CN114315812A - Two alkaloid compounds derived from marine fungi, and their preparation methods and application in preparing antiinflammatory medicine - Google Patents
Two alkaloid compounds derived from marine fungi, and their preparation methods and application in preparing antiinflammatory medicine Download PDFInfo
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Abstract
The invention belongs to the technical field of anti-inflammatory drugs, and discloses two alkaloid compounds derived from marine fungi, a preparation method thereof and application thereof in preparing anti-inflammatory drugs, wherein the structural formulas of the two alkaloid compounds are shown as formulas (I) and (II), and the preparation method comprises the following steps: inoculating marine fungus Penicillium sp.GXNU-Y45 into a seed culture medium, and performing shake cultivation to obtain a seed culture solution; inoculating the seed culture solution into a fermentation culture medium, and standing and culturing to obtain a fermentation product; filtering the fermentation product to obtain mycelium, extracting the mycelium with methanol, concentrating to obtain extract, extracting the extract with ethyl acetate to obtain crude extract, and separating the crude extract by silica gel normal phase chromatography; eluting, collecting fractions, and performing column chromatography to obtain the target compound. The alkaloid compound obtained by the invention has obvious anti-inflammatory activity and clinical application potential of anti-inflammatory treatment.
Description
Technical Field
The invention relates to the technical field of anti-inflammatory drugs, in particular to two alkaloid compounds derived from marine fungi, a preparation method thereof and application thereof in preparing anti-inflammatory drugs.
Background
Metabolites of fungal origin are a challenging area with unlimited possibilities, providing a huge library of novel bioactive lead molecules for drug discovery. Wherein the remarkable diversity of the bioactive secondary metabolites produced by marine fungi has a plurality of potential applications in the biotechnology field. These metabolic molecules are reported to possess a variety of pharmacological activities, including antibacterial, antifungal, cytotoxic, cell proliferative, antioxidant, antiviral, antitubercular, etc. A large number of silent or unexpressed biosynthetic gene clusters integrated in the genome of known fungal species may also have more undeveloped marine fungal secondary metabolites, so that new, safer, eco-friendly and sustainable active natural products are continuously sought as lead compounds for drug development, revealing the importance of marine fungal secondary metabolites on biological activity.
The molecular structure of the metabolism of marine endophytic fungi has diversity, wherein alkaloid is widely distributed, alkaloid is a nitrogen-containing compound with various biological activities, and the alkaloid has various biological activities. Alkaloids have been reported to have significant effects on anti-inflammatory activity, as evidenced by inhibition of the expression of various pro-inflammatory factors, such as cytokines, lipid mediators, and enzymes involved in the inflammatory response. The anti-inflammatory agents on the market also contain alkaloid anti-inflammatory agents such as indomethacin and the like. In the research and development of anti-inflammatory drugs, the metabolic products of marine fungi play an important role in reducing the cost of drugs, improving the curative effect of the drugs and reducing the side effect of the anti-inflammatory drugs. The metabolic product with anti-inflammatory activity is found from marine fungus Penicillium sp.GXNU-Y45, and an important way is provided for marine drug research.
Disclosure of Invention
In response to the needs of the prior art, the primary object of the present invention is to provide two novel alkaloid compounds of marine fungal origin, which have significant anti-inflammatory activity.
The invention also aims to provide a preparation method of the two novel alkaloid compounds derived from the marine fungi.
The invention also aims to provide application of the two novel alkaloid compounds derived from the marine fungi.
The purpose of the invention is realized by the following technical scheme:
two alkaloid compounds derived from marine fungi, the structural formulas of the two novel alkaloid compounds are shown as formulas (I) and (II):
the two alkaloid compounds protected by the invention are obtained by separating from fermentation liquor of marine fungus Penicillium sp.GXNU-Y45; the marine fungus Penicillium sp. GXNU-Y45 was deposited at 9.12.2021 in the culture Collection (GDMCC) with the accession number GDMCC 62111 and was classified and named as Penicillium sp.
The preparation method of the two alkaloid compounds comprises the following steps:
s1, inoculating marine fungus Penicillium sp.GXNU-Y45 into a seed culture medium, and performing shake culture to obtain a seed culture solution;
s2, inoculating the seed culture solution into a fermentation culture medium, and performing static culture to obtain a fermentation product;
s3, filtering the fermentation product to obtain hypha, extracting the hypha for three times by using methanol, concentrating an extracting solution to obtain an extract, extracting the extract by using ethyl acetate to obtain a crude extract, and separating the ethyl acetate crude extract by using silica gel normal phase chromatography; eluting with petroleum ether/ethyl acetate, collecting 20-80% ethyl acetate/petroleum ether fraction, and separating by column chromatography to obtain two novel alkaloids compounds with structural formulas shown in formula (I) and formula (II).
In the present invention, preferably, the seed culture solution of step S1 includes the following components per serving: 30 g of glucose, 1g of yeast extract, 2-5 g of peptone, 1-3 g of agar, 1-5 g of sodium chloride and 1L of water.
In the present invention, preferably, the shaking culture conditions in step S1 are that the shaking rotation speed is 80-150 rpm, and the shaking culture is performed at 25-30 ℃ for 5-15 days.
In the present invention, preferably, the fermentation medium in step S2 is a solid rice fermentation medium consisting of rice and seawater in a ratio of 1 g: 1.2ml of the mixture was mixed at a mass-to-volume ratio.
In the present invention, the static culture condition of S2 is preferably 25-30 ℃ for 25-50 days.
In the present invention, preferably, the column chromatography separation technique of S3 is a silica gel column chromatography technique, a gel column chromatography technique and a C-18 reverse phase column chromatography technique.
Tests prove that the two novel alkaloid compounds prepared by the method have anti-inflammatory activity, and the invention also protects the application of the two novel alkaloid compounds derived from the marine fungi in preparing anti-inflammatory drugs.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the invention provides two novel alkaloid compounds derived from marine fungi, which have obvious anti-inflammatory activity. Therefore, the two novel alkaloid compounds provided by the invention have the clinical application potential of anti-inflammatory treatment.
2. The preparation method for preparing the alkaloid compound by using the marine fungus Penicillium sp.GXNU-Y45 has simple process, and is convenient for industrial mass production of the alkaloid compound.
Detailed Description
In order that the invention may be more clearly expressed, the invention will now be further described by way of specific examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Preparation of compounds
Example 1 isolation and characterization of the marine fungus Penicillium sp. GXNU-Y45
1. Materials: an endophytic fungus sample of acanthus ilicifolius leaves of mangrove forest collected from the river mouth of northern regional Bing of Guangxi city and harbor prevention.
2. Pure endophytic fungus strains are obtained through culture, separation and identification of the strains, and are identified as Penicillium sp. The Penicillium fungi is preserved in Guangdong province microorganism culture Collection (GDMCC) at 9.12 months, has a preservation number of GDMCC 62111, is classified and named as Penicillium sp.GXNU-Y45, and has a preservation unit address of No. 59 building, No. 5 building, Michelia Tokyo 100, Guangzhou City
EXAMPLE 2 preparation of two novel alkaloid Compounds
Two novel alkaloid compounds are separated and prepared from Penicillium sp metabolite by the following steps:
s1, culturing Penicillium sp.GXNU-Y45 seeds:
the seed culture medium comprises the following components in percentage by weight: 30 g of glucose, 1g of yeast extract, 2 g of peptone, 1g of agar, 1g of sodium chloride and 1L of water; preparing a culture medium into a test tube inclined plane, selecting a Penicillium sp.GXNU-Y45 strain, inoculating the strain into the inclined plane, carrying out shake cultivation at 30 ℃ for 5 days at the rotation speed of 150rpm of a shaking table to obtain a seed culture solution;
s2, fermentation culture of Penicillium sp.GXNU-Y45:
inoculating the seed culture solution into a fermentation culture medium, wherein the fermentation culture medium is a solid rice fermentation culture medium and comprises rice and seawater according to the weight ratio of 1: 1.2, and performing static culture for 25 days at the temperature of 25-30 ℃ to obtain a fermentation product;
s3, filtering the fermentation product to obtain fungus hypha, extracting the fungus hypha with methanol for three times, concentrating the extracting solution to obtain an extract, and extracting the extract with ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by using silica gel normal phase chromatography, gradient eluting by using 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% ethyl acetate-petroleum ether respectively, collecting 20% -80% ethyl acetate/petroleum ether eluent, and obtaining two novel alkaloid compounds with structural formulas shown as the formulas (I) and (II) by adopting silica gel column chromatography, gel column chromatography and C-18 reverse phase column chromatography.
EXAMPLE 3 preparation of two novel alkaloid Compounds
Two novel alkaloid compounds are separated and prepared from Penicillium sp metabolite by the following steps:
s1, culturing Penicillium sp.GXNU-Y45 seeds:
the seed culture medium comprises the following components in percentage by weight: 30 g of glucose, 1g of yeast extract, 5 g of peptone, 3 g of agar, 5 g of sodium chloride and 1L of water; preparing a culture medium into a test tube inclined plane, selecting a Penicillium sp.GXNU-Y45 strain, inoculating the strain into the inclined plane, carrying out shake cultivation at 25 ℃ for 15 days at the shaking table rotating speed of 80rpm to obtain a seed culture solution;
s2, fermentation culture of Penicillium sp.GXNU-Y45:
inoculating the seed culture solution into a fermentation culture medium, wherein the fermentation culture medium is a solid rice fermentation culture medium which is prepared by mixing rice and seawater according to the weight ratio of 1 g: mixing 1.2ml of the mixture according to the mass-volume ratio, and performing static culture for 50 days at the temperature of 25-30 ℃ to obtain a fermentation product.
S3, filtering the fermentation product to obtain fungus hypha, extracting the fungus hypha with methanol for three times, concentrating the extracting solution to obtain an extract, and extracting the extract with ethyl acetate to obtain a crude extract; separating the ethyl acetate crude extract by using silica gel normal phase chromatography, gradient eluting by using 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% ethyl acetate-petroleum ether respectively, collecting 20% -80% ethyl acetate/petroleum ether eluent, and obtaining two novel alkaloid compounds with structural formulas shown as the formulas (I) and (II) by adopting silica gel column chromatography, gel column chromatography and C-18 reverse phase column chromatography.
II, confirmation of the Compound
Carrying out structure test analysis on the obtained novel two alkaloid compounds to obtain the following test data:
novel alkaloid compounds I: c20H16N2O5；HRESIMS：363.0981[M-H]-(theoretical calculation 363.0980); one-dimensional nuclear magnetic resonance data:13CNMR 147.6,110.3,178.5,125.1,125.5,133.9,119.2,138.9,123.0,166.4,123.7,123.1,108.9,113.9,202.7,82.4,179.7,159.4,156.4,16.3,14.8。
novel alkaloid compounds II: c21H18N2O5；HRESIMS：377.1139[M-H]-(theoretical calculation 377.1137); one-dimensional nuclear magnetic resonance data:13CNMR 147.6,110.2,178.4,124.9,125.3,133.7,119.1,138.8,122.8,166.2,123.6,123.0,108.8,113.7,202.6,82.3,179.6,159.3,156.3,56.8,16.0,14.7。
through identification, the structural formulas of the novel two alkaloid compounds are shown as the formulas (I) and (II):
anti-inflammatory activity experiment of three or two novel alkaloid compounds
The anti-inflammatory activity tests were carried out on the two novel alkaloid compounds prepared in examples 2 and 3.
1. Materials:
1.1 Experimental materials
The instrument comprises the following steps: the device comprises a cell culture bottle, a cell culture box, an ultra-clean workbench, a microplate reader, an electronic weighing balance and microsamplers with different measuring ranges.
The main reagents are as follows: indomethacin, dimethyl sulfoxide (DMSO), fetal bovine serum, DMEM medium, Lipopolysaccharide (LPS), thiazole blue (MTT).
Test cells: mouse macrophage RAW264.7
Sample preparation: compounds (I) and (II) were dissolved in DMSO, respectively, to prepare sample solutions with a concentration of 10mM for use.
1.2 cell culture
a. Adding 3ml of DMEM high-sugar medium containing 10% fetal calf serum and 1% double antibody into the culture bottles, subpackaging macrophage RAW264.7 cell suspension into each culture bottle, blowing, uniformly mixing, placing in an incubator (the concentration of carbon dioxide is 5%, the temperature is 37 ℃) for culture, and observing the growth condition every day.
b. When the cell growth density is observed to be about 85%, removing the old culture medium in a super clean bench, washing the old culture medium for 3 times by PBS, adding 1-3 ml of DMEM high-sugar culture medium, scraping the cells growing adherent to the wall, slightly blowing off the scraped cells to be separated, and uniformly mixing the cells.
c. The cell density is about 5% -10%, and DMEM high-sugar medium is added to continue passage.
2. Test method
2.1 determination of cell viability of Compounds (I) and (II) by the MTT method
a. And (4) digesting RAW264.7 cells with good growth situation to obtain a suspension, and counting the cells.
b. A volume of 180. mu.L of cell suspension was added to each well of a 96-well plate, and the plate was incubated in a cell incubator at a carbon dioxide concentration of 5% and a temperature of 37 ℃ for 24 hours.
c. The culture solution was poured off, a new volume of 180. mu.L of the medium was added, 10. mu.L of an LPS solution having a concentration of 5mg/mL was pipetted into each well, and the wells were incubated in a cell incubator at a temperature of 37 ℃ and a concentration of carbon dioxide for 2 hours.
d. Indometacin and compound (I) and (II) samples were added to 96-well plates at different concentrations, 3 duplicate wells per concentration. They were cultured in a cell culture chamber at 37 ℃ for 24 hours at a carbon dioxide concentration of 5%.
e. Thiazole blue (MTT) at a concentration of 5mg/mL was pipetted 10. mu.L into each well and allowed to stand in the incubator for 4 hours.
f. The supernatant was removed, 100 μ l of LDMSO was added to each well, and the formazan was shaken until it was completely dissolved.
g. The OD value was determined in a microplate reader (490 nm wavelength was selected). And calculating the cell survival rate.
Calculating the survival rate: percent cell survival rate ═ aSample (I)/ABlank space×100％。
ASample (I): adding the absorbance of the sample; a. theBlank space: absorbance of the non-added sample
2.2 measurement of NO production inhibition Rate
a. 180. mu.L/well of cultured RAW264.7 cells were added to a 96-well plate, and the mixture was placed in an incubator to be cultured for 12 hours.
b. The culture medium is discarded, a new culture medium of 180 mu L/well is added into a 96-well plate, 20 mu L/well of samples (I) and (II) with different concentrations is added, the positive control group is indometacin, a blank group is not added with 20 mu L/well DMEM, 5 parallel multiple wells are arranged, and the culture is carried out for 1h in a constant-temperature incubator.
c. Then, 10. mu.L/well of LPS was added thereto and cultured for 24 hours. The supernatant was collected, centrifuged, and 50. mu.L of the supernatant was pipetted into a new 96-well plate. Measuring NO content, measuring absorbance at 540nm wavelength, calculating inhibition rate according to formula, and calculating IC50The value is obtained. Successful OD value A of inflammation modelingLPS group/A is emptyIs 3 to 8.
NO inhibition (%) - (a)Sample set-ABlank group)/(ALPS group-ABlank group)]100％
3. Test results
The test results show that the effects of the two novel alkaloid compounds on the release inhibition of NO are obvious and shown in table 1.
TABLE 1 anti-inflammatory test results (IC) of two novel alkaloid compounds50/μM)
Therefore, the two alkaloid compounds derived from marine fungi provided by the invention have remarkable anti-inflammatory activity and have clinical application potential of anti-inflammatory treatment.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (10)
1. Two alkaloid compounds of marine fungal origin, characterized in that: the structural formulas of the two novel alkaloid compounds are shown as formulas (I) and (II):
2. the process for the preparation of two alkaloid compounds of marine fungal origin according to claim 1, characterized in that: the two alkaloid compounds are obtained by separating from fermentation liquor of marine fungus Penicillium sp.GXNU-Y45; the marine fungus Penicillium sp.GXNU-Y45 was deposited at the Guangdong province culture Collection (GDMCC) at 9.12.2021 with the accession number GDMCC 62111.
3. The method of claim 2, comprising the steps of:
s1, inoculating marine fungus Penicillium sp.GXNU-Y45 into a seed culture medium, and performing shake culture to obtain a seed culture solution;
s2, inoculating the seed culture solution into a fermentation culture medium, and performing static culture to obtain a fermentation product;
s3, filtering the fermentation product to obtain hypha, extracting the hypha for three times by using methanol, concentrating an extracting solution to obtain an extract, extracting the extract by using ethyl acetate to obtain a crude extract, and separating the ethyl acetate crude extract by using silica gel normal phase chromatography; eluting with petroleum ether/ethyl acetate, collecting 20-80% ethyl acetate/petroleum ether fraction, and separating by column chromatography to obtain two novel alkaloids compounds with structural formulas shown in formula (I) and formula (II).
4. The production method according to claim 3, characterized in that: step S1, wherein each part of the seed culture solution comprises the following components: 30 g of glucose, 1g of yeast extract, 2-5 g of peptone, 1-3 g of agar, 1-5 g of sodium chloride and 1L of water.
5. The production method according to claim 3, characterized in that: and step S1, wherein the shaking table culture conditions are that the shaking table rotation speed is 80-150 rpm, and the culture is carried out for 5-15 days at 25-30 ℃.
6. The production method according to claim 3, characterized in that: step S2, the fermentation medium is a solid rice fermentation medium consisting of rice and seawater in a ratio of 1 g: 1.2ml of the mixture was mixed at a mass-to-volume ratio.
7. The production method according to claim 3, characterized in that: the step S2 is to allow the culture to stand for 25-50 days at 25-30 ℃.
8. The production method according to claim 3, characterized in that: the column chromatography separation technology in the step S3 is silica gel column chromatography, gel column chromatography and C-18 reversed phase column chromatography.
9. Use of two alkaloid compounds of marine fungal origin according to claim 1 for the preparation of an anti-inflammatory medicament.
10. Use of two alkaloid compounds prepared according to the preparation process of any of claims 2-8 for the preparation of an anti-inflammatory agent.
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| WO2008094507A2 (en) * | 2007-01-26 | 2008-08-07 | Cellicon Biotechnologies, Inc. | Novel fusion compounds |
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| WO2008094507A2 (en) * | 2007-01-26 | 2008-08-07 | Cellicon Biotechnologies, Inc. | Novel fusion compounds |
Non-Patent Citations (3)
| Title |
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| 李本超等: "广豆根内生真菌GDG-178 代谢产物研究", vol. 39, no. 2, pages 139 - 143 * |
| 杨超凡等: "海洋生物抗炎活性物质研究进展", vol. 44, no. 11, pages 102 - 113 * |
| 郑穹等: "《药物波谱解析实用教程》", 武汉大学出版社, pages: 278 - 290 * |
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