A chromone and its application in preparing a drug with anti-tumor activity

Abstract

The present invention relates to the technical field of pharmaceutical chemistry, and specifically discloses a method for preparing a novel endophytic fungus from potato. Bipolar disorder A chromone compound Bipolarisketone A isolated from the present invention and its application potential in the preparation of anti-tumor drugs, more specifically, in the preparation of drugs with significant inhibitory activity on tumor cells. The chromone of the present invention can strongly inhibit leukemia HL-60, liver cancer SMMC-7721 and colon cancer SW480 cells.

Description

Technical Field

[0001] The invention relates to the technical field of medicine and chemistry, and in particular to a chromone derived from potato endophytic fungus Bipolaris eleusines and application of the chromone in preparing a drug with anti-tumor activity. Background Art

[0002] A tumor is a new organism formed when local cells lose control of their growth at the genetic level under the influence of various tumorigenic factors, resulting in abnormal cell proliferation. A tumor is a solid mass of tissue formed when abnormal cells gather together. Tumors can affect bones, skin, tissues, organs, and glands. Tumors are divided into cancerous tumors and noncancerous tumors. Malignant or cancerous tumors can spread to nearby tissues, glands, and other parts of the body, and cancerous tumors can recur after treatment (cancer recurrence). These tumors may be life-threatening. Benign tumors do not become cancerous and are rarely life-threatening. They are local, which means that they usually do not affect nearby tissues or spread to other parts of the body. Many noncancerous tumors do not require treatment, but some noncancerous tumors compress other parts of the body and do require medical care. Malignant tumors are complex diseases caused by the combined effects of multiple causes such as social environmental factors and genetic factors. At present, the clinical treatment of cancer mainly relies on surgical resection, radiotherapy and chemotherapy, and traditional Chinese medicine. With the in-depth study of tumor science, the research and development and application of anti-tumor drugs have developed rapidly. New anti-tumor drugs represented by small molecule targeted drugs and large molecule monoclonal antibodies have been widely used in clinical practice, bringing new methods for drug treatment of tumors. However, new anti-tumor drugs have a short time to market, and there are many limitations in clinical research before market launch, such as strict subject screening, limited sample size, and short observation period.

[0003] Potato, also known as potato, ground egg, and sweet potato, is an annual plant of the genus Solanum in the Solanaceae family. As one of the four major food crops in my country, potato is both edible and edible, and occupies an important position in my country's agricultural production. Bipolaris is an important type of plant pathogen that can cause leaf spot, leaf rust, and root rot in corn, wheat, rice, and sorghum, causing serious economic losses. Since the last century, researchers have gradually carried out basic research on the pathogenic substances of Bipolaris, revealing that different types of compounds such as terpenes, ketones, and peptides are the main mycotoxins of this genus of fungi. After that, it is no longer limited to studying its pathogenic substances, but has begun to explore its secondary metabolites extensively, and explore the active application value of different types. Bipolaris belongs to the family of Sporomycetes. So far, a total of 47 species of Bipolaris have been identified, and their taxonomy, geographical distribution, and species synonyms have been clarified. Because of the important role of Bipolaris in plant pathogens, some have been widely used in biotechnology and genetic manipulation. The scientific name of chromone is benzo-γ-pyrone. The derivatives of benzo-γ-pyrone are widely present in plants. Some of them are colored substances, so their parent is called chromone. They have a wide range of pharmacological effects such as anti-inflammatory, antibacterial, and anti-tumor. The applicant isolated a chromone from endophytic fungi in potato and explored its anti-tumor activity. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a chromone and its use in the preparation of a drug with anti-tumor activity.

[0005] A chromone with anti-tumor activity, the structural formula of which is as follows:

[0006]

[0007] Molecular formula: C 16 H 12 O 8

[0008] The use of the chromone in the preparation of a drug for inhibiting tumor cell growth also falls within the protection scope of the present invention.

[0009] Another object of the present invention is to provide a use of the chromone in the preparation of anti-tumor drugs.

[0010] In the present invention, the anti-tumor activity is specifically embodied in: half inhibition of in vitro tumor cell growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW480. The experiments of the present invention prove that the chromone can effectively inhibit tumor cell growth and has the potential use for preparing new anti-tumor drugs.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] 1. The present invention discloses a chromone derived from the potato endophytic fungus Bipolaris eleusines, which can inhibit the growth of tumor cells.

[0013] 2. The present invention provides a chromone with anti-tumor activity, which has the potential use in preparing new anti-tumor drugs.

[0014] 3. The chromones to be protected in the present invention can be obtained by extraction and purification from endophytic fungi in plants, and have the advantages of short culture cycle, high operational feasibility, no chemical pollution, and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The hydrogen spectrum (600 MHz, acetone-d 6 );

[0016] Figure 2 The carbon spectrum (150 MHz, acetone-d 6 ) and DEPT diagram;

[0017] Figure 3 It is the HMQC diagram of the compound prepared in Example 1 in the specific embodiment;

[0018] Figure 4 It is the HMBC diagram of the compound prepared in Example 1 in the specific implementation mode;

[0019] Figure 5 The compound prepared in Example 1 in the specific embodiment 1 H- 1 H COSY diagram;

[0020] Figure 6 ROESY diagram of the compound prepared in Example 1 in the specific embodiment;

[0021] Figure 7 This is a graph showing the tumor cell inhibition of the compound prepared in Example 1 in a specific embodiment. DETAILED DESCRIPTION

[0022] In order to make the invention purpose and invention content of this application more clearly presented, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] Embodiment 1:

[0026] The raw material potato endophytic fungus was isolated from the stem of fresh potato plants in Lincang. The strain was numbered E-19. The ITS sequence was compared with the maximum similarity of the sequence with Bipolaris eleusines, which was as high as 99%. The gene bank accession number was KY909768.1. Therefore, it was identified as Bipolaris eleusines. The strain was deposited in the microbial strain bank of the School of Pharmacy of South-Central University for Nationalities (already published, see: Yang Mansi, Wang Wenxiang, Li Longgen, et al. Isolation, identification and secondary metabolite research of potato endophytic antagonistic fungus E-19 [J]. Natural Product Research and Development, 2015, 27 (10): 1728-1731, 1736).

[0027] The specific isolation process of the above-mentioned Lincang potato endophytic fungus E-19 is as follows: rinse the stems of healthy potato plants under tap water, wipe dry, and cut the stems into 3 cm small segments. Then, under sterile conditions, rinse with 75% ethanol for 1 minute, rinse with sterile water 3 times; disinfect with 2% sodium hypochlorite for 2 minutes, rinse with sterile water 3 times; then place on sterile filter paper to absorb the moisture. Cut off the incisions at both ends of the surface-sterilized stems, split the remaining segments in the middle, and cut into 0.5 cm 3 The small pieces were sterilized on the surface by vertical pressure steam sterilizer (YXQ-LS-100S1, Shanghai Boxun). Then the small pieces were inoculated on PDA medium under sterile conditions and cultured in a constant temperature incubator at 25°C. The top hyphae purification method was used to regularly observe the growth of endophytic fungi every day, and the tip of the newly grown hyphae at the incision was picked and transferred to PDA medium, purified and cultured until a pure strain was obtained, and the purified pure strain was transferred to a test tube containing PDA medium and stored in a 4°C refrigerator.

[0028] The strain E-19 was expanded by wheat solid fermentation: the test tube was taken out of the refrigerator and placed in a sterile environment at 25°C for one night. Then, a bacterial block with a diameter of 0.5 cm was taken from the test tube and inoculated on a flat PDA medium. After growing for about 7 days, the colony covered the PDA and was set aside. The wheat medium ratio: 50 g wheat / bottle, 50 mL distilled water / bottle, placed in a 500 mL culture bottle, sterilized at 120°C for 30 minutes, and after cooling, 0.5 cm was picked from the flat PDA medium in a sterile environment. 3The bacterial blocks were inoculated onto wheat culture medium, a total of 224 bottles, and the culture conditions were: dark culture at 25°C for 40 days.

[0029] The separation and purification process of the compound chromone described in the claims and the invention content of the specification is as follows: the whole wheat solid culture medium after the fermentation is crushed and soaked with a strong polar mixed solvent (dichloromethane: methanol = 1:1, v / v) for 5 times, each time for 12 hours. Centrifuge after each soaking, combine the extracts after 5 centrifugations, concentrate under reduced pressure and evaporate the solvent, dissolve with a small amount of water and ethyl acetate (water: ethyl acetate = 1:1, v / v), and extract with ethyl acetate 5 times, combine the ethyl acetate parts and concentrate under reduced pressure to obtain 166.7g of crude extract. Use 80-100 mesh normal phase silica gel chromatography column chromatography (dichloromethane: methanol = 100:0-0:100, v / v) gradient elution, and use thin layer chromatography (developing solvent is dichloromethane: methanol = 20:1, v / v; thin layer chromatography silica gel plate, Qingdao Ocean Chemical Plant) to detect and develop color, and roughly segment to obtain six components A-F.

[0030] Component D (dichloromethane: methanol = 20:1, v / v, eluent dosage of 10 column volumes, 31.6 g) was subjected to medium pressure liquid chromatography (Biotage SP1, reverse phase filler material: RP-18, 20-45 μm, Fuji Silysia Chemical Ltd., Japan) using methanol and water as eluents (methanol / water = 40:60, 60:40, 80:20, 100:0, v / v, eluted in sequence), gradient elution was performed at a flow rate of 20 mL / min, and the elution time for each ratio was 40 min. Thin layer chromatography (developing solvent was dichloromethane: methanol = 10:1, v / v) was used for detection and color development, and the same or similar components were combined to obtain 10 subcomponents, which were labeled D1-D10 in order from small to large polarity. Fraction D5 (eluted in the first 30 minutes at a methanol / water ratio of 60:40, 1.35 g) was eluted with methanol using a Sephadex LH-20 gel column chromatography (Pharmacia Fine Chemical Co., Ltd., Sweden) in an amount of 5 column volumes of methanol, and detected and developed using thin layer chromatography (developing solvent: dichloromethane: methanol = 10:1, v / v). The same or similar components were combined and separated to obtain thirteen subcomponents, which were labeled D5-1 to D5-13 in order of polarity from small to large. Fraction D5-9 (obtained after elution with methanol for 12 h, 989.0 mg) was gradient eluted with a 200-300 mesh silica gel column (petroleum ether: ethyl acetate = 30:1-0:1, v / v) for 12 hours, and detected and developed by thin layer chromatography (developing solvent: petroleum ether: ethyl acetate = 5:1, v / v; thin layer chromatography silica gel plate, Qingdao Ocean Chemical Plant) to obtain 4 subcomponents, which were labeled D5-9-1 to D5-9-4 in order from small to large polarity. Fraction D5-9-3 (eluted in the first twenty minutes when the eluent is petroleum ether: ethyl acetate at a ratio of 10:1, 18.2 mg) was subjected to high performance liquid chromatography (Agilent 1260; chromatographic column: Agilent Zorbax SB-C18, specification: 9.4 mm×150 mm, 5 μm; acetonitrile-water, 50:50–60:40, v / v; flow rate: 4 mL / min) by gradient elution for 24 min to obtain 1.2 mg of the chromone of the present invention (retention time: 18.5 min).

[0031] Structural identification of the compound: The chromone prepared in Example 1 was dissolved in 0.5 mL of deuterated acetone, transferred to an NMR tube using a 200 μL pipette, and the hydrogen spectrum, carbon spectrum, and two-dimensional spectrum (such as Figure 1-6 ). The structure of the compound was solved by integrating various physical and chemical data and named Bipolarisketone A.

[0032] The NMR data of the obtained chromone:

[0033]

[0034] Other physical and chemical data of chromone:

[0035] Appearance: yellow powder; High resolution mass spectrometry HR-ESI-MS m / z 355.04236[M+Na] + (calcd for C 16 H 12 O 8 Na + ,355.04244).

[0036] According to the above test results, the structural formula of the compound obtained in this example is confirmed to be:

[0037]

[0038] Molecular formula: C 16 H 12 O 8

[0039] Example 2: MTS detection of cell viability

[0040] Principle: MTS is a new MTT analogue, the full name is 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, which is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan compounds. The content of formazan can be measured at 490nm using an enzyme marker. Under normal circumstances, the amount of formazan generated is proportional to the number of living cells, so the number of living cells can be inferred based on the optical density OD value.

[0041] A single cell suspension was prepared with DMEM culture medium containing 10% fetal bovine serum, and 3000 to 15000 cells were inoculated into a 96-well plate with a volume of 100 μL per well. The cells were inoculated and cultured 12 to 24 hours in advance. 100 μL of each gradient concentration drug (compound obtained in Example 1, 40 μM, 8 μM, 1.6 μM, 0.32 μM, 0.064 μM) dissolved in DMSO was added to the above 96-well plate. 20 μL of MTS solution (5 mg / ml, i.e. 0.5% MTS) was added to each well, and the 96-well culture plate was returned to the incubator and incubated at 37°C for 4 hours. Then the culture was terminated, and the absorbance of each well was measured at OD490nm of the enzyme-linked immunosorbent assay.

[0042] Experimental results: The cytotoxicity of the compound of Example 1 after co-culture with leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW480 (all cells are from Kunming Institute of Botany, Chinese Academy of Sciences) was detected by MTS method. The experimental results show that the cell survival rate is less than 30% when the compound is added at a concentration of 40 μM, indicating that the compound has a certain inhibitory effect on the above five tumor cells.

[0043] (OD value indicates the absorbance of a substance at a specific wavelength; the blank group only contains culture medium, MTS, and DMSO, but no cells; the drug concentration in the control cell group is zero)

[0044] Example 3: Antitumor activity assay

[0045] Experimental steps:

[0046] (1) Cell culture: The five tumor cells were prepared into single cell suspensions using DMEM medium containing 10% fetal bovine serum, and 3,000 to 15,000 cells were inoculated into 96-well plates with a volume of 100 μL per well. The cells were inoculated and cultured 12 to 24 hours in advance. The cell concentration was adjusted to 5×10 4 100 μL of cells were inoculated into a new 96-well plate. To avoid edge effects, cells were inoculated only in the middle area of ​​6×10 wells. 100 μL of sterile PBS buffer (pH=7.4, 0.01 M) was added to each well outside the cell wells. 5% CO 2 , and culture at 37°C for 6-12h.

[0047] (2) Drug treatment: Add the test compound solution (the compound prepared in Example 1): the compound was dissolved in DMSO and initially screened at a concentration of 40 μM. The final volume of each well was 200 μL. Three replicate wells were set for each treatment.

[0048] (3) Color development: After culturing at 37°C for 48 hours, discard all the culture medium in the wells of adherent cells (A549, SMMC-7721, MDA-MB-231, SW480), add 20 μL of MTS solution and 100 μL of DMEM culture medium containing 10% fetal bovine serum to each well; discard 100 μL of culture supernatant of suspension cells (HL-60), add 20 μL of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20 μL of MTS solution and 100 μL of DMEM culture medium). Continue incubation for 2 to 4 hours to allow the reaction to proceed fully, and then measure the light absorbance value.

[0049] (4) Colorimetry: Select 492 nm wavelength and use a multifunctional microplate reader (MULTISKAN FC) to read the light absorption value of each well and record the results. After data processing, a cell inhibition rate graph is drawn with the compound number as the horizontal axis and the cell inhibition rate as the vertical axis ( Figure 7 ).

[0050] (OD value indicates the absorbance of a substance at a specific wavelength; the blank group only contains culture medium, MTS, and DMSO, but no cells; the drug concentration in the control group is zero)

[0051] (5) Positive control compounds: Two positive control compounds, cisplatin (DDP) and paclitaxel (Taxol), were set up in each experiment. The solution preparation was the same as step (2). The cell growth curve was drawn with the concentration (0.064, 0.32, 1.6, 8, 40 μM) as the horizontal axis and the cell survival rate as the vertical axis. The IC of the compound was calculated using the two-point method (Reed and Muench method). 50 The results are shown in the following table.

[0052] (OD value indicates the absorbance of a substance at a specific wavelength; the blank group only contains culture medium, MTS, and DMSO, but no cells; the drug concentration in the control cell group is zero)

[0053] Table 1

[0054]

[0055]

[0056] Experimental results:

[0057] The cell viability was detected by MTS test. The results showed that the compound Bipolarisketone A at a concentration of 40 μM reduced the survival rates of HL-60, A549, SMMC-7721, MDA-MB-231, and SW480 tumor cells to less than 30%, indicating that the compound had a half-inhibitory effect on the in vitro growth of these five tumor cells. Therefore, it is meaningful to further study its anti-tumor mechanism. Figure 7 As shown in the in vitro activity test, the chromone has a half-inhibitory effect on the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW480. In the drug administration group, the IC 50 The value is lower than that of cisplatin (as shown in Table 1). These results show that the compound has a highly effective inhibitory effect on some tumor cells.

Claims

1. A chromone having the structural formula: 。 2. Use of the chromone according to claim 1 in the preparation of a drug for inhibiting tumorigenesis.

3. Use of the chromone according to claim 1 in the preparation of a drug for inhibiting the growth of tumor cells HL-60, A549, SMMC-7721, MDA-MB-231 and / or SW480.

4. Use of the chromone according to claim 1 in the preparation of anti-tumor drugs.

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