CN113773216B - Long-chain fatty acid glycerol alcohol compound Rubracin A, preparation method and application thereof - Google Patents
Long-chain fatty acid glycerol alcohol compound Rubracin A, preparation method and application thereof Download PDFInfo
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Abstract
The application relates to a long-chain fatty acid glycerol alcohol compound Rubracina in the technical field of microorganisms, and the structure is shown as the following formula:
the compound is obtained by fermenting and extracting red palm Mao Tongqiang bacteria, the red palm Mao Tongqiang bacteria is named as red brown hair tube cavity bacteria Tubeufia rubra PF02-2, and the preservation unit is as follows: china center for type culture Collection, the preservation number is CCTCC NO: m2019957. The compound has the application of preparing tumor drug resistance reversal agent or tumor drug sensitizer.
Description
Technical Field
The invention relates to the technical field of microorganisms, and in particular relates to a long-chain fatty acid glycerol alcohol compound Rubracin A, a preparation method and application thereof.
Background
Cancer has become one of the serious health and life threatening diseases for human beings. The treatment of tumor mainly comprises chemotherapy, operation, radiotherapy and the like, and the chemotherapy is one of the main means for treating cancer. During chemotherapy, the development of drug resistance by tumor cells is the leading cause of chemotherapy failure. Therefore, the search for a reversal agent with low degree and good activity is the most fundamental way to solve the tumor drug resistance, and has main research value.
P-glycoprotein (P-gp) is one of the most representative proteins of the ABC transporter family, has a molecular weight of 170kD and consists of 1280 amino acid residues. Research shows that P-gp can transport medicine with diverse chemical properties and structures, including part of anticancer medicines, such as adriamycin, taxanes and the like, to cause multidrug resistance (MDR) phenomenon, thereby causing failure of cancer treatment. Thus, the study of P-gp inhibitors and substrates is of great interest for cancer therapy, and the co-administration of P-gp inhibitors with chemotherapeutic agents is an effective strategy to overcome MDR. Currently, several generations of P-gp inhibitors have been developed, the first generation reversal agents including tamoxifen, cyclosporin a, etc., of which verapamil and cyclosporin are typical representatives. However, such drugs often lack the specificity of P-glycoprotein and can cause serious side effects, and the first generation reversal agents are also limited to a large extent clinically (Sato w.et al.1991). Second generation reversal agents stavasporidide (valspodar, PSC 833), dexverapamil (dexverapamil), etc., of which dexamethasone is representative, however, the development of second generation reversal agents is limited due to a series of side effects resulting from high toxicity and drug interactions (Rowinsky E.K. et al 1998; hyafil F.et al 1993; keller R.P.et al 1992). The main representatives of the third generation P-glycoprotein inhibitors are Tariquidar (XR 9576), zosuquidar (LY 335979), S9788, etc., of which Tariquidar (XR 9576) 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 for the development of fourth generation inhibitors.
The natural products from the microorganisms are always important sources for developing innovative drugs, and provide a material basis for developing new drugs. Meanwhile, the microorganism has the advantages of short growth cycle, easy regulation and control of metabolism, easy breeding of strains, realization of industrial production through large-scale fermentation and the like, and further lays an important position in the research and development of new drugs. There are specific reports of the discovery of P-gp inhibitors from natural products of microbial origin, but it is not clear what is specifically the case.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a long-chain fatty acid glycerol alcohol new compound derived from microorganisms, a preparation method thereof and application thereof in preparing a medicine for reversing the activity of drug-resistant tumor cells.
One of the purposes of the invention is to provide a new compound Rubracin A of long-chain fatty acid glycerol, the structure of which is shown as the following formula:
the invention also aims to provide a preparation method of a long-chain fatty acid glycerol new compound Rubracin A, wherein the compound is obtained by fermenting and extracting red palm Mao Tongqiang bacteria, the red palm Mao Tongqiang bacteria is named as red palm pilus fungus Tubeufia Rubra PF02-2, and the preservation unit is as follows: china center for type culture Collection, the preservation number is CCTCC NO: m2019957.
The invention relates to a red palm Mao Tongqiang strain Tubeufia rubra PF02-2 which is obtained by separation from biochemical engineering center of Guizhou university, and the preservation unit is as follows: china center for type culture Collection, addresses: wuhan university, storage day: 2019.11.20 with preservation registration number of CCTCC NO: m2019957.
The source of Tubeufia rubra PF02-2 is as follows:
sampling time: 2016, 5 months, 14 days;
sampling site: a natural protection area of Guangxi Zhuang autonomous region for preventing rain forests at urban shelters and harbors;
sampling mode: collecting rotten wood in natural protection area of urban harbor-preventing city screen rain forest in Guangxi Zhuang autonomous region, and taking plastic sealing bag back to laboratory.
The strain of the red palm Mao Tongqiang strain Tubeufia rubra PF02-2 of the present invention has the following properties:
and (3) colony morphology characteristics: on the natural rotten wood substrate, bacterial colonies are flat, are in a net shape and a point shape, are connected into a sheet shape when the amount of the bacterial colonies is large, are colorless, transparent and white on fresh PF02-2 pure bacterial colonies obtained through separation, and are reddish brown after natural drying of the PF02-2 pure bacterial colonies obtained through separation. Part of the mycelium is buried under the substrate, but mostly is epibiotic, and the mycelium is composed of mycelium with membrane and branch, and is colorless to dark brown. The conidiophores are cylindrical, are single-grown, grow in a curved manner, have membranes, are 50-150 microns long, 4.5-6 microns wide, have tapered tops, are dark brown at the bottoms, are transparent to light brown at the tops, and have smooth surfaces. The spore-forming cells grow singly or multiply, are cylindrical, have cylindrical small odontoid processes, grow coaxially from the middle part to the top part of a molecular spore stalk, are 10-19 microns long and 3-4 microns wide, are colorless, transparent, light brown and have smooth surfaces. The molecular spore is of a spiral type, is single-grown, is top-lateral-grown, is transparent, has a round top end, is curled for 2-3.5 times in a tight spiral manner, has a diameter of 35-50 micrometers, is 3-5 micrometers thick (the average diameter is 45 micrometers, and the thickness is 4.5 micrometers), gradually loosens in water, has an unclear multi-diaphragm, is colorless to light brown, and has a smooth surface. Conidia start germination and growth after 12h in water-agar medium. The colony grows in PDA culture medium at 25-28 deg.C for 2 weeks to reach 16mm, and is brown, round, rough in surface, with obvious protrusions, and with pulse-like wrinkles and complete colony edge.
Specifically, the preparation method comprises the following steps: performing liquid or solid fermentation culture on the red palm Mao Tongqiang strain Tubeufia rubra PF02-2 to obtain a fermented product; extracting the fermentation product, and separating and purifying the obtained extract to obtain the long-chain fatty acid glycerol alcohol compound Rubracin A.
The preparation method specifically comprises the following steps:
s1, strain activation: taking out the preserved strains, inoculating the strains on a basal medium plate, performing static culture for passage to the third generation, and performing amplification culture;
s2, fermentation culture: inoculating the activated strain obtained in the step S1 into a solid culture medium, and standing, fermenting and culturing for a period of time at the temperature of 26-30 ℃;
s3, extraction: taking the thalli and a culture medium, adding ethyl acetate for extraction, and concentrating the extract to obtain a fermentation product;
s4, pretreatment of fermentation products: dissolving a fermentation product by using a methyl =1:1 solvent, uniformly mixing the fermentation product with silica gel according to a mass ratio of 1:1-2, volatilizing the solvent to be used as a primary column sample, then, loading the sample with silica gel powder and a petroleum ether separation column, sequentially and gradiently eluting by using petroleum ether, chloroform, ethyl acetate and methanol, decompressing and recovering an elution solvent by using a rotary evaporator, dissolving the elution solvent by using chloroform, acetone or methanol, spreading the elution solvent by using a developing agent in a thin-layer chromatography dot plate, selecting a liquid with fluorescence of 254nm or 365nm under an ultraviolet visible light analyzer, and developing the color by using an 8% ethanol sulfate vanillin developer; mixing the methanol solvent eluent, and recovering the methanol solvent to obtain methanol extract;
s5, purification and separation: a. dissolving the methanol layer extract by using a methanol solvent, uniformly mixing the methanol layer extract with silica gel according to a mass ratio of 1:1-3, putting the mixture on a pre-column sample when the solvent volatilizes, carrying out balanced reversed-phase medium-pressure column by using 10% methanol water, adding the pre-column containing the sample, sequentially carrying out 10 gradient elutions by using methanol water, recovering the solvent from an eluent by using a rotary evaporator, dissolving the methanol, spreading the eluent by using a thin-layer chromatography dot plate by using a spreading agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining the gray and black components of an 8% ethanol sulfate vanillin developer to obtain a Fr.11 component;
b. dissolving the component Fr.11 in methanol solvent, mixing with silica gel in the mass ratio of 1:1-3, volatilizing the solvent and loading the mixture into a pre-column; adopting 30% methanol water to carry out equilibrium reversed phase medium pressure column, adding a pre-column containing a sample, adopting methanol water to sequentially carry out 8 gradient elution, recovering solvent from eluent by a rotary evaporator, dissolving the methanol, spreading by using a thin layer chromatography dot plate and a developing agent, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining components with 8% ethanol sulfate vanillin color developing agent to develop grey black to obtain Fr.11-6 components;
c. dissolving Fr.11-6 with methanol, uniformly mixing with silica gel according to the mass ratio of 1:1-3, volatilizing the solvent, taking a sample on a column, mixing with silica gel powder and a chlorine A =1:1 solvent, taking a separation column, and adopting chloroform: gradient elution is carried out such as A =1:1, elution liquid is combined by adopting a thin layer chromatography dot plate, and a component Fr.11-6-1 is obtained;
d. performing normal phase silica gel column chromatography on Fr.11-6-1, performing gradient elution with a gradient such as B: A =1:1, and combining TLC spot plates to obtain the compound Rubracin A.
Wherein the solid culture medium in the step S2 is an oat culture medium, and is obtained by mixing 200g of oat and 150mL of double distilled water.
The invention also aims to provide the application of the compound and the medicinal carrier in preparing a tumor drug resistance reversal agent or a tumor drug sensitizer.
Furthermore, the drug or tumor drug is adriamycin.
Further, the tumor includes breast cancer.
Furthermore, the tumor drug resistance reversal agent is a transport pump inhibitor, and the transport pump inhibitor has an inhibiting effect on one or more of drug-resistant protein P-glycoprotein and multidrug-resistant protein.
The fourth purpose of the invention is to provide the application of the compound and a medicinal carrier in preparing a medicament for resisting breast cancer cells.
Drawings
FIG. 1 is a flow chart of the separation and purification of a compound Rubracin A;
FIG. 2 is a mass spectrum of Rubracin A, a compound of the present invention;
FIG. 3 is a diagram of fragment ions of Rubracin A EI compound of the present invention;
FIG. 4 is an infrared spectrum of Rubracin A, a compound of the present invention;
FIG. 5 shows the preparation of Rubracin A, a compound of the present invention 1 H-NMR chart;
FIG. 6 is a DEPT diagram of Rubracin A, a compound of the present invention;
FIG. 7 is an HSQC spectrum of compound Rubracin A of the present invention;
FIG. 8 is an HMBC spectrum of a compound Rubracin A of the present invention;
FIG. 9 shows the cytotoxic activity of Rubracin A, a compound of the present invention.
Detailed Description
The following is further detailed by way of specific embodiments:
1. preparation method of compound Rubracin A
As shown in figure 1 of the drawings, in which,
s1, strain activation
Taking out the strain preserved on the glycerol slant from a refrigerator at minus 80 ℃, digging a strain of a 1-ring strain Tubeufia rubra by using a sterile inoculating loop, cross-streaking and inoculating the strain to a basal medium plate with the diameter of 11cm, standing and culturing at 28 ℃ for 17d, and subculturing to a third generation for amplified culture.
S2, fermentation culture
Fermenting oat solid, subpackaging 200g of oat and 150mL of double distilled water in a 1L Erlenmeyer flask, wherein the inoculation amount of each flask is 1 multiplied by 1cm of the area on a culture plate 2 The amount of the activated strain of (4) was cultured by standing at 28 ℃ for 105 days.
S3, extracting
Adding ethyl acetate into the thallus and the oat culture medium, extracting for three times, shaking and extracting for 24 hours at 160rpm each time, combining the extract liquor, concentrating under reduced pressure at 40 ℃ to obtain a fermentation product, repeating the above operations, and combining the fermentation products to obtain 2027.17g.
S4, pretreatment of fermentation product
Dissolving 2027.17g of fermentation product by using a methyl =1:1 solvent, uniformly mixing the solution with silica gel according to a mass ratio of 1.5 (namely 2027.17g of fermentation product is added with 3041g of silica gel powder with 200-300 meshes), and volatilizing the solvent to obtain a river sand-shaped sample which is used as a primary column sample; weighing 6000g of silica gel powder with 200-300 meshes and a petroleum ether solvent, uniformly mixing (bubbles cannot be generated in the process), loading the silica gel powder and the petroleum ether solvent into a separation column with the length of 1.5m and the inner diameter of 200mm, slowly sinking the silica gel powder until the silica gel powder does not sink, adding a column sample once, sequentially carrying out 4 gradient elutions by adopting petroleum ether, chloroform, ethyl acetate and methanol respectively, carrying out 2-3 (36L-54L of elutant per column volume) column volumes per gradient elutes, collecting one elution solvent per 1000mL, carrying out reduced pressure recovery on each elution sample by a rotary evaporator, dissolving the elution sample by using 10 or 15mL of chloroform, acetone or methanol, transferring the elution sample into a penicillin bottle with the specification of 20mL, carrying out Thin Layer Chromatography (TLC) to dot a plate, and using petroleum ether: chloroform =1:1, petroleum ether: acetone =10, chloroform: acetone =5:1, chloroform: methanol =10, ethyl acetate: developing with methanol =5:1 developing agent, observing whether fluorescence is present at 254nm or 365nm under a conventional ultraviolet visible light analyzer, and developing with 8% sulphuric acid ethanol vanillin developer; the methanol solvent eluents were combined, and the methanol solvent was recovered to obtain 35.77g of methanol extract.
S5, purification and separation
a. Dissolving the methanol layer extract (35.77 g) with methanol solvent, mixing with silica gel at a mass ratio of 1:2 (i.e. adding medium pressure RP-18 reverse phase silica gel into 35.77g of fermentation product), volatilizing the solvent to obtain river sand-like sample, and taking the sample as column sample; adding a pre-column with the length of 10cm and the diameter of 49mm to the upper column sample; the method comprises the steps of (1) balancing a reverse phase medium pressure column (with the column length of 460mm and the diameter of 49 mm) by using 10% methanol water, adding a sample-containing pre-column after balancing about 5-6 column volumes (eluting 5-6L), performing gradient elution by using methanol water (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), sequentially performing 10 gradient elution, eluting 5 column volumes by each gradient, receiving eluent by using a triangular flask with the specification of 500mL, recovering a solvent by using a rotary evaporator for each eluent, dissolving and transferring the eluent into a penicillin bottle with the specification of 20mL by using 10mL methanol, performing TLC (thin layer chromatography) spotting, and using petroleum ether: acetone =2:1, chloroform: acetone =5:1, chloroform: methanol =10: 1. ethyl acetate: developing with methanol =2:1 developing agent, observing whether fluorescence is present at 254nm or 365nm under a conventional ultraviolet visible light analyzer, developing with 8% ethanol sulfate vanillin developer, and combining the components (also the components eluted with 90% methanol water) of 8% ethanol sulfate vanillin which are developed into gray black to obtain Fr.11 component (8.7 g).
b. Dissolving the component Fr.11 (8.7 g) with a methanol solvent, uniformly mixing with silica gel according to a mass ratio of 1:2 (namely adding 17.4g of medium-pressure RP-18 reversed phase silica gel in 8.7g of the component), volatilizing the solvent to obtain a river sand-shaped sample, and taking the sample as a column sample; adding the upper column sample into a pre-column with the length of 10cm and the diameter of 26 mm; using 30% methanol water to balance a reversed phase medium pressure column (the length of the column is 460cm, the diameter is 49 mm), balancing about 5-6 column volumes (eluting 5-6L), adding a pre-column containing samples, using methanol water to perform gradient elution (30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), sequentially performing 8 gradient elution, each gradient elution is 4-5 column volumes, using a triangular flask with the specification of 500mL to receive eluent, using 10mL methanol to dissolve and transfer each eluent into a penicillin bottle with the specification of 20mL after a rotary evaporator to recover solvent, and then using a TLC point plate to perform chromatography, using petroleum ether: acetone =2:1, chloroform: acetone =5:1, chloroform: methanol =10: 1. ethyl acetate: developing with developing agent such as methanol =2:1, observing whether fluorescence is present at 254nm or 365nm under a conventional ultraviolet visible light analyzer, developing with 8% ethanol sulfate vanillin developer, and mixing the components (also the components eluted with 90% methanol water) of 8% ethanol sulfate vanillin with gray black color to obtain Fr.11-6 components (206 mg).
c. Fr.11-6 (206 mg) is dissolved by methanol, and is uniformly mixed with silica gel according to the mass ratio of 1:1.5 (namely, 200-300 silica gel 310mg is added into 206mg components), and a river sand-shaped sample is obtained after the solvent is volatilized and is used as a column sample; weighing 12g of 200-300-mesh silica gel powder and a chlorine A =1:1 solvent, uniformly mixing (no air bubbles can be generated in the process) and putting into a separation column with the length of 260mm and the inner diameter of 15mm, slowly sinking the silica gel powder until the silica gel powder does not sink, adding a sample on the column once, and adopting chloroform: and (3) performing gradient elution such as methyl =1:1, collecting eluent by using a penicillin bottle with the specification of 20mL, and combining the eluent by using a TLC spot plate to obtain a component Fr.11-6-1 (35 mg).
d. Fr.11-6-1 (35 mg) was subjected to normal phase silica gel column chromatography, gradient elution was performed with B =1:1, and TLC plates were combined to give Rubracin A, 10mg.
2. Compound Rubracin A structure identification
Structure of compound Rubracin A
Rubracin A is colorless oil, is easily dissolved in methanol, acetone, DMSO, etc., and has IR spectrum (figure 4) 9) at 3427cm -1 、1736cm -1 1626 shows that the compound has hydroxyl and ester groups. HRESI (detailed in figure 2) shows molecular weight 520.3611[ M ] +Na ]] + Molecular formula is C 28 H 51 NNaO 6 Calculating the degree of unsaturation to be 4; 13 c NMR in combination with DEPT (see FIG. 6 for details) concluded that the compound has two ester groups [ delta ] C 171.8,175.4]One unsaturated fatty chain [ delta ] C 14.4(q),23.6(t),26.0(t),26.5(t),28.1(t),30.2～30.7(t),32.7(t),34.9(t),129.0(d),129.1(d),130.9(d),130.9(d)]5 heteroatom-attached carbon signals [66.5 (t), 68.5 (t), 69.5 (d), 73.3 (t), 77.6 (d)]3 methyl groups attached to a heteroatom [52.3 (q) ]]And finally 1 methylene group [29.0 (t)]； 1 H NMR (FIG. 5) in combination with HSQC (FIG. 7) showed that the compound had one aliphatic chain [0.90 (3H, t, J =6.9 Hz), 1.28-1.32 (m), 1.61 (2H, m), 2.06 (4H, m), 2.35 (2H, t, J = 7.5Hz), 2.77 (2H, t, J = 6.4Hz), 5.28-5.38 (4H, m)]The 8-heteroatom-connected hydrogen proton signals [3.49 (2H, dd, J =13.3, 5.2Hz), 3.53 (1H, td, J =12.6, 4.1Hz), 3.65 (1H, m), 3.75 (1H, dd, J =11.4,2.7 Hz), 3.94 (1H, m), 4.06 (1H, dd, J =11.4, 6.3Hz), 4.16 (1H, dd, J =11.4,4.4 Hz)]1 methylene group [2.22 (1H, m), 2.06 (1H, m)]3 methyl signals [3.20 (9H, s)]。
Compound Rubracin A major HMBC correlation
Detailed 1D NMR data are detailed in Table 1.
TABLE 1 Compound Rubracin A carbon spectra data
On HMBC (see FIG. 8 for details), the hydrogen proton signal delta H [4.06(1H,dd,J＝11.4,6.3Hz),4.16(1H,dd,J＝11.4,4.4)]And delta C 175.4,73.3,69.5; delta. For the preparation of a coating H [3.94(1H,m)]And delta C 66.5,73.3; delta. For the preparation of a coating H [3.94(1H,m)]And delta C 66.5,73.3; delta H [3.49(2H,dd,J＝13.3,5.2Hz)]And delta C 66.5,68.5; the above correlation demonstrates that the compound contains a glycerol moiety and delta C 66.5 (t, C-1) isAttached to the ester group delta C [175.4(s,C-1′)]Upper, delta C 69.5 attachment to C-1, delta C 73.3 attachment to C-2, 68.5 (t, C-4)]With glycerol delta H [3.49 (2H, dd, J =13.3,5.2Hz,73.3 (t, C-3) forms a structural fragment of an ether ] furthermore, the hydrogen proton signal on unsaturated fatty chains [2.35 (2H, t, J =7.5 Hz), 34.9 (t, C-2')]In relation to 175.4,26.0, it was demonstrated that the ester group is attached to an unsaturated fatty chain. Finally, delta H [3.53(1H,td,J＝12.6,4.1Hz),3.65(1H,m),]And delta C 73.3,29.0,77.6; delta H [2.22(1H,m)]And delta C 77.6,171.8; delta H [2.06(1H,m)]And delta C 77.6,171.8, 68.5; delta H 3.75[(1H,dd,J＝11.4,2.7Hz),77.6(d,C-6)]And delta C 171.8,68.5,29.0,52.3; the above correlation demonstrated 77.6 (d, C-6) with the remaining ester group delta C 171.8 (s, C-7) ligation. Combining the above HMBC correlations, a molecular formula C is given in combination with high resolution mass spectrometry 28 H 51 NNaO 6 And finally determining the structure of the compound. Careful analysis 1 H- 1 H COSY, the results confirm that the above presumed structure is correct.
Compound Rubracin A 1 H- 1 H COSY
EI-MS (see FIG. 8 for details) showed fragment ion M/z 438 ([ M-C) 2 H 3 O] - )，117([M-C 2 H 3 O-C 18 H 31 O] - ) The fragment ions are detailed in the figure below. Fragment ion peak m/z 262 proves that the compound has linoleic acid structural fragments, and EI-MS results show that the compound Rubracin A structure is correct.
Compound Rubracin AEI-MS fragment ion diagram
3. Screening of cytotoxic Activity of Compound Rubracin A
3.1 test cell lines: MCF-7/ADR (purchased from Shanghai Meixuan Biotech Co., ltd. At 5 months 2021)
3.2 RPMI1640+10% fetal bovine serum
3.3 cell culture
3.3.1 cell Resuscitation
Taking out the cells from the liquid nitrogen tube, quickly putting the cryopreservation tube into a water bath kettle which is preheated to 37 ℃ for quick thawing, and continuously shaking to quickly thaw the liquid in the tube. After about 1mL of the liquid in the vial was completely dissolved, the cells were taken out under aseptic conditions and inoculated into a cell culture dish (RPMI 1640+10% fetal bovine serum), and placed at 37 ℃ in CO 2 Culturing in incubator, changing culture solution the next day, continuing culturing, and observing growth condition
3.3.2 cell passages
After 80-90% of cells have grown, using a plastic pipette with a 3mL standard under aseptic conditions to aspirate the cell culture medium, adding 1-2mL PBS to rinse 1 time (without calcium and magnesium ions), adding 1mL digestive juice (0.25%/trypsin-0.53 mM EDTA) into the flask, observing the digestion of the cells under an inverted microscope, rapidly returning to the operating table if most of the cells have become round, tapping several times on the flask, and adding 2mL complete medium to stop digestion. In the new culture flask, 4mL of the complete culture medium was added, and 1mL of the complete culture medium containing the cells was added.
3.4 CCK-8 assay for cytotoxic Activity
3.4.1 concentration gradient: 0. 6.25, 12.5, 25, 50, 100, 200, 400ug/mL,3 replicates
Positive control: adriamycin
Negative control: DMSO (dimethylsulfoxide)
3.4.2 Experimental procedure
(1) Cell digestion, cell counting, cell concentration adjustment to 2X 10 4 one/mL.
(2) 100uL of cell suspension was seeded in 96-well plates. The plates were incubated at 5% CO 2 Culturing in an incubator at 37 ℃ for 24h.
(3) According to the grouping, compounds with different concentrations and adriamycin are respectively added, and the mixture is continuously placed in an incubator to be incubated for 48 hours at the temperature of 37 ℃.
(4) After the culture is finished, washing the culture medium for 1 time by PBS (without calcium and magnesium ions), adding 10uL of CCK-8 reagent into each hole, and placing the culture medium in an incubator for incubation for 3 hours.
(5) Absorbance at 490nm was measured with a microplate reader.
3.4 results of the experiment
As shown in FIG. 9, the results show that the compound Rubracin A has no cytotoxic activity to MCF-7/ADR, and can be used for continuously carrying out the screening of the reversal tumor cells.
4. Application of compound Rubracin A in activity of reversing drug resistance of MCF-7/ADR tumor cells
4.1 test cell lines: MCF-7/ADR (purchased from Shanghai Meixuan Biotech Co., ltd. At 5 months 2021)
4.2 RPMI1640+10% fetal bovine serum
4.3 cell culture
4.3.1 cell Resuscitation
Taking out the cells from the liquid nitrogen tube, quickly putting the freezing tube into a water bath kettle preheated to 37 ℃ for quick thawing, and continuously shaking to quickly melt the liquid in the tube. After about 1mL of the liquid in the cryopreservation tube was completely dissolved, the cells were taken out under aseptic conditions and inoculated into a cell culture dish (RPMI 1640+10% fetal bovine serum), and placed in a CO atmosphere at 37 ℃ 2 Culturing in an incubator, replacing the culture solution the next day, continuously culturing, and observing the growth condition.
4.3.2 cell passages
After the cells grow to 80-90%, sucking out the cell culture solution by using a plastic straw with the specification of 3mL under the aseptic operation condition, adding 1-2mL of PBS to wash for 1 time (without calcium and magnesium ions), adding 1mL of digestive juice (0.25%/trypsin-0.53 mM EDTA) into the culture bottle, observing the cell digestion condition under an inverted microscope, if most of the cells become round, quickly taking back the operation table, tapping several times of the culture bottle, and adding 2mL of complete culture medium to stop the digestion. In the new culture flask, 4mL of the complete culture medium was added, and 1mL of the complete culture medium containing the cells was added.
4.4 CCK-8 assay reverses tumor cytotoxic activity
4.4.1 doxorubicin concentration gradient: 0. 6.25, 12.5, 25, 50, 100, 200, 400ug/mL,3 replicates
Rubracin A concentration: 5. 10, 20ug/mL
Positive control: verapamil
Negative control: DMSO (dimethylsulfoxide)
4.4.2 Experimental procedures
(1) Cell digestion, cell counting, cell concentration adjustment to 2X 10 4 one/mL.
(2) 100uL of cell suspension was seeded in 96-well plates. The plates were incubated at 5% CO 2 Culturing in an incubator at 37 ℃ for 24h.
(3) According to the grouping, compounds with different concentrations and adriamycin are added respectively, and the mixture is further placed in an incubator to be incubated for 48 hours at 37 ℃.
(4) After the culture is finished, washing with PBS (without calcium and magnesium ions) for 1 time, adding 10uL of CCK-8 reagent into each hole, and placing in an incubator for incubation for 3h.
(5) Absorbance at 490nm was measured with a microplate reader.
4.4 results of the experiment
The results are shown in Table 2
Table 2: activity of Rubracin A for reversing drug resistance of MCF-7/ADR tumor cells
The results show that: the compound Rubracin A has the activity of reversing drug-resistant MCF-7/ADR at the concentration of 20ug/mL, and the IC thereof 50 The value was 51.35ug/mL, positive control verapamil IC 50 The value was 31.79ug/mL.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (7)
1. A long-chain fatty acid glycerol alcohol compound Rubracin A is characterized in that: the structure is shown as the following formula:
2. a process for preparing a compound according to claim 1, characterized in that: the compound is obtained by fermenting and extracting red palm Mao Tongqiang bacteria, the red palm Mao Tongqiang bacteria is named as red brown hair tube cavity bacteria Tubeufia rubra PF02-2, and the preservation unit is as follows: china center for type culture Collection, the preservation number is CCTCC NO: m2019957.
3. A process for the preparation of a compound according to claim 2, characterized in that: the method comprises the following steps: performing liquid or solid fermentation culture on the Tubeufia rubra PF02-2 of the red palm Mao Tongqiang strain to obtain a fermented product; and extracting the fermentation product, and separating and purifying the obtained extract to obtain the long-chain fatty acid glycerol alcohol compound Rubracin A.
4. A process for the preparation of a compound according to claim 3, characterized in that: the method specifically comprises the following steps:
s1, strain activation: taking out the preserved strains, inoculating the strains on a basal medium plate, performing static culture for passage to the third generation, and performing amplification culture;
s2, fermentation culture: inoculating the activated strain obtained in the step S1 into a solid culture medium, and standing, fermenting and culturing for a period of time at the temperature of 26-30 ℃;
s3, extraction: taking the thalli and a culture medium, adding ethyl acetate for extraction, and concentrating the extract to obtain a fermentation product;
s4, fermentation product pretreatment: dissolving a fermentation product by using a methyl =1:1 solvent, uniformly mixing the fermentation product with silica gel according to a mass ratio of 1:1-2, volatilizing the solvent to be used as a primary column sample, then, loading the sample with silica gel powder and a petroleum ether separation column, sequentially and gradiently eluting by using petroleum ether, chloroform, ethyl acetate and methanol, decompressing and recovering an elution solvent by using a rotary evaporator, dissolving the elution solvent by using chloroform, acetone or methanol, spreading the elution solvent by using a developing agent in a thin-layer chromatography dot plate, selecting a liquid with fluorescence of 254nm or 365nm under an ultraviolet visible light analyzer, and developing the color by using an 8% ethanol sulfate vanillin developer; mixing the methanol solvent eluates, and recovering methanol solvent to obtain methanol extract;
s5, purification and separation: a. dissolving the methanol layer extract with a methanol solvent, uniformly mixing the methanol layer extract with silica gel according to a mass ratio of 1:1-3, loading the mixture on a pre-column when the solvent is volatilized, carrying out balanced reversed-phase medium-pressure column by using 10% methanol water, adding a pre-column containing the sample, sequentially carrying out 10 gradient elutions by using the methanol water, recovering the solvent from an eluent by using a rotary evaporator, dissolving the methanol, then carrying out thin-layer chromatography on a spot plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining 8% ethanol sulfate vanillin color developing agent gray black components to obtain a Fr.11 component;
b. dissolving the component Fr.11 in a methanol solvent, uniformly mixing with silica gel according to the mass ratio of 1:1-3, and performing column sample pre-column loading after the solvent is volatilized; adopting 30% methanol water to carry out equilibrium reversed phase medium pressure column, adding a pre-column containing a sample, adopting methanol water to carry out gradient elution sequentially by 8 times, after the eluent recovers the solvent through a rotary evaporator, dissolving the methanol, spreading the methanol by using a thin layer chromatography dot plate, using a developing agent to develop, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and then combining 8% ethanol sulfate vanillin color developing agent gray black components to obtain Fr.11-6 components;
c. dissolving Fr.11-6 in methanol, uniformly mixing with silica gel 1:1-3 by mass ratio, volatilizing the solvent to obtain a sample, mixing with silica gel powder and a chlorine A =1:1 solvent, and separating with chloroform: gradient elution is carried out such as A =1:1, elution liquid is combined by adopting a thin layer chromatography dot plate, and a component Fr.11-6-1 is obtained;
d. performing normal phase silica gel column chromatography on Fr.11-6-1, performing gradient elution with a gradient such as B: A =1:1, and combining TLC spot plates to obtain the compound Rubracin A.
5. The process for the preparation of a compound according to claim 4, wherein: the solid culture medium in the step S2 is an oat culture medium, and is obtained by mixing 200g of oat and 150mL of double distilled water.
6. The use of a compound according to claim 1 for the preparation of a tumor drug resistance reversal agent or a tumor drug sensitizer, characterized in that: the tumor medicine is adriamycin, and the tumor includes breast cancer.
7. Use according to claim 6, characterized in that: the tumor drug resistance reversal agent is a transport pump inhibitor, and the transport pump inhibitor has an inhibiting effect on one or more of drug-resistant protein P glycoprotein and multidrug-resistant protein.
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