A chloropine diterpenoid compound and application thereof in preparation of anti-ultraviolet eye disease drugs

Abstract

The application discloses a chlorinated pimarane diterpene compound or a medicinal salt thereof, and a chemical structure of the chlorinated pimarane diterpene compound is shown in formula (I). The application provides a new lead compound for researching and developing a new anti-ultraviolet eye disease medicine, and provides a scientific basis for developing and utilizing polar marine medicinal resources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of marine biology and medicine, and particularly relates to a chlorinated pimarane diterpene compound obtained from the mixed fermentation product of two strains of Arctic fungi, Eutypella sp. D-1 and Nectria sp. B-13, through solid medium fermentation, extraction, separation and purification, and the application of the chlorinated pimarane diterpene compound in the preparation of anti-ultraviolet eye disease drugs. BACKGROUND

[0002] Ultraviolet B (UV-B) radiation is the main pathogenic factor of ultraviolet eye disease, and UV-B can induce the occurrence of corneal inflammation, conjunctivitis, cataract, pterygium, macular lesions and other eye diseases by promoting DNA oxidation and apoptosis of human corneal epithelial cells, so finding an effective ultraviolet eye disease treatment drug is an important medical topic. Fungal secondary metabolites have many types, novel structures and strong activities, and are an important source of clinical small molecule drugs. In recent years, with the development of polar exploration, the research on the material basis for adapting to harsh climate and medicinal prospects of polar fungi has attracted widespread attention. The drug source material Eutypella sp. D-1 is isolated from the Arctic and belongs to Ascomycetes, Xylariales, Diatrypaceae, Eutypella, and the literature reports that the secondary metabolites of the genus mainly include sesquiterpenes, diterpenes, steroids, benzopyran derivatives, lactones and cytochalasins, and some compounds show biological activities related to anti-ultraviolet eye disease, such as antioxidant and anti-inflammatory activities; another drug source material Nectria sp. B-13 is also isolated from the Arctic and belongs to Ascomycota, Hypocreales, Nectriaceae and Nectria, and the secondary metabolites isolated from the genus of fungi mainly include chlorinated phenolic sesquiterpenes, monophenols, polyketides, sterols and alkaloids, and some compounds show biological activities such as antibacterial, anti-inflammatory and antioxidant activities. For example, the pimarane diterpene compound eutypellenone A isolated from the fungus of the genus Eutypella has very good anti-inflammatory and antioxidant activities related to the treatment of ultraviolet eye disease (see document: YU H-B, WANG X-L, ZHANG Y-X, et al. Libertellenones O–S and Eutypellenones A and B, Pimarane Diterpene Derivatives from the Arctic Fungus Eutypella sp. D-1 [J]. Journal of Natural Products, 2018, 81(7): 1553-60.).

[0003] So far, there is no report on chlorinated pimarane diterpenoid compounds with UV eye disease treatment effect isolated from the mixed fermentation products of two strains of Arctic fungi D-1 of Eutypella sp. and B-13 of Nectria sp. SUMMARY

[0004] The purpose of the present application is to provide a new chlorinated pimarane diterpenoid compound extracted, isolated and purified from the mixed fermentation products of two strains of Arctic fungi D-1 of Eutypella sp. and B-13 of Nectria sp.

[0005] Another purpose of the present application is to provide an extraction method of the chlorinated pimarane diterpenoid compound.

[0006] Still another purpose of the present application is to provide an application of the chlorinated pimarane diterpenoid compound in preparing anti-UV eye disease drugs.

[0007] In order to achieve the above purposes, the technical solutions adopted by the present application are as follows:

[0008] In the first aspect of the present application, a chlorinated pimarane diterpenoid compound or a pharmaceutically acceptable salt thereof is provided, and the chemical structure thereof is shown as formula (I):

[0009]

[0010] The chlorinated pimarane diterpenoid compound is extracted, isolated and purified from the mixed fermentation products of two strains of Arctic fungi D-1 of Eutypella sp. and B-13 of Nectria sp.

[0011] In the second aspect of the present application, an extraction method of the chlorinated pimarane diterpenoid compound is provided, comprising the following steps:

[0012] Step 1, preparation of total extract:

[0013] The mycelium of Nectria sp. B-13 is picked from the preservation plate, inoculated into a shake flask containing PDB medium, and cultured under the condition of a temperature of 25-30℃ (preferably 28℃) for 3-7 days (preferably 5 days) to obtain a first-stage seed liquid, and the first-stage seed liquid is transferred into a shake flask containing a seed culture medium, and cultured under the same condition for 1-3 days (preferably 2 days) to obtain a second-stage seed liquid;

[0014] The second-stage seed liquid of Eutypella sp. D-1 with the preservation number of CCTCC NO: M 2013144 is obtained by using the same method as above;

[0015] Transfer the secondary seed solution of Nectria sp. B-13 to a shake flask containing fermentation medium and expand the culture. Shake culture at 25-30°C (preferably 28°C) for 1-2 days (preferably 1 day). Transfer the secondary seed solution of Eutypella sp. D-1 to the above shake flask and mix and ferment under the same conditions for 10 days.

[0016] The fermentation broth obtained by the above culture was filtered to obtain bacterial cells and bacterial liquid respectively; the bacterial liquid was extracted with an equal volume of ethyl acetate at least three times, and the extracts were combined and evaporated to dryness to obtain a first extract; the bacterial cells were ultrasonically extracted with dichloromethane and methanol in a volume ratio of 1:1 at least three times, the extracts were concentrated until free of organic solvent, suspended in water, extracted with an equal volume of ethyl acetate at least three times, and the extracts were combined and evaporated to dryness to obtain a second extract; the first extract and the second extract were combined to obtain a total extract;

[0017] The second step is separation and purification:

[0018] ① The total extract was subjected to vacuum liquid column chromatography (VLC) with petroleum ether:ethyl acetate (100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:1, 0:1, v / v) as the solvent for gradient elution. Similar fractions were combined according to TLC thin layer chromatography to obtain 15 components Fr.AO;

[0019] ② Component Fr.K was subjected to normal phase silica gel column chromatography with a gradient elution using petroleum ether:ethyl acetate (25:1, 15:1, 10:1, 5:1, 3:1, 2:1, MeOH, v / v). Similar fractions were combined based on TLC color analysis to obtain five components Fr.K1-K5;

[0020] ③ Purify the component Fr.K4 by reverse-phase high performance liquid chromatography to obtain the compound represented by formula (I).

[0021] The PDB culture medium formula is: 10 g potato extract powder, 20 g glucose, and 1000 mL distilled water.

[0022] The seed culture medium formula is: 125 g / L glucose, 3.3 g / L sodium nitrate, 0.07 g / L potassium hydrogen phosphate trihydrate, 0.4 g / L magnesium sulfate heptahydrate, 0.625 g / L potassium chloride, 0.7 g / L yeast extract, 3.125 mg / L cobalt chloride hexahydrate, 18.75 mg / L ferrous sulfate, 6.5 g / L anhydrous calcium chloride, and 15 g / L L-ornithine hydrochloride.

[0023] The fermentation medium formula is: 51.4 g / L sucrose, 3.3 g / L sodium nitrate, 2.5 g / L urea, 0.7 g / L yeast extract, 0.07 g / L dipotassium hydrogen phosphate trihydrate, 0.4 g / L magnesium sulfate heptahydrate, 0.625 g / L potassium chloride, 18.75 mg / L ferrous sulfate heptahydrate, 6.5 g / L anhydrous calcium chloride, and 3.125 mg / L cobalt chloride hexahydrate.

[0024] The Nectria sp. B-13 secondary seed solution was transferred to a shake flask containing a fermentation medium, and the Nectria sp. B-13 secondary seed solution was inoculated at an inoculum volume of 5% v / v.

[0025] The reverse-phase high-performance liquid chromatography purification conditions for component Fr.K4 are as follows: separation and preparation are carried out using a methanol / water (33:67, 0.1% formic acid) system at a flow rate of 2 mL / min, a detection wavelength of 305 nm, and a retention time of 34 minutes.

[0026] The third aspect of the present invention provides a use of the chloropimarane diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-ultraviolet eye disease drug.

[0027] The anti-ultraviolet eye disease drug refers to an anti-ultraviolet eye disease drug using the chloropimarane diterpene compound or its pharmaceutically acceptable salt as a single active ingredient, or an anti-ultraviolet eye disease drug formed by combining the chloropimarane diterpene compound or its pharmaceutically acceptable salt with other drug carriers and other anti-ultraviolet eye disease drugs.

[0028] The fourth aspect of the present invention provides a use of the chloropimarane diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating corneal damage caused by UVB radiation.

[0029] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0030] The compound obtained in this invention is the first discovered chlorinated pimarane diterpenoid. In vitro activity tests demonstrated that the compound protected human corneal epithelial cells (HCE-T cells) from UVB radiation at a concentration of 20 μM. ABTS antioxidant capacity assays also demonstrated that the compound possessed strong antioxidant capacity, potentially improving cell viability by reducing UVB-induced intracellular oxidative stress. Therefore, the compound could be used to develop novel anti-ultraviolet eye medications for the prevention or treatment of UVB-induced corneal damage.

[0031] The present invention provides a new lead compound for the research and development of new anti-ultraviolet eye disease drugs and provides a scientific basis for the development and utilization of polar marine medicinal resources.

[0032] The chloropimarane diterpene compound of the present invention is obtained by extraction, separation and purification from mixed fermentation products of two Arctic fungi, Curvularia D-1 and Trichoderma B-13.

[0033] Deposit information of biological material samples:

[0034] Depository: China Center for Type Culture Collection

[0035] Address: Wuhan University, Wuhan, China

[0036] Deposit date: April 12, 2013

[0037] Deposit number: CCTCC NO: M2013144

[0038] Classification name: Eutypella sp.D-1

[0039] Deposit information of biological material samples:

[0040] Depository: China Center for Type Culture Collection

[0041] Address: Wuhan University, Wuhan, China

[0042] Deposit date: June 21, 2024

[0043] Accession number: CCTCC M 20241339

[0044] Classification and nomenclature: Nectria sp. B-13 DETAILED DESCRIPTION

[0045] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0046] The raw materials of the two Arctic fungi Curvularia D-1 and Cercospora B-13 used in the present invention are isolated from the high latitudes of the Arctic.

[0047] Example 1: Preparation of the compound of the present invention

[0048] Step 1: Preparation of total extract:

[0049] A small amount of Nectria sp. B-13 mycelium was picked from the seed plate and inoculated into a shake flask containing PDB medium. After incubation at 28°C and 180 rpm for 5 days, a primary seed solution was obtained. Subsequently, 5 mL of the primary seed solution was transferred to a shake flask containing 100 mL of seed medium in a clean bench and incubated under the same conditions for 2 days to obtain a secondary seed solution for future use. Simultaneously, a secondary seed solution of Eutypella sp. D-1, deposited with CCTCC NO: M 2013144, was obtained using the same method. After obtaining the two secondary seed solutions, 25 mL of the Nectria sp. B-13 secondary seed solution was transferred to a shake flask containing 500 mL of fermentation medium for expansion culture. The solution was incubated at 28°C and 180 rpm for 1 day. Subsequently, 25 mL of the Eutypella sp. D-1 secondary seed solution was transferred to the aforementioned shake flask and mixed and fermented under the same conditions for 10 days.

[0050] The PDB culture medium formula is: 10g potato extract powder, 20g glucose, 1000mL distilled water;

[0051] The seed culture medium formula is: 125 g / L glucose, 3.3 g / L sodium nitrate, 0.07 g / L potassium hydrogen phosphate trihydrate, 0.4 g / L magnesium sulfate heptahydrate, 0.625 g / L potassium chloride, 0.7 g / L yeast extract, 3.125 mg / L cobalt chloride hexahydrate, 18.75 mg / L ferrous sulfate, 6.5 g / L anhydrous calcium chloride, and 15 g / L L-ornithine hydrochloride.

[0052] The fermentation medium formula is: 51.4 g / L sucrose, 3.3 g / L sodium nitrate, 2.5 g / L urea, 0.7 g / L yeast extract, 0.07 g / L dipotassium hydrogen phosphate trihydrate, 0.4 g / L magnesium sulfate heptahydrate, 0.625 g / L potassium chloride, 18.75 mg / L ferrous sulfate heptahydrate, 6.5 g / L anhydrous calcium chloride, and 3.125 mg / L cobalt chloride hexahydrate.

[0053] The fermentation broth obtained by the above culture was filtered to obtain bacterial cells and bacterial liquid respectively; the bacterial liquid was extracted three times with an equal volume of ethyl acetate, and the combined extracts were evaporated to dryness to obtain a first extract; the bacterial cells were ultrasonically extracted three times with dichloromethane:methanol = 1:1, 30 minutes each time, the extract was concentrated until free of organic solvent, suspended in water, extracted three times with an equal volume of ethyl acetate, and the combined extracts were evaporated to dryness to obtain a second extract; the first and second extracts were combined to obtain a total extract;

[0054] The second step is separation and purification:

[0055] ① The total extract was subjected to vacuum liquid column chromatography (VLC) with petroleum ether:ethyl acetate (100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:1, 0:1, v / v) as the solvent for gradient elution. Similar fractions were combined according to TLC thin layer chromatography to obtain 15 components Fr.AO;

[0056] ② Component Fr.K was subjected to normal phase silica gel column chromatography with a gradient elution using petroleum ether:ethyl acetate (25:1, 15:1, 10:1, 5:1, 3:1, 2:1, MeOH, v / v). Similar fractions were combined based on TLC color analysis to obtain five components Fr.K1-K5;

[0057] ③ Component Fr.K4 was purified by reverse-phase high-performance liquid chromatography using a methanol / water (33:67, 0.1% formic acid) system at a flow rate of 2 mL / min, a detection wavelength of 305 nm, and a retention time of 34 minutes to obtain the compound represented by formula (I).

[0058] Step 3: Structural Identification

[0059] The chemical structure of the compound represented by formula (I) was determined by various modern spectral techniques such as NMR, HRESIMS, IR, and UV. The structural formula is shown below:

[0060]

[0061]

[0062] The compound represented by formula (I) is 20 H 29 O6Cl, [α]2D5+45.6(c=0.1,MeCN); UV(MeCN)λ max (logε)216(3.44),303(3.89)nm; IR(KBr)v max 3427,2955,2928,2869,1718,1654,1603,1463,1393,1367,1267,1224,1148,1124,1061,1002,957,926,819,785,732,689,542cm -1 ;CD(MeCN)(Δε)296(+19.8),332(-23.3); HRESIMSm / z423.1541[M+Na]+(calcdforC 20 H 29 O6ClNa,423.1545); 1 H and13 The CNMR spectrum data are shown in Table 1.

[0063] Table 1 Nuclear magnetic resonance spectrum data of the compound represented by formula (I)

[0064]

[0065]

[0066] a Measured at 125MHz in Methanol-d; b Measured at 500MHz in Methanol-d.

[0067] Example 2

[0068] In vitro anti-UVB ultraviolet radiation test of the compound represented by formula (I) of the present invention:

[0069] In vitro anti-ultraviolet radiation experiments were conducted on the compound represented by formula (I) of the present invention. The human corneal epithelial cells HCE-T cells and HCE-T cell-specific culture medium used were purchased from Wuhan Punosai Life Science Technology Co., Ltd., UVB PhilipsPL-S9W / 01 was purchased from Philips of the Netherlands, LS125 / UVB-X0 ultraviolet irradiator was purchased from Shenzhen Linshang Technology Co., Ltd., and the total antioxidant capacity detection kit (ABTS rapid method) was purchased from Biyuntian Biotechnology Co., Ltd.

[0070] 1. Effect of the compound represented by formula (I) on the activity of UVB-irradiated HCE-T cells

[0071] The CCK-8 assay was used to detect the effect of the compound represented by formula (I) on the viability of UVB-irradiated HCE-T cells. 4 / mL cell suspension, 100 μL per well was inoculated into a 96-well plate; cultured for 24 hours, and after the cells adhered to the wall, the cells were divided into 4 groups: non-irradiated group, UVB irradiated group, experimental group (UVB irradiation + compound shown in formula (I)), and blank control group.

[0072] UVB irradiation method: After the cells adhered, they were washed once with PBS buffer, and 10 μL of PBS buffer was added to cover the HCE-T cells. The cells were then placed 10 cm below the UVB light source for irradiation (irradiation intensity: 50 μW / cm 2 , irradiation time: 6min, cumulative irradiation dose: 18mJ / cm 2After irradiation, the waste liquid was discarded, 100 μL HCE-T cell culture medium was added to the UVB irradiation group, 20 μM compound of formula (I) was added to the experimental group, and 0.1 μL DMSO was added to each well of the blank control group. After incubation at 37°C for 48 h, 10 μL of the prepared CCK-8 solution was added to each well and incubated at 37°C for 1 h. When obvious changes appeared, the OD value at 450 nm of each well was measured, and the cell survival rate (%) was calculated according to the formula = (OD UVB / 样品 -OD 空白 ) / (OD 未照射 -OD 空白 )×100%.

[0073] 2. Detection of the total antioxidant capacity of the compound represented by formula (I)

[0074] The total antioxidant capacity of the compound represented by formula (I) was detected by the ABTS method. According to the operating instructions of the kit, 20 μL of peroxidase working solution was added to each test well of the 96-well plate. 10 μL of PBS solution was added to the blank control well; 10 μL of Trolox standard solution of different concentrations (0.15, 0.3, 0.6, 0.9, 1.2, 1.5 mM) was added to the standard curve test well; 10 μL of sample was added to the sample test well. 170 μL of ABTS working solution was added to each well, gently mixed, and the OD value at 414 nm was measured after incubation at room temperature for 6 minutes. Calculate ΔOD414 = OD414 for each point of the standard. 空白对照 -OD414 标准品 , use ΔOD414 and Trolox standard solution concentration to make a standard curve. The standard curve can be represented by the formula y=ax+b (y is ΔOD414, x is the concentration of Trolox standard solution, a is the slope of the standard curve, and b is the intercept of the standard curve). 空白对照 -OD414 样品 , calculate how many mM of Trolox standard the sample is equivalent to according to the standard curve formula, and obtain the total antioxidant capacity of the sample, Trolox-Equivalent Antioxidant Capacity (TEAC).

[0075] 3. Experimental results

[0076] The results of CCK-8 test showed that the UVB irradiation dose was 18mJ / cm 2The HCE-T cells can be damaged by UVB irradiation, and the cell viability is 52.14%. The compound shown as formula (I) has UVB irradiation protection effect on the HCE-T cells at an action concentration of 20 μM, and the cell viability can be increased to 71.52%. The ABTS detection compound total antioxidant capacity result shows that the ΔOD414 of the compound shown as formula (I) is equivalent to 0.33 mM Trolox when the compound is detected, and the antioxidant capacity (TEAC) of the compound is 1.65±0.12 mM.

[0077] 4. Conclusion

[0078] The compound shown as formula (I) has UVB irradiation protection effect on the HCE-T cells at an action concentration of 20 μM, and the antioxidant capacity is detected by the ABTS method, which shows that the compound has strong antioxidant capacity, and can improve the cell viability by reducing the oxidative stress caused by UVB in the cells. Therefore, the compound shown as formula (I) can be used as a lead compound of an anti-ultraviolet eye disease drug.

[0079] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as the above preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above-mentioned technical content without departing from the technical solution of the present application, and any simple modification, equivalent change and modification of the above-mentioned embodiment according to the technical essence of the present application still belong to the scope of the present application.

Claims

1. A chloropimarane diterpene compound or a pharmaceutically acceptable salt thereof, characterized in that: Its chemical structure is shown in formula (I):

2. A method for extracting the chloropimarane diterpene compound according to claim 1, characterized in that: The following steps are involved: Step 1: Preparation of total extract: Mycelia of Nectria sp. B-13, deposited with CCTCC NO: M 20241339, were selected from a seed plate and inoculated into a shake flask containing PDB medium. The culture was shaken at 25-30°C for 3-7 days to obtain a primary seed solution. The primary seed solution was transferred to a shake flask containing a seed medium and cultured under the same conditions for 1-3 days to obtain a secondary seed solution. The same method as above was used to obtain the secondary seed solution of Eutypellasp.D-1 with a deposit number of CCTCC NO: M 2013144; Transfer the secondary seed solution of Nectria sp. B-13 to a shake flask containing fermentation medium for expansion culture at 25-30°C with shaking for 1-2 days. Transfer the secondary seed solution of Eutypella sp. D-1 to the above shake flask and mix and ferment under the same conditions for 10 days. The fermentation broth obtained by the above culture was filtered to obtain bacterial cells and bacterial liquid respectively; the bacterial liquid was extracted with an equal volume of ethyl acetate at least three times, and the extracts were combined and evaporated to dryness to obtain a first extract; the bacterial cells were ultrasonically extracted with dichloromethane and methanol in a volume ratio of 1:1 at least three times, the extracts were concentrated until free of organic solvent, suspended in water, extracted with an equal volume of ethyl acetate at least three times, and the extracts were combined and evaporated to dryness to obtain a second extract; the first extract and the second extract were combined to obtain a total extract; The second step is separation and purification: ① The total extract was subjected to vacuum liquid column chromatography (VLC) with petroleum ether and ethyl acetate as solvents for gradient elution. Similar fractions were combined according to TLC thin layer chromatography to obtain 15 components Fr.AO; ② Component Fr.K was subjected to normal phase silica gel column chromatography with petroleum ether and ethyl acetate as solvents for gradient elution. Similar fractions were combined according to TLC color analysis to obtain five components Fr.K1-K5; ③ Purifying the component Fr.K4 by reverse-phase high performance liquid chromatography to obtain the compound represented by formula (I); The reverse-phase high-performance liquid chromatography purification conditions for component Fr.K4 are as follows: a methanol / water system with a volume ratio of methanol to water of 33:67, containing 0.1% formic acid, separation and preparation at a flow rate of 2 mL / min, a detection wavelength of 305 nm, and a retention time of 34 minutes.

3. The method for extracting chloropimarane diterpenoid compounds according to claim 2, wherein The PDB culture medium formula is: 10 g potato extract powder, 20 g glucose, and 1000 mL distilled water.

4. The method for extracting chloropimarane diterpenoid compounds according to claim 2, wherein The seed culture medium formula is: 125 g / L glucose, 3.3 g / L sodium nitrate, 0.07 g / L potassium hydrogen phosphate trihydrate, 0.4 g / L magnesium sulfate heptahydrate, 0.625 g / L potassium chloride, 0.7 g / L yeast extract, 3.125 mg / L cobalt chloride hexahydrate, 18.75 mg / L ferrous sulfate, 6.5 g / L anhydrous calcium chloride, and 15 g / L L-ornithine hydrochloride.

5. The method for extracting chloropimarane diterpenoid compounds according to claim 2, wherein: The fermentation medium formula is: 51.4 g / L sucrose, 3.3 g / L sodium nitrate, 2.5 g / L urea, 0.7 g / L yeast extract, 0.07 g / L dipotassium hydrogen phosphate trihydrate, 0.4 g / L magnesium sulfate heptahydrate, 0.625 g / L potassium chloride, 18.75 mg / L ferrous sulfate heptahydrate, 6.5 g / L anhydrous calcium chloride, and 3.125 mg / L cobalt chloride hexahydrate.

6. The method for extracting chloropimarane diterpenoid compounds according to claim 2, wherein: The Nectria sp. B-13 secondary seed solution was transferred to a shake flask containing a fermentation medium, and the Nectria sp. B-13 secondary seed solution was inoculated at an inoculum volume of 5% v / v.

7. Use of the chloropimarane diterpene compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-ultraviolet eye disease drug.

8. Use of the chloropimarane diterpene compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating corneal damage caused by UVB radiation.

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