CN113005158A - Preparation method and application of azaanthraquinone alkaloid - Google Patents

Preparation method and application of azaanthraquinone alkaloid Download PDF

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CN113005158A
CN113005158A CN202110328855.7A CN202110328855A CN113005158A CN 113005158 A CN113005158 A CN 113005158A CN 202110328855 A CN202110328855 A CN 202110328855A CN 113005158 A CN113005158 A CN 113005158A
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azaanthraquinone
alkaloid
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林生
夏桂阳
夏欢
王敏
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Qinghai Nationalities University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/08—Aza-anthracenes

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Abstract

A preparation method and application of aza-anthraquinone alkaloid, belonging to the technical field of aza-anthraquinone alkaloid preparation and application. The azaanthraquinone alkaloid scorpinone is a derivative of polyketide produced by fungal metabolism, and was first discovered from a strain of marine fungi. Typical structures include benzo [ g ] quinoline-5, 10-dione, pyrido [3, 2-g ] quinoline-5, 10-dione, and the like or derivatives thereof, with the simplest azaanthraquinone alkaloid being cleistophosine. The invention utilizes the endophyte of the red paeony root, cultures, strains ferment, extracts and separates to obtain the high-purity azaanthraquinone alkaloid, has low cost of raw materials, easy culture, simple extraction and high yield, can be suitable for large-scale production, and simultaneously discovers the application of the azaanthraquinone alkaloid in the aspect of anti-inflammatory drugs.

Description

Preparation method and application of azaanthraquinone alkaloid
Technical Field
A preparation method and application of aza-anthraquinone alkaloid, belonging to the technical field of aza-anthraquinone alkaloid preparation and application.
Background
The azaanthraquinone alkaloid Scorpinone, a derivative of polyketide compounds produced by fungal metabolism, was first discovered from a strain of marine fungi (Miljkovic A., Mantle P. G., Williams D. J., Rassing B., Scorpinone, a new Natural azaanthroquinone produced by a Bispora-like nutritional us [ J ]. Journal of Natural Products, 2001, 64, 1251-1253). It has the parent core structure of natural alkaloid with important biological activity, and the common structure comprises benzo [ g ] quinoline-5, 10-diketone, pyrido [3, 2-g ] quinoline-5, 10-diketone and the like or derivatives thereof, wherein the simplest azaanthraquinone alkaloid is Cleistophylline. The planar structural formula of the azaanthraquinone alkaloid is shown as formula (I).
Figure DEST_PATH_IMAGE002
。
Endophytic fungi are a special habitat microorganism, meaning a fungus that exists asymptomatically in The internal tissues below The plant epidermal cell layer at some or all stages of Its life cycle, without causing The host plant to exhibit significant disease symptoms (Wilson D. endogyte: The Evolution of a Term, and Classification of Its uses and Definition [ J ]. Oikos, 1995, 73(2): 274-.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: overcomes the defects of the prior art, and provides a preparation method and application of the azaanthraquinone alkaloid, which has low cost, green and environment-friendly production process and is easy for scale production.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of aza-anthraquinone alkaloid is characterized in that: the method comprises the following steps:
1) separating endophytic fungi from radix Paeoniae Rubra;
2) according to the parts by weight: 200 parts of potato, 8-12 parts of yeast extract, 5-7 parts of peptone, 25-35 parts of glucose and KH2PO41-3 parts of MgSO40.3-0.7 part of the seed culture medium, adding water with the volume of 2.8-3.2 times, adjusting the pH value to 6.8 to prepare a culture solution, sterilizing, inoculating, performing rotary culture at 25-30 ℃ for 3 days, and diluting by 100 times to prepare a seed solution for later use;
3) uniformly mixing rice with water, sterilizing, adding the seed solution obtained in the step 2), and culturing for 40 days, wherein the rice: water: the weight ratio of the seed liquid to the seed liquid is 1.5: 1.5-2.0: 0.18 to 0.22;
4) adding ethyl acetate with the volume of 1.4-1.6 times that of the culture solution obtained in the step 3) into the culture solution, soaking for 50-70 min, performing ultrasonic filtration for 25-35 min to obtain filtrate, repeating the steps for 3 times, performing reduced pressure concentration on the filtrate, removing the solvent to obtain an extract, performing gradient elution on the extract for 7 times through normal-pressure silica gel column chromatography to obtain components F1-F7, performing gradient elution on F7 through a Flash column chromatography for 5 times, sequentially obtaining components F7-1-F7-5, and performing HPLC semi-preparative chromatographic separation on F7-5 to obtain the azaanthraquinone alkaloid.
The azaanthraquinone alkaloid is obtained by a fungus fermentation extraction mode, the fungus culture cost is low, the extraction is simple, the yield is high, and the method is green, environment-friendly and easy to scale.
Preferably, the elution conditions of the normal pressure silica gel column chromatography in the step 4) are as follows: sequentially adopting petroleum: etheracetone volume ratio =100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1: 1. This elution condition elutes more fully with minimal loss.
Preferably, the Flash column chromatography in the step 4) adopts a C18 column, and the elution conditions are that methanol aqueous solution with the volume concentration of 30%, 40%, 50%, 60% and 65% is adopted for elution in sequence. This elution condition elutes more fully with minimal loss.
Preferably, the HPLC semi-preparative chromatographic separation conditions in the step 4) are as follows: an ES-C18 column was used, 50% strength by volume aqueous methanol, the detector wavelength being set at 280 nm.
Preferably, the culture solution before sterilization in the step 2) is subpackaged into 500ml triangular bottles, and each bottle contains 150ml of the culture solution. The strain culture is easier.
Preferably, the sterilization in step 2) and step 3) is performed at 115 ℃ and 68kPa for 30 min. The culture effect is better by matching with the split charging condition.
Preferably, the rotary culture in step 2) is carried out at 155 rpm.
The application of the azaanthraquinone alkaloid is characterized in that: can be used for preparing antiinflammatory drugs.
The pharmaceutically acceptable salts of the azaanthraquinone alkaloid machine can be prepared into oral pharmaceutical preparations, injections and the like according to the prior art.
Compared with the prior art, the invention has the beneficial effects that: the azaanthraquinone alkaloid disclosed by the invention is obtained by fungal fermentation, not only is high in yield, but also is green and environment-friendly, is beneficial to sustainable industrial production, and is low in cost. Meanwhile, the azaanthraquinone alkaloid inhibits the IC of Lipopolysaccharide (LPS) to induce mouse macrophage RAW264.7 to release Nitric Oxide (NO)50Is 11.2μM, significantly stronger than the positive control dexamethasone (22.5)μM) and the azaanthraquinone alkaloid can be used as an anti-inflammatory drug and has good research and development prospects.
Drawings
FIG. 1 is a chromatogram of the product obtained in example 1.
Detailed Description
The present invention is further illustrated by the following examples, example 1 being the best mode of carrying out the invention.
Example 1
A preparation method of azaanthraquinone alkaloid comprises the following steps:
1) separating endophytic fungi from Chinese medicine red peony (Paeonia lactiflora Pall.), and identifying pure strains of the fungi into Ophioplasma tanaceti strains by utilizing NCBI-BlastN 2.0.6 + through alignment of nucleic acid sequences;
2) preparing a strain liquid seed solution: according to the parts by weight: 200g of potato, 10g of yeast extract, 6g of peptone, 30g of glucose and KH2PO4 2g，MgSO40.5g, diluting to 1L, adjusting pH to 6.8 to prepare a culture solution, subpackaging into 500mL triangular bottles, filling 150mL into each bottle, sterilizing at 115 ℃ and 68kPa for 30 minutes, inoculating, performing rotary culture at 28 ℃ for 3 days at a rotating speed of 155 r/min, and diluting by 100 times to prepare a seed solution for later use;
3) solid fermentation of the strain: taking 30 triangular bottles of 1L, bottling 150g of rice and 180ml of purified water in each triangular bottle of 1L, uniformly mixing the rice and the water, sterilizing at 115 ℃ and 68kPa for 30 minutes, adding 20ml of the seed solution obtained in the step 2) into each bottle, and culturing at room temperature for 40 days;
4) extraction and separation: adding 500ml of ethyl acetate into each bottle of solid fermentation product, soaking for 50-70 min, then carrying out ultrasonic filtration for 30min to obtain filtrate, repeating for 3 times, carrying out reduced pressure concentration on the filtrate, removing the solvent to obtain 30g of total extract, carrying out gradient elution on the extract for 7 times by normal-pressure silica gel column chromatography, wherein the elution conditions are as follows: sequentially adopting petroleum: eluting with ether acetone at a volume ratio of =100:1, 50:1, 30:1, 20:1, 10:1, 5:1 and 1:1 to obtain components F1-F7 in sequence, gradient eluting F7 for 5 times through a Flash column chromatography, eluting the Flash column chromatography with a C18 column under the conditions of eluting with 30%, 40%, 50%, 60% and 65% volume concentration methanol aqueous solutions in sequence to obtain components F7-1-F7-5 in sequence, separating F7-5 through HPLC semi-preparative chromatography, wherein the HPLC semi-preparative chromatography conditions are as follows: an ES-C18 column was used, 50% strength by volume aqueous methanol, the detector wavelength being set at 280 nm. The aza anthraquinone alkaloid scorpinone (retention time 19.8 min, 103 mg) was obtained.
Example 2
A preparation method of azaanthraquinone alkaloid, which is based on example 1 and used KH in the step 2)2PO4To 3g, MgSO4The culture temperature was 25 ℃ under the same conditions as in example 1, except that 0.7g was used.
Example 3
A preparation method of azaanthraquinone alkaloid is based on example 1, and 150g of rice, 200ml of purified water and 22ml of seed liquid are adopted in step 3) to carry out monarch solid fermentation, and other conditions are the same as example 1.
Example 4
The application of the azaanthraquinone alkaloid is used for preparing anti-inflammatory drugs.
The ability of the compound to inhibit LPS to induce mouse macrophage RAW264.7 to release NO is examined by adopting a Griess method. Cells that grew well and were in logarithmic phase were harvested, and RAW264.7 cells at a concentration of 1X 106 cell.mL-1 were seeded in a 96-well plate and cultured in a 5% CO2 incubator at 37 ℃ for 24 hours. After 24 h, adding culture medium containing LPS (final concentration 0.5 mug.mL-1) with different drug concentrations, setting blank group and LPS group at 37 deg.C and 5% CO2Culturing for 36 h. After 36 h, the culture medium supernatant was taken, and the Griess reagent was added thereto to measure the absorbance at 540 nm. By usingThe concentrations are respectively 0, 2.5, 5, 10, 25 and 50 mu mol-1NaNO of (2)2Standard curves were plotted and the concentration of NO 2-in the cell culture supernatants and the inhibition of NO release were calculated from the NaNO2 standard curve.
The results show that the azaanthraquinone alkaloid scorpinone and the fermented extract of the Ophiocoumans tanaceti strain containing the azaanthraquinone alkaloid scorpinone can obviously inhibit LPS from inducing macrophage RAW264.7 of a mouse to release NO and IC50The values are respectively 11.2 µM and 14.3µg/mL, the activity of the azaanthraquinone alkaloid scorpinone is stronger than that of the positive control drug dexamethasone (IC)50Value 22.5μM）。
Therefore, the obtained azaanthraquinone alkaloid has pharmacological activity. Mixing with pharmaceutically acceptable carrier and/or excipient according to conventional method, and making into oral preparation or injection, for example, adding pharmaceutically acceptable carrier and/or excipient according to conventional method to make into tablet, capsule, powder, syrup, injection, etc.
Comparative example 1
A process for preparing azaanthraquinone alkaloid from KH2PO4To 3.5g, MgSO4The amount of the mixture was 0.7g, the culture temperature was 25 ℃ and other conditions were the same as in example 1. The strain failed to propagate during the culture process.
Performance testing
The product obtained in example 1 was tested and the character was yellow powder; the nuclear magnetic resonance detection result is as follows:
ESI-MS：[M]+ m/z 283；
1H NMR（600 MHz，DMSO-d 6）δ H: 9.13 (1H, s), 7.77 (1H, s), 7.24 (1H, s), 7.04 (1H, s), 3.97 (3H, s), 3.94 (3H, s), 2.66 (3H, s)；
13C NMR（150 MHz，DMSO-d 6）δ C: 182.8, 179.2, 164.5, 163.7, 162.5, 148.4, 137.2, 136.4, 125.2, 117.0, 114.6, 105.1, 103.9, 56.6, 56.1, 24.4。
the chromatogram is shown in FIG. 1.
The obtained product can be proved to be aza anthraquinone alkaloid scorpinone and the purity is more than 98 percent.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (8)

1. A preparation method of aza-anthraquinone alkaloid is characterized in that: the method comprises the following steps:
1) separating endophytic fungi from radix Paeoniae Rubra;
2) according to the parts by weight: 200 parts of potato, 8-12 parts of yeast extract, 5-7 parts of peptone, 25-35 parts of glucose and KH2PO41-3 parts of MgSO40.3-0.7 part of the seed culture medium, adding water with the volume of 2.8-3.2 times, adjusting the pH value to 6.8 to prepare a culture solution, sterilizing, inoculating, performing rotary culture at 25-30 ℃ for 3 days, and diluting by 100 times to prepare a seed solution for later use;
3) uniformly mixing rice with water, sterilizing, adding the seed solution obtained in the step 2), and culturing for 40 days, wherein the rice: water: the weight ratio of the seed liquid to the seed liquid is 1.5: 1.5-2.0: 0.18 to 0.22;
4) adding ethyl acetate with the volume of 1.4-1.6 times that of the culture solution obtained in the step 3) into the culture solution, soaking for 50-70 min, then carrying out ultrasonic filtration for 25-35 min to obtain filtrate, repeating the process for 3 times, carrying out reduced pressure concentration on the filtrate, removing the solvent to obtain an extract, carrying out gradient elution on the extract through normal-pressure silica gel column chromatography, carrying out gradient elution on the final elution product through a Flash column chromatography, and carrying out HPLC (high performance liquid chromatography) semi-preparative chromatographic separation on the final elution product.
2. A process for the preparation of azaanthraquinone alkaloids according to claim 1, characterized in that: the gradient elution condition of the normal pressure silica gel column chromatography in the step 4) is as follows: sequentially adopting petroleum: etheracetone volume ratio =100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1: 1.
3. A process for the preparation of azaanthraquinone alkaloids according to claim 1, characterized in that: and 4) adopting a C18 column for Flash column chromatography, wherein the gradient elution condition is that methanol aqueous solution with volume concentration of 30%, 40%, 50%, 60% and 65% is adopted for elution in sequence.
4. A process for the preparation of azaanthraquinone alkaloids according to claim 1, characterized in that: the HPLC semi-preparative chromatographic separation conditions in the step 4) are as follows: an ES-C18 column was used, 50% strength by volume aqueous methanol, the detector wavelength being set at 280 nm.
5. A process for the preparation of azaanthraquinone alkaloids according to claim 1, characterized in that: and 2) subpackaging the culture solution before sterilization into 500ml triangular bottles, wherein each bottle contains 150ml of the culture solution.
6. A process for the preparation of azaanthraquinone alkaloids according to claim 5, characterized in that: the sterilization in the steps 2) and 3) adopts 115 ℃ and sterilization is carried out for 30min under 68 kPa.
7. A process for the preparation of azaanthraquinone alkaloids according to claim 1, characterized in that: the rotary culture in the step 2) adopts 155 r/min.
8. Use of an azaanthraquinone alkaloid according to claim 1, characterized in that: can be used for preparing antiinflammatory drugs.
CN202110328855.7A 2021-03-27 2021-03-27 Preparation method and application of azaanthraquinone alkaloid Pending CN113005158A (en)

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