Alkaloid compound Mallochiphilone and its application in preparing neuroprotective drugs

Abstract

The present invention discloses an alkaloid compound Mallochiphilone and its application in the preparation of neuroprotective drugs, belonging to the field of biomedicine. The alkaloid compound Mallochiphilone was isolated from the fungus ACD-5, a co-epiphyte of sea cucumbers in the South China Sea. Experiments have shown that the compound can significantly improve the survival rate of mouse hippocampal neuronal cells HT-22, has no cytotoxicity to them, and has a significant reversal effect on the nerve damage of HT-22 cells induced by Aβ25-35. Therefore, the compound as an active ingredient for the preparation of neuroprotective drugs has a wide range of application prospects. Based on the natural origin of the compound Mallochiphilone and its potential neuroprotective effect, the present invention also strives to provide a safer, more effective and economical solution for the treatment of neurodegenerative diseases, solving the problems of large toxic side effects and high prices of existing drugs for the treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an alkaloid compound Mallochiphilone and an application thereof in the preparation of neuroprotective drugs. Background Art

[0002] Neurodegenerative diseases, including conditions such as Alzheimer's disease (AD), are caused by the gradual loss of neurons in the brain and spinal cord, resulting in symptoms such as memory loss, difficulty moving, and impaired consciousness. With the aging trend of the global population and changes in environmental conditions, the public health burden of such diseases is increasing.

[0003] Alzheimer's disease seriously affects the health and quality of life of the elderly. Studies have shown that one of its main pathological characteristics is the abnormal accumulation of beta-amyloid protein (Aβ) in the brain. The deposition of Aβ between neurons forms senile plaques and neurofibrillary tangles, which damage multiple brain regions including the hippocampus, leading to memory loss and cognitive decline. 25-35 Fragments can be used to establish pathological states similar to AD by inducing cell models, providing an experimental basis for studying disease mechanisms and drug screening.

[0004] Currently, traditional treatments for Alzheimer's disease mainly include the use of acetylcholinesterase inhibitors (such as donepezil, galantamine and rivastigmine) and NMDA receptor antagonists (such as memantine). These drugs are designed to increase acetylcholine levels in the brain or regulate the effects of glutamate, thereby improving cognitive function. Although these drugs have certain therapeutic effects on many AD patients, after long-term use, they can only temporarily relieve symptoms, cannot prevent or reverse the progression of the disease, and may be accompanied by a series of side effects. In view of this, the development of neuroprotective agents with natural origin, low toxicity and high activity is particularly important for delaying the progression of Alzheimer's disease and improving the quality of life of patients.

[0005] Azaphilone compounds are a class of natural small molecule compounds produced by fungi, including two major types: non-alkaloids and alkaloids. Existing patents (such as CN110776518B, CN104311525A, CN102453015B, CN103911407B) disclose the use of such compounds in the preparation of antibacterial agents, antifungal agents, anti-tuberculosis, anticancer and anti-diabetic drugs. However, there are few reports on the neuroprotective effects of such compounds. In particular, up to now, there has been no literature or patent reporting on the research or application of Azaphilone alkaloids in the neuroprotective effect of Aβ-induced nerve damage. Therefore, exploring the potential value of such compounds in the treatment of neurodegenerative diseases, especially their possible neuroprotective effects, is a direction worthy of further research. Summary of the Invention

[0006] The purpose of the present invention is to provide an alkaloid compound Mallochiphilone and its use in the preparation of neuroprotective drugs, so as to solve the problems existing in the above-mentioned prior art and fill the gap in the application of Azaphilone alkaloids in the field of neuroprotection of Aβ-induced nerve damage. The use of the new compound Mallochiphilone of this type of structure as an effective active ingredient in the preparation of neuroprotective drugs has broad prospects. Based on the natural source of the compound Mallochiphilone and its potential neuroprotective effect, the present invention also strives to provide a safer, more effective and economical solution for the treatment of neurodegenerative diseases, and solve the problems of large toxic side effects and high prices of existing drugs for treating neurodegenerative diseases.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an alkaloid compound Mallochiphilone, whose chemical structure is shown in the following formula (I):

[0009]

[0010] The present invention also provides the use of the alkaloid compound Mallochiphilone in any of the following:

[0011] (1) Application in the preparation of neuroprotective drugs;

[0012] (2) Use in the preparation of drugs for alleviating and / or reversing nerve damage;

[0013] (3) Application in the preparation of drugs for increasing the activity of nerve cells;

[0014] Wherein, the alkaloid compound includes a compound represented by formula (I) or a salt or ester thereof;

[0015]

[0016] Preferably, the nerve injury includes Aβ25-35-induced HT-22 cell nerve injury.

[0017] The present invention also provides a neuroprotective drug, the effective active ingredient of which includes a compound represented by the following formula (I) or a salt or ester thereof:

[0018]

[0019] The present invention also provides a drug for alleviating and / or reversing nerve damage, wherein the effective active ingredient comprises a compound represented by the following formula (I) or a salt or ester thereof:

[0020]

[0021] The present invention also provides a drug for increasing the activity of nerve cells, wherein the effective active ingredient comprises a compound represented by the following formula (I) or a salt or ester thereof:

[0022]

[0023] Preferably, the drug further comprises pharmaceutically acceptable carriers and excipients.

[0024] The present invention also provides a method for preparing the alkaloid compound Mallochiphilone, comprising the following steps:

[0025] A solid culture of the ACD-5 strain of the symbiotic fungus of sea cucumbers in the South China Sea was obtained, and the solid culture was extracted with an organic solvent. The extract was concentrated to obtain a crude extract.

[0026] The crude extract is subjected to silica gel column chromatography and detected by silica gel thin layer chromatography, and the eluted fraction containing the target substance is further purified by silica gel column chromatography, gel column chromatography and reverse phase silica gel column chromatography using different elution systems to obtain the alkaloid compound Mallochiphilone.

[0027] Preferably, the organic solvent is selected from one or more of chloroform, dichloromethane, acetone, ethyl acetate, ethanol or methanol;

[0028] The eluent for the silica gel column chromatography is selected from one or more of n-hexane, ethyl acetate, methanol, chloroform and dichloromethane; the eluent for the gel column chromatography is selected from one or more of dichloromethanol, chloroform and methanol; and the eluent for the reversed-phase silica gel column chromatography is selected from one or more of methanol, acetonitrile and water.

[0029] Preferably, the chromatographic characteristics of the alkaloid compound Mallochiphilone are: under the conditions of high performance liquid chromatography analysis using a Kinetex C18 chromatographic column, isocratic elution for 10 minutes with 75% methanol-water as the eluent and a flow rate of 0.6 mL / min, the retention time is 6.28 minutes; on a GF254 thin layer chromatography silica gel plate with dichloromethane and methanol in a volume ratio of 10:1 as the developing solvent, the Rf value is 0.6, appearing as a red substance, and concentrated sulfuric acid-anisaldehyde develops as brown-green spots.

[0030] The present invention discloses the following technical effects:

[0031] The present invention provides a compound Mallochiphilone from the South China Sea sea cucumber symbiotic fungus ACD-5, and experiments have shown that the compound Mallochiphilone can significantly improve the survival rate of mouse hippocampal neuronal cells HT22 and has a significant reversal effect on Aβ25-35-induced HT22 cell nerve damage; in addition, the compound Mallochiphilone is highly effective and has no obvious toxic side effects. Therefore, the compound Mallochiphilone can be applied to the preparation of neuroprotective drugs, drugs that reduce / reverse Aβ25-35-induced nerve cell damage, and drugs that improve nerve cell activity, laying a theoretical foundation for effectively solving the problems of strong toxic side effects and high costs in existing drugs for treating neurodegenerative diseases (especially Alzheimer's disease), showing broad application prospects in the preparation of drugs for preventing / treating neurodegenerative diseases, and providing new technical approaches and treatment methods for effectively responding to neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The key HMBC (arrow) and 1 H- 1 H COSY (bold) correlation;

[0034] Figure 2 HR-ESI-MS spectrum of the compound Mallochiphilone;

[0035] Figure 3 Mallochiphilone1 H NMR spectrum (700 MHz, DMSO-d6);

[0036] Figure 4 Mallochiphilone 13 C NMR spectrum (176 MHz, DMSO-d6)

[0037] Figure 5 Mallochiphilone 1 H- 1 H COSY spectrum (700 MHz, DMSO-d6)

[0038] Figure 6 This is the HMBC spectrum of the compound Mallochiphilone (176MHz, DMSO-d6)

[0039] Figure 7 HSQC spectrum of the compound Mallochiphilone (176MHz, DMSO-d6)

[0040] Figure 8 The cytotoxicity of Mallochiphilone and its effect on Aβ 25-35 induced loss of cell viability. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The South China Sea rough sea cucumber symbiotic fungus (Penicillium mallochii) ACD-5 involved in the present invention has been disclosed in the document "The screening for marine fungal strains with high potential in alkaloids production by in situcolony assay and LC-MS / MS based secondary metabolic profiling" and was deposited in the Guangdong Provincial Microbial Culture Collection on April 24, 2022, with a deposit number of GDMCC No. 62411 and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology.

[0047] Example 1 Preparation of Azaphilone Compound Mallochiphilone

[0048] (1) Fermentation

[0049] The ACD-5 strain of the symbiotic fungus of sea cucumbers in the South China Sea was inoculated into a solid culture medium and fermented for 30 days to obtain a culture; wherein the formula of the solid culture medium is: 1L of pure water contains 20g of artificial sea salt and 1000g of brown rice.

[0050] (2) Rough extraction

[0051] The solid culture obtained in step (1) was extracted with methanol ultrasonically for 3 times and concentrated, each ultrasonication for 30 minutes to obtain a crude extract;

[0052] (3) Separation and purification

[0053] The crude extract obtained in step (2) was crudely separated by silica gel column chromatography (mobile phase: n-hexane, n-hexane-ethyl acetate (v / v=1:1), ethyl acetate, ethyl acetate-methanol (v / v=1:1), methanol, three column volumes each), silica gel column chromatography (mobile phase: n-hexane-ethyl acetate (v / v=15:1, 10:1, 5:1, 3:2, 2:3 and 1:5), ethyl acetate, ethyl acetate-methanol (v / v=1:1), methanol), silica gel column chromatography (mobile phase: dichloromethane-methanol (v / v=80:1, 40:1)), gel column chromatography (mobile phase: dichloromethane-methanol, v / v=1:1), reversed-phase silica gel column chromatography (mobile phase:

[0054] The target substance with specific chromatographic characteristics was gradually purified using methanol-water (v / v = 70:30, 80:20) until it was purified. The target substance was characterized by a red substance with an Rf value of approximately 0.6 on a GF254 TLC plate developed in dichloromethane:methanol (v / v = 10:1). Concentrated sulfuric acid-anisaldehyde developed as brownish-green spots. High-performance liquid chromatography (HPLC) analysis using a Kinetex C18 column (4.6 mm × 100 mm, 5 μm) with an isocratic elution of 75% methanol-water for 10 minutes at a flow rate of 0.6 mL / min yielded a retention time of 6.28 minutes. The target substance was observed as red, needle-shaped crystals that were readily soluble in methanol.

[0055] (4) Identification

[0056] The collected product was subjected to spectral analysis and structural identification. The results showed that the collected product was a pure compound with the molecular formula C 28 H 38 ClNO6, named Mallochiphilone, has a structural formula as shown in formula (I), and is a new structural compound:

[0057]

[0058] In addition, the compound Mallochiphilone has the following physical and chemical properties and spectral characteristics: Mallochiphilone is a red needle-shaped crystal. 1 H. 13 The C NMR data are shown in Table 1, and the relevant spectral information is shown in Figure 1-Figure 7As shown. In the HMBC experiment, correlations can be observed between H-21 and C-22 and C-23, H-22 and C-21, C-23 and C-24, H-23 and C-21, C-22, C-24 and C-25, H-24 and C-22, C-23 and C-25, and H-25 and C-23, C-24, C-26 and C-27. The correlations between H-21 and H-22, H-22 and H-21, H-23, H-23 and H-22, H-24, H-24 and H-23, H-25, H-25 and H-24, H-26 / 27, and H-26 / 27 and H-25 are shown by 1 H- 1 H COSY experiments determined ( Figure 1 ), the compound is a new structural compound, which has a substituent group at the 2-N position that is different from the known Azaphilone compounds. The charge-to-mass ratio in high-resolution mass spectrometry is 520.2468 ([M+H] + , theoretical value 520.2460) confirmed its molecular formula C 28 H 38 ClNO6

[0059] Table 1 Compound Mallochiphilone 1 H. 13 C NMR data (in DMSO-d6, TMS, 1 H NMR is 700 MHz, 13 C NMR is 175 MHz, δ is in ppm)

[0060]

[0061]

[0062] Example 2 Determination of Neuroprotective Activity of Azaphilone Compound Mallochiphilone

[0063] 1. Experimental methods

[0064] (1) Cell viability assay

[0065] To evaluate the cytotoxicity of Mallochiphilone, a cell viability assay was performed. First, HT22 cells were plated at 1.5 × 10 4The cells were seeded into 96-well plates at a density of 100 cells / well. After 24 hours, the cell culture medium was replaced with DMEM medium containing different concentrations of Mallochiphilone (1.92 μM, 9.62 μM, 19.23 μM, 38.46 μM) and cultured for another 48 hours. Afterwards, the culture medium in the wells was discarded, and 100 μL of 5 mg / mL MTT solution was added to each well and incubated in the dark at 37°C and 5% CO2 for 4 hours. Next, the MTT solution in the wells was discarded, 100 μL of DMSO was added, the plates were shaken for 10 minutes (440 rpm), and the absorbance was measured at a wavelength of 570 nm using a microplate reader. Based on the measured absorbance value, the cell viability under the action of Mallochiphilone was calculated.

[0066] (2) Aβ 25-35 Post-injury survival rate test

[0067] To further explore whether Mallochiphilone has neuroprotective effects, a mouse model composed of Aβ 25-35 Induced cell injury model. First, HT22 cells were plated at 1.5×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and treated with DMEM medium containing different concentrations of Mallochiphilone (1.92 μM, 9.62 μM, 19.23 μM, 38.46 μM) for 24 hours. 25-35 (concentration of 20 μM) was added to construct a cell injury model; the control group was added with the same volume of DMEM. After 24 hours of continuous culture, MTT assay was performed according to the above steps, and the absorbance value was used to calculate the effect of Mallochiphilone on Aβ 25-35 induced cell damage alleviation effect.

[0068] The calculation formula of cell viability is as follows: Cell viability (%) = A 570 (Mallochiphilone group) / A 570 (Control group) × 100%.

[0069] The graph was drawn with different compound concentrations as the abscissa and cell viability as the ordinate using statistical software R4.2.3, and the P value was calculated by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test.

[0070] 2. Experimental results

[0071] like Figure 8Figure 2 shows the results of the neuroprotective activity assay of Mallochiphilone. MTT assay results showed that Mallochiphilone had no obvious cytotoxic side effects on HT22 cells within the concentration range of 1.92 μM to 38.46 μM. In addition, Mallochiphilone was able to effectively protect HT22 cells from Aβ 25-35 When the concentration of Mallochiphilone ranged from 1.92μM to 38.46μM, Aβ 25-35 When the concentration was fixed at 20 μM, the cell viability of the treated group was significantly improved compared with the model group, especially at a concentration of 19.23 μM, which showed the best protective effect.

[0072] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An alkaloid compound Mallochiphilone, characterized in that Its chemical structure is shown in the following formula (I): 。 2. The use of the alkaloid compound Mallochiphilone in the preparation of neuroprotective drugs, characterized in that: The alkaloid compound is selected from the compound represented by formula (I) or a salt thereof: 。 3. Use of the alkaloid compound Mallochiphilone in the preparation of a drug for alleviating and / or reversing nerve damage, characterized in that: The alkaloid compound is selected from the compound represented by formula (I) or a salt thereof: 。 4. The use according to claim 3, characterized in that The nerve injury is selected from HT-22 cell nerve injury induced by Aβ25-35.

5. Use of the alkaloid compound Mallochiphilone in the preparation of a drug for increasing nerve cell activity, characterized in that: The alkaloid compound is selected from the compound represented by formula (I) or a salt thereof: 。 6. A drug for protecting nerves, characterized in that: The effective active ingredients include the compound represented by the following formula (I) or its salt: 。 7. A drug for alleviating and / or reversing nerve damage, characterized in that: The effective active ingredients include the compound represented by the following formula (I) or its salt: 。 8. A drug for increasing the activity of nerve cells, characterized in that: The effective active ingredients include the compound represented by the following formula (I) or its salt: 。 9. The drug according to any one of claims 6 to 8, characterized in that The medicine also includes pharmaceutically acceptable carriers and excipients.

10. A method for preparing the alkaloid compound Mallochiphilone according to claim 1, characterized in that: The following steps are involved: (1) The ACD-5 strain of the symbiotic fungus of sea cucumbers from the South China Sea was inoculated into a solid culture medium and allowed to ferment for 30 days to obtain a culture; wherein the formula of the solid culture medium is: 20 g of artificial sea salt and 1000 g of brown rice in 1 L of pure water; the ACD-5 strain of the symbiotic fungus of sea cucumbers from the South China Sea has a deposit number of GDMCC No. 62411; (2) extracting the solid culture obtained in step (1) by ultrasonic extraction with methanol and concentrating to obtain a crude extract; (3) The crude extract obtained in step (2) is subjected to silica gel column chromatography, and after detection by silica gel thin layer chromatography, the eluted fraction containing the target substance is further purified by silica gel column chromatography, gel column chromatography and reverse phase silica gel column chromatography using different elution systems, and the target substance with specific chromatographic characteristics is gradually tracked and purified until a pure compound is obtained, thereby obtaining the alkaloid compound Mallochiphilone; The mobile phases for silica gel column chromatography of the crude extract were n-hexane; n-hexane-ethyl acetate, volume ratio 1:1; ethyl acetate; ethyl acetate-methanol, volume ratio 1:1; methanol, three column volumes each; The mobile phases for the silica gel column chromatography with different elution systems were n-hexane-ethyl acetate in volume ratios of 15:1, 10:1, 5:1, 3:2, 2:3, and 1:5, respectively; ethyl acetate; ethyl acetate-methanol in volume ratio of 1:1; methanol; and dichloromethane-methanol in volume ratios of 80:1 and 40:

1. The mobile phase of the gel column chromatography was dichloromethanol-methanol, with a volume ratio of 1:1; The mobile phase of the reversed-phase silica gel column chromatography was methanol-water with a volume ratio of 70:30 and 80:20, respectively; The chromatographic characteristics of the alkaloid compound Mallochiphilone are as follows: under high-performance liquid chromatography analysis conditions using a Kinetex C18 column, isocratic elution for 10 minutes with 75% methanol-water as the eluent, and a flow rate of 0.6 mL / min, the retention time is 6.28 minutes; on a GF254 thin-layer chromatography silica gel plate using dichloromethane and methanol in a volume ratio of 10:1 as the developing solvent, the Rf value is 0.6, appearing as a red substance, and concentrated sulfuric acid-anisaldehyde develops as brown-green spots.

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