota Wet pulverization, 70-90% ethanol extraction, ultrasonic extraction 1-10 times, 10-30 min each time, pressure 100-130 bar, temperature 35°C-40°C, reduced pressure concentration, butanone extraction, recovery of butanone extract, to obtain crude extract of lanosterane type triterpenoid lactone glycoside compound. Step 2: The crude extract of lanostane-type triterpenoid lactone glycosides was separated by chromatography, and gradient elution was used to obtain different preliminary separated fractions of lanostane-type triterpenoid lactone glycosides. Step 3: Based on the different polarities of the preliminarily separated components of lanostane-type triterpenoid lactone glycosides, the components are separated and purified by gel chromatography, silica gel column chromatography and / or medium-pressure reversed-phase chromatography to obtain lanostane-type triterpenoid lactone glycosides and their derivatives.

[0063] The present invention has the following beneficial effects: This application discloses for the first time three lanostane-type triterpenoid lactone glycoside compounds, including desulfated holothurin A, holothurin B, and desodium holothurin B, which exhibit cytotoxic activity against various tumor cells (MDA-MB-231, LN229, A549) and tumor stem cells (GSC0722, GSC0617). These compounds can serve as novel anticancer agents, providing a new functional drug for cancer treatment. They can also be applied to cancer treatment targeting tumor stem cells, effectively preventing tumor recurrence and metastasis.

[0064] This application also discovered that desulfated holothurin A has good killing effect on drug-resistant tumor cells. Even after tumor cells develop resistance to chemotherapeutic drugs (DDP, ADR, TMZ), they still retain sensitivity to desulfated holothurin A.50 The fact that the value remained almost unchanged or increased significantly less than the degree of resistance to chemotherapy drugs indicates that desulfated holothurin A can bypass the drug resistance mechanism and effectively kill drug-resistant tumor cells. It can be used as a potential candidate drug for the treatment of drug-resistant tumors and is expected to solve the clinical dilemma of no drugs available for patients with drug-resistant tumors.

[0065] This application also analyzes the antitumor activity and structure of lanostane-type triterpenoid lactone glycosides. The results show that the antitumor activity of the lanostane-type aglycone core with the structure 3,12,17-trihydroxy-22,25-epoxylanostane-9(11)-en-18-acid-18,20-lactone is positively correlated with the complexity of the sugar chain at the C-3 position of the aglycone. The tetrasaccharide structure (such as desulfated holothurin A) exhibits significantly better activity than the disaccharide structure. These conclusions provide important molecular modification directions for developing highly active lanostane-type triterpenoid lactone glycoside antitumor drugs. Attached Figure Description

[0066] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 Chemical structural formulas of five sea cucumber-derived compounds; Figure 2 Statistical graph of LN229 cell survival rate relative to DMSO after treatment with 10 μM sea cucumber-derived compound for 72 h; Figure 3 Structural formulas of three lanostane-type triterpenoid lactone glycosides and their corresponding cytotoxic activities are shown in the following graph. Figure 4 Figure showing the results of verifying the killing effect of desulfated holothurin A on LN229 / HepG2 cells using the trypan blue exclusion assay; Figure 5 Figure showing the results of cell necrosis or damage caused by desulfated holothurin A, based on lactate dehydrogenase release rate; Figure 6 A graph showing the results of desulfated holothurin A-induced programmed cell death in flow cytometry apoptosis phenotype analysis. Figure 7 Statistical graph of the cytotoxic activity of desulfated holothurin A, holothurin B and desodium holothurin B against MDA-MB-231 cell line and MDA-MB-231-DDP resistant cell line; Figure 8 Statistical graph of the cytotoxic activities of desulfated holothurin A, holothurin B and desodium holothurin B on LN229 cell line and LN229-TMZ resistant cell line; Figure 9Statistical graph of the cytotoxic activities of desulfated holothurin A, holothurin B and desodium holothurin B against A549 cell line and A549-DDP resistant cell line; Figure 10 Statistical graph of the cytotoxic activities of desulfated holothurin A, holothurin B and desodium holothurin B on HepG2 cell line and HepG2-ADR resistant cell line. Detailed Implementation

[0067] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0068] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0069] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0070] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0071] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0072] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all adopt conventional techniques in the fields of pharmacy, medicine, biochemistry, analytical chemistry, cell culture, and related fields.

[0073] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0074] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0075] The cell lines involved in the following examples: Human glioblastoma cells LN229 were purchased from the American Type Culture Collection (ATCC). Human hepatocellular carcinoma cells HepG2, human non-small cell lung cancer cells A549, and human triple-negative breast cancer cells MDA-MB-231 were purchased from the China Center for Type Culture Collection (CCTCC). Human glioblastoma stem cells GSC0617 and GSC0722 were isolated from glioblastoma tissue of patients, as detailed in the paper: Li K, Zhu H, Sun C, et al. Metabolic Blockade-Based Genome Mining of Saccharopolyspora erythraea SCSIO 07745: Discovery and Biosynthetic Pathway of Aminoquinolinone Alkaloids Bearing 6 / 6 / 5 Tricyclic and 6 / 6 / 6 / 5 Tetracyclic Scaffolds.[J] (Genomic analysis of erythromycin-based streptomyces SCSIO 07745 based on metabolic blockade: discovery and biosynthetic pathway of aminoquinolinone alkaloids with 6 / 6 / 5 tricyclic and 6 / 6 / 6 / 5 tetracyclic structures). Organic Letters, 2024, DOI:10.1021 / ACS.ORGLETT.4C04491. Example 1: Isolation of natural products derived from sea cucumber 1.1 Raw material processing and extraction Take the jade-foot sea cucumber ( Holothuria leucospilota10 kg (wet weight, purchased from Qionghai City, Hainan Province), with internal organs intact, wet-processed by grinding. The sample was extracted with 90% ethanol at room temperature, followed by ultrasonic extraction five times, 20 min each time. The extracts were combined and concentrated under reduced pressure using a rotary evaporator at 120 bar and 37°C to obtain a total extract. The total extract was suspended in water and extracted with methyl ethyl ketone (MEK). After solvent recovery, the MEK extract (i.e., crude extract of sea cucumber saponins) was obtained.

[0076] 1.2 Preliminary Separation The total extract was separated by silica gel column chromatography (100-200 mesh) using a gradient elution system of dichloromethane:methanol (volume ratios 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 80:20, 70:30, 50:50, 0:100), with 2 μL eluted per gradient. Combined with thin-layer chromatography (TLC) and HPLC-DAD fingerprint analysis, adjacent substances were combined according to their polarity distribution from low to high to obtain 10 fractions: HS.A1-HS.A10. The HPLC elution conditions are shown in Table 1.

[0077] 1.3 Subdivision and purification of compounds 1-5 Based on the polarity and compositional distribution of each component, further purification yielded compounds 1-5: Isolation of compound 4: Fractions HS.A5-HS.A6 were combined and separated by silica gel column chromatography, eluting with a dichloromethane:methanol gradient (94:6, 92:8, 90:10, 85:15, 50:50, 0:100 v / v) to obtain six subfractions (HS.J1-HS.J6). Fraction HS.J2 was subjected to medium-pressure reversed-phase chromatography (C18 packing material), eluting with a methanol:water gradient (5%→100%) to obtain compound 4 (28.4 mg, retention time 3.7 min).

[0078] Separation of compounds 2 and 3: Fraction HS.A8 was subjected to medium-pressure reversed-phase chromatography (C18 packing material) with a methanol:water gradient elution (5%→100%) to obtain 17 subfractions (HS.Y1-HS.Y17). Fraction HS.Y11 was purified by Sephadex LH-20 gel chromatography (methanol elution) to obtain 7 subfractions (HS.S1-HS.S7). HS.S4 and HS.S5 were combined and purified by silica gel column chromatography with isocratic elution (dichloromethane:methanol = 50:1) to obtain compound 2 (130 mg, retention time 10.9 min) and compound 3 (3.9 mg, retention time 11.9 min).

[0079] Separation of compounds 1 and 5: The combined fractions HS.A9-HS.A10 were separated and purified by medium-pressure reversed-phase chromatography (C18 packing material) with a methanol:water gradient elution (5%→100%) to obtain compound 1 (149 mg, retention time 9.1 min) and compound 5 (6.9 mg, retention time 17.9 min).

[0080] All the compounds obtained above were isolated by... 1 H-NMR, 13 C-NMR and mass spectrometry (MS) data analysis, and comparison with literature data, identified its structure as desulfated holothurin A, holothurin B, desodium holothurin B, 5-methyl-2'-deoxycytidine, and methyl behenate. The structural formula and information are as follows: Figure 1 As shown.

[0081] Table 1: Elution conditions for analytical HPLC

[0082] Example 2: Initial screening of cytotoxic activity using the CCK-8 assay The sea cucumber obtained in Example 1 (from the jade foot sea cucumber) Holothuria leucospilota Preliminary cytotoxic activity screening was conducted on natural small molecule compounds 1-5, and the inhibitory effects of the compounds on the viability of LN229 tumor cells were detected.

[0083] The CCK8 method generates a water-soluble formazan product through the action of WST-8 on live cell dehydrogenases. The amount of product is directly proportional to the number of live cells, thus it can be used for cell proliferation and toxicity detection.

[0084] (1) Cell plating: LN229 cells were cultured using conventional methods, specifically using DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at a temperature of 37°C (simulating in vivo temperature), a humidity of 95% (with a water tray inside the incubator), and a gas condition of 5% CO2. When the cells reached 80%-90% confluence, the medium in the culture dish was discarded, 2 mL of PBS was added for washing, 1 mL of 0.25% trypsin was added for digestion, digestion was terminated with 1 mL of complete medium containing FBS and the cells were blown back to mix. 20 μL of cell suspension was mixed with 20 μL of 0.4% trypan blue, and 10 μL was added to a cell counting plate. The cells were counted using a cell counter. LN229 tumor cells were diluted to 3000 cells / well and seeded at 100 μL per well in a 96-well plate. The cells were gently mixed and then returned to the incubator for further culture.

[0085] (2) Drug administration: 12-16 h after plating. The 10 mM natural small molecule compound stock solution was diluted to 100 μM with complete culture medium. 10 μL of the solution was added to 100 μL of culture medium to make the final concentration 10 μM. 0.1% DMSO was used as a negative control group.

[0086] (3) After culturing for 72 h, add 10 μL of CCK8 working solution to each well and continue incubation for 3-4 h.

[0087] (4) Measure the absorbance at 450 nm and 600 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0088] (5) Calculate the relative cell viability, and the results are as follows: Figure 2 As shown.

[0089] The formula is as follows: Relative cell viability (%) =

[0090] Figure 2 The results showed that at a concentration of 10 μM, desulfated holothurin A, holothurin B, and desodium holothurin B had an inhibitory activity of up to 90% on human glioblastoma cells LN229, indicating that lanosterane-type triterpenoid lactone glycosides had significant inhibitory activity on human glioblastoma cells.

[0091] Example 3: Cytotoxic activity and structure-activity relationship of three lanostane-type triterpenoid lactone glycosides At a concentration of 10 μM, if the compound inhibits tumor cells by more than 50%, and the cell survival rate in the drug-treated wells shows a statistically significant difference compared to the negative control DMSO group, then it is preliminarily determined to be active. Therefore, based on Example 2, this example further measures the IC50. 50 value.

[0092] The three lanostane-type triterpenoid lactone glycosides screened in Example 2—desulfated holothurin A, holothurin B, and desodium holothurin B—were detected using a CCK8 (Cell Counting Kit-8) assay for IC50 in five cell types, including tumor cells (MDA-MB-231, LN229, A549) and tumor stem cells (GSC0722, GSC0617). 50 .

[0093] The CCK8 method generates a water-soluble formazan product through the action of WST-8 on live cell dehydrogenases. The amount of product is directly proportional to the number of live cells, thus it can be used for cell proliferation and toxicity detection.

[0094] (1) Cell plating: MDA-MB-231, LN229, and A549 cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin, and 1% non-essential amino acids), at a temperature of 37°C (simulating in vivo temperature), a humidity of 95% (with a water tray placed inside the incubator), and a gas condition of 5% CO2. When the cells reached 80%-90% confluence, the medium in the culture dish was discarded, 2 mL of PBS was added for washing, 1 mL of 0.25% trypsin was added for digestion, and digestion was terminated with 1 mL of complete medium containing FBS and the cells were resuspended. GSC0722 and GSC0617 cells were cultured in DMEM / F12 medium (serum-free, penicillin-free, and non-essential amino acid-free), with the other culture conditions as above. When the cells reached 80%-90% confluence, the cell suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant cell culture medium was discarded, and the cells were resuspended with 1 mL of medium. Take 20 μL of cell suspension from each of the above 5 cell types, mix them thoroughly with 20 μL of 0.4% trypan blue, and then add 10 μL of the mixture to a cell counting plate. Use a cell counter to count the cells. Dilute MDA-MB-231, LN229, and A549 tumor cells to 3000 cells / well, and GSC0722 and GSC0617 tumor stem cells to 5000 cells / well. Seed 100 μL of each cell into a 96-well plate, mix gently, and return the plate to the incubator for further culture.

[0095] (2) Drug addition: 12-16 h after plating. Dilute 100 mM natural small molecule compound stock solution to 1 mM with complete culture medium. Then, use this as the highest concentration and perform 3-fold dilutions in sequence for a total of 8 concentration gradients. After thorough mixing, add 10 μL to each well. Use an equal amount of complete culture medium as a blank control group and 0.1% DMSO as a negative control group.

[0096] (3) After culturing for 72 h, add 10 μL of CCK8 working solution to each well and continue incubation for 3-4 h.

[0097] (4) Measure the absorbance at 450 nm and 600 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0098] (5) Calculate the relative cell viability and the half-maximal inhibitory concentration (IC50) of the compound. 50 Value, result as Figure 3 As shown.

[0099] The formula is as follows: Relative cell viability (%) =

[0100] Computing IC 50Using GraphPad Prism software, select the chart type as XY, set cell viability as Y and concentration as X, click Analyze-Transform-Transform concentrations to convert the X-axis concentrations to Log10, select the converted data table, click Analyze, select Nonliner regression (curve fit) for nonlinear fitting, under Dose-response-Inhibition, select log (inhibitor) vs. normalized response-variable slope, and obtain the IC50 in Nonliner regression of Transform X of Data. 50 All data are expressed as Mean ± SD (n ≥ 3). The Unpaired Student's t-test was used to compare data between two groups, and the one-way ANOVA was used to compare data from multiple groups. p<0.05 p<0.01 p<0.001 p<0.0001.

[0101] Figure 3 The results showed that three lanostane-type triterpenoid lactone glycosides exhibited cytotoxic activity against various tumor cells and tumor stem cells. Furthermore, structure-activity relationship analysis revealed that a lanostane-type triterpenoid saponin compound, represented by the general formula [X], where X = 3,12,17-trihydroxy-22,25-epoxylanostane-9(11)-ene-18-acid-18,20-lactone, is characterized by a double bond at position C-9(11) of the parent nucleus and hydroxyl groups at positions C-12 and C-17. Their common core is a 9(11)-dehydrolanostane-type parent nucleus, which is the structural basis for the strong cytotoxic activity of this type of compound. The antitumor activity of these lanosterane-type triterpenoid lactone glycosides is positively correlated with the complexity of the sugar chain at the C-3 position of their aglycone. Tetrasaccharide structures (such as desulfated holothurin A) exhibit significantly better activity than disaccharide structures. Furthermore, the sodium salt form of the terminal sulfate group provides superior efficacy compared to the acidic form. These findings offer important molecular modification directions for developing highly active lanosterane-type triterpenoid lactone glycoside antitumor drugs.

[0102] Example 4 Evaluation of desulfated holothurin A cell activity and cytotoxicity To further confirm the efficacy advantages of desulfated holothurin A, this embodiment uses human glioma cells LN229 and human liver cancer cells HepG2 as models to construct a multi-dimensional cytotoxicity evaluation system.

[0103] The trypan blue exclusion assay can be used to evaluate the immediate killing effect of compounds on tumor cells and the cell proliferation status, and to distinguish between live and dead cells by the principle of dye exclusion.

[0104] (1) Plating: Take 20 μL of LN229 cells or HepG2 cells that were dispersed by pipetting during passage and mix them with 20 μL of 0.4% trypan blue. Take 10 μL and add it to a cell counting plate. Use a cell counter to count the cells. Dilute to 20 w cells / well and seed 2 mL per well into a 6-well plate. Put the plate back into the incubator for further culture.

[0105] (2) Pretreatment: After 12-16 h of plating, prepare complete culture medium containing different concentrations of desulfated holothurin A to change the medium for cells. 2 mL of new culture medium is used for each well. At the same time, cells treated with 0.1% DMSO are set up as a negative control for 12 h.

[0106] (3) Staining treatment: After the desulfated holothurin A treatment, discard the culture medium in each well, wash with 1 mL PBS, and add 1 mL trypsin for digestion. After digestion, blow off the cells with 1 mL of complete culture medium, and mix 20 μL of cell suspension with 20 μL of 0.4% trypan blue solution at a volume ratio of 1:1.

[0107] (4) Microscopic counting: Take 10 μL of the stained cell suspension and add it to the sample of the automated cell counter. Observe the cell counter. Cells whose cytoplasm is stained blue are identified as dead cells, and cells whose cytoplasm is clear, transparent and unstained are identified as live cells.

[0108] (5) Result Calculation: The total number of live and dead cells in each group was counted, and the cell survival rate was calculated: Cell survival rate (%) = (Total number of live cells / Total number of cells) × 100%. This method was used for preliminary screening of the effective concentration range of lanostane-type triterpenoid lactone glycosides, and the results are as follows: Figure 4 As shown.

[0109] Experimental results are as follows Figure 4 As shown, the number of trypan blue positive cells with desulfated holothurin A increased with increasing drug concentration, indicating that it has a dose-dependent killing effect on tumor cells.

[0110] The lactate dehydrogenase (LDH) release assay quantitatively assesses the degree of drug damage to the cell membrane and the intensity of cytotoxicity by detecting the activity of cytoplasmic lactate dehydrogenase (LDH) released into the culture supernatant due to cell membrane damage or rupture.

[0111] (1) Plating: Take 20 μL of LN229 and HepG2 cells dispersed by pipetting during passage and mix them with 20 μL of 0.4% trypan blue. Take 10 μL and add it to a cell counting plate. Use a cell counter to count the cells. Dilute the cells with complete culture medium to 3000 / well and add 100 μL to each well of a 96-well plate. Put the plate back into the incubator for further culture.

[0112] (2) Pretreatment: After 12-16 h of plate preparation, use 1×IC solution respectively. 50 Cells were treated with desulfated holothurin A and 10 μM doxorubicin (a positive control). A negative control was prepared using complete culture medium containing 0.1% DMSO, and an equal volume of complete culture medium served as a blank control. After drug administration, the cells were returned to the incubator for further culture. Cell suspensions were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after drug administration, with three replicates for each treatment. One hour before collecting the supernatant for analysis, 10 μL of LDH release reagent was added to each of the three wells in each group to determine the maximum release rate.

[0113] (3) Preparation and Result Detection of LDH Release Detection Working Solution: Prepare the LDH release detection working solution: According to the experimental dosage, take an appropriate amount of 10×INT solution and dilute it to 1× with INT diluent. Mix the 1×INT solution, lactic acid solution, and enzyme solution in a 1:1:1 ratio. Add 100 μL of the pre-collected cell suspension to a centrifuge tube, centrifuge at 4000 rpm for 5 min, and transfer 80 μL of the supernatant to a new 96-well plate. Add 40 μL of LDH release detection working solution to each well and incubate at room temperature in the dark for 30 min. Use a microplate reader to detect the absorbance of each well at 490 nm. Simultaneously, use 600 nm as a reference wavelength for dual-wavelength measurement.

[0114] (4) Data processing: Based on the measured OD values, the relative LDH release amount of each group was calculated according to the following formula: Relative LDH release amount (%) = [(OD value)] 490 -OD 600 ) Processing Group - (OD) 490 -OD 600 Empty culture medium] / [(OD 490 -OD 600 Maximum release group - (OD) 490 -OD 600 [Empty culture medium] × 100%.

[0115] Experimental results are as follows Figure 5 As shown: Lactate dehydrogenase (LDH) release assay confirmed that desulfated holothurin A induced higher levels of cell membrane damage.

[0116] Flow cytometry detection of apoptosis utilizes Annexin V to specifically recognize phosphatidylserine (PS) residues that indicate early apoptosis, combined with the ability of PI to cross the cell membrane and enter intracellular labeled DNA, enabling precise segmentation of live, apoptotic, and necrotic cells. Annexin is used to identify early apoptotic cells, while PI can enter necrotic or late-stage apoptotic cells.

[0117] (1) Cell plating: In this study, 10-week human glioma cells (LN229) were plated into 6 cm cell culture dishes and cultured for 12 h. There were a total of 7 groups, namely the double-stained negative control group (DMSO), the drug treatment group (1 μM desulfurized holothurin A or 2 μM desulfurized holothurin A), the single-stained Annexin V group, the single-stained PI group, the blank control group, and the double-stained positive group.

[0118] (2) Negative double staining control group and drug treatment group: Add the same volume of DMSO, 1 μM or 2 μM desulfated holothurin A as the maximum drug treatment group for 4 h.

[0119] (3) Cell collection: Collect the culture medium and rinse the cells with 1 mL PBS, add 1 mL trypsin to digest, and finally blow the cells off with complete culture medium. Collect all of the above into a 15 mL centrifuge tube and centrifuge at 3000 rpm for 5 min. Remove the supernatant, resuspend the cells with 1 mL PBS, centrifuge again, and resuspend with 200 μL Binding Buffer.

[0120] (4) Treatment of apoptosis-positive cells: The cells in the positive control group were treated by heating in a 65℃ water bath for 5 min to induce apoptosis. The cells in the single-stained PI and single-stained Annexin V groups consisted of 50% untreated live cells and 50% apoptosis-positive cells.

[0121] (5) Cell staining: Double staining was performed on negative control, positive control, and drug-treated cells using 2 μL of fluorescently labeled Annexin V-FITC (BD556420) and 1 μL of 2 mg / mL PI. Annexin V-FITC-labeled cells were stained on the Annexin V-FITC-labeled cells. PI-labeled cells were stained on the PI-labeled cells. Incubation was carried out at room temperature in the dark for 15 min.

[0122] (6) Flow cytometry detection: The stained cell suspension was injected into a flow cytometer, 10,000 cells were collected and the experiment was recorded. The fluorescent dye was excited by laser and the fluorescence signal was detected. Based on the intensity and distribution of the fluorescence signal, live cells, early apoptotic cells, late apoptotic cells and necrotic cells could be distinguished.

[0123] (7) Data Analysis: The detection results were analyzed and processed using professional flow cytometry data analysis software to obtain information such as the proportion and distribution of apoptosis and necrosis. The results are as follows: Figure 6 As shown.

[0124] Experimental results are as follows Figure 6 As shown: Flow cytometry (Annexin V / PI) analysis further confirmed at the molecular level the significant superiority of desulfated holothurin A in inducing apoptosis.

[0125] This study employed a multidimensional cell biology evaluation system, integrating trypan blue rejection assay, lactate dehydrogenase release analysis, and flow cytometry to detect apoptosis. This consistently demonstrated the excellent antitumor activity of desulfated holothurin A at the levels of human glioma cells LN229 and HepG2 cells.

[0126] Example 5: Effects of three lanostane-type triterpenoid lactone glycosides on the IC50 of drug-resistant malignant tumor cells. 50 Using IC 50 Drug-resistant tumor cell lines were constructed using a dose-direct induction method, with this IC50 as the basis for their development. 50 The dosage, used as the inducing concentration, applies continuous and stable drug pressure to parental sensitive tumor cells, eliminating most of the sensitive tumor cells and screening out surviving cells that can tolerate this drug concentration. These surviving cells gradually develop an acquired drug resistance phenotype by rapidly activating drug resistance-related mechanisms (such as upregulation of drug efflux pumps, enhanced DNA damage repair capacity, inhibition of apoptosis pathways, and enrichment of tumor stem cells). After passage culture and drug-free acclimatization, drug-resistant tumor cell lines with stable drug resistance phenotypes that can be stably passaged are obtained for subsequent drug resistance studies.

[0127] (1) Preliminary experiments to determine IC 50 Dosage: Parental tumor cells in logarithmic growth phase were used, and the half-maximal inhibitory concentration (IC50) of the target chemotherapy drugs (DDP, ADR, TMZ) on parental tumor cells was determined using the CCK8 assay. 50Similar to Example 3, the drug concentrations used for subsequent induction were designed to effectively inhibit the proliferation of 50% of the parental tumor cells while retaining a small number of viable drug-resistant potential cells. The subsequent induction drug concentrations were: MDA-MB-231: DDP (50 μM), LN229: TMZ (200 μM), HepG2: ADR (0.5 μM), and A549: DDP (50 μM).

[0128] (2) IC 50 Dosage-induced culture: Parental tumor cells are routinely seeded into culture dishes. When the cells adhere and grow to a confluence of 70%-80%, a dose-dependent chemotherapy drug (IC50) is added. 50 The cells were continuously treated with a complete culture medium (dose) containing 5% CO2 at 37°C. After 24 hours, the medium was replaced with a complete culture medium without the drug. Cell morphology and survival were closely observed during this period, and the medium was changed every 2-3 days until most of the sensitive cells underwent apoptosis and the remaining few surviving cells showed a proliferative trend. These were recorded as the first generation of drug-resistant cells.

[0129] (3) Stable culture of drug-resistant cells: When the first-generation drug-resistant cells grow to a confluence of 70%-80%, they are passaged using trypsin digestion. After passage, IC40 is added to the culture medium. 50 Complete culture medium containing the prescribed dose of chemotherapy drugs, 5% CO2, 37℃, for 24 hours, then replaced with complete culture medium without the drugs. After stabilization, it is recorded as the second generation. This method is repeated for drug induction and passage culture for 3-5 generations to initially obtain a drug-resistant cell population.

[0130] (4) Drug-free stabilization: Remove the chemotherapy drugs from the culture medium and use drug-free complete culture medium for routine passage culture to eliminate the acute drug stress response of cells, ensure the stable inheritance of drug resistance phenotype, and avoid the decline of drug resistance.

[0131] (5) Identification of drug resistance models: The IC50 of parental tumor cells and constructed drug-resistant cells to the target chemotherapeutic drug was detected by the CCK8 assay. 50 Same as in Example 3, calculate the resistance index (RI), where RI = IC50 of drug-resistant cells. 50 / parental cell IC 50 When the RI ≥ 1.5, the drug-resistant tumor cell line is considered to have been successfully constructed.

[0132] (6) Plating: Four plates of normal tumor cells (LN229, MDA-MB-231, A549 and HepG2) and four plates of their corresponding drug-resistant tumor cells (LN229-TMZ, MDA-MB-231-DDP, A549-DDP and HepG2-ADR) were plated for each compound. The tumor cell lines and their corresponding drug-resistant tumor cell lines were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 1% penicillin antibiotics, and 1% non-essential amino acids) at a temperature of 37°C, a humidity of 95%, and a gaseous condition of 5% CO2. When the cells reached 80%-90% confluence, the medium was discarded, 1 mL of PBS was added for rinsing, and then 1 mL of trypsin was added. After the cells were fully digested, the trypsin was discarded, 1 mL of complete medium was added to stop the digestion, and the cells were blown off and mixed evenly. Mix 20 μL of cell suspension with 20 μL of 0.4% trypan blue solution. Transfer 10 μL of the mixture to a cell counting plate and count the cells using a cell counter. Dilute the tumor cell line and its drug-resistant cell line to 3000 cells / well in a 96-well plate.

[0133] (7) Drug administration: 12-16 h after plating. Dilute 10 μM stock solutions of desulfated holothurin A, holothurin B, or desodium holothurin B 10 μM with complete culture medium 100-fold. Then, use this as the highest concentration and perform 3-fold dilutions sequentially, for a total of 8 concentration gradients. After thorough mixing, add 10 μL to each well. Positive control stock solutions had the following concentrations: MDA-MB-231: DDP (50 μM), LN229: TMZ (200 μM), HepG2: ADR (0.5 μM), and A549: DDP (50 μM). These were first diluted 100-fold with complete culture medium, then used as the highest concentration and performed 3-fold dilutions sequentially, for a total of 8 concentration gradients. After thorough mixing, add 10 μL to each well. A negative control was provided with complete culture medium containing 0.1% DMSO, and a blank control was provided with an equal volume of complete culture medium.

[0134] (8) After culturing for 72 h, add 10 μL of CCK8 working solution to each well and continue incubation for 3-4 h.

[0135] (9) Measure absorbance at 450 nm and 600 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0136] (10) Calculate the relative cell viability and the half-maximal inhibitory concentration (IC50) of the compound. 50 Value, result as Figure 7-10 As shown.

[0137] The formula is as follows: Relative cell viability (%) =

[0138] Computing IC 50Using GraphPad Prism software, select the chart type as XY, set cell viability as Y and concentration as X, click Analyze-Transform-Transform concentrations to convert the X-axis concentrations to Log10, select the converted data table, click Analyze, select Nonliner regression (curve fit) for nonlinear fitting, under Dose-response-Inhibition, select log (inhibitor) vs. normalized response-variable slope, and obtain the IC50 in Nonliner regression of Transform X of Data. 50 All data are expressed as Mean ± SD (n ≥ 3). The Unpaired Student's t-test was used to compare data between two groups, and the one-way ANOVA was used to compare data from multiple groups. p<0.05 p<0.01 p<0.001 p<0.0001.

[0139] Figure 7-10 This indicates that DDP-resistant MDA-MB-231 cells, TMZ-resistant LN229 cells, DDP-resistant A549 cells, and ADR-resistant HepG2 cells were successfully constructed, with resistance indices (RI) of 5.79, 1.79, 3.60, and 2.97, respectively. The IC50 values ​​of three lanostane-type triterpenoid lactone glycosides against the four drug-resistant cell lines were also measured. 50 The results showed that even after tumor cells developed resistance to chemotherapy drugs, they remained sensitive to three lanostane-type triterpenoid lactone glycosides, with an IC50 value of [missing value]. 50 The fact that the values ​​remained almost unchanged or increased significantly less than the degree of resistance to chemotherapy drugs indicates that the three lanostane-type triterpenoid lactone glycosides can circumvent the drug resistance mechanism and effectively kill drug-resistant tumor cells. They can serve as potential candidate drugs for the treatment of drug-resistant tumors and are expected to solve the clinical dilemma of having no drugs available for patients with drug-resistant tumors.

[0140] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. The use of a compound and its derivatives in the preparation of drugs for treating and / or preventing cancer, characterized in that, The compound is a lanostane-type triterpenoid lactone glycoside compound, and the lanostane-type triterpenoid lactone glycoside compound and its derivatives include the following chemical structural formula VII: ... (VII), the cancer in question is a drug-resistant cancer.

2. The application according to claim 1, characterized in that, The treatment and / or prevention of cancer includes reducing cancer recurrence.

3. The application according to claim 1, characterized in that, The treatment and / or prevention of cancer includes inhibiting cancer metastasis.

4. The application according to any one of claims 1-3, characterized in that, The cancers mentioned include one or more of the following: brain cancer, breast cancer, lung cancer, and liver cancer.

5. The application according to claim 4, characterized in that, The brain cancer includes glioblastoma; and / or, the lung cancer includes non-small cell lung cancer.

6. The application according to claim 1, characterized in that, The drug-resistant cancers include platinum-resistant cancers, anthracycline topoisomerase inhibitor-resistant cancers, and / or alkylating agent-resistant cancers.

7. The application according to claim 6, characterized in that, The drug-resistant cancers include cisplatin-resistant cancers, doxorubicin-resistant cancers, and / or temozolomide-resistant cancers.

8. The application according to any one of claims 1-3, characterized in that, Treatment and / or prevention of cancer include killing tumor cells and / or inhibiting the activity of tumor stem cells.

9. A method for preparing a compound and its derivatives, characterized in that, The method includes: Step 1: Take sea cucumber, crush it, extract it with ethanol, concentrate it under reduced pressure, extract it with butanone, and recover the butanone extract to obtain crude extract of lanostane-type triterpenoid lactone glycosides. Step 2: The crude extract of lanostane-type triterpenoid lactone glycosides was separated by chromatography, and gradient elution was used to obtain different preliminary separated fractions of lanostane-type triterpenoid lactone glycosides. Step 3: According to the different polarities of the preliminary separated components of lanostane-type triterpenoid lactone glycosides, purify to obtain any of the compounds and their derivatives as described in claims 1-8, wherein the compounds are lanostane-type triterpenoid lactone glycosides.

Images