Sesterterpenoids as well as preparation method and application thereof

Abstract

The invention discloses a sesterterpene compound and a preparation method and application thereof, the preparation method comprises the following steps: step 1, preparing a strain culture medium of a strain, inoculating an Aspergillus sp. Strain into a seed culture medium, and culturing to obtain a seed culture solution of the strain; step 2, inoculating the seed culture solution into a rice solid fermentation culture medium, and culturing to obtain a fermented product; step 3, extracting the fermented product by using isometric ethyl acetate, combining extract liquids, and concentrating under reduced pressure to obtain an extract; 4, roughly separating the crude extract flow extract through reduced-pressure normal-phase silica gel column chromatography, respectively carrying out gradient elution by using a petroleum ether-ethyl acetate solution and an ethyl acetate-methanol solution, carrying out TLC (Thin Layer Chromatography) analysis, and merging according to polarity to obtain eight components Fr.1-Fr.8; and 5, carrying out compound separation and purification on the Fr.4 component. The sesterterpenoids provided by the invention have good activity of inhibiting benign prostatic hyperplasia and antibacterial activity, and are a potential active ingredient of drugs for inhibiting benign prostatic hyperplasia and antibacterial activity.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a sesquiterpenoid compound and a preparation method and application thereof. Background Art

[0002] Natural products from the ocean are considered to be "a new wave of drugs". Compared with the increasingly scarce terrestrial sources, marine organisms provide a vast resource for the discovery of new antiviral drugs. Mangroves are salt-tolerant plant communities growing in tropical and subtropical intertidal estuaries. Mangroves are one of the most complex ecosystems in the world, consisting of mangrove plants, animals, related microorganisms and abiotic factors, with characteristics such as salt stress, high mineral composition, strong reducibility, and frequent tides. They are mainly distributed at the sea-land junction in tropical and subtropical regions. Mangrove fungi play an important role in mangrove microbial communities. Due to the wide variety of metabolites they produce, their unique structures and strong pharmacological effects, they are also receiving increasing attention in the medical community. Terpenoids are the largest family of natural products. Natural terpenoids are composed of complex multi-line scaffolds and chiral stereochemical structures. Diterpenoids are terpenoids composed of five isoprene units. They are a relatively small subclass of terpenoids. Currently, less than 2,000 members have been discovered, and they have a wide range of biological activities, including antibacterial, anti-inflammatory, cytotoxic and enzyme inhibitory activities. The special habitat creates a rich diversity of endophytic fungi in mangrove plants, and at the same time allows mangrove endophytic fungi to metabolize secondary metabolites with novel structures and significant biological activities, which shows great research potential and development prospects in the research and development of new drugs.

[0003] The diterpenoids produced by mangrove fungi (Aspergillus and Fusarium are the main sources) include 5 / 3 / 7 / 6 / 5, 5 / 7 / (3)6 / 5, and 5 / 8 / 6 / 6 ring systems. So far, only 35 diterpenoids have been identified from mangrove fungi. This unique molecular framework has broad application prospects in drug development and other fields. Benign prostatic hyperplasia (BPH) is a prevalent disease of benign adenomatous hyperplasia of the prostate around the urethra, which is common in the elderly male population and its prevalence increases with age. Its pathogenesis is not fully understood and may be related to androgens, estrogens, insulin, etc. The currently marketed drugs that inhibit BPH cannot meet market demand, and the development of new BPH drugs has become an urgent need for drug development. The special mangrove ecological environment makes mangrove microbial resources rich and diverse, providing a species basis for the discovery of new green and environmentally friendly antibacterial drugs. The structure and mechanism of action of antibacterial substances from mangrove microorganisms may be different from the targets of existing drugs on the market, which is very likely to overcome the defects of antibiotic resistance in the current market. Compared with other marine biological resources, marine microorganisms have unlimited resources, novel and unique structures, and significant activities. Screening precursor compounds with excellent activity from the secondary metabolites of marine microorganisms and developing them into new green and environmentally friendly antibacterial drugs has become an important way to develop new drugs. These active substances will surely make a huge contribution to the development of new drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a diterpenoid compound in view of the technical defects existing in the prior art.

[0005] Another object of the present invention is to provide a method for preparing the diterpenoid compound.

[0006] Another object of the present invention is to provide the application of the diterpenoid compound.

[0007] The technical solution adopted to achieve the purpose of the present invention is:

[0008] A diterpene compound having the structure shown in compound 1-4:

[0009]

[0010] Another aspect of the present invention also includes a method for preparing the diterpenoid compound, comprising the following steps:

[0011] Step 1, preparing a strain culture medium, inoculating the Aspergillus sp. strain into a seed culture medium, and culturing the strain to obtain a seed culture solution;

[0012] Step 2, inoculating the seed culture solution obtained in step 1 into a rice solid fermentation medium to obtain a fermentation product;

[0013] Step 3, extracting the fermented product obtained in step 2 with an equal volume of ethyl acetate, combining the extracts and concentrating under reduced pressure to obtain an extract;

[0014] Step 4, the extract obtained in step 3 is roughly separated by vacuum normal phase silica gel column chromatography, and gradient eluted with petroleum ether-ethyl acetate solution and ethyl acetate-methanol solution respectively, and analyzed by TLC, and 8 components Fr.1 to Fr.8 are obtained by merging according to polarity;

[0015] Step 5, separation and purification of the compound of Fr.4 component:

[0016] Fr.4 was subjected to normal phase silica gel column chromatography using medium pressure preparative chromatography and eluted with a petroleum ether-ethyl acetate gradient. After TLC analysis, 8 components Fr.4A to Fr.4J were obtained. After HPLC gradient analysis, the Fr.4E component was selected and separated and purified by semi-preparative high performance liquid chromatography with methanol-UP aqueous solution as the mobile phase to obtain compounds 1, compound 2, compound 3 and compound 4.

[0017] In the above technical scheme, in step 1, the seed culture medium includes 1.5wt%-3.0wt% of glucose, 0.1wt%-0.5wt% of yeast extract, 0.1wt%-0.5wt% of peptone, 0.11wt%-0.6wt% of coarse sea salt, and the balance is water. After the seed culture medium is prepared, it is sterilized at 120-140°C for 25-30 minutes.

[0018] In the above technical solution, in step 1, the culture temperature is 26-28° C. and the culture time is 3-4 days.

[0019] In the above technical solution, in step 2, the fermentation medium includes rice, coarse sea salt and water, and the mass ratio of the rice, coarse sea salt and water is 100:(0.11-0.6):100; or, the fermentation medium includes 1.5wt%-3.0wt% of glucose, 0.1wt%-0.5wt% of yeast extract, 0.1wt%-0.5wt% of peptone, 0.11wt%-0.6wt% of coarse sea salt, and the balance is water;

[0020] In the above technical solution, in step 2, the fermentation medium is sterilized at 120-140° C. for 25-30 minutes after being prepared.

[0021] In the above technical solution, in step 2, the culture temperature is 26-28°C, static culture is performed, and the culture time is 28-30 days.

[0022] In the above technical scheme, in step 4, during gradient elution, the volume ratios of petroleum ether and ethyl acetate in the petroleum ether-ethyl acetate solution are 10:0, 7:3, 1:1, 7:3 and 0:10, respectively, and the volume ratios of ethyl acetate and methanol in the ethyl acetate-methanol solution are 10:0, 7:3, 1:1, 7:3 and 0:10, respectively.

[0023] In the above technical solution, in step 5, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution is 5:1, 2:1, 1:1, 1:2, 1:3 and 0:1.

[0024] In the above technical solution, in step 5, the volume ratio of methanol to UP water in the methanol-UP aqueous solution is 70:30, and preferably, the flow rate of the methanol-UP aqueous solution is 2.0 mL / min.

[0025] Another aspect of the present invention also includes the use of compound 3 in the diterpenoid compound in the preparation of a drug for inhibiting benign prostatic hyperplasia. The structural formula of compound 3 is as follows:

[0026]

[0027] Another aspect of the present invention also includes the use of compound 1 in the diterpenoid compound in the preparation of antibacterial drugs. The structural formula of compound 1 is as follows:

[0028]

[0029] Another aspect of the present invention is a drug for inhibiting benign prostatic hyperplasia, comprising compound 3 among the sesquiterpenoid compounds or a pharmaceutically acceptable salt thereof and excipients.

[0030] Another aspect of the present invention is an antibacterial drug, comprising compound 1 among the sesquiterpenoid compounds or a pharmaceutically acceptable salt thereof and excipients.

[0031] In the above technical solution, the auxiliary material is a pharmaceutically acceptable carrier, diluent or excipient.

[0032] In the above technical solution, the dosage form of the drug is a solid preparation, a semi-solid preparation or a liquid preparation.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The four sesquiterpenoid compounds of the present invention can effectively inhibit benign prostatic hyperplasia activity and are potential active ingredients of drugs for inhibiting benign prostatic hyperplasia.

[0035] 2. The four sesquiterpenoid compounds of the present invention have good anti-marine Vibrio and anti-Xanthomonas biological activities and are potential active ingredients of antibacterial drugs.

[0036] 3. The method for separating and extracting four sesquiterpenoid compounds of the present invention is convenient and fast, can be widely promoted and applied, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is compound 1 1 H NMR spectra.

[0038] Figure 2 is compound 1 13 C NMR spectrum.

[0039] Figure 3 It is compound 2 1 H NMR spectra.

[0040] Figure 4 It is compound 2 13 C NMR spectrum.

[0041] Figure 5 is compound 3 1 H NMR spectra.

[0042] Figure 6 is compound 3 13 C NMR spectrum.

[0043] Figure 7 is compound 4 1 H NMR spectra.

[0044] Figure 8 is compound 4 13 C NMR spectrum.

[0045] Fig. 9 This is a comparison chart of the calculated CD and tested CD of compound 1.

[0046] Fig.10 This is a comparison chart of the calculated CD and tested CD of compound 2.

[0047] Fig.11 This is a comparison chart of the calculated CD and tested CD of compound 3.

[0048] Fig.12 This is a comparison chart of the calculated CD and tested CD of compound 4.

[0049] Fig.13 is compound 1-4 1 H- 1 H COSY and HMBC. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0051] Example 1

[0052] (1) Cultivation of Aspergillus sp.

[0053] Prepare seed culture medium: 80 g glucose, 8 g peptone, 8 g yeast extract, 12 g coarse sea salt, and 4.0 L water. Divide equally into 8 1000 mL conical flasks and sterilize at 120°C for 25–30 minutes.

[0054] The fungal strain Aspergillus sp.GXIMD 03023 with Genbank accession number MZ976808 was inoculated into the prepared seed culture medium, and cultured at 26-28° C. for 3 days to obtain a seed culture solution;

[0055] (2) Fermentation of Aspergillus sp.

[0056] Prepare fermentation medium: 1.1 kg glucose, 100 g peptone, 100 g yeast extract, 150 g sea salt, 50 L water, evenly distribute into 120 1000 mL conical flasks, sterilize at 120°C for 25-30 minutes.

[0057] An appropriate amount of the seed culture solution obtained in step (1) is inoculated into a conical flask containing a fermentation medium and cultured at 26-28° C. for 30 days.

[0058] (3) Extraction and separation of compounds 1-4

[0059] The fermented product obtained in step (2) was filtered to separate the fermented bacteria. After the filtrate was concentrated, the concentrated filtrate was extracted with an equal volume of ethyl acetate for three times; the extracts were combined, concentrated, and the crude extract was roughly separated by vacuum normal phase silica gel column chromatography. According to the weight of the crude extract, an appropriate amount of normal phase silica gel was weighed in a mortar, and the sample silica gel was ground and evaporated to a fine powder, and the final weight was 46.3g. The sample silica gel: chromatography silica gel = 1:3 was loaded into a vacuum glass column, and the elution system was petroleum ether: ethyl acetate and ethyl acetate: methanol. The gradient elution was performed with petroleum ether: ethyl acetate = 10:0, 7:3, 1:1, 7:3 and 0:10, and the volume ratio of ethyl acetate to methanol in the ethyl acetate-methanol solution was 10:0, 7:3, 1:1, 7:3 and 0:10, respectively. After thin layer chromatography (TLC) analysis, 8 components Fr.1 to Fr.8 were obtained by merging according to polarity. According to the HPLC analysis chart, UV absorption spectrum and sample properties, the compounds in Fr.4 component were separated and purified.

[0060] Fr.4 (5.33 g) was subjected to normal phase silica gel column chromatography using medium pressure preparative chromatography and eluted with a petroleum ether-ethyl acetate gradient. After TLC analysis, 8 components Fr.4A to Fr.4J were obtained. After HPLC gradient analysis, the Fr.4E component was selected and separated and purified by semi-preparative high performance liquid chromatography. The mobile phase was methanol-UP water (MeOH / H 2 O, 70:30, v / v) solution to obtain compound 1, compound 2, compound 3 and compound 4.

[0061] The structural formula of compound 1-4 is shown below:

[0062]

[0063] Compound 1-2 1 H and 13 C-NMR (400 / 100MHz, CD 3 The OD) data are shown in Table 1.

[0064] Table 1

[0065]

[0066]

[0067] Compound 1 is a colorless amorphous powder. According to the HRESIMS ion peak m / z 425.2674 [M+H] + (calcd.for425.2668), its molecular formula is C 25 H 38 O 4 , the unsaturation is 7. 1 Analysis of H NMR spectral data showed that compound 1 (Table 1) had two singlet methyl groups (δ H 0.94,0.77), 2 double peak methyl groups (δ H 0.85,0.77), 8 methylene groups (δ H 3.60, 3.40 / 1.67, 2.59, 2.10 / 1.27, 2.04 / 1.37, 2.02 / 1.95, 1.79 / 1.19, 0.69 / 0.46), 6 methines (δ H 3.58, 2.42, 1.83, 1.69, 1.50, 0.19). 1 A pair of characteristic upfield-shifted proton signals were observed in the H NMR spectrum (Table 1), located at δ H 0.69 (H-8a, dd, J = 8.4, 4.8 Hz) and δ H0.46 (H-8b, t, J = 4.8 Hz), indicating the presence of cyclopropane methylene. In addition, the 13C NMR and HSQC spectra of compound 1 (Table 1) showed 25 carbon signals, including 1 carbonyl carbon (δ C 169.7), 2 olefinic carbons (δ C 158.9, 129.7), 6 methyl carbons (δ C 78.7, 48.9, 47.2, 47.0, 30.3, 28.8), 8 methylene carbons (δ C 62.1, 43.8, 34.2, 33.3, 30.6, 29.5, 27.4, 27.1) and 4 methyl carbons (δ C Based on the observed characteristic UV spectra and NMR data, all of which are very similar to asperterpenoid A, we believe that compound 1 is a diterpenoid derivative.

[0068] The planar structure of compound 1 was determined by detailed analysis of COSY and HMBC spectra. Fig.13 As shown, the existence of three spin systems was confirmed by COSY correlations of H-4 / H-5 / H-6, H-8 / -9 / H-10 / H-15 / H-16 / -17 / H-18 and H-12 / H-13. The key HMBC interactions from H-10 to C-3 / C-6 / C-10 / C-12, H-4 to C-3 / C-2, H-6 to C-3 / C-4, H-10 to C-1 / C-12, H-20 to C-6 / C-8 / C-9, H-21 to C-1 / C-12 and H-22 to C-13 / C-15 / C-18 were then further analyzed. Fig.13 ), thus confirming the same A / B / C / D / E ring system as that of asperterpenoid A. In addition, from the HMBC correlation of H-13 to C-11 / C-15 / C-22, the hydroxyl group was attached to C-13. Therefore, the planar structure of compound 1 was elucidated.

[0069] The relative configuration of compound 1 was inferred from the NOESY spectrum. The correlation between H-9 / H-16 / H-20 and H-16 / H-21 / H-25 showed that H-9, H-16, H-20, H-21 and H-25 had the same orientation. By analyzing the NOESY correlation of H-6 / H-10 / H-24, H-13 / H-22 and H-24 / H-25, the orientations of H-6, H-10, H-13, H-22 and H-24 / H-25 were opposite to the above orientations. Therefore, the relative configuration of compound 1 was determined to be 16S*, while the other orientations (6S, 7R, 9R, 10S, 11R, 13S, 14R and 15S) were the same as those of the diterpenoid derivative asperterpenoid A.

[0070] The structure of the compound asperterpenoid A is shown below:

[0071]

[0072] From a biosynthetic perspective, compared with asperterpenoid A, the absolute configurations of 6S, 7R, 9R, 10S, 11R, 13S, 14R, and 15S are expected to be retained, except for 16S* in compound 1. To further prove the attribution of 16S*, we performed electronic circular dichroism (ECD) calculations on compound 1 dissolved in methanol at the B3LYP(GD3BJ) / 6-31G(dp) theoretical level and using the ωB97XD / def2-TZVP method. The calculated ECD spectra of (6S, 7R, 9R, 10S, 11R, 13S, 14R, 15S, 16S)-1(16S-1) and (6S, 7R, 9R, 10S, 11R, 13S, 14R, 15S, 16R)-1(16R-1)( Fig. 9 ) is consistent with the experimental CD curve. The results of electronic circular dichroism (ECD) spectroscopy indicate the relative configuration of compound 1. Quantum mechanical calculations of nuclear magnetic resonance (NMR) chemical shifts were performed for the 16R-1 and 16S-1 conformations of compound 1 at the B3LYP (GD3BJ) / 6-31G (d, p) theoretical level. At the same time, the chirality of C-16 in compound 1 was further determined by comparing the NMR data of the two isomers. 13The linear regression analysis of C NMR chemical shifts showed that the correlation coefficients (R2) of 16R-1 and 16S-1 were 0.98896 and 0.99178, respectively. In addition, the DP4 calculation and analysis method was used to compare the 13C NMR chemical shifts of 16R-1 and 16S-1 obtained by theoretical calculation with the experimental values ​​of compound 1. The results showed that the probability that compound 1 and 16S-1 are completely identical in structure is 100%. The absolute configuration of compound 1 was finally determined to be 6S, 7R, 9R, 10S, 11R, 13S, 14R, 15S, 16S, and it was named asperterpenoid D.

[0073] Compound 2 is a colorless amorphous powder. According to HRESIMS ion peak m / z 497.2519 [M+H] + (calcd.for497.2515), its molecular formula is C 27 H 38 O 7 , the unsaturation is 9. 1 H CMR and 13 A careful analysis of the C NMR data (Table 1) showed that the structure of compound 2 was very similar to that of compound 1, indicating the presence of the same diterpene skeleton. The key difference was the significant downfield shift (Δδ C +4.1), C-12(Δδ C -2.9) and C-14 (Δδ C -0.6) showed a high-field shift, which indicates that the hydroxyl group at the C-13 position of compound 1 is replaced by the oxalic acid group in compound 2 (δ C 170.1 and 170.6). This assignment was confirmed by the HMBC correlation between H-13 and C-27 observed in compound 2 ( Fig.13 ) was confirmed. Based on the similarity of the optical rotation data and NOESY correlation, the relative configuration of compound 2 was determined. In addition, the electronic circular dichroism (ECD) spectra ( Fig.10 ) and DP4plus methods were used to determine the absolute configuration of compound 2. Therefore, the configuration of compound 2 was determined to be 6S, 7R, 9R, 10S, 11R, 13S, 14R, 15S, 16S, and it was named asperterpenoid E.

[0074] Compound 3-4 1 H and 13 C-NMR (400 / 100MHz, CD 3 The OD) data are shown in Table 1.

[0075] Table 1

[0076]

[0077] Compound 3 is a colorless amorphous powder. According to the HRESIMS spectrum, the molecular formula of the compound is C 28 H 40 O 7 , showing 9 degrees of unsaturation. 1 H NMR and 13 Detailed analysis of C NMR data showed (Table 2) that compound 3 and compound 2 have the same carbon skeleton. The main difference is that there is a methoxy group at the C-19 position of compound 3, which is consistent with the difference of 15 atomic mass units in relative molecular mass. C 168.6) and C-26 (δ C The above results are confirmed by the chemical shift values ​​of H-26 (δ H 3.72) and C-19 (δ C 168.6) Fig.13 ) was confirmed. Therefore, the planar structure of compound 3 was elucidated, and its relative configuration was determined by NOESY correlation spectrum. In addition, by electronic circular dichroism ( Fig.11 )and 13 The C NMR calculation method determined that the absolute configuration of compound 3 was 6S, 7R, 9R, 10S, 11R, 13S, 14R, 15S, 16S. Finally, the structure of compound 3 was determined and named asperterpenoid F.

[0078] Compound 4 is also a white amorphous powder. Its molecular formula was determined to be C by HRESIMS spectrum. 28 H 40 O 6 , showing 9 degrees of unsaturation. 1 H and 13 The C NMR spectrum (Table 2) is similar to that of compound 3, except that compound 4 lacks a hydroxyl group at C-21, which makes its relative molecular mass 16 atomic mass units less than that of compound 3. The HMBC correlation spectrum between H-21 and C-1, C-11 and C-12 verifies the above results, and the results are further confirmed by NOESY correlation spectrum and HRESIMS fragmentation. According to the electronic circular dichroism spectrum ( Fig.12 )and 13 C NMR calculation method was used to determine the absolute configuration of compound 4 as 6S, 7R, 9R, 10S, 11R, 13S, 14R, 15S, and 16S. Finally, the structure of compound 4 was elucidated and named asperterpenoid G.

[0079] Example 2

[0080] (1) Cultivation of Aspergillus sp.

[0081] Prepare seed culture medium (2.0 L): 1.5% (weight percentage, the same below) glucose, 0.5% yeast extract, 0.1% peptone, 0.11% coarse sea salt, and the rest water; divide evenly into 10 500 mL conical flasks and sterilize at 120-140°C for 25-30 minutes.

[0082] The Aspergillus sp. strain was inoculated into the prepared seed culture medium, and cultured at 26-28°C for 4 days to obtain a seed culture solution;

[0083] (2) Fermentation of Aspergillus sp.

[0084] Prepare fermentation medium (10 kg): 100 g rice, 0.11% coarse sea salt, 100 mL water; divide evenly into 100 1000 mL conical flasks and sterilize at 120-140°C for 25-30 minutes.

[0085] An appropriate amount of the seed culture solution obtained in step (1) is inoculated into a conical flask containing a fermentation medium and cultured at 26-28° C. for 30 days.

[0086] (3) Extraction and separation of compounds 1-4

[0087] According to the separation method similar to that in Example 1, compound 1-4 can be obtained, and the structural confirmation data are consistent with that in Example 1.

[0088] Example 3

[0089] (1) Cultivation of Aspergillus sp.

[0090] Prepare seed culture medium (2.0 L): 3.0% glucose (weight percentage, the same below), 0.1% yeast extract, 0.5% peptone, 0.6% coarse sea salt, and the rest is water; divide evenly into 10 500 mL conical flasks (200 mL culture medium each), and sterilize at 120-140° C. for 25-30 minutes.

[0091] The Aspergillus sp. strain was inoculated into the prepared seed culture medium, and cultured at 26-28°C for 4 days to obtain a seed culture solution;

[0092] (2) Fermentation of Aspergillus sp.

[0093] Prepare fermentation medium (30 L): 3.5% (weight percentage, the same below) of glucose, 0.1% of yeast extract, 0.5% of peptone, 0.6% of coarse sea salt, and the rest of water; divide evenly into 100 1000 mL conical flasks (300 mL of medium in each), and sterilize at 120-140° C. for 25-30 minutes.

[0094] An appropriate amount of the seed culture solution obtained in step (1) is inoculated into a conical flask containing a fermentation medium and cultured at 26-28° C. for 28 days.

[0095] (3) Extraction and separation of compounds 1-4

[0096] According to the separation method similar to that in Example 1, compound 1-4 can be obtained, and the structural confirmation data are consistent with that in Example 1.

[0097] Using similar culture, fermentation and extraction and separation methods, GuangPing Cao, GuoQiang Huang, GuangYing Chen, XiangXi Yi, XueSheng Wang, FengJiao Wei, CaiQiong Zhu, ChengHaiGao, YongHong Liu, Meng Bai. Millmerranones G, a meroterpene isolated from amangrove-derived fungus Aspergillus sp. GXIMD 03004, Natural Product Research, DOI: 10.1080 / 14786419.2024.2402460. The Aspergillus sp. GXIMD 03004 disclosed in the paper can isolate compounds 1-4.

[0098] Example 4

[0099] Determination of the inhibitory activity of compounds 1-4 of the present invention on benign prostatic hyperplasia

[0100] Test method: Compounds 1-4 were used to treat RWPE-1 human prostate epithelial cell line.

[0101] The specific method is as follows:

[0102] (1) CCK-8 assay for cell proliferation

[0103] ① Digestion, centrifugation and counting of cells

[0104] Take the logarithmically growing RWPE-1 cells, aspirate the culture medium, add 0.25% trypsin to digest, stop digestion with complete medium and collect the cells, centrifuge for 5 minutes at 1500rpm. Then add new complete medium to resuspend the cells, mix well and count them with a hemocytometer. Adjust the cell concentration to 5×10 3 The cells were seeded in a 96-well plate at 100 μL / well and placed at 37°C with 5% CO. 2 incubator.

[0105] ②Cellular administration and detection

[0106] After incubation for 24 h, different concentrations of compounds were added, and a control group was set up for each concentration. The changes in cell morphology were observed after culturing for 48 h. After adding 10 μL of CCK-8 solution to each well, the cells were incubated at 37°C, 5% CO 2 Incubate in the incubator for 2-4 hours. After the cells are incubated, measure the absorbance (OD value) at 450nm using an enzyme reader and calculate the IC 50 value.

[0107] (2) Cell cycle assay

[0108] ①Cell culture and drug administration

[0109] Take logarithmically growing RWPE-1 cells, digest them, centrifuge them for 5 min at 1500 rpm, remove the supernatant, add new complete medium to resuspend, and inoculate them in 6-well plates at 37°C and 5% CO. 2 After incubation in the incubator for 24 h, different concentrations of compounds were added, and the cells were cultured in the incubator for another 48 h.

[0110] Discard the original drug-containing culture medium, wash the cells with PBS, aspirate the PBS, add EDTA-free trypsin to digest the cells. When the cells become round, add new complete culture medium to terminate digestion, transfer them to a 1.5mL centrifuge tube, centrifuge for 5 minutes at 1500rpm, and discard the supernatant.

[0111] Add 1 mL of pre-cooled PBS to resuspend the cells, centrifuge for 5 min at 1500 rpm, and discard the supernatant. Add 500 μL of pre-cooled PBS to resuspend the cells, and add 1.5 mL of pre-cooled anhydrous ethanol dropwise, shaking gently while adding, to a final concentration of 75%. Place in a refrigerator at -20°C to fix overnight.

[0112] ②Cell staining and detection

[0113] Centrifuge for 5 min at 1500 rpm and discard the supernatant. Resuspend the washed cells with 1 mL PBS, let stand for 15 min, centrifuge for 5 min at 1500 rpm and discard the supernatant. Resuspend the cells with 500 μL PBS, add RNase A to a concentration of 20 μg / mL, incubate in an incubator at 37°C for 30 min, centrifuge for 5 min at 1500 rpm and discard the supernatant. Resuspend the cells with 500 μL PBS, add PI dye to a concentration of 50 μg / mL, and incubate in a 4°C refrigerator away from light for 30 min. Detect the cell cycle using a flow cytometer.

[0114] The results of the inhibitory activity of compounds 1-4 on benign prostatic hyperplasia showed that compound 3 had a certain inhibitory activity on RWPE-1 human prostate epithelial cells, and its IC 50 The value is 3.68μM.

[0115] Example 5

[0116] Determination of antibacterial activity of compounds 1-4 of the present invention

[0117] Test method: Compounds 1-4 were used to test the effects of compounds 1-4 on methicillin-resistant Staphylococcus aureus ATCC 43300, Candida albicans ATCC 10231, Staphylococcus albus ATCC 8799, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 21332, Xanthomonas campestris ATCC 33913, Vibrio parahaemolyticus ATCC 17802, Vibrio alginolyticus ATCC 17749, and Vibrio harveyi ATCC 14126.

[0118] The specific method is as follows:

[0119] (1) Microdilution method to determine the minimum inhibitory concentration (MIC) of the compound against indicator bacteria

[0120] ① Preparation of test drug stock solution and positive drug stock solution: Dissolve the test monomer compound in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mg / mL; dissolve the positive drug chloramphenicol in DMSO to prepare a stock solution with a concentration of 1 mg / mL. Store in a 4°C refrigerator for later use.

[0121] ② Preparation of well plate and inoculation culture: In the clean bench, pour the sterilized LB culture medium into the sterile sample chamber with a volume of 50mL, use a pipette to draw 50μL of bacterial solution from the sterile tube, mix it into the sample chamber, and draw it evenly (the bacterial solution is diluted 1000 times). Use a pipette to add 190μL of the diluted bacterial solution to the first row of wells on the 96-well plate, and then add 100μL of the diluted bacterial solution in equal amounts from top to bottom. Add 1 mg / mL of positive control and 10 μL of the sample to be tested to each well in the first row of wells, and use a pistol to mix them evenly (at this time, the concentrations of the samples and positive controls in the first row of wells are both (100 μg / mL), then use a pistol to draw 100 μL of the mixed solution to the second row, mix them evenly, and then draw 100 μL of the mixed solution to the third row. According to the above steps, operate from top to bottom, and the mixed solution sucked out from the last row of wells is pumped into the waste liquid tank, and then use a washing liquid gun to draw 100 μL of the diluted bacterial solution into each well, so that the final concentrations of the samples in the first to eighth rows are 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, respectively. mL, 3.12μg / mL, 1.56μg / mL, 0.78μg / mL, 0.39μg / mL, 0.19μg / mL. Seal the treated 96-well plate tightly with sealing film and place it in a bacterial constant temperature incubator. Set the temperature to 37°C and culture for 6 hours. Observe once every 2 hours. When the negative control group is full of bacteria, start recording the activity results. Observe each column from bottom to top. When a certain hole remains clear and not turbid, the row number of this hole is the corresponding sample concentration, which is also the minimum inhibitory concentration of the compound. At the same time, record the minimum inhibitory concentration of the positive control. After the observation, sterilize the 96-well plate.

[0122] The antibacterial activity test results of compounds 1-4 showed that compound 1 had certain antibacterial activity against Vibrio harveyi and Xanthomonas campestris, and the MIC values ​​were 3.12 and 6.25 μg / mL, respectively.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A diterpenoid compound, characterized in that: It has the structure shown in compound 1-4:

2. The method for preparing a diterpenoid compound according to claim 1, characterized in that: The following steps are involved: Step 1, preparing a strain culture medium, inoculating the Aspergillus sp. strain into a seed culture medium, and culturing the strain to obtain a seed culture solution; Step 2, inoculating the seed culture solution obtained in step 1 into a rice solid fermentation medium to obtain a fermentation product; Step 3, extracting the fermented product obtained in step 2 with an equal volume of ethyl acetate, combining the extracts and concentrating under reduced pressure to obtain an extract; Step 4, the extract obtained in step 3 is roughly separated by vacuum normal phase silica gel column chromatography, and gradient eluted with petroleum ether-ethyl acetate solution and ethyl acetate-methanol solution respectively, and analyzed by TLC, and 8 components Fr.1 to Fr.8 are obtained by merging according to polarity; Step 5, separation and purification of the compound of Fr.4 component: Fr.4 was subjected to normal phase silica gel column chromatography using medium pressure preparative chromatography and eluted with a petroleum ether-ethyl acetate gradient. After TLC analysis, 8 components Fr.4A to Fr.4J were obtained. After HPLC gradient analysis, the Fr.4E component was selected and separated and purified by semi-preparative high performance liquid chromatography with methanol-UP aqueous solution as the mobile phase to obtain compounds 1, compound 2, compound 3 and compound 4.

3. The preparation method according to claim 2, characterized in that: In step 1, the seed culture medium includes 1.5wt%-3.0wt% of glucose, 0.1wt%-0.5wt% of yeast extract, 0.1wt%-0.5wt% of peptone, 0.11wt%-0.6wt% of coarse sea salt, and the balance is water. After the seed culture medium is prepared, it is sterilized at 120-140° C. for 25-30 minutes; In step 1, the culture temperature is 26-28° C. and the culture time is 3-4 days.

4. The preparation method according to claim 2, characterized in that: In step 2, the fermentation medium comprises rice, coarse sea salt and water, and the mass ratio of the rice, coarse sea salt and water is 100:(0.11-0.6):100; or, the fermentation medium comprises 1.5wt%-3.0wt% of glucose, 0.1wt%-0.5wt% of yeast extract, 0.1wt%-0.5wt% of peptone, 0.11wt%-0.6wt% of coarse sea salt, and the balance is water; In step 2, the fermentation medium is prepared and sterilized at 120-140° C. for 25-30 minutes; In the step 2, the culture temperature is 26-28° C., the culture is statically cultured, and the culture time is 28-30 days.

5. The preparation method according to claim 2, characterized in that: In step 4, during gradient elution, the volume ratios of petroleum ether and ethyl acetate in the petroleum ether-ethyl acetate solution are 10:0, 7:3, 1:1, 7:3 and 0:10, respectively, and the volume ratios of ethyl acetate and methanol in the ethyl acetate-methanol solution are 10:0, 7:3, 1:1, 7:3 and 0:10, respectively; In step 5, in the petroleum ether-ethyl acetate solution, the volume ratio of petroleum ether to ethyl acetate is 5:1, 2:1, 1:1, 1:2, 1:3 and 0:1; In the step 5, the volume ratio of methanol to UP water in the methanol-UP aqueous solution is 70:

30. Preferably, the flow rate of the methanol-UP aqueous solution is 2.0 mL / min.

6. Use of compound 3 among the sesquiterpenoid compounds as claimed in claim 1 in the preparation of a drug for inhibiting benign prostatic hyperplasia.

7. Use of compound 1 among the sesquiterpenoid compounds as claimed in claim 1 in the preparation of antibacterial drugs.

8. A drug for inhibiting benign prostatic hyperplasia, characterized in that: It comprises compound 3 among the sesquiterpene compounds as claimed in claim 1 or a pharmaceutically acceptable salt thereof and excipients.

9. An antibacterial drug, characterized in that: It comprises compound 1 among the sesquiterpenoid compounds as claimed in claim 1 or a pharmaceutically acceptable salt thereof and excipients.

10. The drug according to claim 8 or 9, characterized in that The auxiliary material is a pharmaceutically acceptable carrier, diluent or excipient, and the dosage form of the drug is a solid preparation, a semi-solid preparation or a liquid preparation.

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