A diterpenoid compound against saxitoxin neurotoxicity and application thereof

Abstract

The application discloses a kind of kaurane diterpene compounds or its medicinal salt, its chemical structure is as shown in formula (I). The compound is extracted, isolated from the fermentation product of the fungus (Eutypella sp.D-1) of the north polar arched shell genus (Eutypella sp.D-1) with the preservation number CCTCC NO:M 2013144 for the first time.The application further provides the extraction method of the compound, including the steps of liquid fermentation of fungus, extraction obtains total extract, and separation and purification by vacuum column chromatography, reverse phase ODS column chromatography and high performance liquid chromatography. Through zebra fish and mouse in-vivo pharmacological experiment, the compound of the application can significantly improve the abnormal behavior induced by domoic acid (DA), prolong the latent period of poisoning onset, relieve the symptoms of poisoning, and exhibit good neuroprotective activity. Therefore, the compound of the application can be used as a lead compound for preparing a drug for preventing or treating domoic acid poisoning and memory loss injury caused thereby, 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 pimarane diterpenoid compound obtained from the fermentation product of a fungus D-1 of the genus Eutypella in the Arctic through solid medium fermentation, extraction, separation and purification, and application thereof in the preparation of an anti-domoic acid poisoning drug. BACKGROUND

[0002] Domoic acid (DA) is a neurotoxin produced by marine algae Pseudo-nitzschia, which can accumulate in marine animals through the food chain. Humans may be poisoned after eating marine organisms contaminated with it, causing symptoms such as vomiting, diarrhea, confusion, memory loss, dizziness, and in severe cases, coma, seizures, heart failure, permanent brain damage, and even death. Due to its significant memory loss symptoms, the toxin is also known as "memory loss shellfish poisoning". Therefore, it is an important medical issue to develop effective anti-domoic acid poisoning treatment drugs. Polar extreme environments (low temperature, high pressure, etc.) drive microorganisms to evolve unique metabolic pathways, and their secondary metabolites often have novel structures and biological activities, providing unique resources for the discovery of active lead compounds. Accordingly, exploring the anti-DA poisoning activity of polar microbial secondary metabolites is expected to fill the treatment gap and has important research value. The drug source material Eutypella sp. D-1 was isolated from the Arctic and belongs to the phylum Ascomycetes, order Xylariales, family Diatrypaceae, and genus Eutypella. Literature reports that this species of fungi is widely distributed in various ecosystems, and previous studies have shown that it can produce secondary metabolites of various structural types such as alkaloids, polyketides, and terpenoids, and some compounds exhibit biological activities such as neuroprotection, anti-inflammatory, and antioxidant, providing potential candidate molecules for the treatment of neurodegenerative diseases or toxic nerve damage. For example, the pimarane diterpenoid compound libertellenone Z isolated from the fungus of the genus Eutypella has very good anti-inflammatory activity related to the treatment of neuroinflammatory diseases (see document: Ning, Y.; Zhang, S.; Zheng, T.; Xu, Y.; Li, S.; Zhang, J.; Jiao, B.; Zhang, Y.; Ma, Z.; Lu, X. Pimarane-Type Diterpenes with Anti-Inflammatory Activity from Arctic-Derived Fungus Eutypella sp. D-1. Mar. Drugs 2023, 21, 541.).

[0003] So far, there is no report on isolating a kaurane diterpene compound with anti-dominant toxicity of soft algae acid from the fermentation product of a fungus D-1 of Eutypella sp. in the Arctic. SUMMARY

[0004] The present application aims to provide a new kaurane diterpene compound extracted, separated and purified from the fermentation product of a fungus D-1 of Eutypella sp. in the Arctic.

[0005] Another object of the present application is to provide an extraction method of the kaurane diterpene compound.

[0006] Still another object of the present application is to provide an application of the kaurane diterpene compound in preparing an anti-dominant toxicity of soft algae acid medicine.

[0007] To achieve the above objects, the technical scheme adopted by the present application is as follows:

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

[0009]

[0010] The kaurane diterpene compound is extracted, separated and purified from the fermentation product of a fungus D-1 of Eutypella sp. in the Arctic (preservation unit: China Center for Type Culture Collection; address: Wuhan University, Wuhan, China; preservation date: April 12, 2013; preservation number: CCTCC NO: M2013144; classification and naming: Eutypella sp. D-1 Eutypellasp. D-1).

[0011] In the second aspect of the present application, an extraction method of the above kaurane diterpene compound is provided, comprising the following steps:

[0012] (1) Preparation of total extract: liquid fermentation is carried out by using Eutypella sp. D-1 with the preservation number of CCTCC NO: M 2013144, the fermentation broth is separated into mycelium and broth, and then organic solvent is used for extraction, and the extracts are combined to obtain the total extract;

[0013] (2) Separation and purification: the total extract is sequentially subjected to separation and purification by reduced pressure liquid chromatography column, reverse phase ODS column chromatography and reverse phase high performance liquid chromatography, so as to obtain the kaurane diterpene compound.

[0014] Preferably, in step (1), the liquid fermentation comprises seed liquid culture and scale-up fermentation; wherein the seed liquid culture adopts PDB culture medium, is cultured at 20°C with shaking, and first cultured for 5 days to obtain primary seed liquid, and then subcultured for 3 days to obtain secondary seed liquid; the scale-up fermentation is subculturing the secondary seed liquid into fermentation medium at a 5% (v / v) inoculation amount, and culturing at 20°C with shaking for 12 days.

[0015] More preferably, in the scale-up fermentation process, 0.2% (v / v) of anhydrous ethanol is added to the fermentation broth on the 3rd, 4th and 5th day of fermentation.

[0016] Preferably, the PDB culture medium formula is: potato infusion powder 10 g, glucose 20 g, distilled water 1000 mL.

[0017] Preferably, the fermentation medium formula is: sucrose 51.4 g / L, sodium nitrate 3.3 g / L, urea 2.5 g / L, yeast extract 0.7 g / L, potassium phosphate dibasic 0.07 g / L, magnesium sulfate heptahydrate 0.4 g / L, potassium chloride 0.625 g / L, ferrous sulfate heptahydrate 18.75 mg / L, anhydrous calcium chloride 6.5 g / L, cobalt chloride hexahydrate 3.125 mg / L.

[0018] Preferably, the separation and purification in step (2) specifically comprises:

[0019] ①The total extract is subjected to preliminary separation by a reduced-pressure liquid column chromatography with dichloromethane and methanol as elution solvents, and the flow fractions are combined according to the thin-layer chromatography analysis results to obtain several components including Fr.E;

[0020] ②The component Fr.E is subjected to further separation by a reversed-phase ODS column chromatography with methanol and water as elution solvents, and the flow fractions are combined according to the thin-layer chromatography analysis results to obtain several sub-components including Fr.E2;

[0021] ③The component Fr.E2 is subjected to final purification by reversed-phase high-performance liquid chromatography, and the target fraction is collected to obtain the high-purity pimara-9 (11), 15-diene.

[0022] Preferably, the reversed-phase high-performance liquid chromatography purification condition for the component Fr.E2 in step ③ is: separation and preparation are performed in an acetonitrile / water (40:60, 0.1% formic acid) system at a flow rate of 2 mL / min, the detection wavelength is 317 nm, and the retention time is 26 min.

[0023] In a third aspect, the application provides a use of the pimara-9 (11), 15-diene or a pharmaceutically acceptable salt thereof in the preparation of a drug for resisting soft algal acid poisoning.

[0024] The anti- saxitoxin poisoning drug refers to the use of the above-mentioned sea-mattan diterpene compound or its pharmaceutically acceptable salt as a single active ingredient as an anti- saxitoxin poisoning drug or the use of the above-mentioned sea-mattan diterpene compound or its pharmaceutically acceptable salt in combination with other drug carriers or other anti- saxitoxin poisoning drugs as an anti- saxitoxin poisoning drug.

[0025] In a fourth aspect of the present application, the use of the above-mentioned sea-mattan diterpene compound or its pharmaceutically acceptable salt in the preparation of a drug for preventing or treating memory loss injury caused by saxitoxin poisoning is provided.

[0026] Advantages

[0027] With the above technical solutions, the present application has the following advantages and beneficial effects:

[0028] The compound obtained by the present application is a sea-mattan diterpene compound discovered for the first time. Through in vivo activity tests on model animals, it is shown that the compound (I) can improve DA-induced abnormal behavior of zebrafish at a concentration of 1 μM, improve DA-induced poisoning-like behavior of zebrafish, significantly prolong the latency of poisoning onset of zebrafish, and has a certain neuroprotective effect. Moreover, at a concentration of 5 mg / kg, the severity of poisoning onset of mice can be significantly relieved. Therefore, the compound can be used for developing a new anti- saxitoxin poisoning drug and a drug for preventing or treating memory loss injury caused by saxitoxin poisoning.

[0029] The present application provides a new lead compound for researching and developing a new anti- saxitoxin poisoning drug, and provides a scientific basis for developing and utilizing polar marine medicinal resources.

[0030] Drawings of the specification

[0031] Figure 1 Figure for the effect of compound (I) on DA-induced movement behavior of zebrafish;

[0032] Figure 2 Figure for the effect of compound (I) on DA-induced movement parameters of zebrafish;

[0033] Figure 3 Figure for the effect of compound (I) on DA-induced seizure latency of zebrafish;

[0034] Figure 4 Figure for the effect of compound (I) on DA-induced poisoning behavior severity (Racine score) of mice; DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the present application, the present application will be further described below in combination with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0036] A strain of the Arctic fungus Eutypella sp. D-1 used in the present application is isolated from high latitude areas in the Arctic.

[0037] Example 1: Preparation of the compound of the present application

[0038] First step, preparation of total extract:

[0039] The mycelium of Eutypella sp. D-1 (preserved number CCTCC NO: M2013144) was picked from a preservation plate, inoculated into a flask containing PDB medium, and cultured at 20°C for 5 days to obtain a first-stage seed liquid. The first-stage seed liquid was transferred into a flask containing PDB medium, and cultured under the same conditions for 3 days to obtain a second-stage seed liquid. The second-stage seed liquid of Eutypella sp. D-1 was transferred into a flask containing a fermentation medium, and expanded and cultured at 20°C for 12 days. 0.2% v / v of anhydrous ethanol was added to the fermentation liquid on the 3rd, 4th, and 5th days of fermentation, respectively.

[0040] The PDB medium formula is: potato infusion powder 10 g, glucose 20 g, distilled water 1000 mL.

[0041] The fermentation medium formula is: sucrose 51.4 g / L, sodium nitrate 3.3 g / L, urea 2.5 g / L, yeast extract 0.7 g / L, potassium phosphate dibasic 0.07 g / L, magnesium sulfate heptahydrate 0.4 g / L, potassium chloride 0.625 g / L, ferrous sulfate heptahydrate 18.75 mg / L, anhydrous calcium chloride 6.5 g / L, cobalt chloride hexahydrate 3.125 mg / L.

[0042] The fermentation liquid obtained by the above culture was filtered to obtain the mycelium and the liquid, respectively. The liquid was extracted with an equal volume of ethyl acetate for at least 3 times, and the combined extract was evaporated to dryness to obtain a first part of the extract. The mycelium was ultrasonically extracted with dichloromethane and methanol at a volume ratio of 1:1 for at least 3 times, and the extract was concentrated to remove the organic solvent. The residue was suspended in water and extracted with an equal volume of ethyl acetate for at least 3 times. The combined extract was evaporated to dryness to obtain a second part of the extract. The first part and the second part of the extract were combined to obtain the total extract.

[0043] Second step, separation and purification:

[0044] ①The total extract was subjected to gradient elution with dichloromethane and methanol as solvents by reduced pressure liquid column chromatography VLC, and similar fractions were combined according to TLC thin layer chromatography to obtain 10 fractions Fr. A-J.

[0045] Fr. E2-E6 were obtained by gradient elution with methanol and water as solvent on a reversed-phase ODS column, and then combined according to the TLC analysis.

[0046] Fr. E2 was purified by reversed-phase high performance liquid chromatography to obtain the compound of formula (I).

[0047] Step 3, structure identification:

[0048] The compound of formula (I) was identified by NMR, HRESIMS, IR, UV and other modern spectroscopic techniques, and the chemical structure of the compound of formula (I) was determined. The structural formula is as follows:

[0049]

[0050] The compound of formula (I) is a brown oil, +16.3 (c = 0.1, MeCN); UV (MeCN) λ max (logε) 212 (3.80), 260 (3.62), 317 (3.30) nm; IR (KBr) v max 3378, 2921, 2851, 1714, 1664, 1619, 1602, 1510, 1460, 1414, 1359, 1291, 1253, 1235, 1205, 1170, 1071, 1049, 1026, 1012, 997, 961, 914, 875, 861, 809, 778, 737, 699, 633, 579.46, 435.52 cm -1 ; CD (MeCN) (Δε) 207 (+16.3), 224 (+5.6), 233 (+6.1), 260 (-6.6), 304 (+7.9), 343 (-5.8); HR-ESI-MS m / z 337.1773 [M+Na] + (calcd for C 20 H 26 O3Na, 337.1774). 1 H and 13 The C NMR nuclear magnetic resonance spectral data are shown in Table 1.

[0051] Table 1 Nuclear magnetic resonance spectral data of the compound of formula (I)

[0052]

[0053]

[0054] aMeasured at 125MHz in Methanol-d; b Measured at 500MHz in Methanol-d.

[0055] Example 2

[0056] Improving effect of the compound of formula (I) on DA-induced zebrafish neurotoxicity behavior:

[0057] 1. Construction of DA-induced zebrafish injury model

[0058] Select zebrafish normally developed to 3dpf, anesthetize them with 0.4mg / mL anesthetic, and then place them in an agarose mold for fixation. Under a microscope, use a microinjector to inject 1nL of DA into the pericardial venous plexus, with a concentration of 50μg / ml. After injection, place the zebrafish in zebrafish culture water for culture, and replace the culture medium every 24h. Transfer the zebrafish cultured to 5 or 6dpf to a 48-well plate, with 1 fish per well, and add 1mL of zebrafish culture water. Place them in a Viewpoint zebrafish larva behavior chamber for 10min of adaptation, and then record the behavior trajectory for 10min. Use software to automatically record data every minute, and after the end of the experiment, count the swimming trajectory, total distance, and average speed per minute of the zebrafish within 10min.

[0059] 2. Evaluation of improving effect of the compound of formula (I) on DA-induced zebrafish poisoning-like symptoms

[0060] Select three concentrations of 1μM, 2μM, and 4μM to investigate the improving effect of compound (I) on zebrafish poisoning-like symptoms. The groups are blank control group, model group (DA), positive control Perampanel group, low-dose compound (I) group (1μM), medium-dose compound (I) group (2μM), and high-dose compound (I) group (4μM). To explore the effect of compound (I) and positive control on the movement ability of zebrafish, zebrafish co-treated with DA and compound (I) were subjected to autonomous behavior test. Compared with the blank control group, the movement behavior of zebrafish after DA-induced treatment changed significantly, the swimming trajectory and total swimming distance within 20min increased, and the average swimming speed per minute increased significantly. Compared with the DA-treated group, the positive control can reduce the total swimming distance and average swimming speed per unit time of zebrafish, and can alleviate the poisoning-like behavior of zebrafish. Figure 1 ) Compound (I) at medium and high concentrations co-treated with DA can restore the movement ability of zebrafish, showing a significant decrease in average swimming speed and total movement distance Figure 2

[0061] 3. Experimental results​

[0062] The results show that the compound (I) and the positive control can improve the abnormal behavior of zebrafish induced by DA at a certain concentration, restore the movement ability of zebrafish, improve the DA-induced poisoning behavior of zebrafish, and have a certain neuroprotective effect. Therefore, the compound (I) can be used as a lead compound of an anti-saxitoxin poisoning drug.

[0063] Example 3

[0064] The improvement effect of the compound (I) on the latent period of DA-induced neurotoxicity of zebrafish:

[0065] 1. Construction of DA-induced zebrafish injury model

[0066] Select normal zebrafish developed to 3dpf, anesthetize them with 0.4mg / mL anesthetic, and then place them in an agarose mold for fixation. Under a microscope, 1nL of DA with a concentration of 50μg / ml is injected into the pericardial venous plexus of the zebrafish using a microinjector. After injection, the zebrafish are placed in zebrafish culture water for culture, and the culture solution is replaced every 24h. The zebrafish larvae developed to 5 or 6dpf after microinjection of DA are transferred to a 48-well plate, 1mL of zebrafish culture water is added to each well, and the time for the zebrafish to reach each stage of seizure is observed. The latent time (s) of each group of zebrafish is statistically analyzed according to the grading standard of zebrafish seizure latency, and the zebrafish that do not appear to have seizures are excluded.

[0067] 2. Evaluation of the improvement effect of the compound (I) on the latent period of DA-induced neurotoxicity of zebrafish

[0068] The latent time of the three stages of poisoning onset is monitored. After treatment with 1μM of compound (I), the latent period of the three stages of zebrafish onset is not significantly prolonged. After 24h of treatment with 10μM of compound (I), the latent time of the second and third stages of zebrafish is significantly prolonged. 4μM of compound (I) can significantly prolong the three stages of poisoning onset of zebrafish, but the positive control is not as good as Figure 3 ).

[0069] 3. Experimental results

[0070] The experimental results show that after 24h of treatment of zebrafish with high concentrations of compound (I) and the positive control, the latent time of poisoning onset of zebrafish can be significantly prolonged, and the effect is the most significant. The compound (I) can be used as a lead compound of an anti-saxitoxin poisoning drug

[0071] Example 4

[0072] The improvement effect of the compound (I) on DA-induced neurotoxicity of mice:

[0073] 1. Construction of a mouse injury model induced by DA

[0074] Thirty-six 6-week-old C57BL / 6 mice were used, and were set into a blank control group, a model group, a positive control group, a low-dose compound (I) treatment group, a medium-dose treatment group and a high-dose treatment group. In the model group, the mice were given an equal volume of solvent by gavage, and 30 minutes later, 3 mg / kg of DA was injected intraperitoneally to model. In the positive control treatment group, the mice were given 5 mg / kg of solution by gavage, and in the compound (I) treatment group, the mice were given 2.5, 5 and 10 mg / kg of compound (I) by gavage, and 30 minutes later, 3 mg / kg of DA was injected intraperitoneally to model. After injection, the mice were placed in a cage, and the state of the mice was observed, and the drug was given once every other day.

[0075] 2. Evaluation of the improvement effect of the compound represented by formula (I) on the latent period of DA-induced neurotoxicity in mice

[0076] The epileptiform behavior of the mice after poisoning was evaluated by Racine scoring, and spontaneous occurrence of 4-5 grade seizures was regarded as a successful model: 0 grade: no response; 1 grade: facial muscle spasm, showing rhythmic twitching of the mouth or face; 2 grade: neck muscle spasm, showing nodding movement; 3 grade: forelimb clonus; 4 grade: generalized rigidity; 5 grade: rigidity with falling. The experimental results showed that the normal group of mice had no abnormal behavior. After injection of DA, the mice in the model group had an increased severity of poisoning seizures, indicating that the modeling was successful. After treatment with the positive control, the severity of poisoning seizures was significantly relieved. Figure 4

[0077] 3. Experimental results

[0078] The experimental results showed that after treatment with compound (I), the severity of poisoning seizures in the medium and high concentration groups of mice was relieved, indicating that compound (I) and the positive control could significantly improve the poisoning-like behavior of mice. Therefore, the compound represented by formula (I) can be used as a lead compound for anti-carteric acid poisoning drugs.

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

Claims

1. A pinene diterpene compound or its pharmaceutical salt, characterized in that, Its chemical structure is shown in formula (I): 。 2. A method for extracting the pinene diterpenoid compound according to claim 1, characterized in that, Includes the following steps: (1) Preparation of total extract: using *Cyclospora flexurea* with accession number CCTCC NO: M2013144 Eutypella sp. D-1 was subjected to liquid fermentation. The fermentation broth was separated into cell bodies and cell liquid, and then extracted separately with organic solvents. The extracts were combined to obtain the total extract. The liquid fermentation includes seed culture and scale-up fermentation. The seed culture uses PDB medium and is cultured at 20°C with shaking. The primary seed culture is obtained after 5 days of culture, and then transferred to a secondary seed culture for 3 days. The scale-up fermentation involves transferring the secondary seed culture to the fermentation medium at an inoculation rate of 5% v / v and cultured at 20°C with shaking for 12 days. On the 3rd, 4th, and 5th days of fermentation, 0.2% v / v anhydrous ethanol is added to the fermentation broth. The fermentation medium formula is as follows: sucrose 51.4 g / L, sodium nitrate 3.3 g / L, urea 2.5 g / L, yeast extract 0.7 g / L, dipotassium hydrogen phosphate trihydrate 0.07 g / L, magnesium sulfate heptahydrate 0.4 g / L, potassium chloride 0.625 g / L, ferrous sulfate heptahydrate 18.75 mg / L, anhydrous calcium chloride 6.5 g / L, and cobalt chloride hexahydrate 3.125 mg / L; (2) Separation and purification: The total extract is separated and purified by the following steps: ① The total extract was subjected to reduced pressure liquid column chromatography with gradient elution using dichloromethane and methanol as solvents. Based on thin-layer chromatography analysis, similar fractions were combined to obtain 10 components Fr. AJ; ② The fraction Fr. E was subjected to reversed-phase ODS column chromatography with gradient elution using methanol and water as solvents. Based on thin-layer chromatography analysis, similar fractions were combined to obtain 6 fractions Fr. E1-E6. ③ The component Fr. E2 was purified by reversed-phase high-performance liquid chromatography using a mixed solution of acetonitrile and water as the mobile phase, wherein the volume ratio of acetonitrile to water was 40:60 and the mixed solution contained 0.1% formic acid. The separation was carried out at a flow rate of 2 mL / min and a detection wavelength of 317 nm. The target chromatographic peak with a retention time of 26.2 minutes was collected to obtain the compound shown in formula (I).

3. The method for extracting the diterpenoid compound according to claim 2, characterized in that, The PDB culture medium formula is as follows: 10g potato extract powder, 20g glucose, and 1000mL distilled water.

4. The use of the pinene diterpenoid compound of claim 1 or its pharmaceutical salt in the preparation of a medicament for the prevention and / or treatment of domoic acid poisoning.

5. The application according to claim 4, characterized in that, The drug is used to prevent or treat memory loss caused by domoic acid poisoning.

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