Preparation method of oxaphenanthrene ketone and anti-inflammatory application of oxaphenanthrene ketone

Abstract

The invention discloses a preparation method of oxaphenanthrene ketone and an anti-inflammatory application of the oxaphenanthrene ketone. The oxaphenanthrene ketone is monascinin and monascinol, and the oxaphenanthrene ketone is monascinin and monascinol.

Description

Technical Field

[0001] This invention relates to the field of anti-inflammatory compounds, and more particularly to a method for preparing oxaphenanthone and its anti-inflammatory uses. Background Technology

[0002] Inflammation is a highly conserved defense mechanism of the immune system, designed to protect the host from harmful stimuli. However, abnormal activation of the inflammatory response is closely related to the development of various diseases. Clinically common cardiovascular and cerebrovascular diseases, neurodegenerative diseases, gastrointestinal diseases, and diabetes often induce inflammatory responses due to factors such as infection or local tissue damage, manifesting as local inflammation and a significant increase in the levels of pro-inflammatory cytokines. This complex pattern of inflammatory development and its crucial role in various diseases has attracted widespread attention from researchers. Targeting inflammation-related pathways to assist in the treatment of inflammatory diseases is one of the important directions of current medical research.

[0003] Macrophages play an indispensable role in the body's defense mechanisms. In in vitro experiments, lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7 can produce chemokines and pro-inflammatory factors, thereby triggering an inflammatory response. Besides macrophages, mouse microglia (BV2), as neuroimmune cells in the mouse brain, can also produce various inflammatory factors after LPS induction, such as nitric oxide (NO), TNF-α, IL-6, and IL-1β, thus triggering a neuroinflammatory response. This further demonstrates that inflammatory responses involve multiple cell types and signaling pathways, and their complexity increases the difficulty of treatment while also providing more targets and opportunities for finding new anti-inflammatory drugs.

[0004] Against this backdrop, the discovery of anti-inflammatory small molecules derived from marine microorganisms is of great significance. Marine microorganisms live in unique ecological environments, and their metabolites exhibit rich structural diversity and bioactivity. Discovering and mining anti-inflammatory small molecules from marine microorganisms can provide a new material basis for the development of anti-inflammatory drugs and offer new perspectives for a deeper understanding of the molecular mechanisms of inflammatory responses. Attached Figure Description

[0005] Figure 1 This is a schematic diagram of the structural formulas of oxaphenanthroline monascin and monascinol.

[0006] Figure 2 This is a graph showing the effect of oxaflavin (monascin) on the NO content in the supernatant of LPS-induced Raw264.7 cells.

[0007] Figure 3 This is a graph showing the effect of oxaflavin (monascin) on the NO content in the supernatant of LPS-induced BV2 cells.

[0008] Figure 4 This is a graph showing the effect of oxaflavin (monascinol) on the NO content in the supernatant of LPS-induced Raw264.7 cells.

[0009] Figure 5 This is a graph showing the effect of oxaflavin (monascinol) on the NO content in the supernatant of LPS-induced BV2 cells. Summary of the Invention

[0010] The purpose of this invention is to provide a method for preparing oxapranolone and its novel applications. The preparation method includes the following steps:

[0011] Inoculation: Inoculate Monascus sp. Mr83-1 or its seed liquid into rice and millet solid fermentation medium and then culture by solid fermentation.

[0012] Extraction: After incubating at room temperature for 28±1 days, add ethyl acetate solution to soak the fermentation product. After soaking, pour off the upper layer of ethyl acetate solution. Extract 1-4 times per bottle. Filter the obtained extract with filter paper and concentrate under reduced pressure to solid state to obtain ethyl acetate extract.

[0013] Separation: The ethyl acetate extract was subjected to silica gel column chromatography, sequentially eluted with 0%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethyl acetate / petroleum ether mixtures (e.g., 2% refers to a volume ratio of ethyl acetate to petroleum ether of 2:98), using two column volumes for each gradient. All fractions were collected, and similar fractions were combined by thin-layer chromatography to obtain eight components, named Fr.1-8. Component Fr.3 contained a large amount of yellow precipitate powder, which was filtered to obtain a mixture of monascin and monascinol. This mixture was then purified by preparative high-performance liquid chromatography (HPLC) with acetonitrile-water elution to separate the compounds monascin and monascinol. The structural formulas of monascin and monascinol are shown in formula (A).

[0014]

[0015] Furthermore, the method for preparing the seed liquid is as follows:

[0016] Preparation of the strain: Inoculate activated Monascus sp. Mr83-1 on PDA medium plates and incubate at room temperature;

[0017] Preparation of fermentation seed culture: Select the above single strains and culture them in PDB medium at 180±20 rpm and 28±2℃ for 3±0.5 days. The culture is the seed culture.

[0018] Furthermore, the rice and millet solid fermentation culture medium consists of 35g of rice, 35g of millet, 0.3g of peptone, 0.1g of monosodium glutamate, 100mL of water, and 3g of sea salt per 1L Erlenmeyer flask.

[0019] Furthermore, in the extraction step, the static incubation time at room temperature is 28 days, and each bottle is extracted 3 times.

[0020] Furthermore, in the separation step, the acetonitrile-water gradient elution program is as follows: acetonitrile isocratic at 80%, flow rate at 10 mL / min.

[0021] The present invention also protects the use of the prepared oxafranone or its pharmaceutical salt in the preparation of anti-inflammatory drugs.

[0022] Furthermore, the aforementioned use refers to the use of oxafranone in reducing the levels of inflammatory factors.

[0023] The oxaphenanthrene ketones monascin and monascinol of the present invention can also be prepared by chemical synthesis.

[0024] The embodiments of the present invention verify that both oxaphenone monascin and monascinol can effectively reduce the level of NO released from LPS-induced RAW264.7 cells, and that oxaphenone monascinol can effectively reduce the level of NO released from LPS-induced BV2 cells. Both oxaphenone monascin and monascinol have anti-inflammatory activity.

[0025] Example 1: Isolation and preparation of compounds monascin and monascinol:

[0026] The strain *Monascus* sp. Mr83-1 is a marine *Monascus* sp. strain. It was available from the Marine Microbial Culture Collection Center, Xiamen, China, with the catalog number MCCC 3A02660.

[0027] Preparation of seed culture of Monascus sp. Mr83-1: Commercially available PDA medium was sterilized at high temperature and plated. The plates were then inoculated with activated Monascus sp. Mr83-1 at room temperature as the inoculum. PDB medium was then added to several Erlenmeyer flasks, sterilized at high temperature, and then inoculated with the above inoculum and cultured (at 180±20 rpm and 28±2℃ for 3±0.5 days). This culture was used as the seed culture.

[0028] The strain Monascus sp. Mr83-1 or its seed culture was inoculated into a rice-millet culture medium (culture medium formula: 35g rice, 35g millet, 0.3g peptone, 0.1g monosodium glutamate, 100mL water, 3g sea salt) (using solid-state fermentation, prepare a fermentation bottle, add the rice-millet solid-state fermentation medium, sterilize at high temperature, inoculate with the above seed culture, and culture; the rice-millet solid-state fermentation medium is 35g rice, 35g millet, 0.3g peptone, 0.1g monosodium glutamate, 100mL water, 3g sea salt per 1L Erlenmeyer flask), and incubated statically at 28℃ for 28 days to obtain the fermentation product. The crude extract was then obtained by extraction with ethyl acetate (the fermentation product was soaked in ethyl acetate, and each bottle was extracted three times; the resulting extract was filtered through filter paper and concentrated under reduced pressure to a solid state, yielding the ethyl acetate extract, i.e., the crude extract). The crude extract was then separated by silica gel column chromatography, using 0%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethyl acetate sequentially. A petroleum ether mixture (i.e., ethyl acetate and petroleum ether eluent in volume ratios of 0:100, 2:98, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) was used to flush the column, and all fractions were collected. Thin-layer chromatography analysis was performed to combine similar fractions, ultimately yielding eight components (Fr.1-8). Component Fr.3 contained a large amount of yellow precipitate powder, which was filtered to obtain a mixture of monacin and monacinol. Preparative high-performance liquid chromatography (HPLC) was used for purification, eluting with acetonitrile-water, to separate oxaphenanthone monacin and monacinol (structural formulas shown in (A)).

[0029] Example 2: Structural analysis of compounds monascin and monascinol: The collected monascin was a yellow amorphous powder, and monascinol was an orange-yellow paste; NMR data are shown in Table 1 (measured at 400 MHz).

[0031] Monascin molecular formula C 21 H 26 O5, High-resolution mass spectrometry (HRESIMS) (positive) m / z 381.1674 [M+Na] + (calcd for C 21 H 26 O5Na, 381.1678). Monascinol molecular formula C21 H 28 O5, High-resolution mass spectrometry (HRESIMS) (positive) m / z 383.1840 [M+Na] + (calcd for C 21 H 28 O5Na, 383.1834).

[0032] Ultimately, the compounds were defined as monascin and monascinol, and their structures are shown in [link to structure]. Figure 1 .

[0033] Table 1. NMR data of compounds monascin and monascinol (measured at 400 MHz)

[0034]

[0035]

[0036] Example 3: Anti-inflammatory activity of compound monascin:

[0037] RAW264.7 cells were seeded into 48-well plates and the experiments were conducted according to the following groups:

[0038] A (Control group): Add RAW264.7 cell basal culture medium to the well plates;

[0039] B (LPS group): RAW264.7 cell basal medium was added to the well plate and incubated for 2 hours. Then, LPS and RAW264.7 cell basal medium were added to the well plate to make the final concentration of LPS 200 ng / mL and incubated for 24 hours.

[0040] C (3μM DEX+LPS group): DEX and RAW264.7 cell basal medium were added to the well plates to make the final DEX concentration 3μM, and the mixture was incubated for 2 hours. The supernatant was discarded, and DEX, LPS and RAW264.7 cell basal medium were added to the well plates to make the final DEX concentration 3μM and the final LPS concentration 200ng / mL, and the mixture was incubated for 24 hours.

[0041] D (1μM monascin + LPS group): Add monascin and RAW264.7 cell basal medium to the wells to achieve a final monascin concentration of 1μM, and incubate for 2 hours. Discard the supernatant, add monascin, LPS and RAW264.7 cell basal medium to the wells to achieve a final monascin concentration of 1μM and a final LPS concentration of 200ng / mL, and incubate for 24 hours.

[0042] E (10μM monascin + LPS group): Add monascin and RAW264.7 cell basal medium to the wells to achieve a final monascin concentration of 10μM, and incubate for 2 hours. Discard the supernatant, add monascin, LPS, and RAW264.7 cell basal medium to the wells to achieve a final monascin concentration of 10μM and a final LPS concentration of 200ng / mL, and incubate for 24 hours.

[0043] F (20μM monascin + LPS group): Add monascin and RAW264.7 cell basal medium to the wells to achieve a final monascin concentration of 20μM, and incubate for 2 hours. Discard the supernatant, add monascin, LPS and RAW264.7 cell basal medium to the wells to achieve a final monascin concentration of 20μM and a final LPS concentration of 200ng / mL, and incubate for 24 hours.

[0044] Cells in all groups were cultured at 37℃ and 5% CO2. 50 μL of supernatant was transferred to a new 96-well plate, and 50 μL each of Griess I and Griess II reagents were added sequentially. The mixture was then thoroughly mixed, and the OD value was measured at 540 nm using a microplate reader. The NO content of each group was calculated based on the standard curve.

[0045] like Figure 2 As shown, Figure 2 This is a graph showing the effect of monascin on the NO content in the supernatant of LPS-induced RAW264.7 cells. From... Figure 2 As can be seen, compared with group A (Control group), the NO release level of cells in group B (LPS group) was significantly increased, and the difference was statistically significant (####P<0.0001).

[0046] In group C (3μM DEX+LPS group), after preprotection with 3μM DEX (dexamethasone, positive control), the NO release level of RAW264.7 cells was significantly lower than that in group B (*P<0.05).

[0047] Groups D (1 μM monascin + LPS), E (10 μM monascin + LPS), and F (20 μM monascin + LPS) showed significantly lower NO release levels in RAW264.7 cells compared to group B after pretreatment with different concentrations of monascin. The NO release levels in the 1 μM, 10 μM, and 20 μM monascin groups were statistically significantly different from those in group B (*P < 0.05, ****P < 0.0001). Compared to group C, groups E and F were more effective at reducing NO levels.

[0048] Experimental results showed that LPS-induced RAW264.7 cells produced inflammatory factors such as NO, and the NO release level increased. When different concentrations of monascin were administered, the NO level was significantly reduced, indicating that the compound monascin has anti-inflammatory activity.

[0049] Example 4: Anti-inflammatory activity of compound monascin:

[0050] BV2 cells were seeded in 48-well plates and experiments were conducted according to the following groups:

[0051] Group A (Control group): BV2 cell basal culture medium was added to the well plates;

[0052] Group B (LPS group): After adding BV2 cell basal culture medium to the well plate and incubating for 12 hours, LPS and BV2 cell basal culture medium were added to the well plate to make the final concentration of LPS 500 ng / mL, and incubated for 24 hours.

[0053] Group C (1μM monascin + LPS group): monascin and BV2 cell basal medium were added to the well plates to bring the final concentration of monascin to 1μM, and the mixture was incubated for 12 hours. The supernatant was discarded, and monascin, LPS, and BV2 cell basal medium were added to the well plates to bring the final concentration of monascin to 1μM and the final concentration of LPS to 500ng / mL, and the mixture was incubated for 24 hours.

[0054] Group D (10μM monascin + LPS group): monascin and BV2 cell basal medium were added to the well plates to bring the final monascin concentration to 10μM, and the mixture was incubated for 12 hours. The supernatant was discarded, and monascin, LPS, and BV2 cell basal medium were added to the well plates to bring the final monascin concentration to 10μM and the final LPS concentration to 500ng / mL, and the mixture was incubated for 24 hours.

[0055] Group E (20μM monascin + LPS group): monascin and BV2 cell basal medium were added to the well plates to bring the final monascin concentration to 20μM, and the mixture was incubated for 12 hours. The supernatant was discarded, and monascin, LPS, and BV2 cell basal medium were added to the well plates to bring the final monascin concentration to 20μM and the final LPS concentration to 500ng / mL, and the mixture was incubated for 24 hours.

[0056] Cells in all groups were cultured at 37℃ and 5% CO2. 50 μL of supernatant was transferred to a new 96-well plate, and 50 μL each of Griess I and Griess II reagents were added sequentially. The mixture was then thoroughly mixed, and the OD value was measured at 540 nm using a microplate reader. The NO content of each group was calculated based on the standard curve.

[0057] like Figure 3 As shown, Figure 3 This is a graph showing the effect of monascin on the NO content in the supernatant of LPS-induced BV2 cells. From... Figure 3 As can be seen, compared with group A (Control group), the NO release level of cells in group B (LPS group) was significantly increased, and the difference was statistically significant (####P<0.0001).

[0058] Groups C (1 μM monascin + LPS), D (10 μM monascin + LPS), and E (20 μM monascin + LPS) showed no statistically significant difference in NO release levels between BV2 cells and group B after preprotection treatment with different concentrations of monascin.

[0059] Experimental results showed that LPS-induced BV2 cells produced inflammatory factors such as NO, and the NO release level increased. When different concentrations (1, 10, 20 μM) of monascin were administered, the NO level showed no significant change compared to group B. This indicates that the compound monascin can selectively reduce the NO level in RAW264.7 cells, but has no such effect on BV2 cells.

[0060] Example 5: Anti-inflammatory activity of compound monacinol:

[0061] RAW264.7 cells were seeded into 48-well plates and the experiments were conducted according to the following groups:

[0062] A (Control group): Add RAW264.7 cell basal culture medium to the well plates;

[0063] B (LPS group): After adding RAW264.7 cell basal medium to the well plate and incubating for 2 hours, LPS and RAW264.7 cell basal medium were added to the well plate to make the final concentration of LPS 200 ng / mL, and incubated for 24 hours.

[0064] C (3μM DEX+LPS group): DEX and RAW264.7 cell basal medium were added to the well plates to make the final DEX concentration 3μM, and the mixture was incubated for 2 hours. The supernatant was discarded, and DEX, LPS and RAW264.7 cell basal medium were added to the well plates to make the final DEX concentration 3μM and the final LPS concentration 200ng / mL, and the mixture was incubated for 24 hours.

[0065] D (1μM monascinol + LPS group): Add monascinol and RAW264.7 cell basal medium to the wells to achieve a final monascinol concentration of 1μM, and incubate for 2 hours. Discard the supernatant, add monascinol, LPS, and RAW264.7 cell basal medium to the wells to achieve a final monascinol concentration of 1μM and a final LPS concentration of 200ng / mL, and incubate for 24 hours.

[0066] E (10μM monascinol + LPS group): Add monascinol and RAW264.7 cell basal medium to the wells to achieve a final monascinol concentration of 10μM, and incubate for 2 hours. Discard the supernatant, and add monascinol, LPS, and RAW264.7 cell basal medium to the wells to achieve a final monascinol concentration of 10μM and a final LPS concentration of 200ng / mL, and incubate for 24 hours.

[0067] F (20μM monascinol+LPS group): Add monascinol and RAW264.7 cell basal medium to the wells to a final monascinol concentration of 20μM, and incubate for 2 hours. Discard the supernatant, add monascinol, LPS and RAW264.7 cell basal medium to the wells to a final monascinol concentration of 20μM and a final LPS concentration of 200ng / mL, and incubate for 24 hours.

[0068] Cells in all groups were cultured at 37℃ and 5% CO2. 50 μL of supernatant was transferred to a new 96-well plate, and 50 μL each of Griess I and Griess II reagents were added sequentially. The mixture was then thoroughly mixed, and the OD value was measured at 540 nm using a microplate reader. The NO content of each group was calculated based on the standard curve.

[0069] like Figure 4 As shown, Figure 4 This is a graph showing the effect of monascinol on the NO content in the supernatant of LPS-induced RAW264.7 cells. From... Figure 4As can be seen, compared with group A (Control group), the NO release level of cells in group B (LPS group) was significantly increased, and the difference was statistically significant (####P<0.0001).

[0070] In group C (3μM DEX+LPS group), after preprotection with 3μM DEX (dexamethasone, positive control), the NO release level of RAW264.7 cells was significantly lower than that in group B (*P<0.05).

[0071] Groups D (1 μM monascinol + LPS), E (10 μM monascinol + LPS), and F (20 μM monascinol + LPS) showed significantly lower NO release levels in RAW264.7 cells compared to group B after pretreatment with different concentrations of monascinol. The NO release levels in the 1 μM, 10 μM, and 20 μM monascinol groups were statistically significantly different from those in the LPS group (*P < 0.05, ****P < 0.0001). Compared to group C, groups E and F were more effective at reducing NO levels.

[0072] Experimental results showed that LPS-induced RAW264.7 cells produced inflammatory factors such as NO, and the NO release level increased. When different concentrations of monascinol were administered, the NO level was significantly reduced, indicating that the compound monascinol has anti-inflammatory activity.

[0073] Example 6: Anti-inflammatory activity of compound monascinol:

[0074] BV2 cells were seeded in 48-well plates and experiments were conducted according to the following groups:

[0075] Group A (Control group): BV2 cell basal culture medium was added to the well plates;

[0076] Group B (LPS group): After adding BV2 cell basal culture medium to the well plate and incubating for 12 hours, LPS and BV2 cell basal culture medium were added to the well plate to make the final concentration of LPS 500 ng / mL, and incubated for 24 hours.

[0077] Group C (1μM monascinol + LPS group): monascinol and BV2 cell basal medium were added to the wells to bring the final concentration of monascinol to 1μM, and the mixture was incubated for 12 hours. The supernatant was discarded, and monascinol, LPS, and BV2 cell basal medium were added to the wells to bring the final concentration of monascinol to 1μM and the final concentration of LPS to 500 ng / mL, and the mixture was incubated for 24 hours.

[0078] Group D (10μM monascinol + LPS group): monascinol and BV2 cell basal medium were added to the wells to bring the final concentration of monascinol to 10μM, and the mixture was incubated for 12 hours. The supernatant was discarded, and monascinol, LPS, and BV2 cell basal medium were added to the wells again to bring the final concentration of monascinol to 10μM and the final concentration of LPS to 500ng / mL, and the mixture was incubated for 24 hours.

[0079] Group E (20 μM monascinol + LPS group): monascinol and BV2 cell basal medium were added to the wells to bring the final monascinol concentration to 20 μM, and the mixture was incubated for 12 h. The supernatant was discarded, and monascinol, LPS, and BV2 cell basal medium were added to the wells again to bring the final monascinol concentration to 20 μM and the final LPS concentration to 500 ng / mL, and the mixture was incubated for 24 h.

[0080] Cells in all groups were cultured at 37℃ and 5% CO2. 50 μL of supernatant was transferred to a new 96-well plate, and 50 μL each of Griess I and Griess II reagents were added sequentially. The mixture was then thoroughly mixed, and the OD value was measured at 540 nm using a microplate reader. The NO content of each group was calculated based on the standard curve.

[0081] like Figure 5 As shown, Figure 5 This is a graph showing the effect of monascinol on the NO content in the supernatant of LPS-induced BV2 cells. From... Figure 5 As can be seen, compared with group A (Control group), the NO release level of cells in group B (LPS group) was significantly increased, and the difference was statistically significant (####P<0.0001).

[0082] After preprotection treatment with different concentrations of monascinol, the NO release levels of BV2 cells in groups C (1 μM monascinol + LPS), D (10 μM monascinol + LPS), and E (20 μM monascinol + LPS) were significantly lower than those in group B compared to group D. The NO release levels in the 10 μM and 20 μM monascinol groups were statistically significantly different from those in the LPS group (P < 0.0001).

[0083] Experimental results showed that LPS-induced BV2 cells produced inflammatory factors such as NO, and the NO release level increased. When different concentrations of monascinol were administered, the NO level was significantly reduced, indicating that the compound monascinol has anti-inflammatory activity against BV2 cells and can exert a neuroprotective effect.

[0084] The compound monascinol inhibited LPS-induced NO levels in both RAW264.7 and BV2 cells. Monascinol selectively inhibited LPS-induced NO release from RAW264.7 cells, but had no similar effect on BV2 cells. Figure 1 As shown, compared to monascin, the 3' position of the compound monascinol is hydroxylated, suggesting that derivatization and changes at the 3' position in the structure may affect its anti-inflammatory activity.

[0085] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing oxaphenanthone, characterized in that, Includes the following steps: Inoculation: Inoculate Monascus sp. Mr83-1 or its seed liquid into rice and millet solid fermentation medium and then culture by solid fermentation. Extraction: After incubating at room temperature for 28±1 days, add ethyl acetate solution to soak the fermentation product. After soaking, pour off the upper layer of ethyl acetate solution. Extract 1-4 times per bottle. Filter the obtained extract with filter paper and concentrate under reduced pressure to solid state to obtain ethyl acetate extract. Separation: The ethyl acetate extract was subjected to silica gel column chromatography, with sequential elution using 0%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethyl acetate / petroleum ether mixtures, using two column volumes for each gradient. All fractions were collected, and similar fractions were combined by thin-layer chromatography, ultimately yielding eight components, named Fr.1-8. Component Fr.3 contained a large amount of yellow precipitate powder, which was filtered to obtain a mixture of monascin and monascinol. This mixture was then purified by preparative high-performance liquid chromatography (HPLC) with acetonitrile-water elution to separate the compounds monascin and monascinol. The structural formulas of monascin and monascinol are shown in formula (A).

2. The method for preparing oxaphenanthone as described in claim 1, characterized in that, The method for preparing the seed liquid is as follows: Preparation of the strain: Inoculate activated Monascus sp. Mr83-1 on PDA medium plates and incubate at room temperature; Preparation of fermentation seed culture: Select the above single strains and culture them in PDB medium at 180±20 rpm and 28±2℃ for 3±0.5 days. The culture is the seed culture.

3. The method for preparing oxaphenanthone as described in claim 1, characterized in that, The rice and millet solid fermentation medium consists of 35g of rice, 35g of millet, 0.3g of peptone, 0.1g of monosodium glutamate, 100mL of water, and 3g of sea salt per 1L Erlenmeyer flask.

4. The method for preparing oxaphenanthone as described in claim 1, characterized in that, In the extraction step, the static incubation time at room temperature is 28 days, and each bottle is extracted 3 times.

5. The method for preparing oxaphenanthone as described in claim 1, characterized in that, In the separation step, the acetonitrile-water gradient elution program is as follows: acetonitrile isocratic at 80%, flow rate at 10 mL / min.

6. Use of the oxafranone or its pharmaceutical salt prepared according to claims 1-5 in the preparation of anti-inflammatory drugs.

7. The use as described in claim 6, characterized in that, The aforementioned use refers to the use of oxafranone in reducing the levels of inflammatory factors.

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