Preparation method and application of sphingosine compound

Abstract

The present application relates to the application of a sphingosine compound in the field of biochemical application technology in the preparation of a tumor resistance reversal agent or a tumor drug sensitizer. The structure of the sphingosine compound is shown in the following formula: #imgabs0# The drug resistance or tumor drug is doxorubicin. Studies have shown that the sphingosine compound PF55 has the activity of reversing MCF-7 / ADR when used in combination with doxorubicin at concentrations of 5, 10, and 20 μg / mL, respectively, and its activity is dose-dependent with the concentration of the sphingosine compound PF55.

Description

Technical Field

[0001] The present invention relates to the field of biochemical application technology, and in particular to a preparation method and application of a sphingosine compound. Background Art

[0002] Cancer has become a serious threat to human health and life. Tumor treatments primarily include chemotherapy, surgery, and radiotherapy, with chemotherapy being a primary approach to cancer treatment. During chemotherapy, the development of drug resistance in tumor cells is a major cause of chemotherapy failure. Therefore, the search for low-level, highly active reversal agents is a fundamental approach to addressing tumor drug resistance and holds significant research value.

[0003] P-glycoprotein (P-gp) is one of the most representative proteins in the ABC transporter family, with a molecular weight of 170 kD and composed of 1280 amino acid residues. Studies have shown that P-gp can transport drugs with diverse chemical and structural properties, including some anticancer drugs such as doxorubicin and taxanes, which can cause multidrug resistance (MDR) and lead to the failure of cancer treatment. Therefore, the study of P-gp inhibitors and substrates is of great significance for cancer treatment. Co-administration of P-gp inhibitors with chemotherapeutic drugs can be an effective strategy to overcome MDR. Currently, several generations of P-gp inhibitors have been developed. First-generation reversal agents include tamoxifen and cyclosporine A, with verapamil and cyclosporine being typical representatives. However, these drugs generally lack P-glycoprotein specificity and can cause serious side effects, which has significantly limited the clinical application of first-generation reversal agents (Sato W. et al. 1991). Second-generation reversal agents include sporine analogs such as valspodar (PSC833) and dexverapamil, with dexamethasone being the most prominent. However, the development of second-generation reversal agents is limited by their high toxicity and a range of side effects resulting from drug interactions (Rowinsky EK et al. 1998; Hyafil F. et al. 1993; Keller RP et al. 1992). Representative third-generation P-glycoprotein inhibitors include tariquidar (XR9576), zosuquidar (LY335979), and S9788, with tariquidar (XR9576) and WK-X-34 being the most prominent (Massey PR et al. 2014). The development of P-gp inhibitors from natural products and their derivatives has become a new direction and focus of research and development for fourth-generation inhibitors.

[0004] Natural products derived from microorganisms have long been a crucial source for innovative drug development, providing a material foundation for new drug discovery. Microorganisms also offer advantages such as short growth cycles, easily regulated metabolism, easy strain selection, and the ability to industrialize production through large-scale fermentation, further solidifying their importance in new drug development. While there are specific reports of P-gp inhibitors being developed from natural products derived from microorganisms, the specific substances have not been identified.

[0005] To this end, the applicant has conducted a lot of research and discovered a series of new long-chain fatty acid glycerol compounds such as those disclosed in CN113773216A, CN114014898A, and CN114057811A in the early stage, and found that they have applications in reversing the activity of drug-resistant tumor cells. The applicant continues to study the bacteria in order to discover more compounds that have applications in reversing the activity of drug-resistant tumor cells. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a preparation method and application of a sphingosine compound of biological origin.

[0007] One of the purposes of the present invention is to provide a sphingosine compound for use in the preparation of a tumor resistance reversal agent or a tumor drug sensitizer. The structure of the sphingosine compound (4E,8E)-ND-2′-hydroxyoctadecanoyl-1-O-β-D-glycopyranosyl-9-methyl-4,8-sphingadienine (hereinafter referred to as the sphingosine compound PF55) is shown in the following formula:

[0008]

[0009] Furthermore, the drug-resistant or tumor drug is doxorubicin.

[0010] Furthermore, the tumor is breast cancer.

[0011] Furthermore, the tumor resistance reversal agent is a transporter pump inhibitor, which has an inhibitory effect on one or more of the drug resistance protein P-glycoprotein and multidrug resistance protein.

[0012] A second object of the present invention is to provide a use of a sphingosine compound and a pharmaceutical carrier in the preparation of an anti-tumor cell agent, wherein the tumor cell is an adriamycin-resistant breast cancer cell; the structure of the sphingosine compound is shown in the following formula:

[0013]

[0014] A third object of the present invention is to provide a method for preparing a sphingosine compound. The structure of the sphingosine compound is shown in the following formula:

[0015] The compound is obtained by fermentation and extraction of Tubeufiarubra, and the Tubeufiarubra is named Tubeufiarubra PF02-2, and the preservation unit is China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2019957.

[0016] The red-brown tube fungus Tubeufia rubra PF02-2 described in the present invention was isolated and obtained by the Biochemical Engineering Center of Guizhou University, and the preservation unit is: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, preservation date: 2019.11.20, and the preservation registration number is CCTCC NO: M 2019957.

[0017] The sources of Tubeufia rubra PF02-2 are as follows:

[0018] Sampling time: May 14, 2016;

[0019] Sampling location: Pingfeng Rainforest Nature Reserve, Fangchenggang City, Guangxi Zhuang Autonomous Region;

[0020] Sampling method: Decaying wood was collected from the Pingfeng Rainforest Nature Reserve in Fangchenggang City, Guangxi Zhuang Autonomous Region, and brought back to the laboratory in sealed plastic bags.

[0021] The Tubeufia rubra PF02-2 strain of the present invention has the following properties:

[0022] Colony morphology: On natural decaying wood, the colonies are flat, forming a network or dot pattern, often forming sheets when present. Freshly isolated pure PF02-2 colonies are colorless, transparent, or white, while isolated pure PF02-2 colonies are reddish-brown after natural drying. Mycelium is partially buried beneath the substrate, but mostly superficial, consisting of branched, septate hyphae, colorless to dark brown. Conidiophores are cylindrical, solitary, curved, and septate, 50-150 μm long and 4.5-6 μm wide, tapering to a smooth surface. Conidiophores are solitary or multiple, cylindrical, with columnar denticles. Sympodial cells grow from the middle to the apex of the conidiophore, 10-19 μm long and 3-4 μm wide, and are colorless, transparent, to light brown. The conidia are solitary, apically lateral, transparent, with a rounded apex. When tightly coiled, they curl 2-3.5 times, are 35-50 microns in diameter, and have conidial threads 3-5 microns thick (average diameter 45 microns, thickness 4.5 microns). They gradually unfurl in water, have multiple, indistinct septa, are colorless to light brown, and have a smooth surface. Conidia begin to germinate and grow after 12 hours in water-agar medium. Colonies grown in PDA medium at 25-28°C for two weeks can reach 16 mm. They are brown, rounded, and have a rough surface with distinct protrusions and vein-like wrinkles. The colony margins are intact.

[0023] Furthermore, the preparation method includes the following steps: subjecting Tubeufia rubra PF02-2 to liquid or solid fermentation to obtain a fermentation product; extracting the fermentation product, and separating and purifying the obtained extract to obtain a sphingosine compound.

[0024] The specific steps include:

[0025] S1. Strain activation: Take out the preserved strain, inoculate it on the basal culture medium plate, culture it statically to the third generation and then amplify it;

[0026] S2, fermentation culture: take the activated bacteria from step S1 and inoculate them into solid culture medium, and ferment them at 26-30°C for a period of time;

[0027] S3, extraction: extract the bacterial cells and culture medium, add ethyl acetate for extraction, and concentrate the extract to obtain the fermentation product;

[0028] S4. Pretreatment of fermentation product: dissolve the fermentation product with a solvent of 1:1 chlorine: formaldehyde, and then mix it evenly with silica gel in a mass ratio of 1:1 to 2. After the solvent evaporates, use it as a column sample, then add it to a separation column with silica gel powder and petroleum ether, and use petroleum ether, chloroform, ethyl acetate and methanol in a gradient elution sequence, respectively. After the elution solvent is recovered under reduced pressure on a rotary evaporator, dissolve it with chloroform, acetone or methanol, and then use a thin layer chromatography plate and a developing agent for development. Select the liquid that fluoresces at 254nm or 365nm under an ultraviolet-visible light analyzer, and then use 8% sulfuric acid ethanol vanillin as a color developer to develop the color; combine the ethyl acetate solvent eluate, recover the ethyl acetate solvent, and obtain an ethyl acetate extract;

[0029] S5. Purification and separation: a. Dissolve the ethyl acetate extract in methanol solvent and mix it with silica gel in a mass ratio of 1:1 to 3. After the solvent evaporates, load the sample onto a pre-column. Use 10% methanol water to equilibrate the reverse phase medium pressure column. Add the sample to the pre-column and elute with methanol water in 10 gradients. After the eluate is recycled by rotary evaporation, dissolve it in methanol and then use a thin layer chromatography plate. Use a developing agent to develop the eluate. Select the liquid that fluoresces at 254 nm or 365 nm under a UV-visible spectrometer. Then combine the components that develop black with 8% ethanolic sulfuric acid and vanillin as a color developer to obtain component Fr.14.

[0030] b. Dissolve component Fr.14 in methanol and mix it evenly with silica gel in a mass ratio of 1:1 to 3. After the solvent evaporates, prepare the sample for column loading. Weigh silica gel powder and mix it evenly with a 1:1 ratio of chloroform to methane. Load the mixture into a separation column and add the sample for column loading once more. Gradient elution using a chloroform system is performed. The eluate is recovered by rotary evaporation and dissolved in methanol. The sample is then subjected to TLC and developed with a developing agent. Observe for fluorescence at 254 nm or 365 nm using a UV-visible light analyzer. Combine the components that develop black color using 8% ethanolic sulfate and vanillin as a color developer to obtain Fr.14-9.

[0031] c. Dissolve Fr.14-9 in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and mix it evenly with silica gel in a mass ratio of 1:1 to 3. After the solvent evaporates, prepare the sample for column loading. Weigh silica gel powder and mix it evenly with a solvent of dichloromethane:methanol = 10:1, load it into a separation column, then add the sample for column loading, and use a solvent gradient elution of dichloromethane:methanol = 10:1. The elution solvents are combined using a TLC spot plate to obtain a sphingosine compound PF55.

[0032] The solid culture medium in step S2 is oat culture medium, which is obtained by mixing 200 g of oats and 150 mL of double-distilled water.

[0033] The compound of the present invention can be isolated from the fermentation product of Tubeufia rubra PF02-2, and its content is relatively high, 6 to 20 times that of other similar compounds in the prior art, making it more conducive to industrial application. At the same time, the study found that the sphingosine compound PF55 has the ability to reverse MCF-7 / ADR activity when used in combination with doxorubicin at concentrations of 5, 10, and 20 μg / mL, respectively. Its IC 50 The values ​​were 30.051±1.916, 26.580±2.374 and 21.613±0.632μg / mL respectively. Its activity was dose-dependent with the concentration of sphingosine compound PF55. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a flow chart for the separation and purification of sphingosine compound PF55;

[0035] Figure 2 is the mass spectrum of the sphingosine compound PF55 of the present invention;

[0036] Figure 3 1H-NMR diagram of the sphingosine compound PF55 of the present invention;

[0037] Figure 4 13C and DEPT diagrams of the sphingosine compound PF55 of the present invention;

[0038] Figure 5 This is the original data for screening the cytotoxic activity of the sphingosine compound PF55 in the present invention;

[0039] Figure 6 The original data for screening the reversal activity of the sphingosine compound PF55 on MCF-7 / ADR in the present invention;

[0040] Figure 7 Schematic diagram of the inhibition rate of sphingosine compound PF55 on tumor cells MCF-7 / ADR;

[0041] Figure 8 Figure 5 shows the IC50 values ​​of doxorubicin at different concentrations combined with the sphingosine compound PF55 at concentrations of 5, 10, and 20 μg / mL against MCF-7 / ADM cells. ns: P > 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. DETAILED DESCRIPTION

[0042] The following is further described in detail through specific implementation methods:

[0043] 1. Strain activation

[0044] Remove the strain stored on a glycerol slant from a -80°C freezer, use a sterile inoculating loop to scoop out one loop of the strain Tubeufiarubra PF02-2, inoculate it onto an 11 cm diameter basal medium plate by cross-streaking, and culture it at 28°C for 17 days. Subculture to the third generation before scaling up the culture.

[0045] 2. Fermentation culture

[0046] Oat solid fermentation (1L Erlenmeyer flask filled with 200g oats and 150mL double distilled water), the inoculum size of each bottle was 1×1cm on the culture plate 2 After 105 days of static culture at 28°C, the cells, along with the oatmeal medium, were extracted three times with ethyl acetate, each time with shaking at 160 rpm for 24 hours. The extracts were combined and concentrated under reduced pressure at 40°C to obtain the fermentation product. This process was repeated to obtain 2027.17 g of the combined fermentation product.

[0047] 3. Fermentation product pretreatment

[0048] Dissolve 2027.17 g of fermentation product with acetone solvent in a ratio of 1:1.5 (i.e., add 3041 g of 200-300 mesh silica gel powder to 2027.17 g of fermentation product) and mix them evenly. After the solvent evaporates, a river sand sample is obtained, which is used as a primary column sample. Weigh 6000 g of 200-300 mesh silica gel powder and mix it evenly with petroleum ether solvent (no bubbles can be generated during this process) and load it into a separation column with a length of 1.5 m and an inner diameter of 200 mm. Let the silica gel powder slowly sink until it stops sinking and add a primary column sample. Use petroleum ether, chloroform, ethyl acetate, and methanol for 4 gradient elutions in sequence, with 2 to 3 samples (approximately 100 samples per column) for each gradient elution. The column volume is eluted (36 L to 54 L elution solvent) by column volume, and each 1000 mL elution solvent is collected as a portion. After each elution sample is recovered under reduced pressure on a rotary evaporator, it is dissolved in 10 or 15 mL of chloroform, acetone or methanol and transferred to a 20 mL cillin bottle. Thin layer chromatography (TLC) is performed on a plate using a developing solvent of petroleum ether:chloroform = 1:1, petroleum ether:acetone = 10:1, chloroform:acetone = 5:1, chloroform:methanol = 10:1, or ethyl acetate:methanol = 5:1. The fluorescence at 254 nm or 365 nm is observed under a conventional ultraviolet-visible light analyzer, and then 8% sulfuric acid ethanol vanillin is used as a color developer. The ethyl acetate solvent eluates are combined, and the ethyl acetate solvent is recovered to obtain 61.4 g of ethyl acetate extract.

[0049] 4. Purification and separation

[0050] The ethyl acetate layer extract (61.4 g) was dissolved in methanol solvent and mixed evenly with silica gel at a mass ratio of about 1:1.5 (i.e., medium-pressure RP-18 reverse-phase silica gel was added to 100 g of fermentation product). After the solvent evaporated, a river sand sample was obtained, which was used as the column sample; a pre-column with a length of 10 cm and a diameter of 49 mm was added to the column sample; a reverse-phase medium-pressure column (column length 460 mm, diameter 49 mm) was equilibrated with 10% methanol water, and after equilibration for about 5 to 6 column volumes (approximately elution 5 to 6 L), a sample-containing pre-column was added, and a methanol-water gradient elution (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) was used for 10 steps in sequence. Gradient elution, each gradient elution was 4-5 column volumes, and a 500 mL conical flask was used to receive the eluate. After each eluate was subjected to a rotary evaporator to recover the solvent, it was dissolved in 10 mL of methanol and transferred to a 20 mL penicillin bottle. The plate was then spotted by TLC using petroleum ether: acetone = 2:1, chloroform: acetone = 5:1, chloroform: methanol = 10:1, and ethyl acetate: methanol = 2:1 as developing agents. The fluorescence at 254 nm or 365 nm was observed under a conventional UV-visible analyzer, and then 8% ethanolic sulfate vanillin was used as a color developer for color development. The components that showed black color with 8% ethanolic sulfate vanillin (that is, the components eluted with 80% methanol water) were combined to obtain the 14th component (Fr.14 9.23 g).

[0051] Component Fr.14 (9.23 g) was dissolved in methanol solvent and mixed evenly with 200-300 mesh silica gel at a mass ratio of about 1:1.5 (i.e., 14 g was added to 9.23 g of the component). After the solvent evaporated, a river sand sample was obtained, which was used as the column sample; 300 g of 200-300 mesh silica gel powder was weighed and mixed evenly with a 1:1 solvent of chloroform (no bubbles were generated during this process) and loaded into a separation column with a length of 460 mm and an inner diameter of 100 mm. The silica gel powder was allowed to slowly sink until it stopped sinking, and a column sample was added once. A chloroform system gradient elution (50:1, 30:1, 15:1, 10:1, 5:1, 2:1) was used for each column. The product was eluted with a gradient of 3-4 column volumes (approximately 1.8 L-2.4 L), and the eluate was collected in a 150 mL conical flask. After the solvent was recovered by rotary evaporation, each eluate was dissolved in approximately 10 mL of methanol and transferred to a 20 mL penicillin bottle. The product was then subjected to TLC and developed with chloroform:acetone = 2:1, chloroform:methanol:formic acid = 5:1:1 drops, and ethyl acetate:methanol = 3:1 as a developing agent. The product was observed under a conventional UV-visible analyzer for fluorescence at 254 nm or 365 nm, and then developed with 8% ethanolic sulfate vanillin as a color developer. The fractions that showed black color with 8% ethanolic sulfate vanillin were combined to obtain the ninth fraction (Fr.14-9 186 mg).

[0052] Fr.14-9 (186 mg) was dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then mixed with silica gel in a mass ratio of about 1:1.5 (i.e., 279 mg of 200-300 silica gel was added to 186 mg of the component). After the solvent evaporated, a river sand sample was obtained, which was used as the column sample; 8 g of 200-300 mesh silica gel powder was weighed and mixed with dichloromethane: methanol in a solvent ratio of 10:1 (no bubbles were generated during this process) and loaded into a separation column with a length of 260 mm and an inner diameter of 20 mm. The silica gel powder slowly sank until it stopped sinking. The column sample was added once and eluted with a solvent gradient of dichloromethane:methanol = 10:1. The eluate was collected in a 20 mL penicillin bottle, and the elution solvent was recovered and combined by TLC spot plate to obtain 61.6 mg of compound (4E,8E)-ND-2′-hydroxyoctadecanoyl-1-O-β-D-glycopyranosyl-9-methyl-4,8-sphingadienine.

[0053] 4. PF55 spectral data of sphingosine compounds

[0054]

[0055] Sphingosine compound PF55: HRESIMS m / z 778.5776[M+Na] + , whose molecular weight is C 43 H 81 O9NNa; 1 HNMR (500 MHz, MeOH-d4) δ H5.73(1H,dt,J=15.3,6.0,Hz,H-5),5.47(1H,dd,J=15.4,7.4Hz,H-4),5.14(1H,dd,J=6.8,5.6Hz,H-8),4.26(1H,d,J=7.8Hz,H-1″),4.09-4.15(2H,m,H-1a,3),3.98(2H,td,J=6.4,2.6Hz,H-2,2′),3.86(1H,dd,J=11.8,1.5Hz,H-6″b),3.70(1H,dd,J=10.3,3.5Hz,H-1b),3.63-3.68(1H,m,H-6″a),3.35(1H,t,J=9.0Hz,H-3″),3.26-3.28(2H,m,H-4″,5″),3.18(1H,dd,J=9.1,7.8Hz,H-2″),2.03-2.11(4H,m,H-6,7),1.97(2H,t,J=7.4Hz,H-10),1.66-1.74(1H,m,H-3′a),1.59(3H,s,H-19),1.58-1.52(1H,m,H-3′b),1.39(2H,m,H-4′),1.28-1.39(38H,m,H-12,13,14,15,16,17,5′,6′,7′,8′,9′,10′,11′,12′,13′,14′,15′,16′,17′),0.89(6H,d,J=6.9Hz,H-18,18′); 13C NMR(126MHz,MeOH-d4)δ177.2(s,C-1′),136.8(s,C-9),134.6(d,C-4),1 31.1(d,C-5),124.8(d,C-8),104.7(d,C-1″),78.0(d,C-3″),77.9(d,C-5 ″),75.0(d,C-2″),73.1(d,C-2′),72.9(d,C-3),71.6(d,C-4″),69.7(t,C -1),62.7(t,C-6″),54.6(d,C-2),40.8(t,C-10),35.9(t,C-3′),33.8(t, C-7), 33.1(d,C-16,16′), 30.8-30.4(t,C-12~15,5′~15′), 29.1(t,C-11), 28.7(t,C-6), 26.2(t,C-4′), 23.8(t,C-17′), 23.7(t,C-17), 16.1(q,C-19), 14.5(q,C-18,18′); the above data are consistent with those reported in the literature for the compound (4E,8E)-ND-2′-hydroxyoctadecanoyl-1-O-β-D-glycopyranosyl-9-methyl-4,8-sphingadienine.

[0056] 5. Screening of PF55 Cytotoxicity of Sphingosine Compounds

[0057] 5.1 Test cell line: MCF-7 / ADR (purchased from Shanghai Meixuan Biotechnology Co., Ltd. in May 2021)

[0058] 5.2RPMI1640+10% fetal bovine serum

[0059] 5.3 Cell culture

[0060] 5.3.1 Cell recovery

[0061] Remove the cells from the liquid nitrogen tube and quickly thaw the cryotube in a water bath preheated to 37°C. Shake the tube constantly to melt the liquid in the tube quickly. After about 1 mL of the liquid in the cryotube is completely dissolved, remove the cells under sterile conditions and inoculate them into a cell culture dish (RPMI1640 + 10% fetal bovine serum). Place the dish in a 37°C CO2 incubator and culture. Change the culture medium the next day and continue culturing to observe the growth.

[0062] 5.3.2 Cell Passaging

[0063] After the cells have grown to 80-90%, aseptically remove the cell culture medium using a 3mL plastic pipette. Rinse once with 1-2mL of PBS (calcium- and magnesium-free). Add 1mL of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture flask and observe the cell digestion under an inverted microscope. If the cells are mostly rounded, quickly return the flask to the operating table, tap the culture flask several times, and add 2mL of complete culture medium to terminate the digestion. Add 4mL of new complete culture medium to each new culture flask, followed by 1mL of complete culture medium containing the cells.

[0064] 5.4 CCK-8 cytotoxicity assay

[0065] 5.4.1 Concentration gradient: 0, 1.6, 3.125, 6.25, 12.5, 25, 50, 100 μg / mL, 3 replicates

[0066] Positive control: doxorubicin

[0067] Negative control: DMSO

[0068] 5.4.2 Experimental steps

[0069] (1) Cell digestion, cell counting, and adjusting the cell concentration to 2×10 4 pieces / mL.

[0070] (2) Inoculate 100 μL of the cell suspension in a 96-well plate and incubate the plate in a 5% CO 2 incubator at 37° C. for 24 h.

[0071] (3) According to the groups, different concentrations of compounds and doxorubicin were added and the cells were incubated in an incubator at 37°C for 48 h.

[0072] (4) After the incubation period, the cells were rinsed once with PBS (without calcium and magnesium ions), 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in an incubator for 3 h.

[0073] (5) Measure the absorbance at 490 nm using an enzyme-labeled instrument

[0074] 5.4 Experimental Results

[0075] The results showed that the inhibition rate of PF55, a sphingosine compound, on doxorubicin-resistant breast cancer cells was less than 20% at a concentration of no more than 25 μg / mL, and the next step of tumor cell reversal screening could be continued. Figure 7 .

[0076] 6. Application of sphingosine compound PF55 in reversing the activity of MCF-7 / ADR tumor cells

[0077] 6.1 Test cell line: MCF-7 / ADR (purchased from Shanghai Meixuan Biotechnology Co., Ltd. in May 2021)

[0078] 6.2RPMI1640+10% fetal bovine serum

[0079] 6.3 Cell culture

[0080] 6.3.1 Cell recovery

[0081] Remove the cells from the liquid nitrogen tube and quickly thaw the cryotube in a water bath preheated to 37°C. Shake the tube constantly to melt the liquid in the tube quickly. After about 1 mL of the liquid in the cryotube is completely dissolved, remove the cells under sterile conditions and inoculate them into a cell culture dish (RPMI1640 + 10% fetal bovine serum). Place the dish in a 37°C CO2 incubator and culture. Change the culture medium the next day and continue culturing to observe the growth.

[0082] 6.3.2 Cell Passaging

[0083] After the cells have grown to 80-90%, aseptically remove the cell culture medium using a 3mL plastic pipette. Rinse once with 1-2mL of PBS (calcium- and magnesium-free). Add 1mL of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture flask and observe the cell digestion under an inverted microscope. If the cells are mostly rounded, quickly return the flask to the operating table, gently tap the flask several times, and add 2mL of complete culture medium to terminate the digestion. Add 4mL of new complete culture medium to each new flask, followed by 1mL of complete culture medium containing the cells.

[0084] 6.4 CCK-8 assay for reversal of tumor cytotoxic activity

[0085] 6.4.1 Doxorubicin concentration gradient: 0, 1.6, 3.125, 6.25, 12.5, 25, 50 μg / mL, 3 replicates

[0086] Sphingosine compound PF55 concentration: 5, 10, 20 μg / mL

[0087] Positive control: Verapamil

[0088] Negative control: DMSO

[0089] 6.4.2 Experimental steps

[0090] (1) Cell digestion, cell counting, and adjusting the cell concentration to 2×10 4 pieces / mL.

[0091] (2) Inoculate 100 μL of the cell suspension in a 96-well plate and incubate the plate in a 5% CO 2 incubator at 37° C. for 24 h.

[0092] (3) According to the grouping, different concentrations of compounds and doxorubicin were added and the cells were incubated in an incubator at 37°C for 48 h.

[0093] (4) After the incubation period, the cells were rinsed once with PBS (without calcium and magnesium ions), 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in an incubator for 3 h.

[0094] (5) Measure the absorbance at 490 nm using an enzyme-labeled instrument

[0095] 6.4 Experimental Results

[0096] The results showed that the sphingosine compound PF55 could reverse the MCF-7 / ADR activity when used in combination with doxorubicin at concentrations of 5, 10, and 20 μg / mL. 50 The values ​​were 30.051±1.916, 26.580±2.374 and 21.613±0.632μg / mL respectively. Its activity was dose-dependent with the concentration of sphingosine compound PF55. The results are shown in Figure 8 And Table 1.

[0097] Table 1: Sphingosine compound PF55 reversed the activity of tumor cells MCF-7 / ADR at concentrations of 5, 10, and 20 μg / mL

[0098]

[0099] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Use of a sphingosine compound in the preparation of a tumor resistance reversal agent or a tumor drug sensitizer, wherein the structure of the sphingosine compound is shown in the following formula: The drug in the tumor resistance reversal agent or the drug in the tumor drug sensitizer is doxorubicin, and the tumor is breast cancer.

2. A method for preparing a sphingosine compound, characterized in that: The structure of sphingosine compounds is shown below: The compound is obtained by fermentation and extraction of Tubeufia rubraPF02-2, and the preservation unit is China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2019957.

3. The method for preparing the compound according to claim 2, wherein: The following steps are involved: The red-brown tube fungus Tubeufia rubra PF02-2 is subjected to liquid or solid fermentation culture to obtain a fermentation product; the fermentation product is extracted, and the obtained extract is separated and purified to obtain the sphingosine compound.

4. The method for preparing the compound according to claim 3, wherein: The specific steps include: S1. Strain activation: Take out the preserved strain, inoculate it on the basal culture medium plate, culture it statically to the third generation and then amplify it; S2, fermentation culture: take the activated bacteria in step S1 and inoculate it into solid culture medium, and ferment it at 26-30°C for a period of time; S3, extraction: extract the bacterial cells and culture medium, add ethyl acetate for extraction, and concentrate the extract to obtain the fermentation product; S4. Pretreatment of fermentation product: dissolve the fermentation product in a solvent of dichloromethane:methanol = 1:1, and then mix it evenly with silica gel in a mass ratio of 1:1-2. After the solvent evaporates, use it as a column sample, and then add it to a separation column with silica gel powder and petroleum ether. Use petroleum ether, chloroform, ethyl acetate and methanol in a gradient elution sequence, respectively. After the elution solvent is recovered under reduced pressure on a rotary evaporator, dissolve it in chloroform, acetone or methanol, and then use a thin layer chromatography plate and a developing agent to develop it. Select the liquid that fluoresces at 254 nm or 365 nm under an ultraviolet-visible light analyzer, and then use 8% sulfuric acid ethanol vanillin as a color developer; combine the ethyl acetate solvent eluates, recover the ethyl acetate solvent, and obtain an ethyl acetate extract; S5. Purification and separation: a. Dissolve the ethyl acetate extract in methanol solvent and mix it with silica gel in a mass ratio of 1:1-3. After the solvent evaporates, load the sample onto a pre-column. Use 10% methanol water to equilibrate the reverse phase medium pressure column. Add the sample to the pre-column and elute with methanol water in 10 gradients. After the eluate is recycled by rotary evaporation, dissolve it in methanol and then use a thin layer chromatography plate. Use a developing agent to develop the solution. Select the liquid that fluoresces at 254 nm or 365 nm under a UV-visible spectrometer. Then combine the components that develop black with 8% ethanolic sulfuric acid and vanillin as a color developer to obtain component Fr.

14. b. Dissolve component Fr.14 in methanol and mix it evenly with silica gel in a mass ratio of 1:1 to 3. After the solvent evaporates, prepare the sample for column loading. Weigh silica gel powder and mix it evenly with a solvent of dichloromethane:methanol in a ratio of 1:

1. Load the mixture into the separation column and add the sample for column loading once more. Use a dichloromethane / methanol gradient elution system. After the eluate is solvent-recovered on a rotary evaporator, dissolve it in methanol and perform TLC on a plate. Develop the eluate with a developing agent and observe for fluorescence at 254 nm or 365 nm using a UV-visible light analyzer. Combine the components that develop black with 8% ethanolic sulfuric acid and vanillin as a color developer to obtain Fr.14-9. c. Dissolve Fr.14-9 in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and mix it evenly with silica gel in a mass ratio of 1:1 to 3. After the solvent evaporates, prepare the sample for column loading. Weigh silica gel powder and mix it evenly with a solvent of dichloromethane:methanol = 10:1, load it into a separation column, then add the sample for column loading, and use a solvent gradient elution of dichloromethane:methanol = 10:

1. The elution solvents are combined using a TLC spot plate to obtain sphingosine compounds.

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