Use of an indole diterpene compound in neuroprotection

Abstract

The application provides application of an indole diterpenoid compound in nerve protection. Penicillium The compound can be obtained from fermentation of Penicillium sp. UJNMF0740 with a preservation number of CGMCC No: 40521. Activity test finds that the compound has obvious improvement effect on 6-OHDA-induced PC12 caused cell apoptosis, and has no toxicity, and has good application prospect in development of drugs or drug intermediates with nerve protection effect.

Description

Technical Field

[0001] This invention belongs to the field of biochemical medicine, and specifically relates to a new application of an indole diterpenoid compound. Background Technology

[0002] Neurodegenerative diseases are a group of disease states in which brain and spinal cord nerve cells are lost. With the increasing aging of the global population, the prevalence of neurodegenerative diseases is also rising. Apoptosis refers to the programmed cell death caused by the body's cells through their inherent biochemical and physiological reactions or the activation of certain enzymes. It is a physiological and selective biological phenomenon, widely present in organisms, and plays an important role in vertebrate embryogenesis. Nerve cells generally do not regenerate, so excessive nerve cell apoptosis and the resulting death can be devastating and irreversible to the body. Studies have shown that apoptosis plays a crucial role in the development of neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's syndrome.

[0003] Currently, treatment options for neurodegenerative diseases are very limited. Apoptosis, as a pathological phenomenon that is prevalent in many neurodegenerative diseases, can improve disease symptoms and prevent and treat related diseases caused by neurodegeneration by blocking apoptosis-related signaling networks in neuronal damage and neurodegeneration, such as the MAPKS family and Akt / mTOR signaling.

[0004] Indole diterpenes are an important class of alkaloids mainly produced by fungi. They have a cyclic diterpenoid core derived from geranium diphosphate (GGPP) and an indole moiety derived from tryptophan. They have a wide range of biological activities, such as inhibiting cholesterol acyltransferase activity, inhibiting nitric oxide (NO) production, and inhibiting the growth of bacteria and cancer cells. However, there are currently no reports on the neuroprotective activities of this class of compounds. Summary of the Invention

[0005] In order to provide a preventive or therapeutic agent for neurodegenerative diseases, the present invention provides a neuroprotective activity of an indole diterpenoid compound, which can be used as an active ingredient or parent compound to develop drugs for the treatment of neurodegenerative diseases.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned indole diterpenoid compounds, which can be obtained from Penicillium fungi. Penicillium Extracted from the fermentation broth of sp. UJNMF0740.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] An indole diterpenoid compound, 22,23-dehydro-shearinine A, can be used to produce drugs or drug additives with neuroprotective effects.

[0009] The structure of 22,23-dehydro-shearinine A is shown in formula (I):

[0010]

[0011] Formula (I).

[0012] The preparation method of the above-mentioned indole diterpenoids can be found in You J, 2013 (Shearinines DK, new indole triterpenoids from an endophytic Penicillium The method described in *Sp. (strain HKI0459) with blocking activity on large-conductance calcium-activated potassium channels (Tetrahedron) or Heine D, 2018 (Chemical warfare between leafcutterant symbionts and a co-evolved pathogen, Nature Communications) can also be used to detect *Penicillium* fungi. Penicillium It was isolated from the fermentation product of sp. UJNMF 0740. Specifically, it includes the following steps:

[0013] (1) Penicillium fungi Penicillium Fermentation of sp. UJNMF 0740 yielded fermentation product;

[0014] (2) The crude fermentation extract was obtained from the fermentation product obtained in step (1) by ethanol extraction and ethyl acetate extraction;

[0015] (3) The fermented crude extract in step (2) is separated in the following manner:

[0016] (3-1) The fermentation crude extract was eluted with dichloromethane:methanol at a volume ratio of 1:0-0:1 through a 100-200 mesh normal phase silica gel column to obtain fractions Fr.1-Fr.9;

[0017] (3-2) Fraction Fr.6 was eluted with dichloromethane:methanol at a volume ratio of 1:0-0:1 by gradient elution on a 200-300 mesh normal phase silica gel column to obtain fractions Fr.6.1-Fr.6.8; Fr.6.5 was eluted with methanol by Sephadex LH-20 to obtain fractions Fr.6.5.1-Fr.6.5.6; Fr.6.5.5 was eluted with methanol:water at a volume ratio of 85:15 by semi-preparative high performance liquid chromatography on a C18 column to obtain compound (I).

[0018] Penicillium fungi Penicillium sp. UJNMF 0740, with accession number CGMCC No. 40521, was deposited on March 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located in Beijing, China.

[0019] The present invention also provides a neuroprotective drug comprising a compound of formula (I). The neuroprotective drug further includes medically acceptable excipients. The neuroprotective drug may also include other active ingredients to enhance the neuroprotective effect.

[0020] The present invention has the following advantages:

[0021] The indole diterpenoid compounds of the present invention have neuroprotective activity and have potential applications in the preparation of neuroprotective drugs or pharmaceutical intermediates; they can also serve as lead compounds for the development of drugs for the treatment or delay of neurodegenerative diseases. Attached Figure Description

[0022] Figure 1 The effects of 22,23-dehydro-shearinine A on PC12 cell (A) and 6-OHDA-induced PC12 cell (B) proliferation were investigated.

[0023] Figure 2 The effect of 22,23-dehydro-shearinine A on 6-OHDA-induced apoptosis in PC12 cells;

[0024] Figure 3 The effects of 22,23-dehydro-shearinine A on the expression of key proteins in the PI3K / Akt signaling pathway and ROS expression induced by 6-OHDA in PC12 cells. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0026] Example 1: Preparation of 22,23-dehydro-shearinine A

[0027] (1) Fermentation and crude extract preparation of UJNMF 0740

[0028] Preparation of seed culture medium: 200 mL potato extract, 20 g glucose, 30 g sea salt, and water to a final volume of 1 L. Pour the culture medium into 20 500 mL Erlenmeyer flasks, approximately 150 mL per flask, and autoclave at 121°C for 25 minutes.

[0029] Preparation method of rice fermentation medium: 80g rice, 0.4g yeast extract, 0.4g glucose, 120ml water, 3% sea salt. Place in 1L Erlenmeyer flasks, for a total of 150 flasks. Autoclave at 121℃ for 25 minutes and set aside.

[0030] Use sterile bamboo skewers to pick out an appropriate amount of the preservation number. Penicillium The UJNMF 0740 strain was inoculated into seed culture medium and cultured on a shaker (200 rpm) at 28°C for 3 days to obtain seed liquid. Then, 10 mL of seed liquid was inoculated into a 1 L Erlenmeyer flask containing rice fermentation medium and cultured statically at 28°C for 30 days before the fermentation product was collected.

[0031] After 30 days of fermentation, the rice fermentation medium was transferred out and crushed. It was then soaked in 95% ethanol four times (each soaking lasted 6-7 days). The 95% ethanol was collected after each soaking and concentrated into a solution using a large rotary evaporator. Then, it was extracted with an equal amount of ethyl acetate. After five extractions, 35g of crude fermentation extract was obtained.

[0032] (2) Preparation of 22,23-dehydro-shearinine A

[0033] The above-mentioned crude fermentation extract was subjected to silica gel column chromatography (100-200 mesh) with gradient elution of dichloromethane:methanol (1:0-0:1). During elution, thin-layer chromatography silica gel plates were used for detection and similar fractions of compounds were combined to obtain a total of 9 fractions (Fr.1-Fr.9).

[0034] Fr.6 was purified by silica gel column chromatography (200-300 mesh) with gradient elution of dichloromethane:methanol (1:0-0:1), followed by detection using a silica gel plate for thin-layer chromatography, yielding eight fractions (Fr.6.1-Fr.6.8). Fr.6.5 was purified by Sephadex LH-20 (methanol), yielding six fractions (Fr.6.5.1-Fr.6.5.6). Fr.6.5.5 was purified by semi-preparative high-performance liquid chromatography (methanol / water, v / v 85:15, 3 mL / min), yielding the target product (t). R = 17.8 min, 1.5 mg), and appears as a pale yellow amorphous powder.

[0035] The structure of the isolated target product was identified: based on the quasi-molecular ion peak m / z 582.1 ([M + H]) given by ESIMS mass spectrometry. + And based on NMR data, its molecular formula is deduced to be C. 37 H 43 NO5. By comparing with NMR data from the literature (You J, 2013), this compound was identified as the known compound 22,23-dehydro-shearinine A. .

[0036] Table 1. 22,23-dehydro-shearinine A in CDCl3 1 H (600 MHz) and 13 C (150 MHz) NMR data

[0037] .

[0038] This compound was first reported in 2013 (You J, 2013). It belongs to the class of indole diterpenoid alkaloids and showed no significant inhibitory effect on Candida albicans biofilm formation in tests. In 2018, Heine D et al. also isolated this compound from a fungus isolated from Columbia leafcutter ants, but did not report its activity.

[0039] Example 2: Neuroprotective activity of 22,23-dehydro-shearinine A

[0040] (1) Using 6-OHDA-induced PC12 cell damage as a model, the neuroprotective activity of 22,23-dehydro-shearinine A was evaluated. Cell viability was determined using the MTT assay to assess the neuroprotective activity of the analyte. The results showed that at a test concentration of 50 μM, treatment with 22,23-dehydro-shearinine A increased the survival rate of PC12 cells by 17.9% compared to the control group (6-OHDA), indicating that 22,23-dehydro-shearinine A can reduce 6-OHDA-induced PC12 cell death and has a certain neuroprotective effect.

[0041] (2) Protective effect of 22,23-dehydro-shearinine A against 6-OHDA-induced PC12 cell damage

[0042] Using 6-OHDA-induced PC12 cell injury as a model, the neuroprotective effects of the compound were further evaluated. Cell viability was determined using the MTT assay to assess the cytotoxicity of 22,23-dehydro-shearinine A on PC12 cells and its protective effect against 6-OHDA-induced PC12 cell injury. In many neurological injuries, apoptosis is the main mode of neuronal cell death; the effect of 22,23-dehydro-shearinine A on 6-OHDA-induced apoptosis in PC12 cells was observed using Hoechst 33258 staining, Annexin V-PI double staining, and Western blotting.

[0043] Depend on Figure 1 As can be seen from A, 22,23-dehydro-shearinine A has no toxicity to PC12 cells in the range of 25-100 μM; on the contrary, it has a certain effect on promoting cell proliferation. Figure 1 As can be seen from B, 22,23-dehydro-shearinine A has a good ameliorative effect on the damage of PC12 cells pretreated with 6-OHDA, and its activity is better at lower concentrations (25 μM) within the assay range.

[0044] The results of Hoechst 33258 staining show that ( Figure 2 A) Pretreatment with 6-OHDA induced significant apoptosis in PC12 cells. Incubation with 22,23-dehydro-shearinine A before 6-OHDA treatment significantly improved the apoptosis phenomenon. Flow cytometry Annexin V-PI double staining quantitative analysis of apoptosis was also performed. Figure 2The proportions of apoptotic cells in the 22,23-dehydro-shearinine A pretreatment groups (25 μM, 50 μM, and 100 μM) were 13.79%, 23.96%, and 37.23%, respectively, which were significantly lower than those in the 6-OHDA-only treatment group (41.45%).

[0045] Further analysis was conducted using Western blotting to determine the expression of apoptosis-related proteins. The experimental results are as follows: Figure 2 As shown in CG, compared with the 6-OHDA treatment group, incubation with 22,23-dehydro-shearinine A significantly reduced the expression of cleaved caspase-3, cleaved caspase-9 and Bax, and increased the expression of Bcl-2, further demonstrating that 22,23-dehydro-shearinine A can alleviate 6-OHDA-induced apoptosis in PC12 cells.

[0046] (3) Mechanism of the protective effect of 22,23-dehydro-shearinine A against 6-OHDA-induced PC12 damage

[0047] The phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway plays a crucial role in various cellular processes, including cell metabolism, proliferation, differentiation, migration, and protein synthesis. We used Western blotting to evaluate the effects of 6-OHDA and 22,23-dehydro-shearinine A on the expression of PI3K, p-PI3K, Akt, and p-Akt proteins in PC12 cells. The results are as follows: Figure 3 As shown in AC, compared with the control group, the ratios of p-PI3K / PI3K and p-Akt / Akt were significantly downregulated in the 6-OHDA group. However, compared with the 6-OHDA group, the ratios of p-PI3K / PI3K and p-Akt / Akt were significantly upregulated after pretreatment with 22,23-dehydro-shearinine A (25, 50, and 100 μM), suggesting that the PI3K / Akt pathway is involved in the inhibitory effect of 22,23-dehydro-shearinine A on 6-OHDA-induced PC12 cell damage.

[0048] (4) Effect of 22,23-dehydro-shearinine A on reactive oxygen species (ROS) induced by 6-OHDA in PC12 cells

[0049] Oxidative stress is a significant factor in neuronal damage. 6-OHDA can induce reactive oxygen species (ROS) production, leading to cell damage and apoptosis. Therefore, DCFH-DA and flow cytometry were used to detect the effect of 22,23-dehydro-shearinine A on ROS induced by 6-OHDA in PC12 cells. The results are as follows: Figure 3 As shown in Figure D, compared with the 6-OHDA group, pretreatment of 6-OHDA-induced PC12 cells with 22,23-dehydro-shearinine A significantly reduced intracellular ROS production. Within the assay range, the 25 μM concentration pretreatment showed the best effect.

Claims

1. The use of an indole diterpenoid compound, 22,23-dehydro-shearinine A, in the production of a drug or drug additive with neuroprotective effects.

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