Marine fungus-derived diterpenes diterpenoid compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of marine natural pharmaceutical chemistry, and particularly provides a marine fungus-derived diterpenes diterpenoid compound as well as a preparation method and application thereof. According to the present invention, the diterpenes diterpenes compound is obtained from the marine fungus Penicillium brasilianum HBU-136, and the chemical structural formula of the marine fungus-derived diterpenes diterpenes compound is represented by the formula I, wherein the chemical structural formula of the marine fungus-derived diterpenes diterpenes compound is represented by the formula I, and the chemical structural formula of the marine fungus-derived diterpenes diterpenes compound is represented by the formula II; experiments prove that the compound has strong inhibitory activity on human gastric cancer cells (MGC-803, AGS and HGC-27), pulmonary epithelial cancer cells (A-549), liver cancer cells (HepG2) and colon cancer cells (SW-620), can be used as an antitumor drug, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of marine natural product chemistry, specifically relating to a diterpenoid compound derived from marine fungi (diterpenes), its preparation method, and its application. Background Technology

[0002] Diterpenoids are a class of natural products with a C20 skeleton composed of four isoprene units. They exhibit diverse structures and rich biological activities, making them an important source of drug lead compounds. Existing research has confirmed that diterpenoids demonstrate significant activities in anti-inflammatory, antitumor, antibacterial, antiviral, and immunomodulatory activities, highlighting their core value in pharmaceutical research and development.

[0003] Traditional sources of diterpenoids mainly rely on terrestrial plants. However, terrestrial plants have problems such as long growth cycles, limited resource reserves, and the potential for ecological damage from over-harvesting. Furthermore, the isolation and purification of target compounds from most plants is difficult, making it hard to meet the needs of research and development and production. Therefore, finding novel and efficient sources of diterpenoids has become a research hotspot in the field of natural product chemistry.

[0004] The ocean, covering approximately 71% of the Earth's surface, is the largest repository of biological resources on Earth, providing abundant structural templates and lead compounds for new drug development. Since the mid-20th century, several marine natural products have been successfully developed into clinical drugs, such as cephalosporins and cytarabine, demonstrating the development value of marine pharmaceutical resources. Marine fungi, due to their diverse metabolic systems and convenient fermentation cultivation, have become an important source for research on marine bioactive substances.

[0005] However, the types of diterpenoids reported so far are limited, the metabolic potential of most marine fungal strains has not yet been explored, a large number of diterpenoids have not yet been discovered, and the synthesis mechanism and fermentation regulation strategy of marine fungal diterpenoids in existing studies are still incomplete. Some compounds with excellent activity are difficult to develop for subsequent applications due to low yield and difficulty in isolation.

[0006] Therefore, isolating and identifying novel diterpenoids from marine fungi can not only enrich the structural diversity of diterpenoids, but also provide an important material basis for the development of new drug lead compounds, which has important scientific significance and application value. Summary of the Invention

[0007] The purpose of this invention is to provide a diterpenes compound derived from marine fungi, its preparation method, and its application in antitumor therapy.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A diterpenoid compound derived from marine fungi, *Diterpenes*, has the chemical structural formula shown in Formula I: ; Formula I.

[0009] The preparation method of the above-mentioned diterpenes includes the following steps: (1) Marine fungus Penicillium brasiliensis Penicillium brazilianum HBU-136 strain was inoculated into the culture medium and cultured at 15-35℃ for 3-10 days. The culture medium for the strain is composed of: 1.0-10 wt% glucose, 2.0-4.0 wt% potato extract, 1.0-6.0 wt% agar, 3.0-10 wt% crude sea salt, and the remainder being water; (2) After the strain culture is completed, it is inoculated into the fermentation medium and fermented at 15-35℃ for 30-50 days to obtain the fermented product; each unit of the fermentation medium includes 100-200 g of rice and 100-200 mL of water. (3) Extract the fermentation product with ethyl acetate 2-4 times, combine the ethyl acetate extracts and concentrate under reduced pressure to obtain crude extract; (4) The crude extract was subjected to normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, gel column chromatography and high performance liquid chromatography in sequence to obtain the diterpenes compounds.

[0010] The marine fungus *Penicillium brasiliensis* described in this invention Penicillium brazilianum HBU-136 was deposited on January 25, 2022, with accession number CGMCC No. 40072. The depository is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] Further, in step (4), the normal phase silica gel column chromatography separation is as follows: first, elution is performed using a stationary phase of 100-200 mesh silica gel and a mobile phase of 0-3 vol% methanol / dichloromethane mixed solution, with an elution volume of 3-5 column volumes. The resulting eluent is then concentrated and eluted again using a stationary phase of 200-300 mesh silica gel and a mobile phase of 25-30 vol% ethyl acetate / petroleum ether mixed solution, with an elution volume of 2-3 column volumes.

[0012] Furthermore, in step (4), the stationary phase for gel column chromatography is Sephadex LH-20, and the mobile phase is a mixed solution of petroleum ether / methanol / dichloromethane (PE / CH2Cl2 / MeOH = 2:1:1) for elution, with an elution volume of 3-5 column volumes.

[0013] Furthermore, in step (4), the stationary phase used in the reversed-phase silica column chromatography separation is C2. 18 Silica gel, mobile phase is 60-70 vol% methanol / water mixture, elution volume is 2-3 column volumes.

[0014] Furthermore, in step (4), the chromatographic column used in the high-performance liquid chromatography separation is a semi-preparative C18 column. 18 The chromatographic column was an XBridge OBD, 5 μm, 10 × 250 mm, and the mobile phase was a 70-90 vol% methanol / water mixture.

[0015] The present invention also provides the use of the above-mentioned diterpenes or pharmaceutically acceptable salts thereof in antitumor activity.

[0016] Furthermore, the tumors are human gastric cancer cells (MGC-803, AGS, HGC-27), lung epithelial cancer cells (A-549), liver cancer cells (HepG2), and colon cancer cells (SW-620).

[0017] The present invention also provides an antitumor agent comprising the above-mentioned diterpenes compounds or their pharmaceutically acceptable salts as active ingredients.

[0018] This invention relates to the marine fungus Penicillium brasiliensis. Penicillium brazilianum Diterpenes were obtained from HBU-136, and experiments have shown that they have strong inhibitory activity against human gastric cancer cells (MGC-803, AGS, HGC-27), lung epithelial cancer cells (A-549), liver cancer cells (HepG2), and colon cancer cells (SW-620). They can be used as anti-tumor drugs and have broad application prospects. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0020] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0021] Strain background: The marine fungus Penicillium brasiliensis involved in the following examples Penicillium brazilianum HBU-136 was isolated from marine sediments collected from Huanghua Port in Cangzhou City, Hebei Province. ITS rRNA identification confirmed that this marine fungus belongs to the genus *Penicillium*. Penicillium brazilianum (Fungi, hence the name) Penicillium Brazilian HBU-136.

[0022] Brazilian penicillium Penicillium brazilianum The process of separating, screening, and identifying HBU-136 has been disclosed in Chinese Patent CN 118879505A, and its accession information is as follows: The marine fungus is Penicillium brasilianum strain HBU-136, deposited on January 25, 2022, with accession number CGMCC No.40072. The depository is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] Example 1: Preparation of diterpenes 1. Marine fungi Penicillium brazilianum HBU-136 seed culture Frozen marine fungi Penicillium brazilianum HBU-136 was inoculated into a sterilized culture medium and incubated at 28°C for 5 days. The culture medium contained 1.0 wt% glucose, 2.0 wt% potato starch, 2.0 wt% agar, 3.0 wt% crude sea salt, and the remainder was water. It was prepared as a test tube slant before use.

[0024] 2. Marine fungi Penicillium brazilianum Fermentation culture of HBU-136 Marine fungi Penicillium brazilianum The fermentation medium used for HBU-136 fermentation culture contained 200g of rice and 170mL of water in each 1000mL Erlenmeyer flask. The strain was statically fermented at 28℃ for 40 days to obtain the fermentation product; a total of 200 1000mL Erlenmeyer flasks were used for fermentation.

[0025] 3. Crude extraction and separation of diterpenes Take the fermentation product obtained in step (2), extract it three times with ethyl acetate, combine the ethyl acetate extracts and concentrate under reduced pressure to obtain crude extract. First, perform normal phase silica gel column chromatography separation, stationary phase: 100-200 mesh silica gel, mobile phase: 0 vol% methanol / dichloromethane mixed solution, elute for 5 column volumes, concentrate the eluent and perform normal phase silica gel column chromatography separation again, stationary phase: 200-300 mesh silica gel, mobile phase: 25 vol% ethyl acetate / petroleum ether mixed solution, elute for 2 column volumes.

[0026] After concentration, the eluent was separated by Sephadex LH20 gel column chromatography. The mobile phase was a mixture of petroleum ether / methanol / dichloromethane (PE / CH2Cl2 / MeOH = 2:1:1), eluting for 3 column volumes. After concentration, the eluent was separated by reversed-phase silica gel column chromatography, with C2 as the preferred stationary phase. 18 Silica gel was used, with a preferred mobile phase of 70 vol% methanol / water mixture, eluting for 3 column volumes. The eluent was concentrated and then subjected to high-performance liquid chromatography (HPLC) separation. The stationary phase was semi-preparative C5. 18 A chromatographic column, XBridge OBD, 5 μm, 10 × 250 mm, was used with a mobile phase of 70 vol% methanol / water mixture to prepare and isolate the compound represented by formula I (hereinafter referred to as compound I), with the molecular formula C. 20 H 28 O 5， The structural formula is shown in Equation I, and its structural confirmation data are as follows. The nuclear magnetic resonance spectroscopy data are shown in Table 1. ;

[0027] Formula I.

[0028] Compound I: Yellow solid; α ] D 20- 92.7 ( c 0.1, MeOH); UV (MeOH), λ max (log ε ) 221(2.88) nm; ECD (0.96 mM, MeOH), λ max (Δ ε ) 211 (+0.2), 227 (+2.8), 253 (-0.3),322 (+1.7) nm; IR (KBr) v max 3449,2938,2366,1724,1637 cm -1 ; 1 H and 13 C NMR data are shown in the table below. HRESIMS m / z 371.1822 [M + Na] + (calcd for C 20 H 28 O₅Na, 371.1829 [M + Na] + ). Table 1. Nuclear magnetic resonance spectral data of compound I (600 MHz, DMSO- d 6, TMS, δ ppm)

[0029] Example 2: Preparation of diterpenes 1. Marine fungi Penicillium brazilianum HBU-136 seed culture Frozen marine fungi Penicillium brazilianum HBU-136 was inoculated into a sterilized culture medium and incubated at 35°C for 7 days. The culture medium contained 1.0 wt% glucose, 3.0 wt% potato starch, 6.0 wt% agar, 10.0 wt% crude sea salt, and the remainder was water. It was prepared as a test tube slant before use.

[0030] 2. Marine fungi Penicillium brazilianum Fermentation culture of HBU-136 Marine fungi Penicillium brazilianum The fermentation medium used for HBU-136 fermentation culture contained 100g of rice and 90mL of water in each 1000mL Erlenmeyer flask; the fermentation culture conditions were 35℃ and static culture for 30 days to obtain the fermented product.

[0031] 3. Crude extraction and separation of diterpenes Take the fermentation product obtained in step (2), extract it 4 times with ethyl acetate, combine the ethyl acetate extracts and concentrate under reduced pressure to obtain crude extract. First, perform normal phase silica gel column chromatography separation, stationary phase: 100-200 mesh silica gel, mobile phase: 3 vol% methanol / dichloromethane mixed solution, elute for 3 column volumes, concentrate the eluent and perform normal phase silica gel column chromatography separation again, stationary phase: 200-300 mesh silica gel, mobile phase: 30 vol% ethyl acetate / petroleum ether mixed solution, elute for 3 column volumes.

[0032] After concentration, the eluent was separated by Sephadex LH20 gel column chromatography. The mobile phase was a mixture of petroleum ether / methanol / dichloromethane (PE / CH2Cl2 / MeOH = 2:1:1), and 5 column volumes were eluted. After concentration, the eluent was separated by reversed-phase silica gel column chromatography, with C2 as the preferred stationary phase. 18 Silica gel was used, with a preferred mobile phase of a 60 vol% methanol / water mixture, eluting for 6 column volumes. The eluent was concentrated and then subjected to high-performance liquid chromatography (HPLC) separation. The stationary phase was a semi-preparative C5 solution. 18The chromatographic column was an XBridge OBD, 5 μm, 10 × 250 mm, and the mobile phase was an 80 vol% methanol / water mixture. Compound I was prepared and isolated, and its structural confirmation data were consistent with those in Example 1.

[0033] Other bacterial culture and fermentation conditions not specifically specified in Examples 1 and 2, as well as other experimental operation conditions such as normal-phase silica column chromatography, reversed-phase silica column chromatography, gel column chromatography, and high-performance liquid chiral chromatography, are all conventional experimental operation conditions in the art. Those skilled in the art can make reasonable selections according to actual needs.

[0034] Example 3: Antitumor activity of diterpenes 1. Antitumor activity test The in vitro inhibitory activity of human gastric cancer cells (MGC-803, AGS, HGC-27), lung epithelial cancer cells (A-549), liver cancer cells (HepG2), and colon cancer cells (SW-620) was tested using the MTT assay.

[0035] 2. Activity testing methods According to the cell growth rate, a certain number of cells in the logarithmic growth phase were seeded into 96-well culture plates at a concentration of 90 μL / well. After culturing for 24 h, 10 μL / well of the sample to be tested was added. For each cell line and each concentration gradient, three replicates were performed. After culturing the cells at 37℃ and 5% CO2 for 48 h, 5 mg / mL MTT (Sigma) solution was added and diluted with physiological saline to 20 μL / well. After culturing for another 4 h, 50 μL / well of triple solution (10% SDS-5% isobutanol-0.01 mol / L HCl) was added, and the plates were incubated overnight in a CO2 incubator. Then, the OD was measured using a microplate reader. 570 Value. Cisplatin was used as a positive control.

[0036] 3. Activity test results IC50 inhibitory activity of tumor cell lines 50 The values ​​(μM) are shown in Table 2 below.

[0037] Table 2

[0038] The experimental results showed that compound I had a strong inhibitory effect on human gastric cancer cells (MGC-803, AGS, HGC-27), lung epithelial cancer cells (A-549), liver cancer cells (HepG2), and colon cancer cells (SW-620).

[0039] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention. The purpose of the present invention is to provide a diterpenoid compound derived from marine fungi, its preparation method, and its application in antitumor activity.

Claims

1. A diterpenoid compound derived from marine fungi, characterized in that, Its chemical structural formula is shown in Formula I: ； Formula I.

2. The method for preparing the diterpenes compound according to claim 1, characterized in that, Includes the following steps: (1) Marine fungus Penicillium brasiliensis Penicillium brasilianum HBU-136 strain was inoculated into the culture medium and cultured at 15-35℃ for 3-10 days. The culture medium for the strain is composed of: 1.0-10 wt% glucose, 2.0-4.0 wt% potato extract, 1.0-6.0 wt% agar, 3.0-10 wt% crude sea salt, and the remainder being water; (2) After the strain culture is completed, it is inoculated into the fermentation medium and fermented at 15-35℃ for 30-50 days to obtain the fermentation product; Each unit of the fermentation medium comprises 100-200 g of rice and 100-200 mL of water; (3) Extract the fermentation product with ethyl acetate 2-4 times, combine the ethyl acetate extracts and concentrate under reduced pressure to obtain crude extract; (4) The crude extract was subjected to normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, gel column chromatography and high performance liquid chromatography in sequence to obtain the diterpenes compounds.

3. The method according to claim 2, characterized in that, In step (4), the normal phase silica gel column chromatography separation is performed as follows: first, elution is carried out using 100-200 mesh silica gel as the stationary phase and 0-3 vol% methanol / dichloromethane mixed solution as the mobile phase, with an elution volume of 3-5 column volumes. The resulting eluent is then concentrated and eluted again using 200-300 mesh silica gel as the stationary phase and 25-30 vol% ethyl acetate / petroleum ether mixed solution as the mobile phase, with an elution volume of 2-3 column volumes.

4. The method according to claim 2, characterized in that, In step (4), the stationary phase for gel column chromatography is Sephadex LH-20, and the mobile phase is a mixed solution of petroleum ether / methanol / dichloromethane (PE / CH2Cl2 / MeOH = 2:1:1) for elution, with an elution volume of 3-5 column volumes.

5. The method according to claim 2, characterized in that, In step (4), the stationary phase used in the reversed-phase silica column chromatography separation is C 18 Silica gel, mobile phase is 60-70 vol% methanol / water mixture, elution volume is 2-3 column volumes.

6. The method according to claim 2, characterized in that, In step (4), the chromatographic column used in the high-performance liquid chromatography separation is a semi-preparative C18 column. 18 The chromatographic column was an XBridge OBD, 5 μm, 10 × 250 mm, and the mobile phase was a 70-90 vol% methanol / water mixture.

7. The use of the diterpenes compound of claim 1 or a pharmaceutically acceptable salt thereof in antitumor activity.

8. The application according to claim 7, characterized in that, The tumors described are human gastric cancer cells, lung epithelial cancer cells, liver cancer cells, and colon cancer cells.

9. An antitumor agent, characterized in that, It contains the diterpenes compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

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