A compound extracted from Amomum villosum and its preparation method and application

Abstract

A compound extracted from Amomum villosum and its preparation method and application belong to the field of Chinese medicine extraction, specifically to a compound of podocarpane type diterpenes or hemicarpane type diterpenes or kavalactones as shown in formula (I)-(IV) and its extraction method, wherein the compound has the effect of inhibiting the release of NO activity of mouse macrophages RAW264.7 induced by lipopolysaccharide (LPS) and protecting the oxidative damage of mouse neuromicroglioma cells BV-2 induced by hydrogen peroxide (H2O2), and can be used to prepare anti-inflammatory and antioxidant drugs. The present invention further enriches the structural diversity of the active substances of Amomum villosum, and on this basis, lays the foundation for the subsequent monomer compounds to carry out relevant biological activity tests, provides active lead compounds for new drug development, and also provides a theoretical basis for the in-depth research and development of Amomum villosum medicinal materials.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine extraction, and specifically relates to a podocarpane-type diterpenoids or hemicarpane-type diterpenoids or kavalactone compounds separated from Amomum villosum, a preparation method thereof, and application of the compounds in preparing anti-inflammatory drugs. Background Art

[0002] Amomum villosum is a perennial herb in the Zingiberaceae family, the genus Amomum. It is a traditional Chinese medicine and is concentrated in southern China, such as Yunnan, Guangdong, Guangxi, Hainan, etc. The first record of its use as a medicine was in the Treatise on Materia Medica. It is warm in nature and sweet in taste. It enters the spleen, stomach, and kidney meridians and has the effects of removing dampness and stimulating appetite, warming the spleen and stopping diarrhea, and regulating qi and stabilizing pregnancy (Xia Dongjie, Zhang Guijun, Liu Yang, Fang Li. Overview of the research on the Chinese medicine Amomum villosum in recent years [J]. Information on Traditional Chinese Medicine, 1996, 2:19-20.). Modern pharmacological studies have shown that Amomum villosum has gastrointestinal protective effects (Gao Y, Sun T, Xie Y. Effect of Amomun longiligulare on TFF1 and TFF1 mRNA of gastric mucosa in rats with hepatogenic ulcer [J]. Shanxi Journal of Traditional Chinese Medicine, 2016, 32 (7): 52-54.), antioxidant effects (Tang Jianyang, Liu Fengjiao, Su Mingxing, Huang Sufang, Chen Jingying, Chen Qingxi. Study on the antibacterial and antioxidant effects of Amomum villosum extract [J]. Journal of Xiamen University (Natural Science Edition), 2012, 51 (4): 789-792.), and anti-inflammatory effects (Lee JA, Lee MY, Shin IS, Seo CS, Ha H, Shin H K. Anti-inflammatory effects of Amomum Compactum on RAW 264.7 cells via induction of heme oxygenase-1 [J]. Archives of Pharmacal Research, 2012, 35(4):739-746.) and anti-allergic effects (Kim SH, Lee S, Kim IK, Kwon TK, Moon JY, Park WH, Shin TY. Suppression of mast cell-mediated allergic reaction by Amomum Xanthiodes[J]. Food and Chemical Toxicology, 2007, 45(11):2138-2144.). Summary of the invention

[0003] The primary purpose of the present invention is to provide a compound extracted from Amomum villosum, which is a podocarpane-type diterpenoid or hemiaran-type diterpenoid or kavalactone compound as shown in formula (I)-(IV), or an isomer of the compound, or a pharmaceutically acceptable salt of the compound.

[0004] The second object of the present invention is to provide a method for extracting the podocarpane-type diterpenes or hemerocalactone-type diterpenes or kavalactone compounds.

[0005] The third object of the present invention is to provide a pharmaceutical composition containing podocarpane-type diterpenes or hemerocalactane-type diterpenes or kavalactone compounds.

[0006] The fourth object of the present invention is to provide the use of the podocarpane-type diterpenoids or hemerocalactone-type diterpenoids or kavalactone compounds, or isomers of the compounds, or pharmaceutically acceptable salts of the compounds, or pharmaceutical compositions containing the compounds in the preparation of anti-inflammatory and / or antioxidant drugs.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The general structural formula (I) (II) (III) (IV) is as follows:

[0009]

[0010] In the above general structural formula: R1 is selected from one of hydrogen, hydroxyl, and methoxy; R2 is selected from one of hydrogen, hydroxyl, methoxy, and carbonyl; R3 and R4 are each independently selected from one of methyl, carboxyl, aldehyde, and ester; R5 and R6 are each independently selected from one of ester and 4-methoxydihydropyrone.

[0011] Furthermore, the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds are any one of the compounds shown in the following structural formulas 1-4, or isomers of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds, or pharmaceutically acceptable salts of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds.

[0012]

[0013] The pharmaceutically acceptable salt of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds is a mixture of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds and a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is selected from one or more of sodium salt, potassium salt, ammonium salt, hydrochloride and sulfate.

[0014] The isomers of the podocarpane-type diterpenes, hemiarthane-type diterpenes or kavalactone compounds are selected from optical isomers, cis-trans isomers, racemates and mixtures thereof.

[0015] The present invention also provides a method for extracting the above-mentioned podocarpane-type diterpenoid compound 1, hemerocalcinane-type diterpenoid compound 2, and kavalactone compounds 3 and 4, comprising the following steps:

[0016] (1) Using the stem and leaf parts of Amomum villosum as raw materials, adding ethanol aqueous solution with a volume concentration of 80%-95%, reflux extraction 2-4 times, each extraction for 2-4 hours, combining the obtained extracts, recovering the solvent under reduced pressure, and concentrating to obtain a total extract;

[0017] (2) dispersing the total extract into 5-15 times the mass volume of water, extracting with 5-15 times the mass volume of petroleum ether and ethyl acetate in sequence, recovering the solvent, and obtaining a petroleum ether extract concentrate, an ethyl acetate extract concentrate and an aqueous phase, respectively;

[0018] (3) the ethyl acetate extract concentrate was separated by silica gel column chromatography using a gradient elution of dichloromethane-methanol in a volume ratio of 100:1 to 0:1, and a fraction E3 with a volume ratio of 50:1 was collected;

[0019] (4) After fraction E3 was concentrated, the concentrate E3 was separated by silica gel column chromatography and gradient elution using petroleum ether-acetone in a volume ratio of 100:1-0:1 as the eluent to obtain podocarpane-type diterpenoid compound 1, hemerocalline-type diterpenoid compound 2 and kavalactone compounds 3-4.

[0020] In the above extraction method, the mass times are the mass volume ratios of the added solid substance and the liquid substance.

[0021] In the step (1), the solid-liquid ratio is: Amomum villosum stems and leaves: ethanol aqueous solution = 1 g: (7-15) mL.

[0022] The specific separation and purification process of fraction E3 in step (4) is as follows:

[0023] Fraction E3 was concentrated and separated by silica gel column chromatography, and gradient elution was performed using petroleum ether-acetone with a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1 as eluents, and the fraction with a volume ratio of petroleum ether to acetone of 30:1 was collected and recorded as E33;

[0024] Fraction E33 was concentrated and separated by ODS column chromatography, and gradient eluted with methanol-water in volume ratios of 2:8, 4:6, 5:5, 7:3, and 1:0, to collect fraction E333 in volume ratio of 4:6 and fraction E334 in volume ratio of 5:5;

[0025] Fraction E334 was concentrated and separated by silica gel column chromatography, and gradient elution was performed with petroleum ether-acetone in a volume ratio of 100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 5:2, and 0:1 as the eluent, and the fraction with a volume ratio of petroleum ether to acetone of 30:1 was collected and recorded as E3342, and the fraction with a volume ratio of petroleum ether to acetone of 10:1 was collected and recorded as E3344;

[0026] Fraction E3342 was concentrated and purified by preparative HPLC chromatography using methanol-water with a volume ratio of 80:20 as the mobile phase to obtain podocarpane-type diterpenoid compound 1;

[0027] Fraction E3344 was concentrated and purified by preparative HPLC chromatography using acetonitrile-water with a volume ratio of 75:25 as the mobile phase to obtain the semi-floral diterpenoid compound 2;

[0028] Fraction E333 was concentrated and separated by ODS column chromatography, and gradient eluted with methanol-water in volume ratios of 2:8, 4:6, 5:5, 7:3, and 1:0, and the fraction with a volume ratio of 4:6 was collected and recorded as E3333;

[0029] Fraction E3333 was concentrated and purified by preparative HPLC chromatography using methanol-water with a volume ratio of 80:20 as the mobile phase to obtain fraction E33331;

[0030] E33331 was purified by preparative chiral chromatography using n-hexane-isopropanol in a volume ratio of 45:55 as the mobile phase to obtain kavalactone compounds 3 and kavalactone compounds 4.

[0031] The podocarpane-type diterpenoid compounds, hemiaranane-type diterpenoid compounds and kavalactone compounds are from the extract of the stem and leaf parts of Amomum villosum.

[0032] The application of the extract of the Amomum villosum stem and leaf parts in the preparation of anti-inflammatory and / or antioxidant drugs.

[0033] A pharmaceutical composition, comprising one or more of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds, isomers of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds, and pharmaceutically acceptable salts of the podocarpane-type diterpenes, cymene-type diterpenes or kavalactone compounds; and further comprising one or a combination of pharmaceutically acceptable carriers, excipients, and diluents. The pharmaceutical composition is divided into an oral pharmaceutical composition or an injectable pharmaceutical composition according to the route of administration, and the dosage form of the pharmaceutical composition is selected from one of tablets, capsules, powders, syrups and injections.

[0034] The present invention also provides the use of the podocarpane-type diterpenes or cymene-type diterpenes or kavalactone compounds, isomers of the podocarpane-type diterpenes or cymene-type diterpenes or kavalactone compounds, pharmaceutically acceptable salts of the podocarpane-type diterpenes or cymene-type diterpenes or kavalactone compounds, or the pharmaceutical composition in the preparation of anti-inflammatory and / or antioxidant drugs.

[0035] The advantages of the present invention are:

[0036] In order to maximize the medicinal value of Amomum villosum, the present invention systematically studied the components of the stems and leaves of Amomum villosum, extracted new podocarpane-type diterpenoid compounds, hemiaranthine-type diterpenoid compounds and kavalactone compounds, and confirmed the structures of the compounds by means of nuclear magnetic resonance, mass spectrometry and the like.

[0037] The podocarpane diterpenoids or cypermethrin diterpenoids or kavalactone compounds or their isomers, pharmaceutically acceptable salts or pharmaceutical compositions containing podocarpane diterpenoids or cypermethrin diterpenoids or kavalactone compounds of the present invention can inhibit the NO release activity of mouse macrophages RAW264.7 induced by lipopolysaccharide (LPS). The podocarpane diterpenoid compounds, or pharmaceutically acceptable salts of their isomers or compositions containing podocarpane diterpenoid compounds can protect the mouse neuromicroglioma cells BV-2 from oxidative damage induced by hydrogen peroxide (H2O2), and can be used to prepare anti-inflammatory and antioxidant drugs. And compared with the existing podocarpane diterpenoid compounds extracted from Amomum villosum, it has good anti-inflammatory and antioxidant effects.

[0038] The present invention further enriches the structural diversity of the active substances in Amomum villosum, and on this basis, lays a foundation for the subsequent biological activity tests of the monomer compounds obtained, provides active lead compounds for the development of new drugs, and also provides a theoretical basis for the in-depth research and development of Amomum villosum medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Inhibitory activity of podocarpane-type diterpenoid 1, cypermethrin-type diterpenoid 2, and kavalactones 3 and 4 on LPS-induced NO production in RAW264.7 cells.

[0040] Figure 2 Evaluation of podocarpane-type diterpenoid 1 on H2O2-induced BV-2 cell viability.

[0041] Figure 3 Effect of podocarpane-type diterpenoid compound 1 on the content of total GSH in BV-2 cell lysate induced by H2O2.

[0042] Figure 4Effect of podocarpane-type diterpenoid 1 on ROS content in BV-2 cells induced by H2O2.

[0043] Figure 5 Podocarpane-type diterpenoid 1 protects BV-2 cells from oxidative stress damage by activating the NRF2 / HO-1 signaling pathway. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0045] Example 1

[0046] The method for extracting podocarpane-type diterpenoid compound 1, hemicarpane-type diterpenoid compound 2, and kavalactone compounds 3 and 4 in Amomum villosum comprises the following steps:

[0047] (1) Using the stem and leaf parts of Amomum villosum with a total dry weight of 13.1 kg as raw materials, adding a volume concentration of 90% ethanol aqueous solution (100 L), reflux extraction twice, each extraction for 2 hours, combining to obtain the extract, reducing pressure to recover the solvent, and concentrating to obtain 1348 g of total extract;

[0048] (2) dispersing the obtained total extract into 7.4 times the mass volume of water (10 L), extracting with 5 times the mass volume (6.74 L) of petroleum ether and ethyl acetate in sequence, concentrating the extract to recover the solvent, and obtaining a petroleum ether extract concentrate, an ethyl acetate extract concentrate (149 g) and an aqueous phase, respectively;

[0049] (3) the ethyl acetate extract concentrate was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol with volume ratios of 100:1, 50:1, 20:1, 15:1, 10:1, 5:1, 1:1 and 0:1 as eluents, and fraction E3 with a volume ratio of 50:1 was collected;

[0050] (4) Fraction E3 was concentrated to obtain 25.2 g of concentrated solution, which was separated by silica gel column chromatography and gradient eluted with petroleum ether-acetone in volume ratios of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1, to obtain 3.7 mg of podocarpane-type diterpenoid compound 1, 2.1 mg of cymene-type diterpenoid compound 2, 4.1 mg of kavalactone compound 3 and 3.8 mg of kavalactone compound 4. The specific separation and purification process is as follows:

[0051] Fraction E3 was concentrated and separated by silica gel column chromatography, and the fraction with a volume ratio of petroleum ether to acetone of 30:1 was collected and recorded as E33;

[0052] Fraction E33 was concentrated to obtain 7.8 g of concentrate, which was separated by ODS column chromatography using methanol-water with a volume ratio of 2:8, 4:6, 5:5, 7:3, and 1:0 as the eluent for gradient elution. The fraction with a volume ratio of 4:6 was collected and recorded as E333, and the fraction with a volume ratio of 5:5 was collected and recorded as E334.

[0053] Fraction E334 was concentrated to obtain 1.8 g of a concentrate, which was separated by silica gel column chromatography and gradient eluted with petroleum ether-acetone in a volume ratio of 100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 5:2, and 0:1, to collect a fraction in which the volume ratio of petroleum ether to acetone was 30:1 and recorded as E3342, and a fraction in which the volume ratio of petroleum ether to acetone was 10:1 and recorded as E3344;

[0054] Fraction E3342 was concentrated to obtain 262.2 mg of concentrated solution, which was purified by preparative HPLC chromatography using methanol-water with a volume ratio of 80:20 as the mobile phase to obtain 3.7 mg of podocarpane-type diterpenoid compound 1;

[0055] Fraction E3344 was concentrated to obtain 578.2 mg of concentrated solution, which was purified by preparative HPLC chromatography using acetonitrile-water with a volume ratio of 75:25 as the mobile phase to obtain 2.1 mg of hemi-petane-type diterpenoid compound 2;

[0056] Fraction E333 was concentrated to obtain 1.3 g of concentrate, which was separated by ODS column chromatography, and gradient eluted with methanol-water in volume ratios of 2:8, 4:6, 5:5, 7:3, and 1:0, and the fraction with a volume ratio of 4:6 was collected and recorded as E3333;

[0057] Fraction E3333 was concentrated to obtain 121.5 mg of concentrate, which was purified by preparative HPLC chromatography using methanol-water with a volume ratio of 80:20 as the mobile phase to obtain 7.9 mg of E33331;

[0058] E33331 was purified by preparative chiral chromatography using n-hexane-isopropanol in a volume ratio of 45:55 as the mobile phase to obtain 4.1 mg of kavalactone compound 3 and 3.8 mg of kavalactone compound 4.

[0059] The structures of the extracted podocarpane-type diterpenoid compound 1, hemiaranane-type diterpenoid compound 2, and kavalactone compounds 3 and 4 were identified. The specific physicochemical data are as follows:

[0060] Podocarpane-type diterpenoid compound 1: white amorphous powder, HRESIMS(positive)m / z 285.1833[M+Na]+ (calcd for C 17 H 26 O2Na, 285.1825), it was determined that the diterpenoid compound of podocarpane has a molecular formula C 17 H 26 O2, 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 1.

[0061] Hemi-penetranyl diterpenoid compound 2: yellow oil, HRESIMS(negative)m / z 347.2232[MH] - (calcd for C 21 H 31 O4,347.2227), determine the molecular formula of the semi-floral diterpenoid compound 2 C 21 H 32 O4, 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are shown in Table 2.

[0062] Kavalactone compound 3: white amorphous powder, HRESIMS(positive)m / z 413.1360[M+Na] + (calcd for C 24 H 22 O5Na,413.1359), determine the molecular formula of kavalactone compound 3 C 24 H 22 O5, ECD (CH3OH, c=0.5mg / mL) λnm (Δε) 235 (+18.8), 280 (+15.0), 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are shown in Table 3.

[0063] Kavalactone compound 4: white amorphous powder, HRESIMS(positive)m / z 413.1360[M+Na] + (calcd for C 24 H 22 O5Na,413.1359), determine the molecular formula of kavalactone compound 4 C 24 H 22O5, ECD (CH3OH, c=0.5mg / mL) λnm (Δε) 235 (-18.8), 280 (-15.4), 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are shown in Table 3.

[0064] Table 1 Carbon and hydrogen spectrum data of podocarpane-type diterpenoid compound 1

[0065]

[0066]

[0067] Table 2 C and H spectra data of hemi-penetrane diterpenoid compound 2

[0068]

[0069] Table 3 Carbon and hydrogen spectrum data of kavalactone compounds 3 and 4

[0070]

[0071] Through physical and chemical data and modern spectroscopic methods (HRESIMS and NMR), combined with relevant data from public literature, the structures of the above-mentioned podocarpane-type diterpenes and hemiaranane-type diterpenes and kavalactone compounds were identified, and compounds 1-4 were determined to be new compounds, and their structures are shown below:

[0072]

[0073] Example 2

[0074] The method for extracting podocarpane-type diterpenoid compound 1, hemicarpane-type diterpenoid compound 2, and kavalactone compounds 3 and 4 in Amomum villosum comprises the following steps:

[0075] (1) Using the stem and leaf parts of Amomum villosum with a total dry weight of 13 kg as raw materials, adding a volume concentration of 90% ethanol aqueous solution (110 L), reflux extraction twice, each extraction for 2 hours, combining to obtain extracts, reducing pressure to recover the solvent, and concentrating to obtain 1345 g of total extract;

[0076] (2) dispersing the total extract into 8.6 times the mass volume of water (11.5 L), extracting with 6 times the mass volume of petroleum ether and ethyl acetate in sequence, concentrating the extract to recover the solvent, and obtaining a petroleum ether extract concentrate, an ethyl acetate extract concentrate (154 g) and an aqueous phase, respectively;

[0077] (3) the ethyl acetate extract concentrate was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol with volume ratios of 100:1, 50:1, 20:1, 15:1, 10:1, 5:1, 1:1 and 0:1 as eluents, and fraction E3 with a volume ratio of 50:1 was collected;

[0078] (4) Fraction E3 was concentrated to obtain 26.5 g of concentrated solution, which was separated by silica gel column chromatography, and fraction E33 with a volume ratio of 30:1 was collected by gradient elution using petroleum ether-acetone with a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1 as eluents; fraction E33 was concentrated to obtain 8.1 g of concentrated solution, which was separated by ODS column chromatography, and gradient eluted using methanol-water with a volume ratio of 2:8, 4:6, 5:5, 7:3 and 1:0 as eluents, and further purified to obtain 3.5 mg of podocarpane-type diterpenoid compound 1, 2.6 mg of cymene-type diterpenoid compound 2, 3.6 mg of kavalactone compound 3, and 3.8 mg of kavalactone compound 4. The specific separation and purification process is the same as that in Example 1.

[0079] Example 3

[0080] The method for extracting podocarpane-type diterpenoid compound 1, hemicarpane-type diterpenoid compound 2, and kavalactone compounds 3 and 4 in Amomum villosum comprises the following steps:

[0081] (1) Using the stem and leaf parts of Amomum villosum with a total dry weight of 13.3 kg as raw materials, adding 80% ethanol aqueous solution (150 L) and reflux extraction for 4 times, each extraction for 3 hours, combining the extracts to obtain the extracts, recovering the solvent under reduced pressure, and concentrating to obtain 1353 g of total extract;

[0082] (2) dispersing the total extract into 11.7 times the mass volume of water (15.8 L), extracting with 10 times the mass volume of petroleum ether and ethyl acetate in sequence, concentrating the extract to recover the solvent, and obtaining a petroleum ether extract concentrate, an ethyl acetate extract concentrate (157 g) and an aqueous phase, respectively;

[0083] (3) the ethyl acetate extract concentrate was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol with volume ratios of 100:1, 50:1, 20:1, 15:1, 10:1, 5:1, 1:1 and 0:1 as eluents, and fraction E3 with a volume ratio of 50:1 was collected;

[0084] (4) Fraction E3 was concentrated to obtain 26.9 g of concentrated solution, which was separated by silica gel column chromatography, and fraction E33 with a volume ratio of 30:1 was collected by gradient elution using petroleum ether-acetone with a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1 as eluents; fraction E33 was concentrated to obtain 8.1 g of concentrated solution, which was separated by ODS column chromatography, and gradient eluted using methanol-water with a volume ratio of 2:8, 4:6, 5:5, 7:3 and 1:0 as eluents, and further purified to obtain 3.8 mg of podocarpane-type diterpenoid compound 1, 2.1 mg of cymene-type diterpenoid compound 2, 4.0 mg of kavalactone compound 3, and 4.8 mg of kavalactone compound 4. The specific separation and purification process is the same as that in Example 1.

[0085] Example 4

[0086] The podocarpane-type diterpenoid compound 1, the cypermethrin-type diterpenoid compound 2, and the kavalactone compounds 3 and 4 extracted in the above example have an inhibitory effect on the NO production in the RAW264.7 cells induced by LPS. The specific operation is as follows:

[0087] (1) Cell culture

[0088] Mouse mononuclear macrophage RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin sodium, and incubated and grown in a constant temperature incubator at 37° C. and 5% CO 2 .

[0089] (2) Effects of compounds 1-4 on NO production in RAW264.7 cells

[0090] Mouse mononuclear macrophage RAW 264.7 cells in logarithmic phase were taken and the cell concentration was adjusted to 3.5×10 4cells / well were inoculated in a 96-well plate, and 100 μL of cell suspension was added to each well. In the experiment, a control group (RAW264.7 cells, DMSO), a model group (RAW264.7 cells, DMSO, 0.5 μg / mL of LPS), a positive drug group (RAW264.7 cells, dexamethasone, 0.5 μg / mL of LPS) and a test drug group (RAW264.7 cells, each compound to be tested, 0.5 μg / mL of LPS) were set up at the same time. The cells were cultured and incubated in a 5% CO2, 37°C constant temperature incubator for 24 hours, and then 40 μL of cell supernatant was aspirated and placed in an ELISA plate. 40 μL of an equal volume of Griess reagent was added to each well to mix it with the cell supernatant and react completely. After reacting at room temperature for 10 minutes, the absorbance value of the solution in the well at 540 nm was detected using an ELISA instrument. The inhibition rate (%) of the compound 1-4 treatment group was calculated to evaluate the inhibition of NO release activity. The results are shown in Table 4 and the attached Figure 1 shown.

[0091] Table 4 Inhibitory effect of podocarpane-type diterpenoid compound 1 on RAW264.7 cells

[0092]

[0093]

[0094] From the experimental results, it can be found that the inhibition rates of compounds 1-4 on RAW264.7 cells are 20%, 32%, 30%, and -10%, respectively, indicating that compounds 1-3 of the present invention have in vitro anti-inflammatory effects.

[0095] Example 5

[0096] The podocarpane-type diterpenoid compound 1 extracted from the above example has a protective effect on hydrogen peroxide (H2O2)-induced oxidative damage in mouse microglioma cells BV-2, as follows:

[0097] (1) Cell culture

[0098] Mouse microglioma cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin sodium, and incubated and grown in a constant temperature incubator at 37° C. and 5% CO 2 .

[0099] (2) Evaluation of the antioxidant activity of podocarpane-type diterpenoid compound 1 using the CCK-8 method

[0100] Mouse microglioma BV-2 cells growing in the logarithmic phase were cultured at a concentration of 1×10 4cells / well were seeded in a 96-well plate, and each well contained 100 μL of cell suspension. In the experiment, a control group (BV-2 cells, DMSO), a model group (BV-2 cells, DMSO, 250 μM H2O2) and a test drug group (BV-2 cells, test compound, 250 μM H2O2) were set up at the same time. The drug-treated group was given the podocarpane-type diterpenoid compound 1 and cultured in a 5% CO2, 37°C constant temperature incubator for 24 hours. Then the model group and the test drug group were given 250 μM H2O2. After 0.5 hours of action, 10 μL of CCK-8 reagent was added to each well. The 96-well culture plate was placed in an incubator and incubated for 1-4 hours, and the absorbance at 450 nm was measured using an enzyme reader. The cell survival rate (%) of the podocarpane-type diterpenoid compound 1-treated group was calculated to evaluate its antioxidant activity. The results are shown in Figure 2 As shown: compared with the H2O2 model group, the survival rate of BV-2 cells was significantly increased after pre-protection with the podocarpane-type diterpenoid compound 1, indicating that the compound 1 of the present invention has in vitro antioxidant activity.

[0101] (3) Detection of reduced glutathione GSH by extracting the podocarpane-type diterpenoid compound 1 obtained above

[0102] BV-2 cells in the logarithmic growth phase were inoculated into 6-well plates (5×10 5 cells / well), cells were pre-protected with different concentrations of podocarpane-type diterpenoid compound 1 for 24 h, and then treated with 250 μM H2O2 for 30 min, and then the cells were collected and the GSH content in the cells was detected according to the instructions of the detection kit. Figure 3 As shown in the figure, compared with the control group, the GSH content in the H2O2 group was downregulated. Compared with the H2O2 group, the total GSH content in BV-2 cells was upregulated after pre-protection with compound 1, indicating that compound 1 promoted the increase in the production of antioxidant-related product reduced glutathione GSH in cells.

[0103] (4) Detection of intracellular reactive oxygen species (ROS) using podocarpane-type diterpenoid compound 1

[0104] BV-2 cells in the logarithmic growth phase were inoculated into 6-well plates (5×10 5 cells / well), cells were pre-protected with different concentrations of podocarpane-type diterpenoid compound 1 for 24 h, and then treated with 250 μM H2O2 for 30 min, and then the cells were collected and the ROS content in the cells was detected according to the instructions of the reactive oxygen species detection kit. Figure 4As shown, compared with the control group, the ROS content in the H2O2 group was significantly upregulated, while after administration of different concentrations of podocarpane-type diterpenoid compound 1, the ROS content was significantly downregulated, indicating that podocarpane-type diterpenoid compound 1 can inhibit the generation of ROS during oxidative stress, thereby inhibiting the occurrence of oxidative damage.

[0105] (5) Podocarpane-type diterpenoid compound 1 activated the NRF2 / HO-1 signaling pathway to protect BV-2 cells from oxidative stress damage. The specific operation is as follows:

[0106] BV-2 cells in the logarithmic growth phase were inoculated into 6-well plates (5×10 5 cells / well), cells were pre-protected with different concentrations of podocarpane-type diterpenoid compound 1 for 24 h, and then treated with 250 μM H2O2 for 30 min. The cells were collected and operated according to the western blot experimental method to detect the expression of KEAP1, NRF2, and HO-1 proteins related to the NRF2 / HO-1 signaling pathway. The results are shown in Figure 5 As shown, podocarpane-type diterpenoid 1 promoted the expression of NRF2 and the degradation of KEAP1, further promoting the upregulation of downstream antioxidant-related protein HO-1. This indicates that podocarpane-type diterpenoid 1 protects BV-2 cells from oxidative stress damage by activating the NRF2 / HO-1 signaling pathway.

[0107] The above embodiments are only used to help understand the method and the central idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection of the claims of the present invention.

Claims

1. A podocarpane-type diterpenoid or kavalactone compound extracted from Amomum villosum, characterized in that: It is any one of the compounds shown below; 。 2. A method for preparing a podocarpane-type diterpenoid or kavalactone compound according to claim 1, characterized in that: The steps include: (1) Using the stem and leaf parts of Amomum villosum as raw materials, adding ethanol aqueous solution with a volume concentration of 80%-95%, reflux extraction 2-4 times, each extraction for 2-4 hours, combining to obtain the extract, recovering the solvent under reduced pressure, and concentrating to obtain the total extract; (2) dispersing the total extract into 5-15 times the mass volume of water, extracting with 5-15 times the mass volume of petroleum ether and ethyl acetate in sequence, concentrating the extract to recover the solvent, and obtaining a petroleum ether extract concentrate, an ethyl acetate extract concentrate and an aqueous phase, respectively; (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography using a gradient elution with dichloromethane-methanol in a volume ratio of 100:1 to 0:1, and the fraction E3 with a volume ratio of 50:1 was collected; (4) The concentrated fraction E3 was separated by silica gel column chromatography and gradient elution was performed using petroleum ether-acetone in a volume ratio of 100:1 to 0:1 to obtain the podocarpane-type diterpene compound 1 and the kavalactone compound 3.

3. The method for preparing a podocarpane-type diterpenoid or kavalactone compound according to claim 2, characterized in that: In the step (1), the solid-liquid ratio is: Amomum villosum stems and leaves: ethanol aqueous solution = 1 g: (7-15) mL.

4. The method for preparing a podocarpane-type diterpenoid or kavalactone compound according to claim 2, characterized in that: In step (4), the specific separation and purification process of fraction E3 includes: Fraction E3 was concentrated and separated by silica gel column chromatography, and gradient elution was performed using petroleum ether-acetone with a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1 as eluents, and the fraction with a volume ratio of petroleum ether to acetone of 30:1 was collected and recorded as E33; Fraction E33 was concentrated and separated by ODS column chromatography, and gradient elution was performed using methanol-water with a volume ratio of 2:8, 4:6, 5:5, 7:3, and 1:0 as the eluent, and fraction E333 with a volume ratio of 4:6 was collected; fraction E334 with a volume ratio of 5:5 was collected; Fraction E334 was concentrated and separated by silica gel column chromatography, and gradient eluted with petroleum ether-acetone in a volume ratio of 100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 5:2, and 0:1 as eluents, and fraction E3342 in which the volume ratio of petroleum ether to acetone was 30:1 was collected; Fraction E3342 was concentrated and purified by preparative HPLC chromatography using methanol-water with a volume ratio of 80:20 as the mobile phase to obtain podocarpane-type diterpenoid compound 1; Fraction E333 was concentrated and separated by ODS column chromatography, and gradient eluted with methanol-water in volume ratios of 2:8, 4:6, 5:5, 7:3, and 1:0, and the fraction with a volume ratio of 4:6 was collected and recorded as E3333; Fraction E3333 was concentrated and purified by preparative HPLC chromatography using methanol-water with a volume ratio of 80:20 as the mobile phase to obtain fraction E33331; E33331 was purified by preparative chiral chromatography using n-hexane-isopropanol in a volume ratio of 45:55 as the mobile phase to obtain kavalactone compound 3.

5. A pharmaceutical composition, characterized in that It comprises the podocarpane-type diterpenoid or kavalactone compound as described in claim 1.

6. Use of the compound 1 according to claim 1 in the preparation of antioxidant drugs.

7. Use of compounds 1 and 3 according to claim 1 in the preparation of anti-inflammatory drugs.

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