阅读说明：本技术 一种长链脂肪酸甘油醇类化合物Rubracin A、制备方法及其应用 (Long-chain fatty acid glycerol alcohol compound Rubracin A, preparation method and application thereof ) 是由 康冀川 钱声艳 曾学波 钱一鑫 卢永仲 陈丽庄 何张江 于 2021-09-30 设计创作，主要内容包括：本申请涉及微生物技术领域中的一种长链脂肪酸甘油醇类化合物RubracinA,结构如下式所示：所述化合物由红棕毛筒腔菌发酵提取后获得,所述红棕毛筒腔菌命名为红棕毛筒腔菌Tubeufia rubra PF02-2,保藏单位：中国典型培养物保藏中心,保藏编号为CCTCC NO：M 2019957。该化合物具有制备肿瘤耐药逆转剂或肿瘤药物增敏剂的用途。(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 the phaeosphaeria rubra, the phaeosphaeria rubra is named as phaeosphaeria rubra Tubeufia PF02-2, and the preservation unit is as follows: typical of ChinaThe culture collection center has a collection number of CCTCC NO: m2019957. The compound has the application of preparing tumor drug resistance reversal agent or tumor drug sensitizer.)

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 the preparation of a compound according to claim 1, characterized in that: the compound is obtained by fermenting and extracting the phaeosphaeria rubra, the phaeosphaeria rubra is named as phaeosphaeria rubra Tubeufia 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: carrying out 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 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 solvent of methyl 1:1, uniformly mixing the fermentation product with silica gel according to a mass ratio of 1: 1-2, volatilizing the solvent to obtain a primary column sample, then loading the primary column sample on silica gel powder and a petroleum ether separation column, sequentially carrying out gradient elution by using petroleum ether, chloroform, ethyl acetate and methanol, carrying out reduced pressure recovery on an elution solvent by using a rotary evaporator, dissolving the elution solvent by using chloroform, acetone or methanol, carrying out thin-layer chromatography on a dot plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and developing 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 the mass ratio of 1: 1-3, loading the methanol layer extract on a pre-column when the solvent volatilizes, carrying out balanced reversed-phase medium-pressure column by adopting 10% methanol water, adding a pre-column containing a 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, spreading the eluent by using 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 the components with the developing gray black color of an 8% ethanol sulfate vanillin developer to obtain a Fr.11 component;

b. dissolving the component Fr.11 in a methanol solvent, uniformly mixing the dissolved component Fr.11 with silica gel according to the mass ratio of 1: 1-3, and putting the mixture into a pre-column 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 according to a mass ratio of 1: 1-3, volatilizing the solvent, taking a sample on a column, mixing with silica gel powder and a solvent of which the ratio of chlorine to methyl is 1:1, putting the mixture on a separation column, and adopting chloroform: gradient elution is carried out on the component A1: 1, and the eluent is combined by adopting a thin layer chromatography dot plate to obtain a component Fr.11-6-1;

d. performing normal phase silica gel column chromatography on Fr.11-6-1, performing gradient elution with a ratio of ethyl to methyl being 1:1 and the like, and combining TLC point 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 of 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 of claim 1 and a pharmaceutically acceptable carrier in the preparation of a tumor drug resistance reversal agent or a tumor drug sensitizer.

7. Use according to claim 6, characterized in that: the drug or tumor drug is adriamycin.

8. Use according to claim 6, characterized in that: the tumor comprises breast cancer.

9. 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.

10. Use of a compound of claim 1 and a pharmaceutically acceptable carrier for the preparation of a medicament against breast cancer cells.

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 tumors mainly comprises chemotherapy, surgery, radiotherapy and the like, and the chemotherapy is one of the main means of cancer treatment. 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, PSC833), 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 (XR9576), Zosuquidar (LY335979), S9788, etc., of which Tariquidar (XR9576) and WK-X-34 are representative (Massey P.R. et al.2014). The development of P-gp inhibitors from natural products and their derivatives has become a new direction and focus 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 drug-resistant tumor cell activity.

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 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 rubicun, 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 a 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;

the sampling mode is as follows: 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 phaeophycus erythraea rubella PF02-2 has the following properties:

and (3) colony morphology characteristics: on a 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 bacterial colonies are large in quantity, are colorless, transparent and white on fresh PF02-2 pure bacterial colonies obtained through separation, and are reddish brown after the PF02-2 pure bacterial colonies obtained through separation are naturally dried. Part of the mycelium is buried under the substrate, but mostly is epibiotic, and the mycelium is composed of membrane-branched mycelium 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 and has a diameter of 16mm, brown color, round shape, rough surface, obvious protrusions, pulse-like wrinkles, and complete colony edge.

Specifically, the preparation method comprises the following steps: carrying out 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 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, fermentation product pretreatment: dissolving a fermentation product by using a solvent of methyl 1:1, uniformly mixing the fermentation product with silica gel according to a mass ratio of 1: 1-2, volatilizing the solvent to obtain a primary column sample, then loading the primary column sample on silica gel powder and a petroleum ether separation column, sequentially carrying out gradient elution by using petroleum ether, chloroform, ethyl acetate and methanol, carrying out reduced pressure recovery on an elution solvent by using a rotary evaporator, dissolving the elution solvent by using chloroform, acetone or methanol, carrying out thin-layer chromatography on a dot plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and developing 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 the mass ratio of 1: 1-3, loading the methanol layer extract on a pre-column when the solvent volatilizes, carrying out balanced reversed-phase medium-pressure column by adopting 10% methanol water, adding a pre-column containing a 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, spreading the eluent by using 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 the components with the developing gray black color of an 8% ethanol sulfate vanillin developer to obtain a Fr.11 component;

b. dissolving the component Fr.11 in a methanol solvent, uniformly mixing the dissolved component Fr.11 with silica gel according to the mass ratio of 1: 1-3, and putting the mixture into a pre-column 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 according to a mass ratio of 1: 1-3, volatilizing the solvent, taking a sample on a column, mixing with silica gel powder and a solvent of which the ratio of chlorine to methyl is 1:1, putting the mixture on a separation column, and adopting chloroform: gradient elution is carried out on the component A1: 1, and the eluent is combined by adopting a thin layer chromatography dot plate to obtain a component Fr.11-6-1;

d. performing normal phase silica gel column chromatography on Fr.11-6-1, performing gradient elution with a ratio of ethyl to methyl being 1:1 and the like, and combining TLC point plates to obtain the compound Rubracin A.

Wherein the solid culture medium of step S2 is oat culture medium, and is obtained by mixing 200g oat and 150mL 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 invention¹H-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 Rubracin A, a compound 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 triangular flask, wherein the inoculation amount of each flask is 1 multiplied by 1cm of the area on a culture plate²The amount of the activated strain of (4) was cultured by standing at 28 ℃ for 105 days.

S3, extraction

Adding ethyl acetate into the thallus and the oat culture medium, extracting for three times, performing oscillation extraction for 24h at 160rpm each time, combining the extract, performing reduced pressure concentration at 40 ℃ to obtain a fermentation product, repeating the above operations, and combining the fermentation products to obtain 2027.17 g.

S4 pretreatment of fermentation product

Dissolving 2027.17g of fermentation product by using a solvent of methyl 1:1, uniformly mixing with silica gel according to a mass ratio of about 1:1.5 (namely, 2027.17g of fermentation product is added with 3041g of 200-300-mesh silica gel powder), volatilizing the solvent to obtain a river sand-shaped sample, and taking the river sand-shaped sample as a primary column sample; 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 column sample once, sequentially carrying out 4 gradient elution by using petroleum ether, chloroform, ethyl acetate and methanol, carrying out 2-3 (36L-54L 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 elution sample into a penicillin bottle with the specification of 20mL, carrying out Thin Layer Chromatography (TLC) to spot a plate, and using petroleum ether: 1:1 of chloroform, petroleum ether: acetone 10:1, chloroform: acetone-5: 1, chloroform: methanol 10:1, ethyl acetate: developing with 5:1 methanol developing agent, observing whether fluorescence is present at 254nm or 365nm under conventional ultraviolet visible light analyzer, and developing with 8% ethanol sulfate 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 (35.77g) with methanol solvent, mixing with silica gel at a mass ratio of about 1:2 (i.e. adding medium pressure RP-18 reverse phase silica gel into 35.77g 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 balancing a reversed-phase medium-pressure column (with the column length of 460mm and the diameter of 49mm) by using 10% methanol water, balancing about 5-6 column volumes (eluting 5-6L), adding a pre-column containing a sample, performing gradient elution by using methanol water (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), sequentially performing 10 gradient elution, performing gradient elution to 5 column volumes, 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 (thin layer chromatography) plate, 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 mixing 8% ethanol sulfate vanillin components with gray black color (also 90% methanol water-eluted components) to obtain Fr.11 component (8.7 g).

b. Dissolving the component Fr.11(8.7g) with a methanol solvent, uniformly mixing with silica gel according to a mass ratio of about 1:2 (namely adding 17.4g of medium-pressure RP-18 reverse phase silica gel into 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; the method comprises the steps of balancing a reversed-phase medium-pressure column (with the column length of 460cm and the diameter of 49mm) by 30% methanol water, balancing about 5-6 column volumes (eluting 5-6L), adding a pre-column containing a sample, performing gradient elution by methanol water (30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), sequentially performing 8 gradient elution, eluting 4-5 column volumes by each gradient, receiving eluent by a triangular flask with the specification of 500mL, recovering a solvent from each eluent by 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 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 conventional ultraviolet visible light analyzer, developing with 8% ethanol sulfate vanillin developer, and mixing 8% ethanol sulfate vanillin components (also component eluted with 90% methanol water) to obtain Fr.11-6 components (206 mg).

c. Fr.11-6(206mg) is dissolved by methanol, and is uniformly mixed with silica gel according to the mass ratio of about 1:1.5 (namely, 200-300 silica gel 310mg is added in 206mg components), and a river sand-shaped sample is obtained after the solvent is volatilized and is used as an upper column sample; weighing 12g of 200-mesh 300-mesh silica gel powder and a chlorine-A1: 1 solvent, uniformly mixing (no bubbles can be generated in the process), 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, and adopting chloroform: gradient elution with a ratio of 1:1, collecting the eluate in a 20mL penicillin bottle, and combining by TLC spot plate to obtain fraction Fr.11-6-1(35 mg).

d. Fr.11-6-1(35mg) was chromatographed on normal phase silica gel using a gradient of 1:1 with ethyl to methyl, and the resulting mixture was combined on TLC plates to give the compound Rubracin A10 mg.

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^-11626 shows that the compound has hydroxyl and ester groups. HRESI (see FIG. 2 for details) shows a molecular weight of 520.3611[ M + Na ]]⁺Molecular formula is C₂₈H₅₁NNaO₆Calculating the degree of unsaturation to be 4;¹³c 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)]；¹H NMR (fig. 5) in combination with HSQC (fig. 7) showed that the compound had one aliphatic chain [0.90(3H, t, J ═ 6.9Hz),1.28 to 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 to 5.38(4H, m)]The 8 heteroatom-linked 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 ═ 11.4,2.7Hz),3.94(1H, m),4.06(1H, dd, J ═ 11.4,6.3Hz),4.16(1H, dd, J ═ 11.4,4.4Hz)]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_C175.4,73.3, 69.5; delta_H[3.94(1H,m)]And delta_C66.5,73.3 related; delta_H[3.94(1H,m)]And delta_C66.5,73.3 related; delta_H[3.49(2H,dd,J＝13.3,5.2Hz)]And delta_C66.5,68.5 related; the above correlation demonstrates that the compound contains a glycerol moiety and delta_C66.5(t, C-1) attachment to the ester group delta_C[175.4(s,C-1′)]Upper, delta_C69.5 attachment to C-1, delta_C73.3 attachment to C-2, 68.5(t, C-4)]With glycerol delta_H[3.49 structural fragment where (2H, dd, J ═ 13.3,5.2Hz,73.3(t, C-3) form ether ] furthermore, hydrogen proton signal on unsaturated fatty chain [2.35(2H, t, J ═ 7.5Hz),34.9(t, C-2')]In relation to 175.4,26.0, the ester group was shown to be attached to an unsaturated fatty chain. Finally, delta_H[3.53(1H,td,J＝12.6,4.1Hz),3.65(1H,m),]And delta_C73.3,29.0, 77.6; delta_H[2.22(1H,m)]And delta_C77.6,171.8, are related; delta_H[2.06(1H,m)]And delta_C77.6,171.8, 68.5; delta_H 3.75[(1H,dd,J＝11.4,2.7Hz),77.6(d,C-6)]And delta_C171.8,68.5,29.0, 52.3; the above correlation demonstrates that 77.6(d, C-6) and the remaining ester group delta_C171.8(s, C-7) are linked. Combining the above HMBC correlations, a molecular formula C is given in combination with high resolution mass spectrometry₂₈H₅₁NNaO₆And finally determining the structure of the compound. Careful analysis¹H-¹H COSY, mark of resultsThe above presumed structure is verified to be correct.

Compound Rubracin A¹H-¹H COSY

EI-MS (see FIG. 8 for details) showed fragment ion M/z 438([ M-C)₂H₃O]^-)，117([M-C₂H₃O-C₁₈H₃₁O]^-) 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 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 vial was completely dissolved, the cells were taken out under aseptic conditions and inoculated into a cell culture dish (RPMI1640+ 10% fetal bovine serum), and placed at 37 ℃ in CO₂Culturing in incubator, changing culture solution the next day, continuing culturing, and observing 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 (without calcium and magnesium ions) for washing for 1 time, adding 1mL of digestive juice (0.25% of Trypsin-0.53mM EDTA) into a 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 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 procedures

(1) Digesting the cells, counting the cells, adjusting the cell concentration to 2X 10⁴one/mL.

(2) 100uL of cell suspension was seeded in 96-well plates. Plates were incubated at 5% CO₂Culturing in an incubator at 37 ℃ for 24 h.

(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 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 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.

Fourth, the active application of the compound Rubracin A in reversing MCF-7/ADR tumor cell drug resistance

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 cells from liquid nitrogen tube, quickly putting the freezing tube into water bath kettle preheated to 37 deg.C, and quickly thawingShaking to melt the liquid in the tube rapidly. 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 (RPMI1640+ 10% fetal bovine serum), and placed at 37 ℃ in CO₂Culturing 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 (without calcium and magnesium ions) for washing for 1 time, adding 1mL of digestive juice (0.25% of Trypsin-0.53mM EDTA) into a 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 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 test for reversing 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) Digesting the cells, counting the cells, adjusting the cell concentration to 2X 10⁴one/mL.

(2) 100uL of cell suspension was seeded in 96-well plates. Plates were incubated at 5% CO₂Culturing in an incubator at 37 ℃ for 24 h.

(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 3 h.

(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: rubracin A activity of reversing MCF-7/ADR tumor cell drug resistance

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 thereof₅₀The value was 51.35ug/mL, positive control verapamil IC₅₀The value was 31.79 ug/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.