A pyrrolidine alkaloid compound, its preparation method and application

Abstract

This invention discloses a pyrrolidine alkaloid compound, its preparation method, and its applications, belonging to the field of biotechnology. This invention originates from marine fungi. Trichoderma harzianum A novel pyrrolidine alkaloid compound was extracted and isolated from the fermentation culture of ZN-4. Cell experiments showed that the pyrrolidine alkaloid compound provided by this invention has good cardiomyocyte protective activity and low cytotoxicity, and has development prospects in the preparation of cardiomyocyte protective drugs or functional foods.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for obtaining *Trichoderma harzianum* fungus (… Trichoderma harzianum Pyrrolidine alkaloids extracted from ZN-4 and their applications. Background Technology

[0002] Anthracyclines (ANTs) are a class of cytotoxic drugs produced by Streptomyces spp. with broad-spectrum tumor-killing capabilities. They mainly include doxorubicin and daunorubicin, and are widely used to treat hematologic malignancies and solid tumors, such as acute leukemia, lymphoma, breast cancer, gastric cancer, soft tissue sarcoma, and ovarian cancer. Anthracyclines have a broad antitumor spectrum, strong antitumor effects, and definite efficacy, making them indispensable. However, they can cause a series of adverse reactions, including cardiotoxicity, hepatotoxicity, nephrotoxicity, bone marrow suppression, and hand-foot syndrome. Cardiotoxicity is the most serious adverse reaction, significantly affecting the survival rate and quality of life of cancer patients, thus limiting their clinical application (Li Wei et al. Research progress on cardiotoxicity caused by anthracyclines. Central South Pharmacy, 2024, 22(2):437-444.).

[0003] Multiple clinical observations and laboratory evidences demonstrate that cardioprotective drugs are significantly effective against ANT-induced myocardial injury. For example, dextrazosen, as an adjuvant chemotherapy drug, is highly effective in reducing the cardiotoxicity caused by this class of drugs. Clinically, it is often used in combination with anthracycline chemotherapy drugs to alleviate the cardiotoxicity caused by these anthracyclines. However, dextrazosen also has some adverse reactions, such as skin itching or redness, nausea and vomiting, headache, and fatigue. Therefore, there is a need to find novel cardioprotective drugs with low toxicity and high efficacy.

[0004] 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.

[0005] 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.

[0006] Trichoderma ( Trichoderma 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 biological activities, including antibacterial, antioxidant, anticancer, and antiviral properties (Qingfeng Guo, […]). et al . Structures and BiologicalActivities of Secondary Metabolites from the Trichoderma genus (Covering 2018–2022). J Agric Food Chem , 2023, 71(37):13612-13632), making Trichoderma an important producer of compounds with potential biotechnological applications. Summary of the Invention

[0007] The purpose of this invention is to obtain Trichoderma harzianum ( Trichoderma harzianum Natural active substances with medicinal value are extracted from the secondary metabolites of ZN-4.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention is derived from Trichoderma harzianum ( Trichoderma harzianum A new pyrrolidine alkaloid compound was isolated from the fermentation products of ZN-4. Structural identification revealed the molecular formula of the new compound to be C0. 11 H 17 NO5, with the structural formula shown in formula (Ⅰ), is named harzianopyrrolidone A.

[0010] (I).

[0011] The Trichoderma harzianum ( Trichoderma harzianum ZN-4 was isolated from sediments in Clam Bay, Zhoushan City, Zhejiang Province, and is a publicly available biomaterial. See reference (C. Zhou, et al. Harzianolides BG:Undescribed Butenolides isolated from the fungus Trichoderma harzianum ZN-4. Fitoterapia , 2024,176,106039).

[0012] This invention also provides a method for obtaining Trichoderma harzianum ( Trichoderma harzianum The method for separating and extracting the above-mentioned new compounds from the fermentation products of ZN-4 is described, but the preparation method of the above-mentioned compounds in this invention is not limited to this.

[0013] A method for extracting the pyrrolidine alkaloid compound from fermentation products includes the following steps:

[0014] (1) Trichoderma harzianum ( Trichoderma harzianum ZN-4 was activated and then inoculated into PDB medium for fermentation.

[0015] (2) After the fermentation culture was completed, the fermentation broth was extracted with ethyl acetate to obtain the extract;

[0016] (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 with dichloromethane / methanol mixture at a volume ratio of 9:1 was collected. The fraction was then subjected to reverse-phase silica gel column chromatography. Gradient elution was carried out with methanol / water mixtures with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. Each gradient elution was performed 3 times, and the collected fractions were numbered sequentially from 1 to 27. The fractions numbered 5 to 10 were combined and then separated by high-performance liquid chromatography to obtain the pyrrolidine alkaloid compound.

[0017] In step (1), Trichoderma harzianum ( Trichoderma harzianum ZN-4 was fermented and cultured.

[0018] Trichoderma harzianum ( Trichoderma harzianum ZN-4 is a fungus that can be fermented using conventional PDB medium, which includes the following ingredients: potatoes, glucose and water, with 200 g of potatoes and 20 g of glucose added per 1 L of water.

[0019] The fermentation culture conditions are static culture at 25-32℃ for 15-40 days. The static culture method refers to culture without shaking flasks.

[0020] 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 pyrrolidine alkaloid compound is the highest.

[0021] In step (2), ethyl acetate is used to extract the active ingredients in the fermentation broth to obtain an extract.

[0022] In step (3), the extract is separated by normal-phase silica gel column chromatography, and the resulting fraction is further separated by reversed-phase silica gel column chromatography and high-performance liquid chromatography. Through multi-step separation and purification, pyrrolidine alkaloid compounds with high purity can be obtained.

[0023] Preferably, the high performance liquid chromatography separation method includes: 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 a compound harzianopyrrolidone A with the structural formula shown in formula (I).

[0024] This invention demonstrates that the above method can be used to extract *Trichoderma harzianum* (…). Trichoderma harzianum The pyrrolidine alkaloids isolated from ZN-4 fermentation culture exhibit good cardioprotective activity. Therefore, this invention provides the application of the pyrrolidine alkaloids in the preparation of cardioprotective drugs. This invention uses the compound's anti-Namalwa lymphoma cell activity as its cytotoxicity data, and the results show that it exerts cardioprotective effects while exhibiting low toxicity to human cells, demonstrating biocompatibility.

[0025] Specifically, this invention provides the use of compound harzianopyrrolidone A in the preparation of medicaments for the prevention or treatment of cardiotoxicity caused by anthracyclines.

[0026] Furthermore, the anthracycline drug is doxorubicin. Studies have shown that the compound harzianopyrrolidone A can effectively restore cardiomyocyte damage caused by doxorubicin.

[0027] Furthermore, the manifestations of cardiotoxicity include decreased cardiomyocyte viability.

[0028] The present invention also provides an antitumor pharmaceutical composition comprising a first formulation formed of doxorubicin and a pharmaceutically acceptable carrier, and a second formulation formed of a pyrrolidine alkaloid compound with the structural formula shown in Formula (I) and a pharmaceutically acceptable carrier. The second formulation is used to mitigate the cardiotoxicity caused by the first formulation during antitumor therapy.

[0029] The beneficial effects of this invention are as follows:

[0030] (1) The present invention extracts and isolates a compound with a novel structure, harzianopyrrolidone A, 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.

[0031] (2) Cell experiments showed that the harzianopyrrolidone A compound provided by the present invention has good cardiomyocyte protective activity. Further cytotoxicity tests showed that the harzianopyrrolidone A compound provided by the present invention has low cytotoxicity and has good development prospects in the preparation of myocardial protective drugs and preventive health foods. Attached Figure Description

[0032] Figure 1 For the compound harzianopyrrolidone A 1 H NMR data (in DMSO- d 6,600 MHz).

[0033] Figure 2 For the compound harzianopyrrolidone A 13 C NMR data (in DMSO- d 6,150 MHz).

[0034] Figure 3 For the compound harzianopyrrolidone A 1 H- 1 H-COSY data (in DMSO- d 6).

[0035] Figure 4 HSQC data for compound harzianopyrrolidone A (in DMSO- d 6).

[0036] Figure 5 HMBC data for compound harzianopyrrolidone A (in DMSO- d 6).

[0037] Figure 6 ROESY data for compound harzianopyrrolidone A (in DMSO- d 6).

[0038] Figure 7Electronic circular dichroism chromatogram data for compound harzianopyrrolidone A (in CH3OH).

[0039] Figure 8 Here is the structural formula of the compound harzianopyrrolidone A.

[0040] Figure 9 Analysis of the cardioprotective activity of compound harzianopyrrolidone A. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] Trichoderma harzianum used in the following examples ( Trichoderma harzianum ZN-4 was isolated from marine sediments in previous studies by our research group; see reference (C. Zhou, et al . Harzianolides BG:Undescribed Butenolides isolated from the fungus Trichoderma harzianum ZN-4. Fitoterapia , 2024,176,106039).

[0044] Example 1: Trichoderma harzianum ( Trichoderma harzianum Fermentation culture of ZN-4

[0045] Trichoderma harzianum ( Trichoderma harzianum ZN-4 was activated and inoculated into PDB medium, and then statically fermented at 28°C for 21 days.

[0046] 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.

[0047] Example 2: Preparation of pyrrolidine alkaloid compounds

[0048] Trichoderma harzianum ( Trichoderma harzianumAfter fermentation, 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, diameter 12 mm). Gradient elution was performed 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.

[0049] 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; 5-7: 25-27.

[0050] The fraction was then separated by high performance liquid chromatography. The peak of subfraction 5-3 with a retention time of 14 minutes using a methanol / water mixture with a volume ratio of 40:60 was designated as compound 1.

[0051] Example 3: Structural identification of compound 1

[0052] The purity of the compounds obtained in Example 2 was determined by HPLC. The structures of samples with a purity greater than 98% were determined 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.

[0053] According to the one-dimensional NMR of compound 1 (see Table 1), Figure 1-2 According to the results of mass spectrometry analysis, the molecular formula of compound 1 is C13C12C ... 11 H 17 NO5. Results of analysis by two-dimensional NMR and electronic circular dichroism spectroscopy (see...) Figure 3-7 The structure of the compound was confirmed. By consulting databases and literature, compound 1 was identified as a new compound, harzianopyrrolidone A, with the structure shown below. Figure 8 As shown.

[0054] Table 1. NMR data of compound 1

[0055] Position <![CDATA[ δ C (ppm), type]]> <![CDATA[ δ H (ppm) mult. ( J in Hz)]]> 1 - - 2 170.6, C - 3 <![CDATA[44.1, CH2]]> 2.51, d (16.8) 2.40, d (16.8) 4 107.3, C - 5 69.3, CH 3.77, dd (7.4, 2.6) 6 <![CDATA[32.2, CH2]]> 2.56, dd (14.4, 7.4)1.80, dd (14.4, 2.5) 7 90.7, C - 8 34.0, CH 1.94, p (6.8) 9 <![CDATA[17.4, CH3]]> 0.71, d (7.0) 10 <![CDATA[17.0, CH3]]> 0.73, d (7.0) 11 176.0, C - 12 <![CDATA[27.2, CH3]]> 2.63, s

[0056] Example 4: Analysis of the cardioprotective activity of the compound Harzianopyrrolidone A

[0057] (1) Solution preparation

[0058] DMEM complete medium: 89% DMEM basal medium + 10% FBS + 1% antibiotics;

[0059] DMEM maintenance medium: 98.8% DMEM basal medium + 1% antibiotics;

[0060] PBS (1×) 1 L: NaCl 8 g + KCl 0.2 g + Na2HPO4·12H2O 3.58 g + KH2PO4 0.24 g, filter through a 0.22 μm membrane, tighten the cap to sterilize, and tighten again after natural cooling;

[0061] Cell cryopreservation solution: 80% DMEM basal medium + 10% FBS + 10% DMSO.

[0062] (2) Cell viability determination

[0063] H9c2 cells were treated with 1.0 μM doxorubicin for 24 h to establish a cell model. The protective effect against doxorubicin-induced cardiomyocyte damage was then determined using this cell model in combination with cell survival assays.

[0064] First, take H9c2 cells in the logarithmic growth phase, 7 × 10⁶ cells per well. 3 Cells were seeded in 96-well plates and cultured for 24 h at 37°C and 5% CO2. Cells were divided into three groups: a damage model group, an experimental group, and a positive control group. The model group received 1.0 μM doxorubicin, the drug treatment group received doxorubicin and different concentration gradients of harzianopyrrolidone A, and the positive control group received doxorubicin and the positive control drug dextrorazoxane (20 μM). Each group had three replicates. Cell viability was measured using the MTT assay (10 μL of 5 mg / mL MTT solution was added to each well, incubated for 4 h, the supernatant was discarded, 150 μL of LDMSO was added, and the OD value was measured at 570 nm). The repair effect of the drugs on doxorubicin-induced damage was assessed.

[0065] Survival rate (%) = (mean OD of sample group - mean OD of blank group) / (mean OD of negative group - mean OD of blank group) × 100%.

[0066] (3) Experimental results

[0067] Table 2. Survival rate of H9c2 cardiomyocytes damaged by doxorubicin under the action of the compound

[0068]

[0069] The H9c2 cardioprotective experiment of compound harzianopyrrolidone A yielded the following results: Figure 9 As shown in Table 2, the compound harzianopyrrolidone A significantly improved cell viability at concentrations of 5 μM and 10 μM, with a better effect at 20 μM. This indicates that harzianopyrrolidone A can restore the damage to H9c2 cells caused by doxorubicin at a certain concentration, and this effect is not concentration-dependent. This demonstrates that harzianopyrrolidone A has a good protective effect against cardiomyocytes in vitro.

[0070] Example 5: Cytotoxicity analysis of pyrrolidine alkaloid compounds

[0071] (1) Reagent preparation

[0072] 0.4% SRB solution: Weigh 0.8g SRB, dissolve in 200mL 1% acetic acid, and store at room temperature.

[0073] 50% TCA solution: Weigh 50g of TCA, add water to make up to 100mL, and store at 4℃.

[0074] 10mM Tris-base solution: Weigh 0.6057g Tris-base, add water to a final volume of 500mL, pH 10.5, and store at 4℃.

[0075] (2) Experimental apparatus

[0076] CO2 incubator (Thermo), mini shaker (Kylin-Bell Lab instruments), microplate reader (MD, M5 model).

[0077] (3) Method

[0078] Namalwa cells in logarithmic growth phase were selected, and after centrifugation, the cell concentration was adjusted to 2 × 10⁶ cells / mL using RPMI 1640 medium containing 10% fetal bovine serum. 4Cells were seeded at a rate of 190 μL / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. 10 μL of sample solution (final concentration: compound 20 µM) was added to the drug treatment wells; 20 µM 5-FU was added to the positive control wells; and cultured at 37°C with an equal volume of medium in the control wells for 3 days with 5% CO2. The medium was discarded, and 100 μL of 50% TCA (pre-chilled at 4°C) was gently added to fix the cells. The cells were incubated for 5 min, then transferred to 4°C for 1 h. The fixative was discarded, and the cells were washed 5 times with distilled water to remove TCA, and air-dried for 1 h. 80 μL of 0.4% SRB solution was added to each well, and staining was performed at room temperature for 30 min. The staining solution was discarded, and the cells were washed 5 times with 1% acetic acid to remove unbound SRB, and air-dried. 150 μL of 10 mM Tris-base (pH 10.5) was added to dissolve the cells, and the plates were shaken on a micro-oscillator for 5 min. M5 microplate reader for OD measurement 510nm value.

[0079] (4) Calculation of results

[0080] Tumor cell growth inhibition rate (%) = (OD) 对照 -OD 药物 ) / (OD 对照 -OD 空白 ) × 100%.

[0081] (5) Experimental results

[0082] Table 3. Survival rate of Namalwa cells under the action of the compound

[0083]

[0084] The cytotoxicity of compound 1 to Namalwa cell line at a concentration of 20 µM was further determined. The data showed that the survival rate of Namalwa cell line was 11.2% for the positive control drug 5-Fu and 5.6% for the positive control drug As2O3. Compound 1 showed low cytotoxicity to Namalwa cell line.

Claims

1. A pyrrolizidine alkaloid compound, characterized by, The structural formula of the pyrrolidine alkaloid compound is shown as formula (I), 2. The method for preparing the pyrrolidine alkaloid compound as described in claim 1, characterized in that, The method comprises the following steps: (1) inoculating Trichoderma harzianum ZN-4 into PDB culture medium after activation, and performing fermentation culture; (2) after the fermentation culture is completed, extracting the fermentation liquor by using ethyl acetate to obtain an extraction liquor; (3) concentrating the extraction liquor, and performing normal-phase silica gel column chromatography separation; gradient elution is performed by using dichloromethane / methanol mixed solution 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 in sequence, the fraction eluted by the dichloromethane / methanol mixed solution with a volume ratio of 9:1 is collected, the fraction is subjected to reversed-phase silica gel column chromatography, gradient elution is performed by using methanol / water mixed solution with a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 in sequence, each gradient elution is performed for three times, and the collected fractions are numbered as 1-27 in sequence; the fractions numbered as 5-10 are combined, and high-performance liquid chromatography separation is performed to obtain the pyrrolidine alkaloid compound.

3. The production method according to claim 2, wherein In step (1), the fermentation culture is performed at 25-32 DEG C in a static state for 15-40 days.

4. The production method according to claim 2, wherein In step (3), the method of the high-performance liquid chromatography separation comprises the following steps: under the eluent of methanol / water mixed solution with a volume ratio of 40:60, the peak with a retention time of 14 minutes is the compound harzianopyrrolidone A with the structural formula shown as formula (I).

5. The pyrrolidine alkaloid compound of claim 1 is applied to the preparation of a myocardial protection drug.

6. The pyrrolidine alkaloid compound of claim 1 is applied to the preparation of a drug for preventing or treating anthracycline-induced cardiotoxicity.

7. Use according to claim 6, wherein The anthracycline is doxorubicin.

8. Use according to claim 6 or 7, wherein the compound is ###0002### The cardiotoxicity is manifested in the form of decreased myocardial cell viability.

9. An antitumor pharmaceutical composition, characterized by comprising the compound or a pharmaceutically acceptable salt thereof according to claim 1. The pharmaceutical composition comprises a first preparation formed by doxorubicin and a pharmaceutically acceptable carrier, and a second preparation formed by the pyrrolidine alkaloid compound with the structural formula shown as formula (I) and a pharmaceutically acceptable carrier.

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