CN113773217B - Long-chain fatty acid glycerol alcohol compound Rubracin C, preparation method and application thereof - Google Patents
Long-chain fatty acid glycerol alcohol compound Rubracin C, preparation method and application thereof Download PDFInfo
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Abstract
The application relates to a long-chain fatty acid glycerol alcohol compound RubracinnB in the technical field of microorganisms, the structure of which is shown as the following formula:
the compound is obtained by fermenting and extracting the phaeophycus rubra, the phaeophycus rubra is named as phaeophycus rubra 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 C, 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. Researches show that P-gp can transport medicines with chemical properties and structural diversity, including partial anticancer medicines, such as adriamycin, taxanes and the like, and cause a multidrug resistance (MDR) phenomenon, so that the cancer treatment fails. 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.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 representative drugs of the third-generation P-glycoprotein inhibitors are Tariquidar (XR 9576), zosuquidar (LY 335979), S9788, etc., among which Tariquidar (XR 9576) and WK-X-34 are represented (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 C 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 C, wherein the compound is obtained by fermenting and extracting phaeophycus rubra named as phaeophycus rubra 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 Tubeufia rubra PF02-2 of Erythrocarpium rubrum, which is obtained by separation from the 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, and the preservation registration number is 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 for preventing rain forests on the screen peaks in urban harbors in Guangxi Zhuang autonomous regions;
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 phakopsora rubra Tubeufia rubra PF02-2 strain of the invention has the following properties:
and (3) colony morphology characteristics: on a natural sapwood substrate, bacterial colonies are flat, are in a net shape and a dot shape, are connected into a sheet shape when the amount is large, are colorless, transparent and white on a PF02-2 pure bacterial colony obtained by fresh separation, and are reddish brown after natural drying of the PF02-2 pure bacterial colony obtained by 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 started to germinate and grow 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 phaeophycus rubellus rubella PF02-2 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 C.
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 strain activated 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 solvent A =1, then uniformly mixing the fermentation product with silica gel according to a mass ratio of 1; mixing the ethyl acetate layer solvent eluent, and recovering the ethyl acetate solvent to obtain an ethyl acetate extract;
s5, purification and separation: a. dissolving ethyl acetate extract with a methanol solvent, uniformly mixing the ethyl acetate extract with silica gel according to a mass ratio of 1-3, loading the mixture on a pre-column when the solvent volatilizes, carrying out equilibrium reversed-phase medium-pressure column by adopting 10% methanol water, adding the pre-column containing the sample, sequentially carrying out 10 gradient elutions by adopting the methanol water, recovering the solvent from an eluent by a rotary evaporator, dissolving the methanol, then using a thin-layer chromatography dot plate, developing by using a developing agent, selecting a liquid with fluorescence of 254nm or 365nm under an ultraviolet visible light analyzer, and combining a component with gray and black color development of an 8% ethanol sulfate vanillin color developing agent to obtain a Fr.14 component;
b. dissolving the component Fr.14 in a methanol solvent, uniformly mixing with silica gel according to a mass ratio of about 1; weighing silica gel powder and a chlorine solvent, uniformly mixing and loading the silica gel powder and the chlorine solvent with a ratio of A =1 into a separation column, adding a sample on the column, performing gradient elution by a chloromethyl system, recovering the solvent from an eluent by a rotary evaporator, dissolving the solvent by methanol, spreading the solvent by a thin layer chromatography dot plate by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining a component with 8% ethanol sulfate vanillin developer to develop gray black to obtain a Fr.14-2 component;
c. dissolving Fr.14-2 in methanol, uniformly mixing with silica gel according to a mass ratio of 1-3, taking a sample on a column after the solvent is volatilized, weighing silica gel powder and a chlorine A =20 solvent, uniformly mixing and loading the mixture into a separation column, then adding the sample on the column, performing gradient elution by a chloromethyl system, developing eluent by using a thin layer chromatography dot plate and a developing solvent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining components with a vanillin sulfate reagent of 8% to form gray black to obtain the compound Rubracin C.
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 tumor drug resistance reversal agents or tumor drug sensitizers.
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 C;
FIG. 2 is a graph of the gyrogram of the compound Rubracin C of the present invention;
FIG. 3 is an infrared spectrum of Rubracin C compound of the present invention;
FIG. 4 is a high resolution mass spectrum of Rubracin C of the present invention;
FIG. 5 shows that Rubracin C BETA of the compound of the present invention 1 H NMR chart;
FIG. 6 shows the preparation of Rubracin C of the compound of the present invention 13 C NMR chart;
FIG. 7 is a diagram of HSQC of compound Rubracin C of the present invention;
FIG. 8 shows the preparation of Rubracin C of the present invention 1 H- 1 H COSY spectrogram;
FIG. 9 shows HMBC spectra of Rubracin C of the present invention;
FIG. 10 shows the cytotoxic activity of Rubracin C, a compound of the present invention;
FIG. 11 is a graph showing the proliferation effect of different concentrations of Rubracin C on MCF-7/ADR cells with different concentrations of doxorubicin according to the present invention.
Detailed Description
The following is further detailed by way of specific embodiments:
1. preparation method of compound Rubracin C
As shown in figure 1 of the drawings, in which,
s1, strain activation
Taking out the strain stored on glycerol slant from refrigerator at-80 deg.C, digging out strain of 1-ring strain Tubeufia rubra with sterile inoculating loop, cross-streaking and inoculating on basal medium plate with diameter of 11cm, standing and culturing at 28 deg.C for 17d, and subculturing to third generation for enlarged culture.
S2, fermentation culture
Fermenting oat solid, subpackaging 200g of oat and 150mL of double distilled water in a 1L triangular 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 24h at 160rpm each time, combining the extract, 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 products
Dissolving 2027.17g of fermentation product by using a methyl chloride =1 solvent, dissolving the fermentation product by using an acetone solvent, uniformly mixing the fermentation product and silica gel according to a mass ratio of 1; weighing 6000g of 200-300-mesh silica gel powder and a petroleum ether solvent, uniformly mixing (bubbles cannot be generated in the process), loading the mixture 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 first upper column sample, sequentially carrying out 4 gradient elution by using petroleum ether, chloroform, ethyl acetate and methanol respectively, carrying out 2-3 (about 36L-54L of elution solvent per column volume) column volumes per gradient elution, collecting one elution solvent per 1000mL, carrying out reduced pressure recovery on each elution sample by using a rotary evaporator, dissolving the elution sample by using 10 or 15mL of chloroform, acetone or methanol, transferring the solution into a bottle with the specification of 20mL, carrying out Thin Layer Chromatography (TLC) on a point plate, and using the petroleum ether: chloroform =1, petroleum ether: acetone =10, chloroform: acetone =5, chloroform: methanol =10: 1. ethyl acetate: developing with a developing agent of methanol =5, observing whether fluorescence is generated at 254nm or 365nm under a conventional ultraviolet visible light analyzer, and developing with a vanillin developer of 8% sulfuric acid ethanol; and combining the ethyl acetate layer solvent eluent, and recovering the ethyl acetate solvent to obtain 61.4g of ethyl acetate layer extract.
S5, purification and separation
a. Dissolving ethyl acetate layer extract (61.4 g) with methanol solvent, mixing with silica gel according to the mass ratio of 1.5 (namely 100g fermentation product added with medium pressure RP-18 reverse phase silica gel) uniformly, volatilizing the solvent to obtain river sand-shaped 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 pre-column containing a sample after balancing about 5-6 column volumes (about 5-6L of elution), performing gradient elution by using methanol water (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), sequentially performing 10 gradient elution, performing 4-5 column volumes of each gradient elution, receiving eluent by using a triangular flask with the specification of 500mL, recovering a solvent from each eluent by using a rotary evaporator, dissolving and transferring the eluent into a penicillin bottle with the specification of 20mL by using 10mL of methanol, performing TLC point chromatography, and performing petroleum ether: acetone =2, chloroform: acetone =5, chloroform: methanol =10: 1. ethyl acetate: developing with methanol =2, 1 developing agent, observing whether fluorescence is generated at 254nm or 365nm under a conventional ultraviolet visible light analyzer, developing with 8% ethanol sulfate vanillin developer, combining the components of 8% ethanol sulfate vanillin developing gray black color (namely the components eluted with 80% methanol water), and obtaining 14 th component (Fr.14.9.23 g).
b. Dissolving the component Fr.14 (9.23 g) in a methanol solvent, uniformly mixing with 200-300-mesh silica gel according to a mass ratio of about 1.5 (namely adding 14g to 9.23g of the component), volatilizing the solvent to obtain a river sand-shaped sample, and taking the sample as a column sample; weighing 300g of silica gel powder of 200-300 mesh and a solvent of chloro: a =1 (no bubble can be generated in the process) and loading into a separation column of 460mm in length and 100mm in inner diameter, allowing the silica gel powder to slowly sink until no more sink, adding a sample to the column, gradient eluting with a chloro system (50: developing with a developing agent of methanol =3 and 1, observing whether fluorescence is generated at 254nm or 365nm under a conventional ultraviolet visible light analyzer, developing with a vanillin developer of 8% ethanol sulfate, and combining the components of 8% ethanol sulfate vanillin developing gray black to obtain a component 2 (Fr.14-2.28g).
c. Fr.14-2 (1.28 g) is dissolved by methanol, and is uniformly mixed with 200-300 mesh silica gel according to the mass ratio of 1.5 (namely 2g is added in 1.28g of components), and a river sand-shaped sample is obtained after the solvent is volatilized and is used as an upper column sample; weighing 60g of 200-300 mesh silica gel powder and a chlorine: a =20 solvent, uniformly mixing (no air bubbles can be generated in the process) the silica gel powder with the chlorine: a =1 solvent, loading the silica gel powder into a separation column with a length of 240mm and an inner diameter of 30mm, allowing the silica gel powder to slowly sink until the silica gel powder does not sink, adding a sample to the column once, performing gradient elution by a chlorine chloride system (20.
2. Identification of compound Rubracin C structure
Rubracin C is colorless oil, and is easily dissolved in solvents such as methanol, acetone, DMSO, etc.
See figure 2 for details; IR spectrum (see attached figure 3 for details) at 1737cm -1 、1681cm -1 、1660cm -1 Has an absorption peak, which indicates that the compound contains an ester group. HRESI (detailed in figure 4) shows molecular weight 730.55695[ M ] +Na ]] + Molecular formula is C 42 H 77 NNaO 7 Calculating the unsaturation degree to be 5;1D NMR combined with HSQC (see FIGS. 5, 6, and 7 for details) concluded that the compound has two long-chain fatty chains [ delta ] C 14.1(q),22.6(t),22.7(t),24.9(t),25.0(t),25.6(t),27.2(t),29.2～29.7(t),31.5(t),31.9(t),34.2(t),34.3(t),127.9(d),128.1(d),130.8(d),131.0(d)，173.1,173.6](ii) a Bonding of 1 Hydrogen signal delta on HNMR spectra H [0.86(6H,t,J＝7.0Hz),2.76(2H,t,J＝6.5Hz),27.2(4H,m),5.31(2H,m),5.34(2H,m)]One of the two long-chain fatty chains is an unsaturated fatty chain containing two double bonds, and the other is a saturated fatty chain; the remaining fragment consists of three methyl groups [52.3 (q), 3.28 (9H, s)]Tetramethylene [29.1 (t), 2.01 (1H, m), 2.24 (1H, m); 62.4 (t), 4.12 (1h, dd, j =11.7,6.4 hz), 4.33 (1h, dd, j =11.8, 3.9hz); 67.7 (t), 3.70 (2H, m); 69.6 (t), 3.56 (1H, dd, J =10.5, 4.9Hz), 3.62 (1H, dd, J =10.6, 5.4Hz)]Two methines [69.9 (d), 5.19 (1H, m); 77.0 (d), 3.75 (1H, m)]And a quaternary carbon atom.
Detailed one-dimensional nuclear magnetic data are detailed in table 1.
TABLE 1 carbon spectra data of Rubracin C compound
1 H- 1 The connection of the compounds H-1, H-2 and H-3 can be deduced on the H COSY spectrogram (see the attached figure 8 in detail); h-4, H-5 and H-6 are connected; h-2 'and H-3' are connected; h-8', H-9', H-10', H-11', H-12', H-13' and H-14'; h-15 'and H-16' are connected; h-2 'and H-3' are connected; h-15 'and H-16' are connected.
On HMBC (see FIG. 9 for details), the hydrogen proton signal delta H [4.12(1H,dd,J＝11.7,6.4Hz),4.33(1H,dd,J＝11.8,3.9Hz),H-1)]And delta C 173.6 (s, C-1 '), 69.9 (d, C-2), 69.6 (t, C-3), demonstrating that C-1 is attached to the ester group C-1'; hydrogen proton signal delta H [5.19(1H,m,H-2)]And delta C 173.1 (s, C-1 '), 62.4 (t, C-1), 69.6 (t, C-3), demonstrating that C-2 is attached to the ester group C-1'; hydrogen proton signal delta H [3.56(1H,dd,J＝10.5,4.9Hz,H-3),3.62(1H,dd,J＝10.6,5.4Hz,H-3)]And delta C 62.4 (t, C-1), 69.9 (d, C-2), 67.7 (t, C-4) relative to the combined hydrogen proton signal δ H [3.70(2H,m,H-4)]In connection with 69.6 (t, C-3), 29.1 (t, C-5), 77.0 (d, C-6), it can be concluded that C-3 and C-4 are linked via an oxygen atom to form the ether moiety.
1D-NMR, HSQC, HMBC, HPLC, are carefully analyzed in conjunction with high resolution mass spectrometry, 1 H- 1 H, COSY data, and finally determining the structure of Rubracin C.
3. Screening of cytotoxic Activity of Compound Rubracin C
3.1 test cell lines: MCF-7/ADR (purchased from Shanghai Meixuan Biotech Co., ltd. In 5 months 2021)
3.2RPMI1640 Dairy serum 10%
3.3 cell culture
3.3.1 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.
3.3.2 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-0.53mM 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.
3.4CCK-8 assay for cytotoxic Activity
3.4.1 concentration gradient 0, 6.25, 12.5, 25, 50, 100, 200, 400. Mu.g/mL, 3 replicates
Positive control: adriamycin
Negative control: DMSO (dimethylsulfoxide)
3.4.2 Experimental procedures
(1) Digesting the cells, counting the cells, adjusting the cell concentration to 2X 10 4 one/mL.
(2) 100 μ L of cell suspension was seeded in a 96-well plate. Plates were incubated in 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 continuously 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 10 μ L 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.
3.4 results of the experiment
The result shows that the compound Rubracin C has no cytotoxic activity to MCF-7/ADR, and can be continuously used for reversing tumor cell screening (the result is shown in figure 10 in detail).
4. Application of compound Rubracin C in 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)
4.2RPMI1640+10% fetal calf serum
4.3 cell culture
4.3.1 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 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 culture solution the next day, continuing culturing, and observing growth conditions.
4.3.2 cell passages
After the cells grow to 80-90%, using a plastic pipette with a specification of 3mL under aseptic conditions to aspirate the cell culture solution, adding 1-2mL of PBS to wash for 1 time (without calcium and magnesium ions), adding 1mL of digestive juice (0.25% trypsin-0.53mM EDTA) into the culture flask, 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, 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.
4.4CCK-8 assay to reverse tumor cytotoxic Activity
4.4.1 Adriamycin concentration gradient 0, 6.25, 12.5, 25, 50, 100, 200, 400. Mu.g/mL, 3 replicates
Rubracin C concentration: 5. 10, 20. Mu.g/mL
Positive control: verapamil
Negative control: DMSO (dimethylsulfoxide)
4.4.2 Experimental procedures
(1) Digesting the cells, counting the cells, adjusting the cell concentration to 2X 10 4 one/mL.
(2) 100uL of cell suspension was seeded in 96-well plates. 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, washing with PBS (without calcium and magnesium ions) for 1 time, adding 10 μ L of CCK-8 reagent into each well, and incubating for 3h in an incubator.
(5) Absorbance at 490nm was measured with a microplate reader.
4.4 results of the experiment
The results are shown in FIG. 11 and Table 2.
TABLE 2 Compound Rubracin C reverses the Activity of MCF-7/ADR at different concentrations
The results show that: the compound Rubracin C concentration is respectively 5 mug/mL, 10 mug/mL and 20 mug/mL, has the activity of reversing drug resistance MCF-7/ADR and IC 50 The values are 47.33. Mu.g/mL, 40.29. Mu.g/mL and 33.46. Mu.g/mL respectively, and the IC of the positive control verapamil at the same drug concentration 50 The values were 46.55. Mu.g/mL, 38.45. Mu.g/mL, and 31.79. Mu.g/mL, respectively.
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 C is characterized in that: the structure is shown as the following formula:
2. a process for the preparation of a compound according to claim 1, characterized in that: the compound is obtained by fermenting and extracting the phaeophycus rubra, the phaeophycus rubra is named as phaeophycus rubra 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 preparing a compound according to claim 2, characterized in that: the method comprises the following steps: performing liquid or solid fermentation culture on the phaeophycus rubellus rubella PF02-2 to obtain a fermented product; and (3) extracting the fermentation product, and separating and purifying the obtained extract to obtain the long-chain fatty acid glycerol alcohol compound Rubracin C.
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 strain activated 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 solvent A =1, then uniformly mixing the fermentation product with silica gel according to a mass ratio of 1; mixing the ethyl acetate layer solvent eluent, and recovering ethyl acetate solvent to obtain ethyl acetate extract;
s5, purification and separation: a. dissolving ethyl acetate extract with a methanol solvent, uniformly mixing the ethyl acetate extract with silica gel according to a mass ratio of 1-3, loading the mixture on a pre-column when the solvent volatilizes, carrying out equilibrium reversed-phase medium-pressure column by adopting 10% methanol water, adding the pre-column containing the sample, sequentially carrying out 10 gradient elutions by adopting the methanol water, recovering the solvent from an eluent by a rotary evaporator, dissolving the methanol, then using a thin-layer chromatography dot plate, developing by using a developing agent, selecting a liquid with fluorescence of 254nm or 365nm under an ultraviolet visible light analyzer, and combining a component with gray and black color development of an 8% ethanol sulfate vanillin color developing agent to obtain a Fr.14 component;
b. dissolving the component Fr.14 in a methanol solvent, uniformly mixing with silica gel according to the mass ratio of 1; weighing silica gel powder and a chlorine solvent, uniformly mixing and loading the silica gel powder and the chlorine solvent with a ratio of A =1 into a separation column, adding a sample on the column, performing gradient elution by a chloromethyl system, recovering the solvent from an eluent by a rotary evaporator, dissolving the solvent by methanol, spreading the solvent by a thin layer chromatography dot plate by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining a component with 8% ethanol sulfate vanillin developer to develop gray black to obtain a Fr.14-2 component;
c. dissolving Fr.14-2 in methanol, uniformly mixing with silica gel according to a mass ratio of 1-3, taking a sample as an upper column after the solvent is volatilized, weighing silica gel powder, uniformly mixing with a chlorine: A =20 solvent which is 1, loading into a separation column, adding the sample into the upper column, performing gradient elution by a chloromethyl system, developing eluent by using a thin-layer chromatography dot plate and a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining components with a vanillin sulfate color developing agent of 8% to obtain the compound Rubracin C.
5. The method for preparing the 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, wherein: the drug resistant or tumor drug is adriamycin, and the tumor comprises breast cancer.
7. Use according to claim 6, characterized in that: the tumor drug resistance reversal agent is a transfer pump inhibitor, and the transfer pump inhibitor has an inhibiting effect on one or more of drug-resistant protein P-glycoprotein and multidrug-resistant protein.
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