Preparation method and application of glycolipid compound PF60

Abstract

The application relates to application of glycolipid compound PF60 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 PF60 is shown in the following formula: the drug or the tumor drug is adriamycin. Research shows that the glycolipid compound PF60 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] This invention relates to the field of biochemical application technology, specifically to a method for preparing and applying a glycerol glycolipid compound PF60. Background Technology

[0002] Malignant tumors have become one of the most serious threats to human life. Drug resistance in tumor cells is a major cause of chemotherapy failure during treatment. Tumor cells can develop resistance through various mechanisms, such as resistance-related proteins, DNA damage and repair dysfunction; autophagy; reduced drug accumulation and increased drug output; metabolic detoxification; and alterations in drug targets and signal transduction molecules. Among these, P-gp protein is one of the main pathways for tumor cell resistance. Encoded by the MDR-1 gene, P-gp is an ATP-driven drug efflux pump that can bind to a wide range of drugs, including many commonly used anti-tumor drugs such as paclitaxel (PTX, Taxol), adriamycin (ADR), and vincristine. Therefore, developing P-gp protein inhibitors for combined use with anti-tumor drugs can increase the sensitivity of tumor cells to these drugs, thereby reversing the activity of drug-resistant tumor cells. Co-administration of P-gp inhibitors with chemotherapy drugs can be an effective strategy to overcome MDR. Several generations of P-gp inhibitors have been developed. First-generation reversal agents include tamoxifen and cyclosporine A, with verapamil and cyclosporine being typical examples. However, these drugs typically lack specificity for P-glycoproteins, leading to severe side effects, and their clinical application has been significantly limited (Sato W. et al. 1991). Second-generation reversal agents include sporin analogs such as valspodar (PSC833) and dexverapamil, with dexamethasone being a representative example. However, the development of second-generation reversal agents has been limited by high toxicity and a series of side effects caused by drug interactions (Rowinsky EK et al. 1998; Hyafil F. et al. 1993; Keller RPetal. 1992). The main representative drugs of third-generation P-glycoprotein inhibitors include Tariquidar (XR9576), Zosuquidar (LY335979), and S9788, among which Tariquidar (XR9576) and WK-X-34 are representative (Massey Pharmaceutical, 2014). Developing P-gp inhibitors from natural products and their derivatives has become a new direction and focus in the research and development of fourth-generation inhibitors.

[0003] Microbial-derived natural products have long provided high-quality source molecules for new drug development. However, as the probability of discovering novel active natural products from ordinary environments decreases while the probability of repeatedly discovering known natural products increases, research has turned to microorganisms from specific habitats. These microorganisms, adapted to extreme environments, are more likely to develop new biosynthetic pathways, thus producing more structurally novel natural products. Currently, there are specific reports of developing P-gp inhibitors from microbial-derived natural products, but the specific substances involved are not clearly identified.

[0004] To this end, the applicant has conducted extensive research and has previously discovered a series of novel long-chain fatty acid glycerol compounds disclosed in CN113773216A, CN114014898A, and CN114057811A, and found that they have applications in reversing the activity of drug-resistant tumor cells. The applicant continues to study this bacterium in order to discover more compounds with applications in reversing the activity of drug-resistant tumor cells. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying PF60, a biologically derived glycerol glycolipid compound.

[0006] One objective of this invention is to provide an application of glycerol glycolipid compound PF60 in the preparation of tumor drug resistance reversal agents or tumor drug sensitizers. The structure of glycerol glycolipid compound PF60(4E,8E)-ND-2′-hydroxyoctadecanoyl-1-O-β-D-glycopyranosyl-9-methyl-4,8-sphingadienine (hereinafter referred to as glycerol glycolipid compound PF60) is shown in the following formula:

[0007]

[0008] Furthermore, doxorubicin is a drug that is resistant to or associated with tumors.

[0009] Furthermore, the tumor in question is breast cancer.

[0010] Furthermore, the tumor resistance reversal agent is a transport pump inhibitor, which has an inhibitory effect on one or more of the drug resistance protein P-glycoprotein and multidrug resistance protein.

[0011] A second objective of this invention is to provide the application of the glycerol glycolipid compound PF60 and its pharmaceutical carrier in the preparation of antitumor cell agents, wherein the tumor cells are doxorubicin-resistant breast cancer cells; the structure of the glycerol glycolipid compound PF60 is shown in the following formula:

[0012]

[0013] A third objective of this invention is to provide a method for preparing a glycerol glycolipid compound PF60, the structure of which is shown in the following formula:

[0014] The compound was obtained by fermentation and extraction of Tubeufia rubra, which was named Tubeufia rubraPF02-2 and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2019957.

[0015] The red-brown tubercle bacterium Tubeufia rubra PF02-2 described in this invention was isolated by the Biochemical Engineering Center of Guizhou University. It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on November 20, 2019, with accession number CCTCC NO: M 2019957.

[0016] The source of Tubeufia rubra PF02-2 is as follows:

[0017] Sampling time: May 14, 2016;

[0018] Sampling location: Pingfeng Rainforest Nature Reserve, Fangchenggang City, Guangxi Zhuang Autonomous Region;

[0019] Sampling method: Rotten wood was collected from Pingfeng Rainforest Nature Reserve in Fangchenggang City, Guangxi Zhuang Autonomous Region, and brought back to the laboratory in sealed plastic bags.

[0020] The *Tubeufia rubra* PF02-2 strain of the present invention has the following properties:

[0021] Colony morphology: On natural decaying wood substrate, colonies are flat, reticulate, or dotted, sometimes forming sheets when abundant. Freshly isolated pure PF02-2 colonies are colorless to white, while naturally dried pure PF02-2 colonies turn reddish-brown. Some mycelium is embedded beneath the substrate, but most is superficial. The mycelium consists of septate, branched hyphae, ranging in colorless to dark brown. Conidiophores are cylindrical, solitary, curved, septate, 50-150 μm long and 4.5-6 μm wide, tapering gradually at the apex, dark brown at the base and transparent to light brown at the apex, with a smooth surface. Conidiophores are solitary or multiple, cylindrical, with columnar teeth, sympodially growing from the middle to the apex of the conidiophore, 10-19 μm long and 3-4 μm wide, colorless to light brown, with a smooth surface. Molecular spores are spiral-shaped, solitary, apical and lateral, transparent, with a rounded tip, and coiled 2-3.5 times when tightly coiled, with a diameter of 35-50 micrometers. Conidiophores are 3-5 micrometers thick (average diameter 45 micrometers, thickness 4.5 micrometers), gradually unwinding in water. They have 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℃ for 2 weeks can reach 16 mm in size, are brown, round, with a rough surface, obvious protrusions, vein-like wrinkles, and intact colony edges.

[0022] Furthermore, the preparation method includes the following steps: liquid or solid fermentation culture of Tubeufia rubra PF02-2 to obtain fermentation product; extraction of fermentation product, and separation and purification of the obtained extract to obtain glycerol glycolipid compound PF60.

[0023] Specifically, the following steps are included:

[0024] S1. Activation of the strain: Take out the preserved strain, inoculate it on a basic culture medium plate, and incubate it statically until the third generation before scale-up culture.

[0025] S2. Fermentation culture: Take the activated strain from step S1 and inoculate it into a solid culture medium, and let it ferment at 26-30℃ for a period of time.

[0026] S3. Extraction: Take the bacterial cells along with the culture medium, add ethyl acetate for extraction, and concentrate the extract to obtain the fermentation product.

[0027] S4. Pretreatment of fermentation products: Dissolve the fermentation products in a 1:1 ratio of chloroform to methyl ether, then mix them with silica gel at a mass ratio of 1:1 to 2. After the solvent evaporates, use this as the first sample for column loading. Then, load the sample onto a separation column with silica gel powder and petroleum ether. Elute sequentially with petroleum ether, chloroform, ethyl acetate, and methanol. After recovering the eluent under reduced pressure using a rotary evaporator, dissolve the eluent in chloroform, acetone, or methanol. Develop the sample using a thin-layer chromatography plate with a developing solvent. Select the liquid that fluoresces at 254 nm or 365 nm under UV-Vis light and develop the color with 8% sulfuric acid-ethanol-vanillin reagent. Combine the ethyl acetate eluents and recover the ethyl acetate solvent to obtain ethyl acetate extract.

[0028] S5. Purification and separation: a. Dissolve the ethyl acetate extract in methanol solvent, mix it with silica gel at a mass ratio of 1:1 to 3, and wait for the solvent to evaporate before loading the sample onto the pre-column. Use 10% methanol-water to balance the reversed-phase medium-pressure column, add the sample-containing pre-column, and elute with methanol-water in 10 gradients. After recovering the solvent from the eluent using a rotary evaporator, dissolve it in methanol and then use a thin-layer chromatography plate. Develop the solution using the developing solvent, and select the liquid that fluoresces at 254 nm or 365 nm under a UV-Vis spectrophotometer. Combine the components that turn black with 8% sulfuric acid-ethanol-vanillin colorimetric reagent to obtain the Fr.14 component.

[0029] b. Dissolve component Fr.14 in methanol solvent, then mix it with silica gel at a mass ratio of 1:1 to 3. After the solvent evaporates, load the sample onto the column. Weigh the silica gel powder and mix it with a 1:1 ratio of chloroform to formaldehyde solvent, then load it into the separation column. Use a chloroform system for gradient elution. After recovering the solvent from the eluent using a rotary evaporator, dissolve it in methanol and then use a thin-layer chromatography plate. Develop the eluent using the developing solvent. Select the liquid that fluoresces at 254 nm or 365 nm under a UV-Vis spectrophotometer. Combine the components that turn black with 8% sulfuric acid ethanol vanillin colorimetric reagent to obtain component Fr.14-8.

[0030] c. Fr.14-8 was dissolved in a mixed solvent of dichloromethane and methanol, and then mixed with silica gel at a mass ratio of 1:1 to 3. After the solvent evaporated, the sample was loaded onto a pre-column. The sample was then loaded onto a reverse-phase medium-pressure column using 75% methanol-water. The pre-column containing the sample was added, and the sample was eluted sequentially with methanol-water at five gradients. The eluent was recycled by rotary evaporator, dissolved in methanol, and then developed using a thin-layer chromatography plate. The fractions that showed a gray-black color with 8% sulfuric acid, ethanol, and vanillin were combined to obtain the glycerol glycolipid compound PF60.

[0031] In step S2, the solid culture medium is oat culture medium, which is obtained by mixing 200g of oats with 150mL of double-distilled water.

[0032] The compounds of this invention can be isolated from the fermentation products of *Tubeufia rubra* PF02-2 in high concentrations, making them more suitable for industrial application. Simultaneously, studies have found that the glycerol glycolipid compound PF60, at concentrations of 5, 10, and 20 μg / mL, when used in combination with doxorubicin, exhibits MCF-7 / ADR reversal activity, IC50... 50 The values ​​were 29.913±4.829, 26.092±2.717, and 22.609±2.476 μg / mL, respectively. Its activity was dose-dependent on the concentration of the glycerol glycolipid compound PF60. Attached Figure Description

[0033] Figure 1 Flowchart for the separation and purification of PF60, a glycerol glycolipid compound;

[0034] Figure 2 This is the mass spectrum of PF60, a glycerol glycolipid compound in this invention;

[0035] Figure 3 The glycerol glycolipid compound PF60 in this invention 1 H-NMR spectrum;

[0036] Figure 4 DEPT of PF60, a glycerol glycolipid compound in this invention, and 13 C-NMR spectrum;

[0037] Figure 5 This provides the initial screening data for the cytotoxic activity of the glycerol glycolipid compound PF60 in this invention.

[0038] Figure 6 This is the original screening data for the reversal of MCF-7 / ADR activity of the glycerol glycolipid compound PF60 in this invention;

[0039] Figure 7 This is a schematic diagram illustrating the inhibition rate of the glyceroglycolipid compound PF60 on tumor cells MCF-7 / ADR in this invention;

[0040] Figure 8 The IC50 values ​​of doxorubicin at different concentration gradients, when combined with the glycerol glycolipid compound PF60 at concentrations of 5, 10, and 20 μg / mL, were compared with those of MCF-7 / ADM. 50 Values. ns: P > 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Detailed Implementation

[0041] The following detailed description illustrates the specific implementation method:

[0042] 1. Activation of the strain

[0043] Take the bacterial strain preserved on a glycerol slant from a -80℃ freezer, use a sterile inoculation loop to scoop out one loopful of strain Tubeufiarubra PF02-2, and streak it onto an 11cm diameter basal medium plate. Incubate at 28℃ for 17 days, and then subculture to the third generation for scale-up.

[0044] 2. Fermentation culture

[0045] Oat solid-state fermentation (200g of oats and 150mL of double-distilled water were dispensed into 1L Erlenmeyer flasks), with each flask containing an inoculum of 1×1cm² on a culture plate. 2 The activated bacterial culture was cultured statically at 28°C for 105 days. The bacterial cells, along with the oat culture medium, were extracted three times with ethyl acetate, each extraction being performed 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. The above operation was repeated, and the combined fermentation product yielded 2027.17 g.

[0046] 3. Pretreatment of fermentation products

[0047] Dissolve 2027.17g of fermentation product in acetone using a chloroform:methanol (dichloromethane:methanol) solvent at a ratio of 1:1. Mix this solution with silica gel at a mass ratio of approximately 1:1.5 (i.e., 3041g of 200-300 mesh silica gel powder to 2027.17g of fermentation product). After the solvent evaporates, a sand-like sample is obtained; this sample is used as the first column loading sample. Weigh 6000g of 200-300 mesh silica gel powder and mix it thoroughly with petroleum ether solvent (avoiding the generation of air bubbles during this process). Pack this mixture into a 1.5m long, 200mm inner diameter separation column. Allow the silica gel powder to slowly settle until it stops settling. Add the first column loading sample. Elute with four gradients: petroleum ether, chloroform, ethyl acetate, and methanol, with 2-3 elutions per gradient (approximately every...). Elution volume was 36-54 mL of elution solvent per column. Each 1000 mL elution solvent was collected as a fraction. Each elution sample was recovered under reduced pressure using a rotary evaporator, dissolved in 10 or 15 mL of chloroform, acetone, or methanol, and transferred to a 20 mL vial. 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, or ethyl acetate:methanol = 5:1 developing solvents. Fluorescence was observed at 254 nm or 365 nm under standard UV-Vis spectrophotometry. The sample was then developed with 8% sulfuric acid ethanol vanillin reagent. The ethyl acetate eluents were combined, and the ethyl acetate solvent was recovered to obtain 61.4 g of ethyl acetate.

[0048] 4. Purification and separation

[0049] Ethyl acetate extract (61.4 g) was dissolved in methanol and mixed with silica gel at a mass ratio of approximately 1:1.5 (i.e., RP-18 reversed-phase silica gel at medium pressure per 100 g of fermentation product). After the solvent evaporated, a sand-like sample was obtained, which was used as the column loading sample. A pre-column with a length of 10 cm and a diameter of 49 mm was added to the pre-column. The reversed-phase medium-pressure column (column length 460 mm, diameter 49 mm) was equilibrated with 10% methanol-water solution for approximately 5-6 column volumes (approximately 5-6 L of elution). Then, the pre-column containing the sample was added, and gradient elution with methanol-water solution (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) was performed sequentially for 10 cycles. Gradient elution was performed, with each gradient elution consisting of 4-5 column volumes. The eluent was collected in 500 mL Erlenmeyer flasks. After solvent recovery from each eluent fraction using a rotary evaporator, it was dissolved in 10 mL of methanol and transferred to 20 mL vials. The eluent 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 solvents. Fluorescence was observed at 254 nm or 365 nm under standard UV-Vis spectrophotometry. The fraction that showed a black color with 8% sulfuric acid ethanol vanillin was then combined (this is the fraction eluted with 80% methanol and water) to obtain fraction 14 (Fr.14 9.23 g).

[0050] Component Fr.14 (9.23 g) was dissolved in methanol and then mixed with 200-300 mesh silica gel at a mass ratio of approximately 1:1.5 (i.e., 14 g of the 9.23 g component was added). After the solvent evaporated, a sand-like sample was obtained, which was used as the column sample. 300 g of 200-300 mesh silica gel powder was weighed and mixed thoroughly with a 1:1 ratio of dichloromethane to methanol (no air bubbles should be generated during this process). This mixture was then loaded into a 460 mm long, 100 mm inner diameter separation column. The silica gel powder was allowed to slowly settle until it stopped settling. The column sample was then added once, and a gradient elution system of dichloromethane to methanol (50:1, 30:1, 15:1, 10:1, 5...) was used. Elute using a 1:1 or 2:1 ratio, for each gradient, elute 3-4 column volumes (approximately 1.8 L-2.4 L). Collect the eluent in a 150 mL Erlenmeyer flask. After recovering the solvent from each eluent using a rotary evaporator, dissolve it in approximately 10 mL of methanol and transfer it to a 20 mL vial. Develop the eluent by TLC using chloroform:acetone = 2:1, chloroform:methanol:formic acid = 5:1:1 drops, or ethyl acetate:methanol = 3:1 as the developing solvent. Observe for fluorescence at 254 nm or 365 nm under standard UV-Vis spectrophotometry. Then develop the eluent with 8% sulfuric acid ethanol vanillin. Combine the fractions that show a black color with 8% sulfuric acid ethanol vanillin to obtain fraction 8 (Fr. 15-8 104 mg).

[0051] Fr.14-8 (104 mg) was dissolved in a 1:1 mixture of dichloromethane and methanol. This solution was then mixed with RP18 reversed-phase silica gel at a mass ratio of approximately 1:1.5 (i.e., 156 mg of reversed-phase silica gel for every 104 mg of the component). After solvent evaporation, a sand-like sample was obtained, which was used as the loading sample for column chromatography. A 10 cm long, 20 mm diameter pre-column was added to the pre-column. The reversed-phase medium-pressure column (260 mm long, 20 mm diameter) was equilibrated using 75% methanol-water for approximately 5-6 column volumes (approximately 400-500 mL of elution). The pre-column containing the sample was then added, and a methanol-water gradient elution was performed (75%, 80%, 85%, 90%, 95%, 100%), with 5 gradients eluted sequentially, each gradient for 4-5 column volumes. The eluent was collected in 100 mL Erlenmeyer flasks. Each eluent was then subjected to a rotary evaporator to recover the solvent. After being dissolved in 10 mL of methanol and transferred to a 20 mL vial, the solution was spotted by TLC and developed using chloroform:acetone = 5:1, chloroform:methanol = 30:1, and ethyl acetate:methanol = 25:1. The mixture was combined with 8% sulfuric acid ethanol vanillin reagent to produce a gray-black color, yielding compound (2S)-2,3-O-dioctadeca-9Z,12Z-dienoylglyceryl-6'-O-(α-D-galactopyranosyl)-β-D-galactopyranosid e (53 mg).

[0052] IV. PF60 Spectral Data of Glycerol Glycolipids

[0053]

[0054] Glycerol glycolipid compound PF60:

[0055] (2S)-2,3-O-dioctadeca-9Z,11Z-dienoylglyceryl-6′-O-(α-D-galactopyranoside(PF60):HRESIMS m / z 963.59937[M+Na] + Its molecular weight is C 51 H 88 O 15 Na; 1 HNMR (500MHz, CD3OD)δ H5.30-5.37(8H,m,H-9′″,9″″,10′″,10″″,12′″,12″″,13′″,13″″),5.24(1H,m,H-2),4.86(1H,br s,H-1″),4.43(1H,dd,J=12.1,2.9Hz,H-3β),4.23(1H,d,J=7.2Hz,H-1′),4.22(1H,dd,J=12.0,6.6Hz,H-3α),3.93(1H,dd,J=10.9,5.4Hz,H-6′β),3.81-3.91(4H,overlap,H-1β,2″,4″,5″),3.78(1H,dd,J=10.1,3.7Hz,H-4′),3.69-3.74(5H,overlap,H-5′,3″,6′α,6″),3.67(1H,dd,J=10.0,6.2Hz,H-1α),3.46-3.51(2H,overlap,H-2′,3′),2.77(4H,t,J=6.5Hz,H-11′″,11″″),2.32(2H,t,J=7.1Hz,H-2′″),2.31(2H,t,J=7.2Hz,H-2″″),2.06(8H,m,H-8′″,14′″,8″″,14″″),1.60(4H,m,H-3′″,3″″),1.29-1.37(28H,m,H-4′″~7′″,15′″~17′″,4″″~7″″,15″″~17″″),0.90(6H,t,J=6.8Hz,H-18′″,18″″); 13 C NMR(126MHz,CD3OD)δ C175.0(s,C-1″″),174.7(s,C-1′″),131.0(d,C-9′″,9″″),130.9(d,C-13′″,13″″),129.1 (d,C-10′″,10″″),129.0(d,C-12′″,12″″),105.3(d,C-1′),100.6(d,C-1″),74.7(d,C-3′ ),74.6(d,C-5′),72.5(d,C-5″),72.4(d,C-2′),71.7(d,C-2),71.5(d,C-3″),71.1(d,C-4 ″),70.2(d,C-4′),70.0(d,C-2″),68.7(t,C-6′),67.8(t,C-1),64.0(t,C-3),62.8(t,C-6 ″),35.1(t,C-2″″),35.0(t,C-2′″),32.7(t,C-16′″,16″″),30.8-30.2(t,C-4′″~7′″,15′ ″,4″″~7″″,15″″),28.2(t,C-8′″,8″″,14′″,14″″),26.6(t,C-11′″,11″″),26.0(t,C-3′″ ,3″″),23.6(t,C-17′″,17″″),14.5(q,C-18′″,18″″); The above data are consistent with the data of compound (2S)-2,3-O-dioctadeca-9Z,12Z-dienoylglyceryl-6'-O-(α-D-galactopyranosyl)-β-D-galactopyranosid e reported in the literature.

[0056] V. Screening for PF60 cytotoxic activity of glycerol glycolipid compounds

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

[0058] 5.2 RPMI 1640 + 10% fetal bovine serum

[0059] 5.3 Cell Culture

[0060] 5.3.1 Cell resuscitation

[0061] Remove the cells from the liquid nitrogen tube and quickly thaw them in a preheated 37°C water bath, shaking constantly to ensure rapid thawing. Once approximately 1 mL of liquid in the cryovial has completely thawed, aseptically remove the cells and seed them into cell culture dishes (RPMI 1640 + 10% fetal bovine serum). Incubate at 37°C in a CO2 incubator. Change the culture medium the next day and continue culturing, observing the growth.

[0062] 5.3.2 Cell passage

[0063] Once the cells have grown to 80-90% confluence, under aseptic conditions, use a 3mL plastic pipette to aspirate the cell culture medium. Wash once with 1-2mL of PBS (free of calcium and magnesium ions). Add 1mL of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture flask. Observe the cell digestion under an inverted microscope. If most of the cells become rounded, quickly return the flask to the work surface, gently tap it a few times, and add 2mL of complete culture medium to stop the digestion. Add 4mL of fresh complete culture medium to each new culture flask, and then add 1mL of complete culture medium containing the cells.

[0064] 5.4 CCK-8 assay for cytotoxic activity

[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 Procedure

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

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

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

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

[0073] (5) Measure the absorbance at 490 nm using an ELISA reader.

[0074] 5.4 Experimental Results

[0075] The inhibitory effect of compound (2S)-2,3-O-dioctadeca-9Z,11Z-dienoylglyceryl-6′-O-(α-D-galactopyranoside (PF60)) on MCF-7 / ADR was tested using the CCK-8 assay. The results showed that at concentrations not exceeding 25 μg / mL, the inhibition rate against doxorubicin-resistant breast cancer cells (MCF-7 / ADR) was less than 20%, allowing for further screening to reverse tumor cell reversal. For detailed results, please refer to [link to results]. Figure 7 .

[0076] VI. Application of PF60, a glycerol glycolipid compound, in reversing the activity of MCF-7 / ADR tumor cells.

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

[0078] 6.2 RPMI 1640 + 10% fetal bovine serum

[0079] 6.3 Cell Culture

[0080] 6.3.1 Cell resuscitation

[0081] Remove the cells from the liquid nitrogen tube and quickly thaw them in a preheated 37°C water bath, shaking constantly to ensure rapid thawing. Once approximately 1 mL of liquid in the cryovial has completely thawed, aseptically remove the cells and seed them into cell culture dishes (RPMI 1640 + 10% fetal bovine serum). Incubate at 37°C in a CO2 incubator. Change the culture medium the next day and continue culturing, observing the growth.

[0082] 6.3.2 Cell passage

[0083] Once the cells have reached 80-90% confluence, under aseptic conditions, use a 3mL plastic pipette to aspirate the cell culture medium. Wash once with 1-2mL of PBS (free of calcium and magnesium ions). Add 1mL of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture flask. Observe the cell digestion under an inverted microscope. If most of the cells become rounded, quickly return the flask to the work surface, gently tap it a few times, and add 2mL of complete culture medium to stop the digestion. Add 4mL of fresh complete culture medium to each new culture flask, followed by 1mL of complete culture medium containing the cells.

[0084] 6.4 CCK-8 assay for reversing tumor cytotoxicity

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

[0086] (2S)-2,3-O-dioctadeca-9Z,11Z-dienoylglyceryl-6′-O-(α-D-galactopyranoside (PF60) concentration: 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, and adjustment of cell concentration to 2×10⁻⁶ 4 per mL.

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

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

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

[0094] (5) Measure the absorbance at 490 nm using an ELISA reader.

[0095] 6.4 Experimental Results

[0096] (2S)-2,3-O-dioctadeca-9Z,11Z-dienoylglyceryl-6′-O-(α-D-galactopyranoside(PF60) reverses MCF-7 / ADM activity. Figure 8 And as shown in Table 1. The IC50 values ​​of this drug in combination with doxorubicin at three concentrations were 29.913±4.829, 26.092±2.717, and 22.609±2.476, respectively. Compared with the negative control, its IC50 values ​​were... 50 The values ​​were all lower than those of the negative control (47.637±5.157), and the one-way ANOVA results showed statistically significant differences (P<0.001 and P<0.0001, respectively), revealing that the compound had significant reversal activity at all three concentrations. Compared with the positive control, the IC50 values ​​of the positive control at concentrations of 5, 10, and 20 μg / mL were significantly lower. 50The values ​​were 26.766±1.687, 23.322±1.571, and 20.645±1.491, respectively, and their IC values ​​were... 50 The comparison results showed that the IC50 values ​​of the compound and the positive control were similar when used in combination with doxorubicin under the same concentration conditions. 50 The values ​​were not as low as those of the positive control, revealing that the compound's activity was not as good as the positive control under the same concentration conditions.

[0097] Table 1: Compound PF60 reversed the A549 / ADR activity of tumor cells at concentrations of 5, 10, and 20 μg / mL.

[0098]

[0099] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of a glycerol glycolipid compound PF60 as the sole active ingredient in the preparation of tumor drug resistance reversal agents or tumor drug sensitizers, wherein the structure of the glycerol glycolipid compound PF60 is shown in the following formula: The drug resistance or tumor drug is doxorubicin, and the tumor is breast cancer.

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