Terpenoids Derived from Fungi, Their Preparation Methods and Applications

Abstract

The present invention discloses terpene compounds derived from polar fungi, their preparation methods and applications, and respectively provides a calamenane-type sesquiterpene compound eutyacorane and a pimarane-type diterpene compound libertellenone Z derived from polar fungi. The structures are shown in the following formulas I and II respectively: The experimental results show that the two compounds have a better inhibition rate on the release of NO in the LPS-induced inflammatory model of mouse macrophages RAW264.7, providing a new type of choice for anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine organisms and medicine, and relates to terpene compounds derived from polar fungi, their preparation methods and applications. Specifically, it relates to a calamenane-type sesquiterpene compound eutyacorane and a pimarane-type diterpene compound libertellenone Z isolated from the secondary metabolites of polar-derived Curvularia fungi, as well as their applications in the preparation of anti-inflammatory drugs. Technical Background

[0002] Due to the extreme living environment of polar fungi, they can produce numerous natural active products with novel structures. Eutypella sp. D-1 is a polar fungus that mainly produces terpene compounds. In previous studies, the secondary metabolites of Eutypella sp. D-1 were found to have good biological activities. Experiments showed that most of the compounds had good anti-tumor cytotoxic activities, especially terpene compounds. Among them, the pimarane-type diterpene compound libertellenone H showed varying degrees of cytotoxic activities against pancreatic cancer cell line SW-1990, gastric cancer cell line SG7901, cervical cancer cell line HeLa, and glioma cell line U251, etc., and the IC 50 value ranged from 3 - 50 μM [1] ; libertellenone O-Q showed significant cytotoxic activities against HeLa, MCF-7, HCT-116, K562, and SW1990 tumor cell lines, and the IC 50 value ranged from 0.26 - 6.02 μM [2] .

[0003] Most of the compounds isolated from Eutypella sp. D-1 are diterpene compounds, and sesquiterpene compounds are relatively few. This type of compound shows significant antibacterial activities. For example, the eudesmane-type sesquiterpene compound eut-Guaianesesquiterpene showed significant antibacterial activities against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus strains [3] . Thus, it can be seen that the terpene secondary metabolites produced by Eutypella sp. D-1 are important sources of a series of drug lead compounds such as anti-tumor and antibacterial agents. In previous studies, calamenane-type sesquiterpene compounds were not found in Eutypella sp. D-1; at the same time, due to the good activities of the libertellenone series compounds, it is also of great significance to continue to explore this series of compounds.

[0004] In recent years, due to their novel structures, diverse activities, and low side effects, natural products have also become a new source for the development of anti-inflammatory drugs. So far, the anti-inflammatory activity data of calamenane-type sesquiterpenoids and libertellenone-type compounds are shown in Table 1 below. [4] The activity was measured by determining the inhibition rate of NO release at a drug concentration of 10 μM in an LPS-induced RAW264.7 macrophage inflammation model in mice, with dexamethasone as the positive control.

[0005] Table 1 Anti-inflammatory activity data of compounds

[0006]

[0007] The structures of the compounds in the above table are as follows:

[0008] Summary of the Invention

[0009] Based on the above research, the present invention continues to explore the secondary metabolites of Eutypella sp. D-1 and provides two novel terpene structures: a calamenane-type sesquiterpenoid compound and a pimarane-type diterpenoid compound libertellenone Z.

[0010] The first object of the present invention is to provide the structures of the above two compounds; the second object is to provide two compounds isolated by a fermentation method; the third object is to provide the use of the two compounds in the preparation of anti-inflammatory drugs.

[0011] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0012] In the first aspect of the present invention, a calamenane-type sesquiterpenoid compound eutyacorane derived from a polar fungus is provided, and its chemical structure is shown in Formula I below:

[0013]

[0014] In the second aspect of the present invention, a pimarane-type diterpenoid compound libertellenone Z derived from a polar fungus is provided, and its chemical structure is shown in Formula II below:

[0015]

[0016]

[0017] The polar fungus is Eutypella sp.D-1 isolated from the root soil of Arctic dryas, and was deposited in China Center for Type Culture Collection (CCTCC) on April 12, 2013, located at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO:M 2013144.

[0018] The third aspect of the present invention provides a method for preparing the above-mentioned acorane-type sesquiterpene compound eutyacorane and pinmarane-type diterpene compound libertellenone Z, comprising the following steps:

[0019] a) Strain fermentation

[0020] Use a sterile inoculation loop to pick up an appropriate amount of Eutypella sp.D-1 mycelium in a 50 mL volume of sterile PDB liquid culture medium (24 g / L), seal it with a sealing film, and place it in a shaker at 28°C and 180 rpm for 3 days to obtain seed liquid for use. Add the seed liquid to 500 mL of PDB culture medium at a ratio of 5% (V / V) of the inoculation amount, seal it with a sealing film, and place it in a shaker at 24°C and 180 rpm for 9 days.

[0021] b) Fermentation product collection

[0022] The fermentation liquid was filtered through 8 layers of gauze, the filtrate was fully mixed with an equal volume of ethyl acetate, and the ethyl acetate layer was collected after stratification, and this process was repeated three times; the ethyl acetate was evaporated to dryness using a rotary evaporator; at the same time, the filtered mycelium was immersed in a mixed solvent of dichloromethane and methanol (1:1), and ultrasonically crushed for 30 minutes, and this process was repeated three times; the mixed liquid was filtered through 8 layers of gauze, the solvent was evaporated to dryness using a rotary evaporator, and the fermentation products were combined to obtain the total fermentation product of Eutypella sp.D-1.

[0023] c) Separation and purification of fermentation products

[0024] Firstly, the total fermentation product was eluted by MPLC using methanol and water as mobile phases, and the eluted compounds were analyzed by TLC; the normal phase silica gel chromatography column was eluted using petroleum ether and ethyl acetate as mobile phases, and the eluted compounds were then purified by HPLC. The preparation conditions of the acorane-type sesquiterpene compound eutyacorane were 60% methanol / water, flow rate 2mL / min, and retention time 52min; the preparation conditions of the pinmarane-type diterpene compound libertellenone Z were 75% methanol / water, flow rate 2mL / min, and retention time 32min.

[0025] In the fourth aspect of the present invention, there is provided the use of the above-mentioned calamenane sesquiterpene compound eutyacorane and pimarane diterpene compound libertellenone Z in the preparation of anti-inflammatory drugs.

[0026] The experimental results show that in the LPS-induced inflammation model of mouse macrophages RAW264.7, the calamenane sesquiterpene compound eutyacorane and the pimarane diterpene compound libertellenone Z exhibit significant inhibitory activities on NO release. Taking the model group as a control, the inhibition rates on NO release are 65% and 60% respectively, only slightly lower than the inhibition rate of 72% of the positive drug dexamethasone, and they can be used as a supplement to current anti-inflammatory drugs for the preparation of anti-inflammatory drugs.

[0027] The beneficial technical effects of the present invention are as follows:

[0028] The present invention continues to explore the secondary metabolites of Eutypella sp. D-1, and provides two novel terpene structures: the calamenane sesquiterpene compound and the pimarane diterpene compound libertellenone Z. The experimental results show that the two compounds have better inhibition rates on the release of NO in the LPS-induced inflammation model of mouse macrophages RAW264.7, providing new types of choices for anti-inflammatory drugs. Detailed implementation manners

[0029] The following further elaborates the present invention through examples, aiming to better understand the content of the present invention. The examples given do not limit the protection scope of the present invention.

[0030] Example 1: Strain fermentation and product collection

[0031] (1) Strain fermentation

[0032] Use a sterile inoculation loop to pick an appropriate amount of Eutypella sp. D-1 mycelium into a 50 mL volume of sterile PDB liquid medium (24 g / L), seal it with a sealing film, and place it in a shaker for culturing at 28 °C and 180 rpm for 3 d to obtain a seed solution for standby. Add the seed solution to the PDB medium with a volume of 500 mL at a ratio of 5% (V / V), seal it with a sealing film, and place it in a shaker for culturing at 24 °C and 180 rpm for 9 d.

[0033] (2) Fermentation product collection

[0034] The fermentation broth was filtered using eight layers of gauze. The filtrate was thoroughly mixed with an equal volume of ethyl acetate. After phase separation, the ethyl acetate layer was collected, and this process was repeated three times. The ethyl acetate was evaporated to dryness using a rotary evaporator. Meanwhile, the filtered mycelium was immersed in a mixed solvent of dichloromethane and methanol (1:1), and ultrasonicated for 30 min, and this process was repeated three times. The mixture was filtered using eight layers of gauze, and the solvent was evaporated to dryness using a rotary evaporator. The fermentation products were combined to obtain the total fermentation products of Eutypella sp. D-1.

[0035] Example 2: Isolation and purification of the calamenane sesquiterpene compound eutyacorane

[0036] First, the total fermentation products were eluted by MPLC using a mobile phase of methanol and water, and the eluted compounds were analyzed by TLC. The components were eluted from a normal-phase silica gel column using a mobile phase of petroleum ether and ethyl acetate, and then the eluted compounds were purified by HPLC under the conditions of 60% methanol / water, a flow rate of 2 mL / min, and a retention time of 52 min. Finally, a new calamenane sesquiterpene compound eutyacorane was obtained, and its structure is shown below:

[0037]

[0038] eutyacorane, a colorless oil, with the molecular formula C 15 H 26 O 2 , a molecular weight of 238; high-resolution mass spectrometry 261.18

[0039] [M+Na] + , and the structural core of eutyacorane belongs to the calamenane sesquiterpene. 1 H and 13 C nuclear magnetic resonance spectrum data are shown in Table 2:

[0040] Table 2 Nuclear magnetic resonance data of eutyacorane

[0041]

[0042] Example 3: Isolation and purification of the pimarane diterpene compound libertellenone Z

[0043] First, the total fermentation products were eluted by MPLC using the mobile phase of methanol and water, and the eluted compounds were analyzed by TLC. The components were eluted by normal-phase silica gel column chromatography using the mobile phase of petroleum ether and ethyl acetate, and then the eluted compounds were purified by HPLC under the conditions of 75% methanol / water, a flow rate of 2 mL / min, and a retention time of 32 min. Finally, a new pimarane-type diterpenoid compound, libertellenone Z, was obtained.

[0044] libertellenone Z, a yellow oil, with the molecular formula C 20 H 28 O 4 , a molecular weight of 332; high-resolution mass spectrometry 355.18 [M+Na] + , and the structural core of libertellenone Z belongs to the pimarane-type diterpenoid. 1 The H and 13 C nuclear magnetic resonance spectrum data are shown in Table 3.

[0045]

[0046] Table 3 Nuclear magnetic resonance data of libertellenone Z

[0047]

[0048]

[0049] Example 4: Anti-inflammatory activity experiment of the compound

[0050] Mouse macrophages RAW264.7 in the logarithmic growth phase were diluted with DMEM high-glucose medium containing 10% serum to a concentration of 1 - 2×10 5 cells / mL and inoculated into a 96-well plate at 100 μL per well, and cultured in a cell incubator for 24 h. The test compound was dissolved in DMSO and formulated into a 10 μM solution using DMEM medium. LPS (TLR4 activator) was added to the above-mentioned concentration solution to make the final concentration of LPS 1 μg / mL, and positive control dexamethasone group, model group, and blank control group were set. The original medium in the 96-well plate was aspirated, and 100 μL of the prepared sample solution was added, and cultured in a cell incubator for 24 h. 50 μL of the supernatant in the 96-well plate was aspirated, and according to the method described in the NO assay kit instruction manual, the absorbance of the supernatant at 540 nm was measured using an enzyme-linked immunosorbent assay reader. The calculation formula for the inhibition rate of NO release is: Inhibition rate = (NO release in the model group - NO release in the compound group) / NO release in the model group × 100%.

[0051] The results showed that under the LPS-induced inflammatory model of mouse macrophages RAW264.7, the calamenane sesquiterpene compound eutyacorane and the pimarane diterpene compound libertellenone Z showed significant inhibitory activities on NO release. Taking the model group as the control, the inhibition rates on NO release were 65% and 60% respectively, only slightly lower than the inhibition rate of 72% of the positive drug dexamethasone, and they can be used to prepare anti-inflammatory drugs.

[0052] The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Although the present invention has been disclosed in the above embodiments, it does not limit the scope of use of the present invention. Any person skilled in the art of this patent can make some changes or modifications by using the technical content prompted above within the scope of the technical solution of the present invention, and it should be regarded as equivalent embodiments. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the present invention.

Claims

1. A eutyacorane-type sesquiterpene compound derived from polar fungi, It is characterized in that Its chemical structure is shown in the following formula I:

2. A pinmarane-type diterpene compound libertellenone Z derived from polar fungi, It is characterized in that Its chemical structure is shown in the following formula II:

3. The compound according to claim 1 or 2, characterized in that: in, The polar fungus is Eutypellasp.D-1 of the genus Curvularia isolated from the soil at the base of the Arctic dryas, and the preservation number is CCTCC NO:M 2013144.

4. A method for preparing the compound according to claim 1 or 2, It is characterized in that The steps include: a) Strain fermentation Use a sterile inoculation loop to pick up an appropriate amount of Eutypella sp.D-1 mycelium in a 50 mL volume, 24 g / L sterile PDB liquid culture medium, seal it with a sealing film, and place it in a shaker at 28°C and 180 rpm for 3 days to obtain seed liquid for use; add the seed liquid to 500 mL of PDB culture medium at a ratio of 5% of the inoculation amount, seal it with a sealing film, and place it in a shaker at 24°C and 180 rpm for 9 days; b) Fermentation product collection The fermentation liquid was filtered through 8 layers of gauze, and the filtrate was fully mixed with an equal volume of ethyl acetate. After stratification, the ethyl acetate layer was collected, and this process was repeated three times, and the ethyl acetate was evaporated to dryness using a rotary evaporator; at the same time, the filtered mycelium was immersed in a mixed solvent of dichloromethane and methanol with a volume ratio of 1:1, and ultrasonically crushed for 30 minutes. After the process was repeated three times, the mixed solution was filtered through 8 layers of gauze, and the solvent was evaporated to dryness using a rotary evaporator; the fermentation products were combined to obtain the total fermentation product of Eutypella sp.D-1; c) Separation and purification of fermentation products Firstly, the total fermentation product was eluted by MPLC using methanol and water as mobile phases, and the eluted compounds were analyzed by TLC; the normal phase silica gel chromatography column was eluted using petroleum ether and ethyl acetate as mobile phases, and the eluted compounds were then purified by HPLC. The preparation conditions of the acorane-type sesquiterpene compound eutyacorane were 60% methanol / water, flow rate 2mL / min, and retention time 52min; the preparation conditions of the pinmarane-type diterpene compound libertellenone Z were 75% methanol / water, flow rate 2mL / min, and retention time 32min.

5. Use of the acorane-type sesquiterpene compound eutyacorane according to claim 1 or the pimarane-type diterpene compound libertellenone Z according to claim 2 in the preparation of anti-inflammatory drugs.

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