Furanopyrrole alkaloid compound, preparation method and application thereof
By extracting and isolating the furanylpyrrolidone B compound from the marine fungus Trichoderma harziano, the problem of the lack of effective regulation of angiogenesis in the existing technology has been solved, and the inhibitory effect on angiogenesis has been achieved, which has good potential for drug development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SECOND AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
- Filing Date
- 2025-02-21
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies lack effective compounds for regulating angiogenesis, making it difficult to achieve precise treatment of diseases such as tumor growth and diabetic retinopathy.
A new compound, harzianopyrrolidone B, was extracted and isolated from the marine fungus *Trichoderma harzianum* ZN-4. The furanylpyrrolidone alkaloid compound, which inhibits angiogenesis, was obtained through fermentation, extraction, and chromatography.
harzianopyrrolidone B exhibits good anti-angiogenic activity and low cytotoxicity, showing promising potential for development into drugs related to angiogenesis.
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Figure CN120058716B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of marine fungal active ingredient analysis technology, specifically to furanylpyrrole alkaloid compounds extracted from Trichoderma harzianum ZN-4 and their applications. Background Technology
[0002] Angiogenesis is a biological process in which new blood vessels are formed through the proliferation and migration of existing vascular endothelial cells. It plays a crucial role in tissue development, wound repair, and pathological conditions. Its mechanism involves precise multi-factor regulation. When tissues are hypoxic or damaged, pro-angiogenic factors such as VEGF and FGF activate endothelial cells, which then degrade the basement membrane via matrix metalloproteinases, promoting endothelial cell migration and the formation of luminal structures. Physiological angiogenesis is essential for embryonic development and wound healing, while pathological angiogenesis is closely related to tumor growth, diabetic retinopathy, and other conditions. Regulating angiogenesis to treat diseases has always been a hot topic and a challenge in the medical field. Researchers are dedicated to discovering compounds in nature that promote and inhibit angiogenesis, hoping to combine biomaterials and gene editing technologies in the future to develop spatiotemporally controllable vascularization therapies and achieve breakthroughs in precision medicine for vascular diseases.
[0003] Compared to terrestrial microorganisms, marine microorganisms are able to tolerate the extreme conditions unique to the ocean, such as high salinity, high pressure, low oxygen, and low light. This unique living environment leads to diversity in species, genetic composition, and ecological functions among marine microorganisms. The special characteristics of the marine environment, coupled with advancements in marine microbial resource acquisition technologies, have brought unprecedented opportunities for the research of natural medicinal compounds derived from marine microorganisms.
[0004] Marine fungi are a rich source of bioactive secondary metabolites. With the rapid development and improvement of biology, organic chemistry, separation and identification techniques, and screening methods, researchers have discovered many compounds from marine organisms exhibiting diverse activities in the study of marine bioactive substances, such as anticancer, antidiabetic, antiviral, anticoagulant, and anti-inflammatory pharmacological activities. Discovering natural products with specific structural types using marine fungi as raw materials is of great significance for the development of marine drugs.
[0005] Trichoderma is a ubiquitous genus of filamentous fungi that has garnered significant attention as a biocontrol agent or plant pathogen. They are widely distributed within plants, in the surrounding air, soil, and decaying vegetation. Trichoderma contains a variety of chemical components, such as polyketides, peptides, and diketopiperazines, which possess broad bioactivities, including antibacterial, antioxidant, anticancer, and antiviral properties, making it a significant producer of compounds with potential biotechnological applications. Therefore, this paper proposes a furanylpyrrole alkaloid compound, its preparation method, and its applications. Summary of the Invention
[0006] The purpose of this invention is to extract natural active substances with medicinal value from Trichoderma harzianum.
[0007] To achieve the above objectives, this invention isolated a new compound from the fermentation product of Trichoderma harzianum ZN-4. After 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 strain was isolated from sediments in Chengzi Bay, Zhoushan City, Zhejiang Province. 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-mentioned new compounds from the fermentation products of Trichoderma harzianum ZN-4, but the preparation method of the above-mentioned compounds in the present invention is not limited thereto.
[0011] A method for extracting the furanylpyrrole alkaloid compound from fermentation products includes the following steps:
[0012] (1) Trichoderma harzianum ZN-4 was activated and inoculated into PDB medium for fermentation culture;
[0013] (2) After the fermentation culture was completed, the extract was obtained by extraction with ethyl acetate;
[0014] (3) After concentrating the extract, normal phase silica gel column chromatography was performed. Gradient elution was carried out 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, and 0:10. The fraction eluted from the dichloromethane / methanol mixture with a volume ratio of 9:1 was collected and then separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the furanpyrrole alkaloid compound.
[0015] In step (1), Trichoderma harzianum ZN-4 was fermented and cultured.
[0016] Trichoderma harzianum ZN-4 is a fungus that can be fermented using conventional PDB medium. The PDB medium consists of the following ingredients: potatoes, glucose, and water, with 200 g of potatoes and 20 g of glucose added per 1 L of water.
[0017] The fermentation culture conditions are static culture at 25-32℃ for 15-40 days. The static culture method refers to culture without shaking flasks.
[0018] Preferably, the fermentation culture temperature is 28-30°C. More preferably, the culture is carried out at 28°C for 21 days, under which the yield of the furanylpyrrole alkaloid compound is the highest.
[0019] In step (2), the furanpyrrole alkaloid compound is extracted and separated from the fermentation product.
[0020] In step (3), the separation and purification method is as follows: the extract is separated by normal phase silica gel column chromatography, and the obtained fraction is then separated by reverse phase silica gel column chromatography and high performance liquid chromatography. Through multi-step separation and purification, high purity furanpyrrole alkaloid compounds can be obtained.
[0021] Preferably, the fractions are subjected to reversed-phase silica gel column chromatography, with gradient elution using methanol / water mixtures at volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, eluting three times for each gradient, resulting in the collection of 27 fractions, numbered sequentially from 1 to 27. Fractions numbered 5 to 10 are combined and then separated by high-performance liquid chromatography. The peak with a retention time of 14 minutes using a methanol / water mixture at a volume ratio of 40:60 is the compound harzianopyrrolidone B, as shown in formula (I).
[0022] The present invention demonstrates that the furanylpyrrole alkaloid compound isolated from the fermentation culture of Trichoderma harzianum ZN-4 using the above method has good anti-angiogenic activity. Therefore, the present invention provides the application of the furanylpyrrole alkaloid compound in the preparation of angiogenesis-related drugs.
[0023] Specifically, this invention provides the use of the compound harzianopyrrolidone B, with the structural formula shown in formula (I), in the preparation of drugs related to angiogenesis.
[0024] The beneficial effects of this invention are:
[0025] (1) The present invention extracts and isolates a compound with a novel structure from the fermentation culture of marine fungi. The method is simple to operate, has a high extraction yield and high product purity, and is suitable for large-scale production.
[0026] (2) The zebrafish angiogenesis inhibition experiment showed that the harzianopyrrolidone B compound provided by the present invention has good angiogenesis inhibition activity. Further cytotoxicity test showed that the harzianopyrrolidone B compound provided by the present invention has low cytotoxicity and has good development prospects in the preparation of angiogenesis-related drugs. Attached Figure Description
[0027] Figure 1 This is the structural formula of the furanpyrrole alkaloid compound of the present invention.
[0028] Figure 2 It is the compound harzianopyrrolidone B. 1 H NMR data (in DMSO-d6, 600 MHz).
[0029] Figure 3 It is the compound harzianopyrrolidone B. 13C NMR data (in DMSO-d6, 150 MHz).
[0030] Figure 4 This is an activity analysis of the compound harzianopyrrolidone B.
[0031] Figure 5 This is a statistical graph showing the activity analysis of compound harzianopyrrolidone B. Detailed Implementation
[0032] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.
[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.
[0034] Example 1: Fungal Isolation
[0035] The sediments were collected from the Clam Bay in Zhoushan City, Zhejiang Province. After the samples were brought back to the laboratory, they were suspended in a small amount of sterile seawater. 0.1 mL of the suspension was spread on Martin's medium (containing 8 U / L gentamicin) plates. After incubation at room temperature (20℃) for 10 days, single colonies were picked, purified by streak plating, and then transferred to slant plates for storage at 4℃ for later use.
[0036] Example 2: Identification of Trichoderma fungi
[0037] The isolated fungus was cultured on a PDA, and the ITS sequence of the strain was determined. The ITS sequence of the strain is shown in SEQ ID No. 1.
[0038] Based on the morphological characteristics and ITS sequence analysis, this strain was identified as a fungus belonging to the genus *Trichodermas*. 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.
[0039] Example 3: Fermentation culture of Trichoderma fungi
[0040] The Trichoderma harzianum ZN-4 isolated and identified in Example 2 was activated and inoculated into PDB medium, and then statically fermented at 28°C for 21 days.
[0041] The PDB culture medium formula is: potatoes, glucose, and water, with 200 g of potatoes and 20 g of glucose added per 1 L of water. It is then autoclaved at 121℃ for 20 min.
[0042] Example 4: Preparation of furanylpyrrole alkaloid compounds
[0043] After fermentation, Trichoderma harzianum ZN-4 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 performed with dichloromethane / methanol mixtures at 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, and 0:10. The fraction eluted from the dichloromethane / methanol mixture at a volume ratio of 9:1 was collected.
[0044] The fraction was subjected to reversed-phase silica gel column chromatography with methanol / water (1:9-9:1) as the eluent. Gradient elution was performed sequentially with methanol / water mixtures of volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, with 1.5 L eluted per gradient. Fractions were collected in 0.5 L increments using an automated collector, resulting in 27 fractions, numbered 1 to 27. Based on the similarity of their components, the fractions were combined to form seven subfractions, 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; and 5-7: 25–27.
[0045] The fractions were then separated by high performance liquid chromatography. The peak of subfraction 5-3 with a retention time of 20 minutes using a methanol / water mixture with a volume ratio of 40:60 was designated as compound 1.
[0046] Example 5: Structural identification of furanpyrrole alkaloid compounds
[0047] The purity of the obtained compounds was determined by HPLC. The structures of samples with a purity greater than 98% were identified by mass spectrometry and nuclear magnetic resonance (NMR). NMR was measured using a JEOL 600 MHz spectrometer with TMS as an internal standard. High-resolution mass spectrometry was performed using an AB Sciex 5500 Q-TRAP.
[0048] According to the NMR of compound 1 (see Table 1, ...), Figure 2-3 The analysis results show that the molecular formula of compound 1 is C16. 11 H 17 NO5, compound 1 was identified as a new compound, harazianopyrrolidone B, with the structure shown below. Figure 1 As shown in (I).
[0049] Table 1. NMR data of compound 1
[0050]
[0051] Example 6: Analysis of the anti-angiogenic activity of the Harzianopyrrolidone B compound
[0052] At 16:00, female and male transgenic fluorescent zebrafish (flia:EGFP) were placed in spawning areas at a 2:1 ratio. The spawning was carried out between 8:00 and 10:00 the following day, and the collected eggs were recorded as 0hpf. The entire process was carried out using E3+PTU culture medium. After 24 hours, a sufficient number of fluorescent zebrafish eggs were picked out under a fluorescence microscope.
[0053] Fluorescent zebrafish at 48 hpf were used in 24-well plates to set up a blank control, a positive control (0.25 μg / mL PTK787), and a target compound experimental group (40 μL). A total of 8 fluorescent zebrafish were used per well. Except for the blank control group, the corresponding compound was added to each well. After 24 h (72 hpf), the angiogenesis of the zebrafish was observed under a fluorescence microscope. After 48 h (96 hpf), methylcellulose was used as a fixative to observe the repair of vascular damage under a microscope, and each fish was photographed. Figure 4 ).
[0054] ImageJ was used to analyze and statistically analyze the ISV vessel length of each group of zebrafish and found that ( Figure 5 PTK787 showed no statistically significant difference from Harzianopyrrolidone B, while both showed significant statistically significant differences from the control group, indicating that harzianopyrrolidone B has a good inhibitory effect on angiogenesis.
[0055] The scope of protection of this invention is not limited to the above embodiments and their variations. Conventional modifications and substitutions made by those skilled in the art based on the content of these embodiments are all within the scope of protection of this invention.
Claims
1. A furanopyrroloid alkaloid compound, characterized in that, The structural formula of the furanylpyrrole alkaloid compound is shown in formula (I). (I)。 2. The method for preparing the furanylpyrrole alkaloid compound as described in claim 1, characterized in that, Includes the following steps: (1) Trichoderma harzianum fungus Trichoderma harzianum ) ZN-4 was inoculated in PDB medium after activation, and fermentation culture was carried out; (2) After the fermentation culture was completed, the fermentation broth was extracted with ethyl acetate to obtain the extract; (3) After concentrating the extract, normal phase silica gel column chromatography was performed. Gradient elution was carried out 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, and 0:
10. The fraction eluted from the dichloromethane / methanol mixture with a volume ratio of 9:1 was collected and then separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the furanpyrrole alkaloid compound.
3. The production method according to claim 2, wherein In step (1), the PDB culture medium includes the following raw materials: potatoes, glucose and water, with 200 g of potatoes and 20 g of glucose added per 1 L of water.
4. The production method according to claim 2, wherein In step (1), the fermentation culture conditions are static culture at 25-32℃ for 15-40 days.
5. The production method according to claim 2, wherein In step (3), the fractions are subjected to reversed-phase silica gel column chromatography, with gradient elution using methanol / water mixtures of volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, eluting three times for each gradient. The collected fractions are numbered 1 to 27. Fractions numbered 5 to 10 are combined and then separated by high-performance liquid chromatography. The peak with a retention time of 14 minutes using a methanol / water mixture of volume ratio of 40:60 is the compound harzianopyrrolidone B, which has the structural formula shown in formula (I).
6. A furanopyrrolidine alkaloid compound of structural formula (I) Use of a furanopyrrolidine alkaloid compound of structural formula (I) shown in the preparation of a drug related to neovascularization.
7. Use according to claim 6, characterized in that Furanpyrrole alkaloids with the structural formula shown in formula (I) have angiogenic activity.