Furan pyrrole alkaloid compound as well as preparation method and application thereof

Abstract

The invention discloses a furan pyrrole alkaloid compound as well as a preparation method and application thereof, and belongs to the technical field of active ingredient analysis of marine fungi. The furopyrrole alkaloid compound with a novel structure is extracted and separated from a fermentation culture of marine fungi Trichoderma harzianum ZN-4, and an in-vitro zebra fish angiogenesis inhibition test shows that the furopyrrole alkaloid compound provided by the invention has relatively good angiogenesis inhibition activity, and can be used for preparing an anti-angiogenesis drug. The compound has a development prospect in the aspect of preparing medicines or functional foods related to angiogenesis.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis of active ingredients of marine fungi, and particularly relates to a furan pyrrole alkaloid compound obtained by extraction from the fungus Trichoderma harzianum ZN-4 and its application. Background Art

[0002] Angiogenesis is a biological process in which new blood vessels are formed by the proliferation and migration of original vascular endothelial cells, and plays a key role in tissue development, wound repair and pathological conditions. Its occurrence mechanism involves precise regulation by multiple factors. When tissues are hypoxic or damaged, angiogenic factors such as VEGF and FGF activate endothelial cells, degrade the basement membrane through matrix metalloproteinases, and promote the migration of endothelial cells and the formation of lumen structures. Physiological angiogenesis is crucial for embryonic development and wound healing, while pathological angiogenesis is closely related to tumor growth, diabetic retinopathy, etc. Treating diseases by regulating angiogenesis has always been a research hotspot and difficulty in the medical field. Researchers are committed to discovering compounds with angiogenic and anti-angiogenic effects in nature, with the expectation of combining biomaterials and gene editing technologies in the future to develop spatio-temporally controllable vascularization treatment plans and achieve breakthroughs in precision medicine in the field of vascular diseases.

[0003] Compared with terrestrial microorganisms, marine microorganisms can tolerate extreme conditions unique to the ocean, such as high salinity, high pressure, low oxygen, low light, etc. The specificity of the living environment leads to the diversity of marine microorganisms in terms of species, gene composition and ecological functions. The particularity of the marine environment and the progress of marine microorganism resource acquisition technologies have brought unprecedented opportunities to the research of natural medicinal compounds derived from marine microorganisms.

[0004] Marine fungi among marine microorganisms are a rich source of active secondary metabolites. With the rapid development and improvement of biology, organic chemistry, separation and identification technologies, and screening methods, etc., many compounds from marine organisms have been found to exhibit different activities in the research of marine active substances, such as anti-cancer, anti-diabetic, anti-viral, anti-coagulant, anti-inflammatory and other pharmacological activities. Discovering natural products with specific structural types from marine fungi is of great significance for the research and development of marine drugs.

[0005] The genus Trichoderma is a widespread genus of mitosporic fungi that has received extensive attention as a biocontrol agent or plant pathogen. They are widely present in plants, the surrounding air, soil, and decaying vegetation. The genus Trichoderma contains various chemical components such as polyketides, peptides, and diketopiperazines, which have a wide range of biological activities, including antibacterial, antioxidant, anticancer, and antiviral activities, making Trichoderma an important producer of compounds with potential biotechnological application value. For this reason, a furanopyrrole alkaloid compound, its preparation method, and its application are proposed. Summary of the Invention

[0006] The object of the present invention is to extract natural active substances with medicinal value from Trichoderma harzianum.

[0007] To achieve the above object, the present invention isolated 1 new compound from the fermentation products of Trichoderma harzianum ZN-4. Through structural identification, the specific structural formula of the new compound is shown in formula (I), and it is named harzianopyrrolidone B.

[0008] (I).

[0009] The Trichoderma harzianum ZN-4 was isolated from the sediment of Razor Clam Bay, Zhoushan City, Zhejiang Province, and the strain has been published: see Zhou, C.; Ge, Y.; Lan, D.; Zhao, M.; Wu, B., Harzianolides B–G: Undescribed Butenolides isolated from the fungus Trichoderma harzianum ZN-4. Fitoterapia 2024, 176, 106039. DOI: 10.1016 / j.fitote.2024.106039.

[0010] The present invention also provides a method for isolating and extracting the above new compound from the fermentation products of Trichoderma harzianum ZN-4, but the preparation method of the above compound in the present invention is not limited thereto.

[0011] The method for extracting the furanopyrrole alkaloid compound from the fermentation products includes the following steps: (1) Trichoderma harzianum ZN-4 is activated and then inoculated into a PDB medium for fermentation culture; (2) After the fermentation culture is completed, ethyl acetate is used for extraction to obtain an extract. (3) After the extract is concentrated, it is separated by normal-phase silica gel column chromatography, and gradient elution is carried out successively with dichloromethane / methanol mixed solutions with volume ratios of 100:1, 50:1, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10. The fractions eluted with the dichloromethane / methanol mixed solution with a volume ratio of 9:1 are collected, and then reverse-phase silica gel column chromatography and high-performance liquid chromatography separation are carried out to obtain the furan pyrrole alkaloid compound.

[0012] In step (1), Trichoderma harzianum ZN-4 is subjected to fermentation culture.

[0013] Trichoderma harzianum ZN-4 is a fungus, and a conventional PDB medium can be used for fermentation culture. The PDB medium includes the following raw materials: potatoes, glucose and water. 200 g of potatoes and 20 g of glucose are added to every 1 L of water. The conditions for fermentation culture are static culture at 25 - 32 °C for 15 - 40 days. The static culture method means that shaking flask culture is not carried out.

[0014] Preferably, the temperature for fermentation culture is 28 - 30 °C. More preferably, it is cultured at 28 °C for 21 days, and the yield of the furan pyrrole alkaloid compound is the highest under this condition.

[0015] In step (2), the furan pyrrole alkaloid compound is extracted and separated from the fermentation product.

[0016] In step (3), the separation and purification method is: the extract is separated by normal-phase silica gel column chromatography, and the obtained fractions are then subjected to reverse-phase silica gel column chromatography and high-performance liquid chromatography separation. Through multi-step separation and purification, a furan pyrrole alkaloid compound with a higher purity can be obtained.

[0017] Preferably, the fractions are subjected to reverse-phase silica gel column chromatography, and gradient elution is carried out successively with methanol / water mixed solutions with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. Each gradient is eluted 3 times, and a total of 27 fractions are collected, numbered 1 - 27 in sequence; the fractions numbered 5 - 10 are combined, and then separated by high-performance liquid chromatography. Under the eluent of a methanol / water mixed solution with a volume ratio of 40:60, the peak with a retention time of 14 minutes is the compound harzianopyrrolidone B with the structure shown in formula (I).

[0018] The research of the present invention shows that the furan pyrrole alkaloid compound isolated from the fermentation culture of Trichoderma harzianum ZN-4 by the above method has good angiogenesis inhibitory activity. Therefore, the present invention provides the application of the described furan pyrrole alkaloid compound in the preparation of drugs related to angiogenesis.

[0019] Specifically, the present invention provides the application of the compound harzianopyrrolidone B with the structural formula shown in formula (I) in the preparation of drugs or functional foods related to angiogenesis.

[0020] Advantages of the present invention: (1) The present invention extracts and isolates a compound with a novel structure from the fermentation culture of marine fungi. This method is simple in operation, high in extraction yield, and high in product purity, and is suitable for large-scale production.

[0021] (2) The angiogenesis inhibition experiment on zebrafish shows that the compound harzianopyrrolidone B provided by the present invention has good angiogenesis inhibitory activity. Further cytotoxicity tests show that the compound harzianopyrrolidone B provided by the present invention has low cytotoxicity and has good development prospects in the preparation of drugs related to angiogenesis and preventive health foods. Description of the drawings

[0022] Figure 1 It is the structural formula of the furan pyrrole alkaloid compound of the present invention.

[0023] Figure 2 It is of the compound harzianopyrrolidone B 1 1H NMR data (in DMSO-d6, 600 MHz).

[0024] Figure 3 It is of the compound harzianopyrrolidone B 13 13C NMR data (in DMSO-d6, 150 MHz).

[0025] Figure 4 It is the activity analysis of the compound harzianopyrrolidone B.

[0026] Figure 5 It is the statistical chart of the activity analysis of the compound harzianopyrrolidone B. Detailed implementation manners

[0027] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0028] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0029] Example 1: Fungal isolation The sediment was collected from Razor Clam Bay, Zhoushan City, Zhejiang Province. After the sample was brought back to the laboratory, it was suspended with a small amount of sterile seawater, and 0.1 mL was taken and spread on a Martin's medium (containing 8 U / L of gentamicin) plate; after culturing at room temperature of 20 °C for 10 d, single colonies were picked and purified by streaking and then transferred to a slant and stored at 4 °C for later use.

[0030] Example 2: Identification of Trichoderma fungi The isolated fungus was cultured on PDA, and the ITS sequence of the strain was determined. The ITS sequence of this strain is shown in SEQ ID No. 1.

[0031] According to the morphological characteristics and ITS sequence analysis results of the strain, this strain was identified as a Trichoderma sp. fungus. And it was named Trichoderma harzianum ZN-4, see Zhou, C.; Ge, Y.; Lan, D.; Zhao, M.; Wu, B., Harzianolides B–G: Undescribed Butenolides isolated from the fungus Trichoderma harzianum ZN-4. Fitoterapia 2024, 176, 106039. DOI: 10.1016 / j.fitote.2024.106039.

[0032] Example 3: Fermentation culture of Trichoderma fungi The Trichoderma harzianum ZN-4 isolated and identified in Example 2 was inoculated into PDB medium after activation and statically fermented at 28 °C for 21 days.

[0033] Among them, the formula of PDB medium is: potatoes, glucose and water, adding 200 g of potatoes and 20 g of glucose per 1 L of water. Autoclave at 121 °C for 20 min.

[0034] Example 4: Preparation of Furan Pyrrole Alkaloid Compounds After fermentation and culture of Trichoderma harzianum ZN-4, it was extracted and concentrated with ethyl acetate, and then separated by normal-phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 50 mm, Ø 12 mm). Gradient elution was carried out successively with dichloromethane / methanol mixtures with volume ratios of 100:1, 50:1, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, and the fractions eluted with the dichloromethane / methanol mixture with a volume ratio of 9:1 were collected.

[0035] The fraction was subjected to reverse-phase silica gel column chromatography, and the eluent was methanol / water (1:9 - 9:1). Gradient elution was carried out successively with methanol / water mixtures with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. Each gradient was eluted with 1.5 L, and collected in units of 0.5 L using an automatic collector, for a total of 27 fractions, numbered 1 - 27 in sequence. According to the similarity of the fraction components, they were combined to form 7 sub-components, named 5-1 to 5-7. Specifically, 5-1: 1 - 3; 5-2: 4; 5-3: 5 - 10; 5-4: 11 - 12; 5-5: 13 - 18; 5-6: 19 - 24; 5-7: 25 - 27.

[0036] Subsequently, separation by high-performance liquid chromatography was carried out. For sub-component 5-3, the peak with a retention time of 20 minutes under the eluent of methanol / water mixture with a volume ratio of 40:60 was designated as compound 1.

[0037] Example 5: Structure Identification of Furan Pyrrole Alkaloid Compounds HPLC was used to identify the purity of the prepared compound. Samples with a purity greater than 98% were subjected to structure identification using mass spectrometry and nuclear magnetic resonance techniques. Nuclear magnetic resonance was measured with a JEOL 600 MHz spectrometer, using TMS as the internal standard; high-resolution mass spectrometry was measured with an AB Sciex 5500 Q-TRAP.

[0038] According to the NMR analysis results of compound 1 (see Table 1, Figure 2-3 ), it can be known that the molecular formula of compound 1 is C 11 H 17 NO 5 , and compound 1 was identified as the new compound harzianopyrrolidone B, and its structure is as shown in (I) of Figure 1 .

[0039] Table 1. NMR Data of Compound 1 Position <![CDATA δ C (ppm), type]]> <![CDATA δ H (ppm) mult. ( J in Hz)]]> 1 - 2 171.1, C - 3 <![CDATA[44.5, CH 2 > a 2.61, d (16.8) b 2.42, d (16.8) 4 107.5, C - 5 69.2, CH 3.71, d (5.4) 6 <![CDATA[33.9, CH 2 > a 2.46, d (13.8) b 1.98, dd (13.8, 5.4) 7 90.1, C - 8 35.3, CH 1.89, p (7.0) 9 <![CDATA[18.3, CH 3 > 0.85, d (7.0) 10 <![CDATA[17.8, CH 3 > 0.84, d (7.0) 11 175.0, C - 12 <![CDATA[27.0, CH 3 > 2.51, s Example 6: Analysis of the angiogenesis inhibitory activity of Harzianopyrrolidone B compound At 16:00, female and male transgenic fluorescent zebrafish (flia: EGFP) were set to spawn at a ratio of 2:1. The plates were removed to collect eggs the next day from 8:00 to 10:00, and the collected eggs were recorded as 0 hpf. The whole process was cultured in E3 + PTU culture medium. After 24 h, a sufficient number of fluorescent zebrafish eggs were selected under a fluorescence microscope.

[0040] Take the fluorescent zebrafish at 48 hpf, set up blank control, positive control (0.25 μg / mL PTK787), and test groups of the target compound at 40 μM / L in a 24-well plate, with a total of 8 fluorescent zebrafish in each well. Except for the blank control group, the corresponding compounds were added to each well. After 24 h (72 hpf), the angiogenesis of zebrafish was observed under a fluorescence microscope. After 48 h (96 hpf), methylcellulose was used as a fixative to observe the blood vessel injury repair under a microscope and take pictures of each fish ( Figure 4 )

[0041] Analysis and statistics of the ISV blood vessel length of zebrafish in each group using Image J found that ( Figure 5 ), there was no statistical difference between PTK787 and Harzianopyrrolidone B, and both had significant statistical differences from the blank group, indicating that Harzianopyrrolidone B has a good effect on inhibiting angiogenesis.

[0042] The protection scope of the present invention is not limited to the above examples and their variations. Routine modifications and substitutions made by those skilled in the art based on the content of this example all fall within the protection scope of the present invention.

Claims

1. A furanpyrrole alkaloid compound, characterized in that: The structural formula of the furanpyrrole alkaloid compound is as shown in formula (I), (I)。 2. The method for preparing a furanpyrrole alkaloid compound according to claim 1, wherein The following steps are involved: (1) Trichoderma harzianum Trichoderma harzianum ) After activation, ZN-4 was inoculated into PDB medium for fermentation culture; (2) After the fermentation culture is completed, the fermentation liquid is extracted with ethyl acetate to obtain an extract; (3) After the extract is concentrated, it is separated by normal phase silica gel column chromatography, and gradient elution is performed with a dichloromethane / methanol mixture with a volume ratio of 100:1, 50:1, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, and the fractions eluted with the dichloromethane / methanol mixture with a volume ratio of 9:1 are collected, and then the fractions are separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the furanpyrrole alkaloid compound.

3. The preparation method according to claim 2, characterized in that: In step (1), the PDB culture medium comprises the following raw materials: potatoes, glucose and water, with 200 g potatoes and 20 g glucose added to every 1 L of water.

4. The preparation method according to claim 2, characterized in that: In step (1), the fermentation culture conditions are static culture at 25-32° C. for 15-40 days.

5. The preparation method according to claim 2, characterized in that: In step (3), the fraction is subjected to reverse phase silica gel column chromatography, and gradient elution is performed with a methanol / water mixture with a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively, each gradient elution is performed 3 times, and the collected fractions are numbered 1 to 27 in sequence; the fractions numbered 5 to 10 are combined and then separated by high performance liquid chromatography. Under the eluent of a methanol / water mixture with a volume ratio of 40:60, the peak with a retention time of 14 minutes is the compound harzianopyrrolidone B with a structural formula as shown in formula (I).

6. Use of the furanpyrrole alkaloid compound having the structural formula (I) in the preparation of angiogenesis-related drugs or functional foods.

7. The use according to claim 6, characterized in that The compound harzianopyrrolidone B has angiogenesis inhibitory activity.

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