Preparation method and application of glycolipid compound PF35

Abstract

The application relates to application of glycolipid compound PF35 in preparation of a tumor drug resistance reversing agent or a tumor drug sensitizer, and belongs to the technical field of biochemical application. The structure of the glycolipid compound PF35 is shown in the following formula: the drug or the tumor drug is adriamycin. Research shows that the glycolipid compound PF35 has the activity of reversing MCF-7 / ADR when being combined with adriamycin at concentrations of 5, 10 and 20 mu g / mL respectively.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biochemical application, in particular to a preparation method and application of glycolipid compound PF35. BACKGROUND

[0002] Malignant tumor has become one of the diseases that seriously threaten human life safety. In the process of tumor treatment, drug resistance of tumor cells is one of the main reasons for the failure of chemotherapy. Tumor cells can produce drug resistance through various mechanisms such as drug resistance related proteins, DNA damage and repair dysfunction, autophagy, reduction of drug accumulation and increase of drug output, metabolic detoxification, changes of drug targets and signal transduction molecules. Among them, P-gp protein is one of the main pathways of drug resistance of tumor cells. P-gp is expressed by MDR-1 gene and is an ATP-driven drug efflux pump that can bind to a wide range of drugs including paclitaxel (PTX, Taxol), adriamycin (ADR) and vinblastine and many commonly used antitumor drugs. Therefore, the development of P-gp protein inhibitors combined with tumor drugs can increase the sensitivity of tumor drugs to tumor cells, thereby reversing the activity of drug-resistant tumor cells, and the co-administration of P-gp inhibitors and chemotherapy drugs can be used as an effective strategy to overcome MDR. At present, several generations of P-gp inhibitors have been developed, the first generation of reversal agents including tamoxifen, cyclosporin A, etc., among which verapamil and cyclosporin are typical representatives. However, such drugs usually lack specificity of P-glycoprotein and can produce serious side effects, and the first generation of reversal agents is also greatly limited in clinical use (Sato W. et al. 1991). The second generation of reversal agents such as the fungicidin analogue valspodar (PSC833), dexverapamil, etc., among which dexamethasone is a representative, however, the development of the second generation of reversal agents is limited due to a series of side effects caused by high toxicity and drug interactions (Rowinsky E.K. et al. 1998; Hyafil F. et al. 1993; Keller R.P. et al. 1992). The main representative drugs of the third generation of P-glycoprotein inhibitors are Tariquidar (XR9576), Zosuquidar (LY335979), S9788, etc., among which Tariquidar (XR9576) and WK-X-34 are representative (Massey P.R. et al. 2014). The development of P-gp inhibitors from natural products and their derivatives has become a new direction and focus of the research and development of the fourth generation of inhibitors.

[0003] Microbial-derived natural products have always been a good source of molecules for new drug development. However, as the development of microbial drugs from ordinary environments decreases the probability of discovering active new natural products, the probability of repeatedly discovering known natural products increases. Therefore, people turn their attention to microorganisms from special habitats. Microorganisms from special habitats are more likely to produce new biosynthetic pathways and thus more likely to produce structurally novel natural products. There are specific reports on the development of P-gp inhibitors from microbial-derived natural products, but it is not clear what specific substances are.

[0004] To this end, the applicant has made a lot of research, and in the early stage, a series of long-chain fatty acid glycerol new compounds such as CN113773216A, CN114014898A, and CN114057811A are disclosed, and it is found that they have application in reversing drug-resistant tumor cell activity. The applicant continues to study the bacteria in order to find more compounds with application in reversing drug-resistant tumor cell activity. SUMMARY

[0005] The present application aims at the deficiencies of the prior art and provides a preparation method and application of a glycolipid compound PF35 of biological origin.

[0006] One of the purposes of the present application is to provide an application of a glycolipid compound PF35 in preparing a tumor drug resistance reversing agent or a tumor drug sensitizer. The structure of the glycolipid compound PF35 (4E, 8E)-N-D-2'-hydroxyoctadecanoyl-1-O-β-D-glycopyranosyl-9-methyl-4, 8-sphingadienine (hereinafter referred to as glycolipid compound PF35) is shown in the following formula:

[0007]

[0008] Further, the drug resistance or tumor drug is doxorubicin.

[0009] Further, the tumor is breast cancer.

[0010] Further, the tumor drug resistance reversing agent is a transport pump inhibitor, and the transport pump inhibitor has an inhibitory effect on one or several of the drug resistance proteins P-glycoprotein and multidrug resistance protein.

[0011] The second purpose of the present application is to provide an application of a glycolipid compound PF35 and a pharmaceutical carrier in preparing an anti-tumor cell agent, wherein the tumor cell is a doxorubicin-resistant breast cancer cell; the structure of the glycolipid compound PF35 is shown in the following formula:

[0012]

[0013] The third object of the present application is to provide a preparation method of glycolipid compound PF35, and the structure of glycolipid compound PF35 is shown in the following formula:

[0014] The compound is obtained after fermentation and extraction of the red-brown tube cavity fungus, and the red-brown tube cavity fungus is named Tubeufia rubra PF02-2, and the preservation unit is China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2019957.

[0015] The red-brown tube cavity fungus Tubeufia rubra PF02-2 in the present application is isolated and obtained by Guizhou University Biochemical Engineering Center, and is preserved by China Center for Type Culture Collection, and the address is Wuhan University, Wuhan, China, and the preservation date is November 20, 2019, and the preservation registration number is CCTCC NO: M 2019957.

[0016] The source of the red-brown tube cavity fungus Tubeufia rubra PF02-2 is as follows:

[0017] Sampling time: May 14, 2016;

[0018] Sampling site: Pingfeng rainforest nature reserve in Fangchenggang City, Guangxi Zhuang Autonomous Region;

[0019] Sampling method: collecting rotten wood in Pingfeng rainforest nature reserve in Fangchenggang City, Guangxi Zhuang Autonomous Region, and taking back to the laboratory in a plastic sealed bag.

[0020] The red-brown tube cavity fungus Tubeufia rubra PF02-2 strain in the present application has the following properties:

[0021] Colony morphological characteristics: on natural rotten wood substrate, the colony is flat, net-like, dotted, and connected into a sheet when in large quantity. The pure colony of PF02-2 obtained by fresh separation is colorless and transparent to white, and the pure colony of PF02-2 obtained by separation is red-brown after natural drying. Part of the mycelium is buried under the substrate, but most of it is superfacial. The mycelium is composed of septum-banded branched hyphae, colorless to dark brown. The conidial stalk is cylindrical, single, curved, septate, 50-150 microns long, 4.5-6 microns wide, tapered at the top, dark brown at the bottom, transparent to light brown at the top, and smooth in surface. The sporulating cell is single or multiple, cylindrical, with columnar denticles, and grows in the middle to the top of the conidial stalk, 10-19 microns long, 3-4 microns wide, colorless and transparent to light brown, smooth in surface. The conidial spore is single, top-side, transparent, round at the top, 2-3.5 times coiled when tightly coiled, 35-50 microns in diameter, 3-5 microns thick (average diameter 45 microns, thickness 4.5 microns), gradually loose in water, with unclear multiple septa, colorless to light brown, smooth in surface. The conidial spore starts to germinate after 12 hours in water-agar medium. The colony grows to 16 mm in PDA medium at 25-28°C for 2 weeks, brown, round, rough in surface, with obvious protrusions, vein-like wrinkles, and complete colony edge.

[0022] Further, the preparation method comprises the following steps: liquid or solid fermentation culture of Tubeufia rubra PF02-2 to obtain a fermentation product; extraction of the fermentation product, and separation and purification of the obtained extract to obtain glycolipid compound PF35.

[0023] Specifically comprising the following steps:

[0024] S1, strain activation: take out the preserved strain, inoculate on a basic medium plate, and culture at rest to the third generation for amplification culture;

[0025] S2, fermentation culture: inoculate the strain activated in step S1 in a solid culture medium, and culture at rest at 26-30°C for a period of time;

[0026] S3, extraction: take the mycelium together with the culture medium, add ethyl acetate for extraction, concentrate the extract to obtain a fermentation product;

[0027] S4, fermentation product pretreatment: the fermentation product is dissolved with a chloro:methyl = 1:1 solvent, then mixed with silica gel at a mass ratio of 1:1-2, and after the solvent is volatilized, the mixture is used as a sample for column chromatography, then mixed with silica gel powder and petroleum ether, and then column chromatography is performed, and petroleum ether, chloroform, ethyl acetate and methanol are used for gradient elution, respectively, the elution solvent is recovered by a rotary evaporator under reduced pressure, then dissolved with chloroform, acetone or methanol, and then TLC point plate is used, and the eluent is developed with a developing agent, and then the liquid with fluorescence under 254nm or 365nm of ultraviolet-visible light is selected, and then 8% sulfuric acid ethanol vanillin color developing agent is used for color development; the ethyl acetate eluent is combined, and the ethyl acetate solvent is recovered, to obtain an ethyl acetate extract;

[0028] S5, purification and separation: a, the ethyl acetate extract is dissolved with a methanol solvent, mixed with silica gel at a mass ratio of 1:1-3, and after the solvent is volatilized, the mixture is used as a sample for column chromatography, and 10% methanol water is used for equilibrium reversed-phase medium-pressure column chromatography, and then the sample is added, and then methanol water is used for gradient elution, and then the eluent is recovered by a rotary evaporator, and then dissolved with methanol, and then TLC point plate is used, and the eluent is developed with a developing agent, and then the liquid with fluorescence under 254nm or 365nm of ultraviolet-visible light is selected, and then the light blue component obtained by combining 8% sulfuric acid ethanol vanillin color developing agent is obtained, to obtain a Fr.5 component;

[0029] b, the component Fr.5 is dissolved with a dichloromethane:methanol = 1:1 mixed solvent, then mixed with silica gel at a mass ratio of 1:1-3, and after the solvent is volatilized, the mixture is used as a sample for column chromatography, silica gel powder is weighed and mixed with dichloromethane:methanol:formic acid = 200:20:1 solvent, and then loaded into a separation column, and then ethyl acetate:methanol:formic acid = 200:20:1 is used for gradient elution, and then the eluent is collected in a 50mL triangular flask, and then the elution solvent is combined by TLC point plate, to obtain a component Fr.5-2;

[0030] c, the component Fr.5-2 is subjected to gel column chromatography, and then glycolipid compound PF35 is obtained by elution with methanol.

[0031] In the step S2, the solid culture medium is oat medium, and 200g of oat and 150mL of double-distilled water are mixed to obtain the solid culture medium.

[0032] The compound can be separated from the fermentation product of Tubeufia rubra PF02-2, and the content is high, which is more conducive to industrial application. It is found that glycolipid compound PF35 has the activity of reversing MCF-7 / ADR when used in combination with adriamycin at concentrations of 5, 10 and 20 μg / mL, and IC 50The values are 31.954±2.698, 29.908±3.911 and 25.839±3.184 μg / mL respectively. The activity is in dose-dependent relationship with the concentration of glycolipid compound PF35. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Flow chart for isolation and purification of glycolipid compound PF35;

[0034] Figure 2 Mass spectrum of glycolipid compound PF35 in the present application;

[0035] Figure 3 H-NMR spectrum of glycolipid compound PF35 in the present application; 1 H-NMR spectrum;

[0036] Figure 4 DEPT and C-NMR spectrum of glycolipid compound PF35 in the present application; 13 C-NMR spectrum;

[0037] Figure 5 Original data of cytotoxic activity screening of glycolipid compound PF35 in the present application;

[0038] Figure 6 Original data of reverse MCF-7 / ADR activity screening of glycolipid compound PF35 in the present application;

[0039] Figure 7 Inhibition rate diagram of glycolipid compound PF35 on tumor cell MCF-7 / ADR in the present application;

[0040] Figure 8 IC50 values of MCF-7 / ADM when different concentration gradients of doxorubicin are combined with glycolipid compound PF35 at concentrations of 5, 10 and 20 μg / mL respectively. 50 ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. DETAILED DESCRIPTION

[0041] The following will be further explained in detail through specific embodiments:

[0042] 1. Activation of strain

[0043] The strain Tubeufiarubra PF02-2 was taken out from the glycerol slant in a -80℃ refrigerator, and 1 loop of the strain was taken out with a sterile inoculation loop, and was crosswise inoculated on a basic medium plate with a diameter of 11 cm. The plate was incubated at 28℃ for 17 days, and was subcultured to the third generation for amplification.

[0044] 2. Fermentation culture

[0045] Oat solid fermentation (1L flask, 200g oat and 150mL double distilled water) 2 After 105 days of incubation at 28℃, the bacterial cells were mixed with the oat medium and extracted with ethyl acetate for three times, each time for 24h with 160rpm shaking. The extracts were combined and concentrated under reduced pressure at 40℃ to obtain the fermentation product. The above procedure was repeated and the fermentation products were combined to obtain 2027.17g.

[0046] 3. Pretreatment of fermentation product

[0047] The 2027.17g fermentation product was dissolved in acetone and mixed with silica gel in a mass ratio of about 1:1.5 (i.e. 2027.17g fermentation product plus 3041g 200-300 mesh silica gel powder) to obtain a river sand-like sample. The sample was used as the first column sample. 6000g 200-300 mesh silica gel powder was mixed with petroleum ether (no bubbles were generated during the process) and loaded into a separation column with a length of 1.5m and an inner diameter of 200mm. The silica gel powder was allowed to slowly sink until it stopped sinking, and then the first column sample was added. Petroleum ether, chloroform, ethyl acetate, and methanol were used to elute the sample in four gradients, each gradient was eluted for 2-3 column volumes (about 36L-54L elution solvent per column volume). Each 1000mL elution solvent was collected as one sample. After the elution samples were recovered by rotary evaporation under reduced pressure, they were dissolved in 10 or 15mL chloroform, acetone, or methanol and transferred to 20mL sinter bottles. Thin layer chromatography (TLC) was performed using petroleum ether:chloroform=1:1, petroleum ether:acetone=10:1, chloroform:acetone=5:1, chloroform:methanol=10:1, and ethyl acetate:methanol=5:1 as the developing agent. The samples were observed under a conventional ultraviolet-visible light analyzer for fluorescence at 254nm or 365nm, and then developed with 8% sulfuric acid ethanol vanillin color reagent. The ethyl acetate eluate was combined and the ethyl acetate solvent was recovered to obtain 61.4g ethyl acetate.

[0048] 4. Purification and separation

[0049] (1) The ethyl acetate layer extract (61.4 g) was dissolved in methanol and mixed with silica gel at a mass ratio of about 1:1.5 (i.e. 61.4 g of the fermentation product plus 93 g of medium-pressure RP-18 reversed-phase silica gel), and after the solvent was evaporated, a river sand-like sample was obtained, which was used as the sample for column chromatography. The sample was added to a pre-column with a length of 10 cm and a diameter of 49 mm. A 10% methanol water solution was used to equilibrate the reversed-phase medium-pressure column (460 mm in length and 49 mm in diameter), and after about 5-6 column volumes (about 5-6 L) were equilibrated, the sample pre-column was added. A methanol water gradient elution (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) was used, and each gradient elution was performed for 4-5 column volumes. A 500 mL triangular flask was used to collect the eluate, and after the solvent was recovered by a rotary evaporator, 10 mL of methanol was added to dissolve the eluate, which was then transferred to a 20 mL vial. TLC spotting was performed, and the sample was developed with petroleum ether:acetone = 2:1, chloroform:acetone = 5:1, chloroform:methanol = 10:1, and ethyl acetate:methanol = 2:1 as the developing agent. Whether there was fluorescence at 254 nm or 365 nm was observed under a conventional ultraviolet-visible light analyzer, and then 8% sulfuric acid ethanol vanillin color developing agent was used for color development. The light blue components (i.e. the components eluted by 90% methanol water) were combined, and the fifth component (Fr. 5, 767 mg) was obtained.

[0050] (2) Fr. 5 (767 mg) was dissolved in dichloromethane and methanol mixed at a volume ratio of 1:1, and then mixed with silica gel at a mass ratio of about 1:1.5 (i.e. 767 mg of the component plus 1.2 g of 200-300 mesh silica gel). After the solvent was evaporated, a river sand-like sample was obtained, which was used as the sample for column chromatography. 72 g of 200-300 mesh silica gel powder was mixed with dichloromethane:methanol:formic acid = 200:20:1 to obtain a mixture (no bubbles were generated during this process), which was loaded into a separation column with a length of 260 mm and an inner diameter of 20 mm. The silica gel powder was allowed to slowly sink until it stopped sinking, and then the sample was added. An ethyl acetate:methanol:formic acid = 200:20:1 gradient elution was used, and a 50 mL triangular flask was used to collect the eluate. After the elution solvent was recovered, methanol was used to dissolve and transfer the eluate to a 20 mL vial. TLC spotting was performed, and the component Fr. 5-2 (28 mg) was obtained.

[0051] (3) Fraction Fr.5-2 (28 mg) was subjected to column chromatography on Sephadex with methanol as eluent to give compound (2S)-1-O-(9Z,12Z-octadecadienoyl)-3-O-[a-D-galactopyranosyl-(1"→6")-O-β-D-galactopyranosyl-(1"'→6")-O-β-D-galactopyranosyl]glycerol (PF35) 17 mg.

[0052] Four, glycolipid compound PF35 spectral data

[0053]

[0054] Unsaturated long-chain glycolipid compound PF35:

[0055] (2S)-1-O-(9Z,12Z-octadecadienoyl)-3-O-[a-D-galactopyranosyl-(1"→6")-O-β-D-galactopyranosyl-(1"'→6")-O-β-D-galactopyranosyl]glycerol (PF35): HRESIMS m / z 863.42169 [M+Na] + with a molecular weight of C 39 H 68 O 19 Na; 1 H NMR (500 MHz, CD3OD) δ H5.28-5.38 (4H, m, H-9', 10', 12', 13'), 4.85 (1H, br s, H-1""), 4.30 (1H, d, J = 7.5 Hz, H-1'''), 4.27 (1H, d, J = 7.6 Hz, H-1"), 4.16 (1H, dd, J = 11.4, 4.4 Hz, H-1β), 4.13 (1H, dd, J = 11.4, 6.3 Hz, H-1α), 4.06 (1H, t, J = 6.1 Hz, H-3β), 3.94-4.01 (4H, m, H-2, 6"β, 6"'β, 4""), 3.85-3.90 (2H, m, H-2"", 6"'α), 3.80-3.82 (2H, m, H-4"', 5""), 3.78 (1H, dd, J = 7.2, 3.3 Hz, H-6""β), 3.69-3.78 (6H, m, H-6""α, 4"', 5"', 5"", 3α, 3""'), 3.66 (1H, dd, J = 10.6, 5.0 Hz, H-6"α), 3.50-3.53 (3H, m, H-2", 2"", 3""), 3.46 (1H, dd, J = 9.7, 3.3 Hz, H-3"), 2.77 (2H, t, J = 6.5 Hz, H-11'), 2.35 (2H, t, J = 7.5 Hz, H-2'), 2.06 (4H, m, H-8', 14'), 1.61 (2H, m, H-3'), 1.28-1.36 (14H, m, H-4' ~ 7', 15' ~ 17'), 0.90 (3H, t, J = 6.9 Hz, H-18'); 13 C NMR (126 MHz, CD3OD) δ C175.6 (s, C-1'), 130.9 (d, C-9', 13'), 129.1 (d, C-10'), 129.0 (d, C-12'), 105.3 (d, C-1'), 105.3 (d, C-1"), 100.8 (d, C-1"'), 76.7 (d, C-5"), 74.9 (d, C-3'"), 74.6 (d, C-3"), 74.5 (d, C-5'"), 72.6 (d, C-2", 2'"), 72.1 (t, C-3), 71.4 (d, C-3""), 71.2 (d, C-4""), 70.8 (d, C-5""), 70.3 (d, C-4'"), 70.2 (d, C-2""), 70.1 (d, C-4"), 69.7 (t, C-6"), 69.6 (d, C-2), 68.1 (t, C-1), 62.5 (t, C-6""), 34.9 (t, C-2'), 32.7 (t, C-16'), 30.7-30.2 (t, C-4'~7', 15'), 28.2 (t, C-8', 14'), 26.6 (t, C-11'), 26.0 (t, C-3'), 23.7 (t, C-17'), 14.5 (q, C-18'); the above data are consistent with the reported data of (2S)-1-O-(9Z, 12Z-octadecadienoyl)-3-O-[α-D-galactopyranosyl-(1"→6'')-O-β-D-galactopyranosyl-(1"'→6"')-O-β-D-galactopyranosyl]glycerol.

[0056] V. Cytotoxic activity screening of glycolipid compound PF35

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

[0058] 5.2 RPMI1640 + 10% fetal bovine serum

[0059] 5.3 Cell culture

[0060] 5.3.1 Cell recovery

[0061] Cells were taken out from the liquid nitrogen tube, and the frozen tube was quickly put into a water bath preheated to 37 degrees for quick thawing, and the tube was constantly shaken to quickly melt the liquid. After about 1 mL of liquid in the frozen tube was completely dissolved, the cells were taken out under sterile conditions and inoculated into a cell culture dish (RPMI1640 + 10% fetal bovine serum), and cultured in a 37 degree CO2 incubator. The next day, the culture medium was replaced and the cells were continuously cultured and observed for growth.

[0062] 5.3.2 Cell passage

[0063] After the cells grew to 80-90%, the cells were taken out under sterile conditions using a 3 mL plastic pipette, 1-2 mL PBS was added for washing once (without calcium and magnesium ions), 1 mL of digestion solution (0.25% trypsin-0.53 mM EDTA) was added to the culture bottle, and the cell digestion was observed under an inverted microscope. If most of the cells were rounded, the culture bottle was quickly taken back to the operation platform, and 2 mL of complete culture medium was added to terminate the digestion. New complete culture medium was added to the new culture bottle, and 1 mL of complete culture medium containing cells was added.

[0064] 5.4 CCK-8 test cell toxicity activity

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

[0066] Positive control: doxorubicin

[0067] Negative control: DMSO

[0068] 5.4.2 Experimental steps

[0069] (1) Cell digestion, cell counting, and adjustment of cell concentration to 2 x 104 / mL.

[0070] (2) Inoculate 100 μL of cell suspension in a 96-well plate. Incubate the plate in a 5% CO2 incubator at 37°C for 24 h.

[0071] (3) According to the grouping, add different concentrations of compounds and doxorubicin, respectively, and continue to incubate in the incubator at 37°C for 48 h.

[0072] (4) After incubation, wash once with PBS (without calcium and magnesium ions), add 10 μL of CCK-8 reagent to each well, and incubate in the incubator for 3 h.

[0073] (5) Measure the absorbance at 490 nm with a microplate reader

[0074] 5.4 Experimental results

[0075] The activity screening results show that the inhibition rate of compound (2S)-1-O-(9Z, 12Z-octadecadienoyl)-3-O-[a-D-galactopyranosyl-(1””→6”')-O-β-D-galactopyranosyl-(1”'→6″)-O-β-D-galactopyranosyl]glycerol (PF35) on Adriamycin-resistant breast cancer tumor cells (MCF-7 / ADR) is less than 20% at a concentration not higher than 25 μg / mL, and the next step of reversing tumor cell screening can be continued. The results are shown in detail in Figure 7 .

[0076] Six, application of glycolipid compound PF35 in reversing MCF-7 / ADR tumor cell activity

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

[0078] 6.2 RPMI1640 + 10% fetal bovine serum

[0079] 6.3 Cell culture

[0080] 6.3.1 Cell recovery

[0081] Take the cells out of the liquid nitrogen tube, quickly put the frozen tube into the water bath pot which has been preheated to 37 degrees for rapid thawing, and constantly shake to quickly melt the liquid in the tube. After about 1 mL of liquid in the frozen tube is completely dissolved, take out the cells under sterile conditions and inoculate them into a cell culture dish (RPMI1640 + 10% fetal bovine serum), and place them in a 37-degree CO2 incubator for culture. The next day, replace the culture medium and continue to culture, and observe the growth

[0082] 6.3.2 Cell passage

[0083] After the cells grow to 80-90%, use a 3mL plastic pipette to aspirate the cell culture solution under aseptic operation conditions, add 1-2mL PBS for flushing once (without calcium and magnesium ions), add 1mL digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture bottle, and observe the cell digestion under an inverted microscope. If most of the cells are rounded, quickly take back to the operation table, gently knock the culture bottle a few times, and then add 2mL complete culture medium to terminate digestion. Add new complete culture medium 4mL to the new culture bottle, and then add 1mL complete culture medium containing cells.

[0084] 6.4 CCK-8 test of reversing tumor cell toxicity activity

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

[0086] Compound (2S)-1-O-(9Z, 12Z-octadecadienoyl)-3-O-[a-D-galactopyranosyl-(1””→6”')-O-β-D-galactopyranosyl-(1”'→6")-O-β-D-galactopyranosyl]glycerol (PF35) concentrations: 5, 10, 20 μg / mL

[0087] Positive control: verapamil

[0088] Negative control: DMSO

[0089] 6.4.2 Experimental procedure

[0090] (1) Cell digestion, cell counting, adjusting the cell concentration to 2 x 104 / mL.

[0091] (2) Inoculate 100 μL of cell suspension in a 96-well plate. Incubate the plate in a 5% CO2 incubator at 37°C for 24 h.

[0092] (3) According to the grouping, add different concentrations of compounds and doxorubicin respectively, and continue to incubate in the incubator at 37°C for 48 h.

[0093] (4) After incubation, rinse once with PBS (without calcium, magnesium ions), add 10 μL of CCK-8 reagent per well, and incubate in the incubator for 3 h.

[0094] (5) Measure the absorbance at 490 nm with a microplate reader

[0095] 6.4 Experimental results

[0096] The IC50 values of compound (2S)-1-O-(9Z, 12Z-octadecadienoyl)-3-O-[a-D-galactopyranosyl-(1””→6”')-O-β-D-galactopyranosyl-(1”'→6")-O-β-D-galactopyranosyl]glycerol (PF35) at three concentrations were 31.954 ± 2.698, 29.908 ± 3.911 and 25.839 ± 3.184 μg / mL, respectively. The IC50 value of the negative control doxorubicin was 47.637 ± 5.157, indicating that the compound was combined with doxorubicin at concentrations of 5, 10 and 20 μg / mL, respectively. 50The values of all groups were lower than that of the negative control, and the single factor variance analysis had significant statistical difference (P < 0.001 and P < 0.0001), which indicated that the compound had significant reverse activity at the three concentration gradients, and the reverse multiples were 1.49, 1.59 and 1.84 respectively. The IC 50 values of the positive control combined with ADR at 5, 10 and 20 μg / mL were 26.766 ± 1.687, 23.322 ± 1.571 and 20.645 ± 1.491 μg / mL respectively, and the IC 50 values of the compound at the three concentrations were all lower than that of verapamil. The results are shown in Figure 8 and Table 1.

[0097] Table 1: Reverse tumor cell A549 / ADR activity of compound PF35 at concentrations of 5, 10 and 20 μg / mL

[0098]

[0099] The above only is the embodiment of the present application, and the common knowledge of specific structure and characteristics in the scheme is not described too much. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be regarded as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. Use of glycolipid compound PF35 as the only active ingredient in the preparation of a tumor drug resistance reversing agent or a tumor drug sensitizer, the structure of the glycolipid compound PF35 being shown in the following formula: ###0001### wherein R1 is H or CH3; R2 is H or CH3; R3 is H or CH3; R4 is H or CH3; R5 is H or CH3; R6 is H or CH3; R7 is H or CH3; R8 is H or CH3; R9 is H or CH3; R10 is H or CH3; R11 is H or CH3; R12 is H or CH3; R13 is H or CH3; R14 is H or CH3; R15 is H or CH3; R16 is H or CH3; ， ​

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