Preparation method and application of atestin diterpenoid compound Agallochaol Q

Abstract

The invention provides a preparation method and application of an atesane diterpenoid compound Agallochaol Q. The atesane diterpenoid compound Agallochaol Q is extracted and separated from stems and branches of sea lactuca collected from Branchiostoma City, Hainan Province. According to the present invention, the prepared atesane diterpenoid compound Agallochaol Q provides significant inhibition activity for influenza A virus and influenza B virus, such that the atesane diterpenoid compound has great development prospects and great research values in the anti-influenza virus aspect; and a new choice can be provided for research screening and clinical application of novel anti-influenza virus drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a preparation method of an athysenane diterpenoid compound Agallochaol Q and its application. Background Art

[0002] Influenza is an infectious respiratory disease caused by influenza virus infection. It has the characteristics of short incubation period, rapid onset, strong infectiousness and rapid spread. Its clinical manifestations include fever, cough, inflammation and general weakness. Influenza can cause many fatal diseases such as alveolar hemorrhage, pulmonary edema, acute bronchopneumonia, acute respiratory distress syndrome, etc. Therefore, there is an urgent need to explore new safe and effective antiviral drugs.

[0003] Influenza virus belongs to the family Orthomyxoviridae. It is an enveloped RNA virus with a single-stranded negative-strand genome. As one of the pathogens that induce acute respiratory diseases, it is mainly transmitted through droplets in the air or contact with infected people, which seriously threatens human health. According to the difference in the antigenicity of the viral nucleoprotein, influenza viruses can be divided into four types: influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV) and influenza D virus (IDV). The influenza that is prevalent in the human population is mainly caused by IAV and IBV. Among them, IAV poses the greatest threat to human health and can cause seasonal epidemics and sporadic epidemics.

[0004] At present, the anti-influenza virus treatment strategies include vaccines and small molecule drugs. Based on the structural characteristics and life cycle of influenza viruses, the anti-influenza virus drugs that have been marketed mainly include M2 ​​ion channel inhibitors (such as adamantane and rimantadine) and neuraminidase inhibitors (such as oseltamivir phosphate, zanamivir, and laninamivir). However, there are still many limitations in the treatment of influenza viruses. Although vaccines are an effective means of preventing influenza outbreaks, they need to be reformulated every year to adapt to antigenic variations. Their research and development cycle is long and the cost is high. These shortcomings make small molecule drugs the main means of preventing and treating influenza. For example, influenza vaccines face huge challenges due to their adverse reactions and poor broad-spectrum. They develop resistance to M2 ion channel inhibitors, and adamantane and rimantadine are not recommended for self-use by patients due to their high neurotoxicity. In addition, due to the continuous mutation of the virus, it develops resistance to existing neuraminidase inhibitors. Therefore, there is an urgent need to find highly effective anti-influenza drugs. Summary of the invention

[0005] In order to overcome the problems of rapid mutation of influenza virus and drug resistance of existing anti-influenza virus drugs in clinic, the present invention provides a preparation method of an athisanane diterpenoid compound Agallochaol Q and application thereof. The athisanane diterpenoid compound Agallochaol Q is extracted and separated from stems and branches of Lacquer oleraceus collected in Wenchang City, Hainan Province. The athisanane diterpenoid compound has a significant inhibitory effect on influenza A and B viruses, and can provide a new choice for the research, screening and clinical application of novel anti-influenza virus drugs.

[0006] The technical solution adopted by the present invention to achieve the technical purpose is:

[0007] The present invention provides a method for preparing an athisanane diterpenoid compound Agallochaol Q, comprising the following steps:

[0008] S1. Dry the stems and branches of the lacquer tree and crush them into coarse powder;

[0009] S2, soaking the crude powder obtained in step S1 with anhydrous ethanol and stirring for extraction, concentrating under reduced pressure to obtain an extract, and extracting the obtained extract with a water-n-hexane solvent to obtain a n-hexane layer extract;

[0010] S3, subjecting the n-hexane layer extract obtained in step S2 to normal phase silica gel column chromatography, selecting petroleum ether-ethyl acetate as the elution system, performing gradient elution thereto, and collecting fractions 90 to 100;

[0011] S4, combining Fr.90 to Fr.100 collected in step S3, separating and purifying the combined sample through an ODS (octadecylsilane bonded silica gel filler) column, using acetone-water as an elution system for gradient elution, and collecting a component with an acetone volume fraction of 65%;

[0012] S5. Separate and purify the component with acetone volume fraction of 65% obtained in step S4 by semi-preparative high performance liquid chromatography to obtain the atissenane diterpene compound Agallochaol Q.

[0013] The structure of the atissenane diterpenoid compound Agallochaol Q is shown in formula (I):

[0014]

[0015] Furthermore, in step S1, the stems and branches of the Aconitum carmichaelii were collected from Wenchang City, Hainan Province.

[0016] Furthermore, in step S2, the volume ratio of water to n-hexane in the water-n-hexane solvent is 1:3.

[0017] Furthermore, in step S3, gradient elution is performed with a petroleum ether-ethyl acetate mixed solvent with volume ratios of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:100, in sequence.

[0018] Furthermore, in step S4, gradient elution is performed with acetone-water solvents having volume ratios of 40:60, 55:45, 65:35, 75:25, 85:15, and 100:0, respectively.

[0019] Furthermore, in step S5, the mobile phase of the semi-preparative HPLC is 60% acetonitrile-water solution.

[0020] Furthermore, in step S5, the flow rate of the semi-preparative HPLC is 3 mL / min, and the detection wavelength is 195 nm.

[0021] The present invention also provides the use of the athisanane diterpenoid compound Agallochaol Q and its pharmaceutically acceptable salts, solvates or stereoisomers in the preparation of anti-influenza virus drugs.

[0022] Among them, the athisanane diterpenoid compound is preferably obtained by the above preparation method.

[0023] Furthermore, the influenza virus includes influenza A virus and / or influenza B virus.

[0024] Furthermore, the influenza A virus includes H1N1 A / PR / 8 / 34 and H1N1 ZX / 1109.

[0025] Furthermore, the influenza B virus includes B / Florida / 78 / 2015.

[0026] The present invention also provides an anti-influenza virus drug, comprising any one or more of the athisanane diterpenoid compound Agallochaol Q, its pharmaceutically acceptable salt, its solvate, and its stereoisomer.

[0027] Among them, the athisanane diterpenoid compound Agallochaol Q is preferably obtained by the above preparation method.

[0028] Furthermore, the anti-influenza virus drug also includes pharmaceutically acceptable excipients.

[0029] Furthermore, the anti-influenza virus drug is an oral agent, an injection, an external agent or an inhalation agent.

[0030] Preferably, the oral dosage form includes tablets, capsules, pills, granules or oral liquids.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The athysenane diterpenoid compound Agallochaol Q prepared by the present invention exhibits significant inhibitory activity against both influenza A virus and influenza B virus, wherein the half effective concentration (EC 50) of influenza A virus strain (H1N1 A / PR / 8 / 34) and influenza B virus resistant strain (B / Florida / 78 / 2015) is 2.34%. 50 ) is superior to the positive control drug oseltamivir, so the artesanane diterpenoid compound has great development prospects and research value in anti-influenza virus, and is expected to be further designed and developed into a new anti-influenza virus drug in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The obtained athysenane diterpenoid compound Agallochaol Q in Example 1 H NMR spectrum.

[0034] Figure 2 The obtained athysenane diterpenoid compound Agallochaol Q in Example 13 C NMR spectrum.

[0035] Figure 3 This is the HRESIMS spectrum of the athisanane diterpenoid compound Agallochaol Q obtained in Example. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Example

[0039] 1. Preparation of Agallochaol Q, an Artisanane Diterpenoid

[0040] The specific steps include:

[0041] S1. Raw material processing: drying and crushing the stems and branches of the lacquerwood collected from Wenchang City, Hainan Province;

[0042] S2. Preparation and extraction of extract: soak the obtained sea lacquer sample with 95% ethanol and stir to extract, and the extract is concentrated under reduced pressure to obtain sea lacquer extract; suspend the obtained extract in water, and extract with a water-n-hexane mixed solvent with a volume ratio of 1:3 to obtain a n-hexane layer extract;

[0043] S3, column chromatography separation: the hexane layer extract of the sea lacquer was separated by a normal phase silica gel column, and the elution system was selected as petroleum ether-ethyl acetate. The volume ratio of petroleum ether-ethyl acetate was 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:100, and one fraction was collected for every 1L. A total of 314 fractions, i.e., Fr.1 to Fr.314, were collected. Subsequently, high performance liquid chromatography was used to separate the extract. The similar fractions Fr.90 to Fr.100 were combined according to the TLC analysis results, and the combined fractions were separated and purified by ODS (octadecylsilane bonded silica gel filler) column chromatography, acetone-water was selected as the elution system, and the acetone-water solution was gradient eluted with volume fractions of 40%, 55%, 65%, 75%, 85%, and 100%, respectively, to obtain 51 sub-fractions (Fr.90-100-1 to Fr.90-100-51);

[0044] S4. Preparation of monomer compounds: Component Fr.90-100-14 (65% acetone aqueous solution) was separated and purified by semi-preparative high performance liquid chromatography with a mobile phase of 60% acetonitrile / water at a flow rate of 3 mL / min, a detection wavelength of 195 nm, and a retention time of 59 min to obtain the atissenane diterpene compound Agallochaol Q shown in formula (I).

[0045]

[0046] 2. Structural confirmation of atisanane diterpenoids

[0047] The athysenane diterpenoid compound Agallochaol Q prepared in step 1 was subjected to 1 H NMR spectrum, 13 CNMR spectrum and HR-ESIMS spectrum are measured, and the corresponding spectra are Figures 1 to 3 As can be seen from the results, a series of spectrum data and NMR data of the obtained compound all correspond to its structural formula, and it can be confirmed that its structure is the structure shown in formula (I).

[0048] Test case: Anti-influenza virus activity test

[0049] 1. Cell lines and virus strains

[0050] Cell lines: Canine kidney cells (MDCK cells) were purchased from the American Type Culture Collection (ATCC);

[0051] Influenza virus:

[0052] (1) Influenza A viruses: H1N1 A / PR / 8 / 34; H1N1 ZX / 1109;

[0053] (2) Influenza B virus: B / Florida / 78 / 2015.

[0054] 2. Preparation of reagents

[0055] DMEM medium containing 10% fetal bovine serum: 500 mL of basic DMEM medium, 50 mL of fetal bovine serum (FBS) and 5 mL of penicillin-streptomycin double antibody are fully mixed, the bottle is marked with information, and stored at 4°C for use;

[0056] 2μg / mL trypsin (TPCK-treated) in D / F-12: 500mL D / F-12 basal medium, 125μL 8mg / ml trypsin (TPCK-treated) and 5mL penicillin-streptomycin double antibody were mixed thoroughly and stored at 4℃ until use.

[0057] 3. Cell viability detection

[0058] Cell Viability Luminescent Cell Viability Assay.

[0059] The specific experimental steps are as follows:

[0060] (1) MDCK cells were inoculated into 96-well white-bottom plates (1.5×10 4 cells / mL, 100 μL / well), incubated overnight in a 37°C, 5% CO2 incubator;

[0061] (2) Discard the original culture medium and add 100 μL of 2 μg / mL trypsin (TPCK-treated) in D / F-12 to the control well, sample well, and virus control well respectively;

[0062] (3) The TCID 50 The influenza virus supernatant was diluted to 100× TCID with 2 μg / mL trypsin (TPCK treated) in D / F-12. 50 , the compound was diluted to 4-fold working concentration with 2 μg / mL trypsin (TPCK-treated) in D / F-12;

[0063] (4) Add the diluted virus solution and the test compound (50 μL / well) to a 96-well plate;

[0064] (5) The 96-well plate was placed in a cell culture incubator (37°C, 5% CO2) and cultured for 3 days. The cell status and CPE process were observed under a microscope every day.

[0065] (6) When the virus wells show changes in cell morphology, cell death, or loss of cell function, discard the original culture medium and add 7:3 diluted PBS. The detection solution was shaken and lysed for 5 minutes in the dark, and the luminescent signal was allowed to stand for 3 minutes to stabilize, and the luminescent value of each well was detected by an enzyme-labeled instrument;

[0066] (7) The CPE inhibition rate was calculated according to the following formula:

[0067] %Inhibition=(Sample-Aver(virus)) / (Aver-Aver(virus))

[0068] (Sample represents the luminescence value of the sample well, and Aver represents the average luminescence value)

[0069] EC was calculated using OriginPro 9.0 software 50 When the EC 50 When the value cannot be calculated, it is expressed as >100 μM (i.e., greater than the maximum working concentration);

[0070] (8) The cytotoxicity of the compound was determined in the same system as above (without adding virus). The cytotoxicity was calculated as follows:

[0071] %Cell toxicity=[1-Sample / (Aver(cell))]×100%

[0072] (Sample represents the luminescence value of the sample well, and Aver represents the average luminescence value)

[0073] When the cytotoxicity was less than 10%, the compound was considered non-toxic to cells. CC was calculated using OriginPro 9.0 software. 50 value; when the CC 50 When the value cannot be calculated, it is expressed as >100 μM (i.e., greater than the maximum working concentration);

[0074] (9) The selectivity index (SI) is calculated as follows:

[0075] SI=CC 50 / EC 50

[0076] When the SI of a compound cannot be calculated, it is indicated by “-”.

[0077] 4. Test results

[0078] Table 1 Test results of anti-influenza virus activity of athisanane diterpenoids

[0079]

[0080] The anti-influenza virus test results of the athysenane diterpenoid compound Agallochaol Q of the present invention are shown in Table 1, indicating that the compound has a significant inhibitory effect on influenza A virus (H1N1 A / PR / 8 / 34, H1N1 ZX / 1109) and influenza B virus (B / Florida / 78 / 2015), among which the EC 50 The values ​​were 2.82±0.13μM and 0.54±0.42μM, respectively, both better than the positive control drug oseltamivir.

[0081] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing an athisanane diterpenoid compound Agallochaol Q, comprising the following steps: S1. Dry the stems and branches of the lacquer tree and crush them into coarse powder; S2, soaking the crude powder obtained in step S1 with anhydrous ethanol and stirring for extraction, concentrating under reduced pressure to obtain an extract, and extracting the obtained extract with a water-n-hexane solvent to obtain a n-hexane layer extract; S3, subjecting the n-hexane layer extract obtained in step S2 to normal phase silica gel column chromatography, selecting petroleum ether-ethyl acetate as the elution system, performing gradient elution thereto, and collecting fractions 90 to 100; S4, combining Fr.90 to Fr.100 collected in step S3, separating and purifying the combined sample through an ODS column, performing gradient elution using acetone-water as an elution system, and collecting a component with an acetone volume fraction of 65%; S5. Separate and purify the component with acetone volume fraction of 65% obtained in step S4 by semi-preparative high performance liquid chromatography to obtain the atissenane diterpene compound Agallochaol Q.

2. The preparation method according to claim 1, characterized in that: In step S2, the volume ratio of water to n-hexane in the water-n-hexane solvent is 1:

3.

3. The preparation method according to claim 1, characterized in that: In step S3, gradient elution is performed with a petroleum ether-ethyl acetate mixed solvent with volume ratios of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:100, in sequence.

4. The preparation method according to claim 1, characterized in that: In step S4, gradient elution is performed with acetone-water solvents having volume ratios of 40:60, 55:45, 65:35, 75:25, 85:15, and 100:0, respectively.

5. The preparation method according to claim 1, characterized in that: In step S5, the mobile phase of the semi-preparative HPLC is 60% acetonitrile-water solution.

6. The preparation method according to claim 1, characterized in that: In step S5, the flow rate of the semi-preparative HPLC is 3 mL / min, and the detection wavelength is 195 nm.

7. Use of the athysenane diterpenoid compound Agallochaol Q and its pharmaceutically acceptable salts, solvates or stereoisomers in the preparation of anti-influenza virus drugs.

8. The use according to claim 7, characterized in that: The athisanane diterpenoid compound Agallochaol Q is obtained by the preparation method according to any one of claims 1 to 6.

9. An anti-influenza virus drug comprising any one or more of the athysenane diterpenoid compound Agallochaol Q, its pharmaceutically acceptable salts, its solvates, and its stereoisomers.

10. An anti-influenza virus drug according to claim 9, characterized in that: The athisanane diterpenoid compound Agallochaol Q is obtained by the preparation method according to any one of claims 1 to 6.

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