Aromatic polyketide compound, and preparation method and application thereof
By isolating and extracting aromatic polyketide compounds from Crystallomyces hypericus fungus, the shortcomings of existing technologies in the development of multi-target antitumor drugs have been overcome, achieving effective inhibition of various cancer cells and demonstrating the potential for anticancer drug development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- KUNMING UNIVERSITY
- Filing Date
- 2026-02-10
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies are insufficient to effectively utilize fungal-derived aromatic polyketide compounds to develop multi-target anti-tumor drugs, and lack inhibitory effects on various cancer cells.
Two aromatic polyketide compounds were isolated and extracted from the fungus Crystallomyces hypericus. Compound I and compound II were prepared by specific culture medium and extraction method, and then separated by gradient reversed-phase column chromatography and silica gel column chromatography to obtain compounds with anticancer activity.
Compounds I and II showed significant inhibitory effects on human liver cancer, cervical cancer, neurocarcinoma, and pancreatic cancer cells, with IC50 values ranging from 19 to 31 μmol/L, demonstrating their potential for development into anticancer drugs.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to an aromatic polyketide compound, its preparation method, and its application. Background Technology
[0002] Cancer is now the second leading cause of death worldwide, after cardiovascular disease. With the continued rise in global cancer incidence and mortality rates, the threat of cancer to population health is becoming increasingly serious. According to statistics and estimates from the World Health Organization (WHO), incidence and mortality rates are increasing year by year.
[0003] Fungi, acting as "biosynthetic factories" of natural products, can produce structurally diverse polyketide compounds via the polyketide synthase (PKS) pathway. These compounds exhibit broad-spectrum antitumor activity due to their unique ring structures and functional group modifications. Studies have shown that fungal-derived aromatic polyketides exert their antitumor effects through multiple mechanisms, including inducing tumor cell apoptosis, arresting the cell cycle, inhibiting tumor angiogenesis, and regulating signaling pathways, providing an important foundation for the development of multi-target antitumor drugs. The antitumor mechanisms of fungal polyketides are being continuously elucidated: from traditional targets such as inhibition of Hsp90 and protein tyrosine kinases, to innovative mechanisms discovered in recent years, such as targeting PRDX1 to induce copper death, regulating DCTPP1 to intervene in metabolic reprogramming, and activating AMPK to regulate energy metabolism, highlighting the enormous potential of these compounds in targeted therapy. This study focuses on... Crystallomyces hypericus The study aims to explore the polyketide metabolites of fungi, elucidate their structural characteristics and antitumor activities, and provide a theoretical basis and material foundation for the discovery and development of novel antitumor lead compounds.
[0004] The present invention aims to provide an aromatic polyketide compound that can effectively inhibit a variety of cancer cells. Summary of the Invention
[0005] To address the problems existing in the prior art, one objective of this invention is to provide an aromatic polyketide compound, the structure of which is shown in Formula I or Formula II:
[0006] Formula I;
[0007] Formula II.
[0008] A second objective of this invention is to provide a method for preparing aromatic polyketide compounds, wherein the aromatic polyketide compounds are derived from... Crystallomyces hypericus The following steps were taken to separate the samples:
[0009] (1) Crystallomyces hypericus First, the seed culture medium is used to culture the seed culture solution, which is then used to obtain the seed solution.
[0010] (2) The seed liquid was inoculated into rice culture medium to obtain culture medium with attached bacteria. The culture medium with attached bacteria was extracted by cold soaking with methanol. After solid-liquid separation, methanol extract was obtained. The methanol extract was concentrated to obtain methanol concentrate.
[0011] (3) Ethyl acetate is added to the methanol concentrate for extraction to obtain ethyl acetate layer extract, and the ethyl acetate layer extract is concentrated to obtain ethyl acetate extract paste.
[0012] (4) The ethyl acetate extract was separated by gradient reversed-phase column chromatography using methanol-water solution, and the eluent obtained by methanol-water solution was collected at 80%.
[0013] (5) The eluent obtained in step (4) is separated by silica gel column chromatography. The eluent is a mixture of dichloromethane and methanol in a volume ratio of 2:1, which can obtain compound I.
[0014] (6) The eluent obtained in step (4) is separated by silica gel column chromatography. The eluent is a mixture of petroleum ether and acetone in a volume ratio of 1:1, which can obtain compound II.
[0015] Preferably, the Crystallomyces hypericus It was deposited on August 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42087. The suggested classification name is... Crystallomyces hypericus .
[0016] Preferably, the seed culture medium in step (1) is liquid potato culture medium.
[0017] Preferably, the temperature for seed culture in step (1) is 25-28℃ and the culture time is 7-10 days.
[0018] Preferably, the fermentation temperature in step (2) is 25-28℃ and the culture time is 30-40 days.
[0019] Preferably, in step (2), the cold extraction with methanol is performed at least 3 times, and each extraction lasts at least 12 hours.
[0020] Preferably, the mass concentration of industrial methanol in step (2) is 95%.
[0021] Preferably, in step (3), the amount of ethyl acetate used is 2 times or more the volume of the methanol concentrate.
[0022] Preferably, the mass fraction of the methanol solution in step (4) is 10~100%.
[0023] Preferably, the packing material in the column during the reversed-phase column chromatography separation in step (4) is RP-18.
[0024] Preferably, the flow rate of silica gel column chromatography separation in steps (4) and (5) is 1 mL / min, and the eluent after column chromatography with a retention volume of 20-50 mL is collected.
[0025] A third objective of this invention is to provide an application of the aforementioned aromatic polyketide compound in the preparation of anticancer drugs.
[0026] Preferably, the cancer is liver cancer, cervical cancer, neurogenic cancer, or pancreatic cancer, and is used in the drug.
[0027] Compared with the prior art, the present invention provides an aromatic polyketide compound, its preparation method and application, which has the following beneficial effects:
[0028] This invention is the first to use rice culture medium-cultured Crystallomyces hypericus Two novel aromatic polyketide compounds were isolated from the samples. Experimental verification showed that compounds I and II obtained in this invention exhibited significant inhibitory effects on human liver cancer cells, human cervical cancer cells, human neurocarcinoma cells, and human pancreatic cancer cells. Compound I showed a relatively high IC50 value. 50 The values were 19, 21, 23, 31, and 25 μmol / L, respectively; the IC50 of compound II was... 50 The values were 19, 11, 13, 25, and 23 μmol / L, respectively, therefore the two new aromatic polyketide compounds can be used to prepare anticancer drugs. Attached Figure Description
[0029] Figure 1 This is a schematic diagram of the chemical structure of compound I in this invention.
[0030] Figure 2 This is a high-resolution mass spectrum of compound I in this invention.
[0031] Figure 3 Nuclear magnetic resonance of compound I in this invention 1 H NMR spectrum.
[0032] Figure 4 Nuclear magnetic resonance of compound I in this invention 13 C10 NMR spectrum.
[0033] Figure 5 This is the HSQC nuclear magnetic resonance spectrum of compound I in this invention.
[0034] Figure 6 This is the nuclear magnetic resonance HMBC spectrum of compound I in this invention.
[0035] Figure 7This is the infrared spectrum of compound I in this invention.
[0036] Figure 8 This is a schematic diagram of the chemical structure of compound II in this invention.
[0037] Figure 9 This is a high-resolution mass spectrum of compound II in this invention.
[0038] Figure 10 Nuclear magnetic resonance of compound II in this invention 1 H NMR spectrum.
[0039] Figure 11 Nuclear magnetic resonance of compound II in this invention 13 C10 NMR spectrum.
[0040] Figure 12 This is the HSQC nuclear magnetic resonance spectrum of compound II in this invention.
[0041] Figure 13 This is the nuclear magnetic resonance HMBC spectrum of compound II in this invention.
[0042] Figure 14 This is the infrared spectrum of compound II in this invention. Detailed Implementation
[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0044] The instruments, equipment, and materials used in this invention are shown below. Reagents not specifically described are conventional reagents in the art, and methods not specifically described are conventional methods in the art.
[0045] Jasco-P-1020 polarimeter; Horiba SEPA-300 polarimeter; Bruker Tensor 27 FT-IR spectrometer; Bruker AV-600MHz NMR spectrometer; Waters Autospec Primier P776 spectrometer; APIQSTAR pulsar L spectrometer; silica gel (200-300 mesh, Qingdao Ocean Chemical Co., Ltd., China); Sephadex LH-20 gel (Amerson Biosciences, Sweden); RP-18 silica gel (40-75 μm, Fuji Silesia Chemical Co., Ltd.). Fractions were monitored by TLC, and spots were visualized using silica gel plates with sulfuric acid (5% sulfuric acid) as a colorimetric reagent.
[0046] The preparation method of the culture medium used in this invention:
[0047] (1) Liquid potato culture medium: Cut 200g of potatoes into pieces about 1cm thick. 3 Add 500mL of water to the potato chunks and boil until the potatoes are soft. Filter the liquid through gauze and add 20g of glucose to the liquid. Add distilled water to make up to 1L and sterilize at 121℃ for 15 minutes.
[0048] (2) Rice solid culture medium: Add 70 mL of water and 30 g of rice to a 500 culture flask and sterilize at 121 °C for 30 minutes.
[0049] Unless otherwise specified, all reagents used in this invention are commercially available analytical grade reagents.
[0050] The strains used in this invention Crystallomyces hypericus The strain was isolated from the flower buds of Hypericum genus (family Clusium) in Cangshan, Dali, Yunnan, China. The nucleotide sequence of the 16S rRNA of this bacterium is shown in SEQ ID NO:1. This strain was deposited on August 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42087.
[0051] Example 1
[0052] Crystallomyces hypericus The specific steps for the extraction and separation of aromatic polyketide metabolites from fermentation and ethyl acetate extract are as follows:
[0053] (1) Dip the bacterial solution into a 1L flask containing 400mL of seed culture medium and incubate at 25℃ for 7 days at a speed of 180rpm / min to obtain the seed culture solution.
[0054] (2) 5 mL of seed culture solution was inoculated into a 500 mL culture bottle containing rice solid culture medium and cultured statically in an incubator at 25 °C for 30 days to obtain a culture medium with attached bacteria. After the culture was completed, an industrial methanol with a mass concentration of 95% was added to the culture bottle with an equal volume of the culture medium with attached bacteria for 12 h of cold soaking extraction. After the extraction was completed, solid-liquid separation was performed with gauze and the methanol soaking solution was collected. Methanol extraction was performed 3 times in total. The methanol soaking solutions collected each time were mixed and concentrated by vacuum concentration to obtain methanol concentrate for later use.
[0055] (3) Add 2 times the volume of ethyl acetate to the methanol concentrate and extract. Collect the ethyl acetate extract. Extract three times in total. Combine the extracts obtained each time and concentrate the extracts using a vacuum method to obtain ethyl acetate extract paste.
[0056] (4) The ethyl acetate extract was separated by reversed-phase column chromatography (column type: 10×50cm; packing material: RP-18). The elution process was as follows: the extract was eluted by a methanol-water gradient with mass fractions of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%, and the eluent obtained by eluting 80% methanol-water was collected.
[0057] (5) Separate the eluent obtained in step (4) by silica gel column chromatography (column type: 2×30cm, packing material: 200-300 mesh silica gel powder). The eluent is a mixture of dichloromethane and methanol in a volume ratio of 2:1, and the flow rate is 1mL / min. Collect 20-50mL of the eluent after column chromatography to obtain compound I.
[0058] (6) Separate the eluent obtained in step (4) by silica gel column chromatography (column type: 2×30cm, packing material: 200-300 mesh silica gel powder). The eluent is a mixture of petroleum ether and acetone in a volume ratio of 1:1, and the flow rate is 1mL / min. Collect the eluent after passing through the column with a retention volume of 20-50mL to obtain compound II.
[0059] Example 2
[0060] Structural identification of compounds I and II
[0061] 1. Structural identification of compound I
[0062] Mass spectrometry and nuclear magnetic resonance analysis were performed on the compound. +2.66( c 0.03,MeOH); HR-ESI-MS m / z:265.1065 [M+H] + (calcd. for C 14 H16 O5, 265.1071); IR (KBr) ν max : 3414,1707,1645,1453,1313,1262,1169,1110,1076cm –1 , Figure 7 The high-resolution mass spectrum of compound I is shown below. Figure 2 As shown; its nuclear magnetic resonance 1 H NMR spectrum as shown Figure 3 As shown, its nuclear magnetic resonance 13 The C NMR spectrum is as follows Figure 4 As shown in Table 1, the data indicates that this compound contains one methyl group (δ-methyl group). C 20.9), 4 methylene groups (δ C 20.9, 36.7, 42.8, 51.8), 2 ketone groups (δ C (172.5, 209.5), this compound I is an aromatic polyketide compound, named Crystalloketide A.
[0063] Table 1 Compound I 1 H and 13 C NMR data (δ in ppm, J (in Hz)
[0064] Simultaneously, by measuring the two-dimensional spectrum of this compound, the HSQC nuclear magnetic resonance spectrum of this compound is shown below. Figure 5 As shown, the nuclear magnetic resonance HMBC spectrum is as follows: Figure 6 As shown, the signal assignments of all carbon and hydrogen atoms in the compound and its chemical structure are known. A schematic diagram of the compound's chemical structure is shown below. Figure 1 As shown.
[0065] Note: δ Chemical shift is measured in ppm. 1 H-NMR and 13 The C-NMR test solvent was deuterated methanol (CD3OD), and the carbon and hydrogen signals of the compound were assigned by HSQC and HMBC spectra.
[0066] 2. Structural identification of compound II
[0067] Mass spectrometry and nuclear magnetic resonance analysis were performed on compound II. +17.20( c 0.05,MeOH); HR-ESI-MS m / z:487.1963 [M+H] + (calcd. for C 26 H30 O8, 487.1964), such as Figure 9 As shown; IR (KBr) ν max :3448,2931,1714,1644,1623,1453,1315,1264,1172,1110cm –1 , Figure 14 Compound II 1 H and 13 The C NMR data are shown in Table 2 below. 1 H NMR spectrum as shown Figure 10 As shown, its nuclear magnetic resonance 13 The C NMR spectrum is as follows Figure 11 As shown, compound II is an aromatic polyketide compound containing two methyl groups (δ-methyl groups). C 21.7, 20.2), 7 methylene groups (δ C 51.8, 42.4, 37.9, 37.0, 36.3, 31.2, 20.3), 3 ketone groups (δ C 210.5, 172.6, 171.3), named CrystallomycinD.
[0068] Table 2 Compound II 1 H and 13 C NMR data (δ in ppm, J (in Hz)
[0069]
[0070] Simultaneously, by measuring the two-dimensional spectrum of this compound, the HSQC nuclear magnetic resonance spectrum of this compound is shown below. Figure 12 As shown, the nuclear magnetic resonance HMBC spectrum is as follows: Figure 13 As shown, the signal assignments of all carbon and hydrogen atoms in the compound and its chemical structure are known. A schematic diagram of the compound's chemical structure is shown below. Figure 8 As shown.
[0071] Note: δ Chemical shift is measured in ppm. 1 H-NMR and 13 The C-NMR test solvent was deuterated methanol (CD3OD), and the carbon and hydrogen signals of the compound were assigned by HSQC and HMBC spectra.
[0072] Example 3
[0073] Antitumor activity tests of compounds I and II
[0074] This experiment used MTT assay to detect cytotoxic activity. The principle is as follows: the dehydrogenase present in the mitochondria of living cells can reduce the yellow thiazolyl blue MTT to insoluble blue-purple formazan. In dead cells, this enzyme disappears, and MTT is not reduced. The formazan was dissolved in DMSO (dimethyl sulfoxide), and the absorbance (OD) was measured at 570 nm using a microplate reader. The OD value is directly proportional to the number of living cells.
[0075] The tumor cell lines used in this invention are: human cervical cancer cells (HeLa), human liver cancer cells (HEPG2), human pancreatic cancer cells (Miapaca), human neurocellular cells (Sy5y), and human pancreatic cancer cells (Panc-1).
[0076] Experimental methods: Tumor cells in the logarithmic growth phase were digested with trypsin, then pipetted into single-cell suspensions, and diluted with culture medium to a concentration of 1×10⁻⁶. 5 Cells were seeded at 100 μL / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. Different concentrations of the test samples, compounds I and II, were added to the experimental groups at concentrations of 10, 3.3, 1.1, and 0.33 mmol / L, resulting in final concentrations of 100, 33, 11, and 3.3 μmol / L, respectively. The blank control group received 1 μL of DMSO solution diluted 1000 times with 1640 medium to cell-free wells. Each group had three replicates. After incubation at 37°C with 5% CO2 for 48 hours, 10 μL of freshly prepared MTT solution containing 5 mg / mL was added to each well, and the plates were incubated at 37°C for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well to dissolve the formazan. The absorbance (OD) was measured at 570 nm using a microplate reader. 570 ), calculate the inhibition rate (%). Calculation formula: Inhibition rate (%) = (1 - OD) Sample / OD Control ) × 100%. IC 50 The value represents the drug concentration at which the inhibition rate is 50%. The IC50 was calculated using Probit regression analysis in SPSS 29.0 software. 50 value.
[0077] The results showed that compound I had a significant inhibitory effect on human liver cancer cells, human cervical cancer cells, human neurocarcinoma cells, and human pancreatic cancer cells, with an IC50 concentration of [missing value]. 50 The concentrations were 19, 21, 23, 31, and 25 μmol / L, respectively; Compound II showed significant inhibitory effects on human liver cancer cells, human cervical cancer cells, human neurocarcinoma cells, and human pancreatic cancer cells, with IC50 values of 19, 21, 23, 31, and 25 μmol / L. 50 The concentrations were 19, 11, 13, 25, and 23 μmol / L, respectively, as shown in Table 3 below.
[0078] Table 3. Cytotoxic activities of compounds I and II (μmol / L)
[0079]
[0080] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. An aromatic polyketide compound, characterized in that: The structure of the aromatic polyketide compound is shown in Formula II: Formula II.
2. The method for preparing the aromatic polyketide compound according to claim 1, characterized in that: Specifically, the steps include the following: (1) Name the category Crystallomyces hypericus The bacteria are first cultured in a seed culture medium to obtain a seed culture solution, which is then used to obtain a seed solution. (2) The seed liquid was inoculated into rice culture medium to obtain a culture medium with attached bacteria. The culture medium with attached bacteria was extracted by cold soaking with industrial methanol. After solid-liquid separation, a methanol extract was obtained. The methanol extract was concentrated to obtain a methanol concentrate. (3) Ethyl acetate was added to the methanol concentrate for extraction to obtain an ethyl acetate layer extract. The ethyl acetate layer extract was then concentrated to obtain an ethyl acetate extract paste. (4) The ethyl acetate extract was separated by gradient reversed-phase column chromatography using methanol-water solution, and the eluent with 80% methanol-water solution was collected. (5) The eluent obtained in step (4) is separated by silica gel column chromatography. The eluent is a mixture of dichloromethane and methanol in a volume ratio of 2:1 to obtain compound I. (6) The eluent obtained in step (4) is separated by silica gel column chromatography. The eluent is a mixture of petroleum ether and acetone in a volume ratio of 1:1 to obtain compound II. The Crystallomyces hypericus Its accession number is CGMCC No. 42087, and its taxonomic name is Crystallomyces hypericus ; The seed culture medium mentioned in step (1) is liquid potato culture medium.
3. The method for preparing the aromatic polyketide compound according to claim 2, characterized in that: In step (4), the mass fraction of the methanol aqueous solution is 10~100%.
4. The method for preparing the aromatic polyketide compound according to claim 2, characterized in that: In steps (5) and (6), the flow rate of silica gel column chromatography is 1 mL / min, and the eluent after column chromatography with a retention volume of 20-50 mL is collected.
5. The use of the aromatic polyketide compound of claim 1 in the preparation of anticancer drugs; wherein the cancer is one of liver cancer, cervical cancer, neurogenic cancer, and pancreatic cancer.