CN115043748B - Long-chain fatty acid glycerol alcohol compound, preparation method and application thereof - Google Patents
Long-chain fatty acid glycerol alcohol compound, preparation method and application thereof Download PDFInfo
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Abstract
The application relates to a long-chain fatty acid glycerol alcohol compound Rubracin F in the technical field of microorganisms, and the structure is shown as the following formula:the compound is obtained after fermentation and extraction of the Caulophyllum reesei, wherein the Caulophyllum reesei is named as Caulophyllum reesei Tubeufia rubra PF02-2, and the preservation unit is as follows: china Center for Type Culture Collection (CCTCC) NO: m2019957. The compound has the application of preparing tumor drug resistance reversal agent or tumor drug sensitizer.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a long-chain fatty acid glycerol alcohol compound, a preparation method and application thereof.
Background
Cancer has become one of the serious health and life threatening diseases for humans. The treatment of tumors mainly comprises chemotherapy, operation, radiotherapy and the like, and the chemotherapy is one of the main means for treating cancers. During chemotherapy, the development of drug resistance by tumor cells is the leading cause of chemotherapy failure. Therefore, the searching of the reversing agent with low degree and good activity is the most fundamental way for solving the drug resistance of the tumor, and has main research value.
The P-glycoprotein (P-gp) is one of the most representative proteins of the ABC transporter family, having a molecular weight of 170kD and consisting of 1280 amino acid residues. Studies have shown that P-gp is capable of transporting drugs of chemical nature and structural diversity, including some anticancer drugs, such as doxorubicin, taxanes, etc., causing multi-drug resistance (MDR) phenomena, leading to failure of cancer treatment. Thus, the study of P-gp inhibitors and substrates is of great interest for cancer treatment, and P-gp inhibitors can be co-administered with chemotherapeutic agents as an effective strategy to overcome MDR. Several generations of P-gp inhibitors have been developed, the first generation of reversal agents including tamoxifen, cyclosporin A, etc., of which verapamil and cyclosporin are typical. However, such drugs often lack the specificity of P-glycoprotein and cause serious side effects, and the first generation of reversal agents are also limited in clinical practice to a great extent (Sato W.et al 1991). The second generation reversers, the cephalosporin analogs valspodada (valspodar, PSC 833), dextro verapamil (dexverapamil), etc., are represented by dexamethasone, however, the development of second generation reversers 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 of P-glycoprotein inhibitors are Tariquidar (XR 9576), zosuquidar (LY 335979), S9788, etc., wherein 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 in the development of fourth generation inhibitors.
Microbial natural products are all the 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 strain breeding, and the like, and can realize industrial production through large-scale fermentation, thereby further laying the important role 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.
For this reason, the applicant has made a great deal of research, found a series of new long-chain fatty acid glycerol alcohol compounds as disclosed in CN113773216A, CN114014898A, CN114057811a, and found that they have application in reversing the activity of drug-resistant tumor cells, but the above compounds are all low in quantity after separation and purification, so how to obtain compounds which have large quantity after separation and purification and also have application in reversing the activity of drug-resistant tumor cells is the key of industrialization.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides a long-chain fatty acid glycerol alcohol new compound from microorganism sources, a preparation method thereof and application thereof in preparing medicines for reversing drug-resistant tumor cell activities.
The application aims to provide a novel long-chain fatty acid glycerol alcohol compound, which has a structure shown in the following formula:
the second purpose of the application is to provide a preparation method of a new long-chain fatty acid glycerol alcohol compound, wherein the compound is obtained by fermenting and extracting Caulophyllum reesei, and the Caulophyllum reesei is named Caulophyllum reesei Tubeufia rubra PF-2, and the preservation unit is: china Center for Type Culture Collection (CCTCC) NO: m2019957.
The red palm hair cylinder fungus Tubeufia rubra PF02-2 is obtained by separation from the chemical engineering center of Guizhou university and has a preservation unit: china center for type culture Collection, with the addresses: university of martial arts, preservation day: 2019.11.20, accession number CCTCC NO: m2019957.
The sources of the cylindrical bore fungus Tubeufia rubra PF02-2 are as follows:
sampling time: 2016, 5 months and 14 days;
sampling site: the Guangxi Zhuang autonomous region is protected against urban harbor city screen peak rain forest natural protection areas;
sampling mode: and (3) collecting sapropel in natural protection areas of urban and harbor city peak prevention rain forest in Guangxi Zhuang autonomous areas, and taking the sapropel back to a laboratory in a plastic sealing bag.
The strain Tubeufia rubra PF02-2 of the application has the following properties:
colony morphology characterization: on the natural saprophyte substrate, the colony is flat, net-shaped and dot-shaped, and is connected into a sheet shape when the amount is large, the pure colony of the PF02-2 obtained by separation is colorless, transparent and white, and the pure colony of the PF02-2 obtained by separation is naturally dried and is reddish brown. Part of the mycelium is buried under the matrix, but mostly emerges, the mycelium consists of mycelium with branches of the septum, colorless to dark brown. Conidiophores are cylindrical, single, grown in a bending manner, have a diaphragm, are 50-150 microns long, 4.5-6 microns wide, taper at the top, are dark brown at the bottom, are transparent to light brown at the top, and have smooth surfaces. The spore-producing cells are single or multiple, cylindrical, with columnar small dentate, and the synthetic axis grows from the middle part to the top of the molecular spore peduncles, 10-19 microns long, 3-4 microns wide, colorless transparent to light brown, and smooth in surface. The molecular spore is coiled, single, top side is transparent, top end is round, coiled for 2-3.5 times when being tightly coiled, diameter is 35-50 microns, conidium filaments are 3-5 microns thick (average diameter is 45 microns, thickness is 4.5 microns), and the conidium filaments are gradually loosened and dispersed in water, have unclear multi-diaphragm, are colorless to light brown and have smooth surfaces. Conidia began to germinate and grow after 12h in water-agar medium. Colonies were grown in PDA medium at 25-28deg.C for 2 weeks up to 16mm, brown, round, rough, with obvious protrusions, pulse wrinkles, and complete colony edges.
Specifically, the preparation method comprises the following steps: carrying out liquid or solid fermentation culture on the red palm fiber cylindrical fungus Tubeufia rubra 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 F.
The preparation method specifically comprises the following steps:
s1, strain activation: taking out the preserved strain, inoculating the strain on a basal medium plate, standing, culturing and passaging to the third generation, and then amplifying and culturing;
s2, fermenting and culturing: inoculating the strain activated in the step S1 into a solid culture medium, and standing, fermenting and culturing for a period of time at 26-30 ℃;
s3, extracting: extracting thallus and culture medium with ethyl acetate, concentrating the extractive solution to obtain fermentation product;
s4, pretreatment of fermentation products: dissolving a fermentation product by using a chlorine methyl=1:1 solvent, uniformly mixing the fermentation product with silica gel according to the mass ratio of 1:1-2, taking the mixture as a primary column sample after the solvent volatilizes, sequentially and gradiently eluting with petroleum ether, chloroform, ethyl acetate and methanol respectively on a silica gel powder and a petroleum ether separation column, recovering the eluting solvent by a rotary evaporator under reduced pressure, dissolving the eluting solvent by using chloroform, acetone or methanol, spreading the eluting solvent by using a spreading agent, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and developing by using an 8% sulfuric acid ethanol vanillin color developing agent; combining the methanol solvent eluates, and recovering the methanol solvent to obtain a methanol layer extract;
s5, purifying and separating: a. dissolving the methanol layer extract with methanol solvent, uniformly mixing the methanol layer extract with silica gel according to the mass ratio of 1:1-3, carrying out equilibrium reverse phase compression on an upper column sample after solvent volatilization, adding 10% methanol water to carry out equilibrium reverse phase compression on the column, adding the pre-column containing the sample, sequentially carrying out 10 gradient elution with the methanol water, recovering the solvent from eluent by a rotary evaporator, dissolving the methanol, then spreading the eluent by a thin layer chromatography dot plate, using a developing agent, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and then combining components with black color development of 8% sulfuric acid ethanol vanillin developer to obtain Fr.11 components;
b. after the component Fr.11 is dissolved by a methanol solvent, the mixture is uniformly mixed with silica gel according to the mass ratio of 1:1-3, and after the solvent volatilizes, the mixture is used as a pre-column on a column sample; performing balanced counter-phase column pressing by 30% methanol water, balancing 5-6 column volumes, adding a pre-column containing a sample, adding an upper column sample, sequentially performing 8 gradient elution by using methanol water, recovering a solvent from eluent by a rotary evaporator, dissolving the methanol, then using a thin layer chromatography point plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and then combining 8% sulfuric acid ethanol vanillin developer grey-black components to obtain Fr.11-13 components;
c. dissolving Fr.11-13 with methanol, uniformly mixing with silica gel according to a mass ratio of 1:1-3, taking an upper column sample after the solvent volatilizes, weighing silica gel powder, uniformly mixing with an ethyl methyl=1:1 solvent, loading into a separation column, adding the upper column sample, adopting an ethyl methyl system for gradient elution, using a thin layer chromatography point plate for eluent, using a developing agent to develop, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and then combining 8% sulfuric acid ethanol vanillin developer grey-black components to obtain the long-chain fatty acid glycerol alcohol compound.
Wherein the solid medium in the step S2 is oat medium, and 200g of oat and 150mL of double distilled water are mixed to obtain the oat culture medium.
The research shows that when the fermentation product is 2027.17g, the quality of the separated and purified long-chain fatty acid glycerol alcohol compound is 68mg, and compared with other separated compounds in the prior art, the quality of the compound is 6-20 times that of other similar compounds in the prior art, thereby being more beneficial to industrialized application.
The application also aims to provide the application of the compound and the medicinal carrier in preparing tumor drug resistance reversal agents or tumor drug sensitizers.
Further, the drug resistant or neoplastic drug is doxorubicin.
Further, the tumor includes breast cancer, lung cancer or leukemia.
Furthermore, the tumor drug resistance reversing agent is a transport pump inhibitor, and the transport pump inhibitor has an inhibiting effect on one or more of drug resistance protein P-glycoprotein and multi-drug resistance protein.
The application also provides application of the compound and a medicinal carrier in preparing antitumor cell medicaments, wherein the tumor cells comprise adriamycin-resistant breast cancer cells, adriamycin-resistant lung cancer cells or adriamycin-resistant leukemia.
Drawings
FIG. 1 is a flow chart for separating and purifying a compound Rubracin F;
FIG. 2 is a spectrum of the optical spectrum of the compound Rubracin F of the application;
FIG. 3 is an infrared spectrum of a compound Rubracin F in the application;
FIG. 4 is a UV spectrum of compound Rubracin F of the present application;
FIG. 5 is a high resolution mass spectrum of the compound Rubracin F of the application;
FIG. 6 is a schematic diagram of the compound Rubracin F according to the application 1 H-NMR chart;
FIG. 7 shows the compound Rubracin F in the present application 13 C-NMR and DEPT maps;
FIG. 8 is a HSQC spectrum of the compound Rubracin F of the present application;
FIG. 9 is a schematic representation of the compound Rubracin F of the application 1 H- 1 H COSY profile;
FIG. 10 is a HMBC spectrum of the compound Rubracin F of the present application;
FIG. 11 is an EI-MST diagram of the compound Rubracin F of the present application;
FIG. 12 is a schematic representation of the cytotoxic activity of Rubracin F against drug resistant tumor cells MCF-7/ADM;
FIG. 13 is a schematic representation of the cytotoxic activity of Rubracin F against drug resistant tumor cells A549/ADM;
FIG. 14 is a schematic representation of the cytotoxic activity of Rubracin F against drug resistant tumor cells K562/ADM.
Detailed Description
The following is a further detailed description of the embodiments:
1. preparation method of compound Rubracin F
As shown in figure 1 of the drawings,
s1, strain activation
Taking out the strain stored on glycerol slant from refrigerator at-80deg.C, inoculating strain of strain Tubeufia rubra of 1 ring with sterile inoculating loop, standing at 28deg.C for 17d, and subculturing to third generation of amplification culture.
S2, fermenting and culturing
Oat solid fermentation (1L Erlenmeyer flask split charging 200g oat and 150mL double distilled water), each bottle of inoculum size is 1X 1cm of culture plate upper area 2 Is subjected to stationary culture at 28 ℃ for 105 days.
S3, extracting
Extracting thallus and oat culture medium with ethyl acetate for three times, oscillating at 160rpm for 24 hr each time, mixing extractive solutions, concentrating under reduced pressure at 40deg.C to obtain fermentation product, repeating above steps, and mixing fermentation products to obtain 2027.17g.
S4, pretreatment of fermentation products
Dissolving 2027.17g of fermentation product by using methyl chloride=1:1 solvent and acetone solvent, uniformly mixing the fermentation product with silica gel according to the mass ratio of about 1:1.5 (namely 2027.17g of fermentation product is added with 3041g of 200-300 mesh silica gel powder), and volatilizing the solvent to obtain a river sand sample which is taken as a primary column sample; the method comprises the steps of weighing 6000g of 200-300 mesh silica gel powder, uniformly mixing the 6000g with petroleum ether solvent (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 is no longer sinking, adding an upper column sample, respectively adopting petroleum ether, chloroform, ethyl acetate and methanol, sequentially carrying out 4 gradient elution, collecting 2-3 gradient elution columns (about 36L-54L of eluting solvent per column volume) each with one 1000mL of eluting solvent, recovering each eluting sample under reduced pressure by a rotary evaporator, dissolving the eluting sample with 10 or 15mL of chloroform, acetone or methanol, transferring the eluting sample into a penicillin bottle with the specification of 20mL, and carrying out Thin Layer Chromatography (TLC) on a petroleum ether: chloroform = 1:1, petroleum ether: acetone=10:1, chloroform: acetone=5:1, chloroform: methanol=10: 1. ethyl acetate: developing with methanol=5:1 developing agent, observing whether fluorescence exists at 254nm or 365nm under a conventional ultraviolet-visible light analyzer, and developing with 8% sulfuric acid ethanol vanillin color developing agent; the methanol solvent eluates were combined and the methanol solvent was recovered to give 35.77g of methanol layer extract.
S5, purifying and separating
a. Dissolving the methanol layer extract (35.77 g) with methanol solvent, uniformly mixing with silica gel according to a mass ratio of about 1:2 (namely adding medium-pressure RP-18 reverse phase silica gel into 35.77g fermentation product), and volatilizing the solvent to obtain a river sand sample which is taken as an upper column sample; adding a pre-column with the length of 10cm and the diameter of 49mm into the upper column sample; after balancing about 5-6 column volumes (about eluting 5-6L) by using 10% methanol water to perform balanced counter-phase compression column (column length of 460mm, diameter of 49 mm), adding a pre-column containing a sample, performing gradient elution (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) by using methanol water, performing gradient elution sequentially by 10, performing gradient elution by 4-5 column volumes, receiving eluent by using a 500mL Erlenmeyer flask, recovering solvent from each eluent by using a rotary evaporator, dissolving and transferring the eluent into a 20mL penicillin bottle by using 10mL methanol, and performing TLC (thin layer chromatography) plate by 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% sulfuric acid ethanol vanillin developer, combining 8% sulfuric acid ethanol vanillin grey black colored components (i.e., 90% methanol water eluted components) to give 11 th component (fr.11.7 g).
b. After the component Fr.11 (8.7 g) is dissolved by a methanol solvent, the mixture is uniformly mixed with silica gel according to the mass ratio of about 1:2 (namely, 17.4g of medium-pressure RP-18 reverse phase silica gel is added into 8.7g of the component), and a river sand sample is obtained after the solvent volatilizes, and is taken as a column sample; adding a pre-column with the length of 10cm and the diameter of 26mm into the sample on the column; after balancing about 5-6 column volumes (about eluting 5-6L) by using 30% methanol water to perform balanced counter-phase compression column (column length of 460cm, diameter of 49 mm), adding a pre-column containing a sample, performing gradient elution (30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) by using methanol water, performing gradient elution by 8 in sequence, performing gradient elution by using 4-5 column volumes, receiving eluent by using an Erlenmeyer flask with a specification of 500mL, recovering solvent from each eluent by using a rotary evaporator, performing dissolution transfer to a xilin bottle with a specification of 20mL by using 10mL methanol, and performing TLC (time-point plate) by 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 exists at 254nm or 365nm under conventional ultraviolet-visible light analyzer, developing with 8% sulfuric acid ethanol vanillin developer, combining 8% sulfuric acid ethanol vanillin black-colored components (i.e. 90% methanol water eluted components), and obtaining components (Fr.11-13 650 mg).
c. Fr.11-13 (650 mg) is dissolved by methanol and is uniformly mixed with 200-300 mesh silica gel according to the mass ratio of about 1:1.5 (namely, 1g is added into 650mg of components), and a river sand sample is obtained after the solvent volatilizes and is taken as a column sample; 60g of 200-300 mesh silica gel powder is weighed and evenly mixed with a solvent of methyl=1:1 (bubbles cannot be generated in the process), the mixture is filled into a separation column with the length of 240mm and the inner diameter of 30mm, the silica gel powder is slowly submerged until a column loading sample is added when the silica gel powder is no longer submerged, gradient elution (1:1) is adopted by an ethyl methyl system, each gradient elution is carried out for 4-5 column volumes (about 480mL-600 mL), an eluent is collected by a penicillin bottle with the specification of 20mL, a component is eluted by a TLC spot plate, chloroform: acetone=1:1, chloroform: methanol=8:1 and ethyl acetate: methanol=3:1 developing agent, whether fluorescence exists at 254nm or 365nm is observed under a conventional ultraviolet-visible light analyzer, color development is carried out by using an 8% sulfuric acid ethanol aldehyde developing agent, and a component with black gray color is developed by 8% sulfuric acid ethanol vanillin (namely ethyl methyl=1:1) is combined, so that a compound rubrin F68 mg is obtained.
2. Structural identification of compound Rubracin F
The Rubracin F is colorless oil, and is easily dissolved in solvents such as methanol, acetone, DMSO, etc.See figure 2 for details; IR spectrum (see FIG. 3 for details) at 1738cm -1 、1627cm -1 Has an absorption peak indicating that the compound contains an ester group. UV (MeOH) lambda max 197 (4.03), 258 (2.33) demonstrates that the compound has a double bond (see FIG. 4 for details); HRESI (see FIG. 5 for details) shows a molecular weight of 760.60583[ M+Na ]] + Molecular formula C 44 H 83 NNaO 7 Calculating the unsaturation degree to be 4;1D NMR, DEPT combined with HSQC (see FIGS. 6, 7, 8 for details) deduced that the compound has two long chain fatty chains [ delta ] C 14.5(q),23.8(t),26.1(t),28.2(t),30.2～30.9(t),33.1(t),35.0(t),35.1(t),130.8(d),130.9(d),174.5,174.9]The method comprises the steps of carrying out a first treatment on the surface of the Bonding of 1 Hydrogen signal delta on H NMR spectrum H [0.89(6H,t,J＝7.0Hz),28.2(2H,m),5.34(2H,m)]It can be further deduced that one of the two long chain fatty chains is an unsaturated fatty chain containing one double bond, and the other is a saturated fatty chain; the remaining fragment was composed of three methyl groups [52.4 (q), 3.20 (9H, s)]Four methylene groups [29.1 (t), 2.06 (1H, m), 2.21 (1H, m); 63.9 (t), 4.16 (1 h, dd, j=12.0, 6.9 hz), 4.41 (1 h, dd, j=12.0, 3.1 hz); 70.5 (t), 3.60 (2 h, dd, j=5.2, 4.0 hz); 68.7 (t), 3.53 (1 h, td, j=9.2, 4.5 hz), 3.68 (1 h, m)]Two methines [71.5 (d), 5.21 (1H, m); 77.5 (d), 3.70 (1H, m)]The composition of one quaternary carbon atom and detailed one-dimensional nuclear magnetic data are shown in table 1.
1 H- 1 The H-1, H-2 and H-3 linkages of the compound can be deduced on the H COSY spectrum (see FIG. 9 for details); h-4, H-5 and H-6; h-2 'and H-3' are linked; h-8', H-9', H-10 'and H-11' are linked; h-17 'and H-18' are linked; h-2 'and H-3' are linked; h-15 'and H-16' are linked.
TABLE 1 Rubracin F1D NMR data for the compounds
On HMBC (see fig. 10 for details), hydrogen proton signal δ H [4.16(1H,dd,J＝12.0,6.9Hz,H-1),4.41(1H,dd,J＝12.0,3.1Hz),H-1)]And delta C 174.9 (s, C-1 '), 71.5 (d, C-2), 70.5 (t, C-3) correlate, demonstrating that C-1 is linked to an ester group C-1'; hydrogen proton signal delta H [5.21(1H,m,H-2)]And delta C 174.5 (s, C-1 '), 63.9 (t, C-1), 70.5 (t, C-3), demonstrating that C-2 is linked to the ester group C-1'. Hydrogen proton signal delta H [3.60(2H,dd,J＝5.2,4.0Hz,H-3)]And delta C 63.9 (t, C-1), 68.7 (t, C-4), 71.5 (t, C-2) and binding to hydrogen proton signal delta H [3.53(1H,td,J＝9.6,4.5Hz,H-4),3.68(1H,m,H-4)]In relation to 70.5 (t, C-3), 29.1 (t, C-5), 77.0 (d, C-6), it can be inferred that C-3 and C-4 are linked by an oxygen atom to form a structural fragment of an ether; hydrogen proton signal delta H [3.70(1H,m,H-6)]And 171.6 (s, C-7), 68.7 (t, C-4), 52.4 (q, -NMe) 2 ) 29.1 (t, C-5) to confirm that C-6 is linked to C-7; the signal proton 2.30 (2 h, t, j=5.2 hz, h-2 ') correlated with 174.9 (s, C-1'), 26.1 (2 h, m, h-3 '), 30.2-30.9 (2 h,1.28-1.32, h-4'), demonstrated that C-1 is linked to one fatty chain; another fatty acid is attached to C-2, as evidenced by the association of 2.33 (2H, t, J=5.2 Hz, H-2 ') with 174.5 (s, C-1'), 26.1 (2H, m, H-3 '), 30.2-30.9 (2H, 1.28-1.32, H-4').
Careful analysis of 1D-NMR, HSQC, HMBC in combination with high resolution mass spectrometry, 1 H- 1 H COSY data finally determine the structure of the Rubracin F, and the detailed formula is shown in the specification.
The detailed fragment ion characteristics of the compound are shown in the following formula. The rationality of the cleavage of the compound fragment ions justifies the structural resolution of Rubracin F.
3. Compound Rubracin F cytotoxic Activity Screen
3.1 test cell lines: MCF-7/ADM, A549/ADM, K562/ADM
3.2 RPMI1640+10% foetal calf serum
3.3 cell culture
3.3.1 cell resuscitation
The cells were removed from the liquid nitrogen tube, and the frozen tube was quickly placed into a water bath that had been preheated to 37 degrees for quick thawing, and continuously shaken to quickly thaw the liquid in the tube. After about 1mL of the liquid in the freezing tube is completely dissolved, cells are taken out under aseptic conditions and inoculated into a cell culture dish (RPMI 1640+10% fetal bovine serum), the cells are placed into a 37-DEG CO2 incubator for culture, the culture solution is replaced the next day, the culture is continued, and the growth condition is observed.
3.3.2 passage of cells
After the cells grow to 80-90%, sucking out the cell culture solution by using a plastic suction pipe with the specification of 3mL under aseptic operation condition, adding 1-2mL of PBS for washing 1 time (without calcium and magnesium ions), adding 1mL of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture flask, observing the digestion condition of the cells under an inverted microscope, if the cells are mostly rounded, quickly taking back to an operating table, tapping a few culture flasks, and adding 2mL of complete culture medium to terminate the digestion. 4mL of fresh complete medium was added to each new flask, followed by 1mL of complete medium containing cells.
3.4 CCK-8 assay for cytotoxic Activity
3.4.1 concentration gradients were set for MCF-7/ADR (0, 6.25, 12.5, 25, 50, 100, 200. Mu.g/mL), K562/ADR and A549/ADR (0, 1.6, 3.125, 6.25, 12.5, 25, 50, 100), 3 replicates, doxorubicin as positive control, DMSO as negative control, all data calculated using SPSS software.
3.4.2 Experimental procedure
(1) Cell digestion, cell count, and cell concentration adjustment to 2×10 4 And each mL.
(2) 100. Mu.L of the cell suspension was seeded in 96-well plates. The plates were incubated at 5% CO 2 Culturing in incubator at 37deg.C for 24 hr.
(3) According to the grouping, the compound and the doxorubicin with different concentrations are respectively added, and the mixture is placed in an incubator to be incubated for 48 hours at 37 ℃.
(4) After the incubation, PBS (without calcium and magnesium ions) was washed 1 time, 10. Mu.L of CCK-8 reagent was added to each well, and incubated in an incubator for 3 hours.
(5) Measurement of absorbance at 490nm with an ELISA reader
3.4 experimental results
The results show that the compound Rubracin F has no cytotoxic activity on MCF-7/ADM, A549/ADM and K562/ADM, and can be used for further reverse tumor cell screening (the results are shown in FIGS. 12, 13 and 14).
4. Application of compound Rubracin F in reversing MCF-7/ADR tumor cell drug resistance activity
4.1 test cell lines: MCF-7/ADR (2021, 5 month purchased from Shanghai Meixuan Biotechnology Co., ltd.)
4.2 RPMI1640+10% foetal calf serum
4.3 cell culture
4.3.1 cell resuscitation
The cells were removed from the liquid nitrogen tube, and the frozen tube was quickly placed into a water bath that had been preheated to 37 degrees for quick thawing, and continuously shaken to quickly thaw the liquid in the tube. After complete dissolution of about 1mL of the liquid in the cryopreservation vessel, cells were removed under aseptic conditions and inoculated into a cell culture dish (RPMI 1640+10% fetal bovine serum) and placed in 37℃CO 2 Culturing in an incubator, replacing the culture solution the next day, continuing culturing, and observing the growth condition.
4.3.2 passage of cells
After the cells grow to 80-90%, sucking out the cell culture solution under aseptic operation condition by adopting a plastic suction pipe with the specification of 3mL, adding 1-2mL of PBS for washing 1 time (without calcium and magnesium ions), adding 1mL of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture flask, observing the digestion condition of the cells under an inverted microscope, if the cells are mostly rounded, quickly taking back to an operating table, tapping the culture flask, and adding 2mL of complete culture medium to terminate the digestion. 4mL of fresh complete medium was added to each new flask, followed by 1mL of complete medium containing cells.
4.4CCK-8 assay to reverse tumor cytotoxic Activity
4.4.1 concentration gradients were set for MCF-7/ADR (0, 6.25, 12.5, 25, 50, 100, 200. Mu.g/mL), K562/ADR and A549/ADR (0, 1.6, 3.125, 6.25, 12.5, 25, 50, 100), 3 replicates, respectively; the concentration of the Rubracin F is 5, 10 and 20 mug/mL; verapamil was positive control, DMSO was negative control, and all data were calculated using SPSS software.
4.4.2 Experimental procedures
(1) Cell digestion, cell count, and cell concentration adjustment to 2×10 4 And each mL.
(2) 100uL of the cell suspension was seeded in 96-well plates. The plates were incubated at 5% CO 2 Culturing in incubator at 37deg.C for 24 hr.
(3) According to the grouping, the compound and doxorubicin with different concentrations are added respectively, and the mixture is placed in an incubator to be incubated for 48 hours at 37 ℃.
(4) After the incubation, PBS (without calcium and magnesium ions) was washed 1 time, 10. Mu.L of CCK-8 reagent was added to each well, and incubated in an incubator for 3 hours.
(5) Measurement of absorbance at 490nm with an ELISA reader
4.4 experimental results
The result shows that the compound Rubracin F has reverse drug-resistant MCF-7/ADM activity at the concentration of 10 mug/mL and 20 mug/mL, and the IC thereof 50 Values were 91.849 μg/mL, 50.156 μg/mL, respectively; the compound Rubracin F has reverse drug resistance A549/ADM and K562/ADM activities at concentrations of 5 μg/mL, 10 μg/mL and 20 μg/mL respectively, and the reverse drug resistance tumor cell activity increases with increasing Rubracin F concentration, its detailed IC 50 See table 2.
Table 2 IC of the compound Rubracin F in combination with ADM against three resistant tumor cells 50 Value of
Remarks: A. b, C the compound concentrations were 5. Mu.g/mL, 10. Mu.g/mL and 20. Mu.g/mL, respectively
The foregoing is merely exemplary embodiments of the present application, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present application, and these should also be considered as the scope of the present application, which does not affect the effect of the implementation of the present application and the utility of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (2)
1. A preparation method of a long-chain fatty acid glycerol alcohol compound is characterized by comprising the following steps of: the structure is shown as follows:
the compound is obtained after fermentation and extraction of the Caulophyllum reesei, wherein the Caulophyllum reesei is named as Caulophyllum reesei Tubeufia rubra PF02-2, and the preservation unit is as follows: china Center for Type Culture Collection (CCTCC) NO: m2019957; the preparation method specifically comprises the following steps:
s1, strain activation: taking out the preserved strain, inoculating the strain on a basal medium plate, standing, culturing and passaging to the third generation, and then amplifying and culturing;
s2, fermenting and culturing: inoculating the strain activated in the step S1 into a solid culture medium, and standing, fermenting and culturing for a period of time at 26-30 ℃;
s3, extracting: extracting thallus and culture medium with ethyl acetate, concentrating the extractive solution to obtain fermentation product;
s4, pretreatment of fermentation products: dissolving a fermentation product by using a chlorine methyl=1:1 solvent, uniformly mixing the fermentation product with silica gel according to the mass ratio of 1:1-2, taking the mixture as a primary column sample after the solvent volatilizes, sequentially and gradiently eluting with petroleum ether, chloroform, ethyl acetate and methanol respectively on a silica gel powder and a petroleum ether separation column, recovering the eluting solvent by a rotary evaporator under reduced pressure, dissolving the eluting solvent by using chloroform, acetone or methanol, spreading the eluting solvent by using a spreading agent, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and developing by using an 8% sulfuric acid ethanol vanillin color developing agent; combining the methanol solvent eluates, and recovering the methanol solvent to obtain a methanol layer extract;
s5, purifying and separating: a. dissolving the methanol layer extract with methanol solvent, uniformly mixing the methanol layer extract with silica gel according to the mass ratio of 1:1-3, carrying out equilibrium reverse phase compression on an upper column sample after solvent volatilization, adding 10% methanol water to carry out equilibrium reverse phase compression on the column, adding the pre-column containing the sample, sequentially carrying out 10 gradient elution with the methanol water, recovering the solvent from eluent by a rotary evaporator, dissolving the methanol, then spreading the eluent by a thin layer chromatography dot plate, using a developing agent, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and then combining components with black color development of 8% sulfuric acid ethanol vanillin developer to obtain Fr.11 components;
b. after the component Fr.11 is dissolved by a methanol solvent, the mixture is uniformly mixed with silica gel according to the mass ratio of 1:1-3, and after the solvent volatilizes, the mixture is used as a pre-column on a column sample; performing balanced counter-phase column pressing by 30% methanol water, balancing 5-6 column volumes, adding a pre-column containing a sample, adding an upper column sample, sequentially performing 8 gradient elution by using methanol water, recovering a solvent from eluent by a rotary evaporator, dissolving the methanol, then using a thin layer chromatography point plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and then combining 8% sulfuric acid ethanol vanillin developer grey-black components to obtain Fr.11-13 components;
c. dissolving Fr.11-13 with methanol, uniformly mixing with silica gel according to a mass ratio of 1:1-3, taking an upper column sample after the solvent volatilizes, weighing silica gel powder, uniformly mixing with an ethyl methyl=1:1 solvent, loading into a separation column, adding the upper column sample, adopting an ethyl methyl system for gradient elution, using a thin layer chromatography point plate for eluent, using a developing agent to develop, selecting liquid with fluorescence at 254nm or 365nm under an ultraviolet-visible light analyzer, and then combining 8% sulfuric acid ethanol vanillin developer grey-black components to obtain the long-chain fatty acid glycerol alcohol compound.
2. A process for the preparation of a compound according to claim 1, characterized in that: the solid medium in the step S2 is oat medium, and 200g of oat and 150mL of double distilled water are mixed to obtain the oat culture medium.
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