A method for preparing N1-reverse isopentenylindole alkaloids

Abstract

This invention discloses a method for preparing N1-reverse isopentenylindole alkaloids, belonging to the fields of natural product chemistry and microbial fermentation technology. Using *Purple Ear fungus* (accession number GDMCC NO:62288) as the strain, two N1-reverse isopentenylindole alkaloids (compound 1: C) were obtained by liquid fermentation, ethyl acetate extraction, and reversed-phase silica gel-dextran gel-normal-phase silica gel column chromatography with directional separation. 15 H 19 NO4; Compound 2: C 14 H 15 NO2). Activity verification showed that both compounds had no significant cytotoxicity at concentrations of 1-200 μM and exhibited significant anti-inflammatory activity. This invention is the first to obtain active N1-reverse isopentenyl indole alkaloids from *Auricularia auricula-judae*, and the preparation method is green, stable, and scalable, providing new lead compounds and technical support for the development of anti-inflammatory drugs and functional foods.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry, microbial fermentation engineering and preparation of active natural products, and specifically relates to a method for preparing N1-reverse isopentenyl indole alkaloids. Background Technology

[0002] Indole natural products are among the most widely distributed and biologically active heterocyclic compounds in nature. Indole-3-carboxylic acid N1-isopentenyl substituted derivatives are a class of natural products with unique structures and outstanding activities. Their parent core is indole, with a carboxylic acid linked at position 3 and an isopentenyl side chain directly attached at position 1 (nitrogen atom). Based on the linkage method, they are divided into two categories: normal isopentenene (-CH2CH=C(CH3)2) and reverse isopentenene (-C(CH3)2CH=CH2). N1-isopentenyl indole alkaloids possess anti-inflammatory, neuroprotective, antioxidant, and antitumor activities, and exhibit good water solubility and high membrane permeability, making them highly valuable lead structures in the field of natural drug development.

[0003] Currently, the main methods for obtaining N1-reverse isopentenyl indole alkaloids rely on natural extraction and chemical synthesis, but both methods face significant technical bottlenecks. Regarding natural extraction, these compounds exist only in small quantities in a few terrestrial plants, marine microorganisms, and fungi, with extremely low accumulation in organisms, making separation difficult, recovery rates low, and hindering large-scale preparation. In terms of chemical synthesis, the field of synthetic chemistry currently struggles to establish efficient and highly selective synthetic routes, especially given the long-standing lack of N1-reverse isopentenylation technology for indole. Traditional synthesis requires multiple protection and deprotection steps, uses large amounts of toxic reagents, produces numerous byproducts, has low yields, and makes it difficult to obtain naturally occurring products, significantly reducing their biological activity. Therefore, there is an urgent need to develop a green, efficient, and scalable preparation method.

[0004] Recent studies have shown that edible and medicinal fungi are an important source of natural active compounds, and the genus *Auricularia* (*Auricularia*) is a significant source of such compounds. Auricularia Fungi are rich in secondary metabolites, but no utilization of purple ear fungus (Auricularia auricula-judae) has been observed. Auricularia thailandica Research on the preparation of indole-3-carboxylic acid N1-reverse isopentenyl substituted derivatives by fermentation and their anti-inflammatory activities shows a significant gap in the high-value utilization of related resources. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of high difficulty, limited raw materials, and low purity in the preparation of natural N1-reverse isopentenyl indole alkaloids in the prior art, and to fill the gap in the research on secondary metabolites of *Auricularia auricula-judae*. This invention provides a method for preparing N1-reverse isopentenyl indole alkaloids, specifically using a *Auricularia auricula-judae* strain (preservation number GDMCC NO:62288) to prepare two high-purity N1-reverse isopentenyl indole alkaloid monomer compounds, and using the prepared N1-reverse isopentenyl indole alkaloid monomer compounds as anti-inflammatory drugs.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A method for preparing N1-reverse isopentenyl indole alkaloids involves using *Pseudomonas aeruginosa* strain with accession number GDMCC NO: 62288 as raw material, and obtaining two N1-reverse isopentenyl indole alkaloids through liquid fermentation, organic extraction, and separation and purification. The N1-reverse isopentenyl indole alkaloids are both N1-substituted indole-3-carboxylic acid alkaloids, including compound 1 and compound 2, wherein compound 1 has the molecular formula C1. 15 H 19 NO4, compound 2 has the molecular formula C 14 H 15 NO2 has the following structural formulas: or .

[0007] Furthermore, the above method specifically includes the following steps: (1) Liquid fermentation: After activating the purple ear fungus strain, a liquid fermentation seed liquid was obtained. Then, the liquid fermentation seed liquid was inoculated into a liquid fermentation medium and cultured in a shaker at a constant temperature to obtain the fermentation broth. (2) Organic extraction: The fermentation broth is concentrated to 1 / 4 to 1 / 6 of its original volume, extracted with an equal volume of organic solvent, the organic phase is collected, and after dehydration and concentration, the crude organic extract is obtained. (3) Separation and purification: After dissolving the crude organic extract, reversed-phase silica gel column chromatography, Sephadex LH20 dextran gel column chromatography, or normal-phase silica gel column chromatography were performed sequentially to obtain pure products of compound 1 and compound 2, respectively.

[0008] Furthermore, the liquid fermentation medium mentioned in step (1) is potato glucose liquid medium, and the culture conditions are 25-28℃, shaker 160-280 rpm, and constant temperature culture for 15-20 days.

[0009] Furthermore, the activation described in step (1) involves inoculating the Purple Ear strain onto potato dextrose agar solid slant medium and culturing it at a constant temperature of 25-30℃ for 5-9 days. Subsequently, the bacterial blocks are cut and transferred into potato liquid medium and continuously cultured at 25-30℃ and 200-250 rpm for 12-20 days as liquid fermentation seed liquid. The inoculation amount is 1-10% of the culture medium volume.

[0010] Furthermore, the organic solvent in step (2) is one or more organic solvents that are not miscible with water, such as ethyl acetate and ethyl butyrate.

[0011] Further, the reversed-phase silica gel column chromatography conditions in step (3) are as follows: gradient elution is performed using organic solvent-water of different concentrations as eluents, wherein the organic solvent is methanol, acetone or acetonitrile, with a volume ratio of 30-100%, and finally detected by thin-layer chromatography. The developing solvent is a mixture of chloroform and methanol in a volume ratio of 10:1, and the colorimetric reagent includes iodine or 10 wt% sulfuric acid ethanol solution. The eluents under the gradient are combined with 50-70% organic solvent-water mixture. When the volume ratio of organic solvent is 50%, the eluent is collected as the component containing compound 1, and when the volume ratio of organic solvent is 70%, the eluent is collected as the component containing compound 2.

[0012] Furthermore, in step (3), the Sephadex LH20 dextran gel column chromatography conditions are as follows: methanol or acetone is used as the eluent, the flow rate is 15-20 s / drop, and 5 mL is collected from each tube; the fractions with an elution volume of 800-1200 mL are combined, of which the elution volume of 1000-1200 mL is the fraction containing compound 1, and the elution volume of 800-1000 mL is the pure product of compound 2.

[0013] Furthermore, in step (3), the normal phase silica gel column chromatography conditions are as follows: using dichloromethane-methanol with a volume ratio of 600:1 to 300:1 as the eluent, combining similar components, and concentrating under reduced pressure to dryness to obtain the pure compound; wherein the eluents under the dichloromethane-methanol elution gradient with a volume ratio of 400:1 are combined, and concentrated under reduced pressure to dryness to obtain the pure compound 1.

[0014] The N1-reverse isopentenyl indole alkaloid prepared by the above method can be used in the preparation of anti-inflammatory drugs or anti-inflammatory functional foods. The structural formula of the N1-reverse isopentenyl indole alkaloid is as follows: or .

[0015] Furthermore, the anti-inflammatory drug or anti-inflammatory functional food also includes pharmaceutically or food-grade excipients.

[0016] Compared with the prior art, the present invention has the following advantages: (1) The purple ear fungus strain GDMCC NO: 62288 provided by the present invention has stable genetic traits, strong liquid fermentation ability, and can stably synthesize indole secondary metabolites. The culture conditions are simple and controllable, making it suitable for large-scale fermentation production. It provides an excellent starting strain for the high-value utilization of purple ear fungus resources.

[0017] (2) The preparation method provided by the present invention uses the liquid fermentation product of purple ear fungus as raw material. Through the combination separation process of organic extraction + reversed-phase silica gel column chromatography + dextran gel column chromatography + normal-phase silica gel column chromatography, two high-purity indole monomer compounds can be prepared by directional separation. The process steps are controllable, the separation efficiency is high, and the product purity is good. This solves the problem that natural N1-reverse isopentenyl indole alkaloids are difficult to prepare on a large scale in the prior art.

[0018] (3) This invention is the first to isolate two novel natural N1-reverse isopentenyl indole alkaloids (compound 1 and compound 2) from the fermentation products of *Auricularia auricula-judae*, enriching the compound library of natural indole alkaloids. Both compounds 1 and 2 exhibit significant anti-inflammatory activity: compound 1 significantly inhibited LPS-induced NO production in RAW264.7 cells at concentrations of 100 μM to 200 μM (P<0.01); compound 2 significantly inhibited NO release at concentrations of 50 μM and ~200 μM (P<0.05), with a highly significant difference at 200 μM (P<0.001). These compounds can serve as new lead compounds for the development of novel natural anti-inflammatory drugs, expanding the application scope of *Auricularia auricula-judae* resources and possessing significant research value and industrialization potential. Attached Figure Description

[0019] Figure 1 Compound 1 of the present invention 1 H-NMR spectrum.

[0020] Figure 2 Compound 1 of the present invention 13 C-NMR spectrum.

[0021] Figure 3 Compound 2 of the present invention 1 H-NMR spectrum.

[0022] Figure 4 Compound 2 of the present invention 13 C-NMR spectrum.

[0023] Figure 5 The results show the toxicity of compound 1 to RAW264.7 cells. *** indicates a highly significant difference compared to the Normal group (P<0.001).

[0024] Figure 6 The results show the toxicity of compound 2 to RAW264.7 cells. *** indicates a highly significant difference compared to the Normal group (P<0.001).

[0025] Figure 7 The inhibitory effect of compound 1 on LPS-induced NO production in RAW264.7 cells is shown in Figure 1. *** indicates a highly significant difference compared to the normal control group (P < 0.001); ## indicates a significant difference compared to the LPS model group (P < 0.01); and ### indicates a highly significant difference compared to the LPS model group (P < 0.0001).

[0026] Figure 8 The inhibitory effect of compound 2 on LPS-induced NO production in RAW264.7 cells is shown in Figure ***, which indicates a highly significant difference compared to the normal control group (P < 0.001); # indicates a significant difference compared to the LPS model group (P < 0.05); and ### indicates a highly significant difference compared to the LPS model group (P < 0.001). Detailed Implementation

[0027] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0028] Unless otherwise specified, all materials and reagents used in the following embodiments of the present invention are commercially available conventional reagents; and all experimental methods used are conventional operations in the field unless otherwise specified.

[0029] The purple ear fungus strain described in the following embodiments of the present invention is a high-temperature type of wood ear fungus strain (Thai wood ear fungus) with accession number GDMCC NO: 62288. Auricularia thailandica It has been named Purple Ear Fungus (or simply Purple Ear Fungus). This strain has been disclosed in the patent "A High-Temperature Fungus Strain and Its Fruiting Body Cultivation Method".

[0030] Thin-layer chromatography detection method: Take samples during elution, concentrate them and spot them on silica gel thin-layer plates, develop them with chloroform-methanol = 10:1 as the developing solvent, spray with 10wt% sulfuric acid ethanol colorimetric reagent after development, heat at 105℃ until the spots are clear, and combine components with consistent Rf values ​​and similar spot characteristics.

[0031] The composition of potato glucose liquid culture medium is: 200 g / L potato, 20 g / L glucose, and the balance being water, with natural pH; it is sterilized at 0.1 MPa and 121℃ for 20 min and then cooled before use.

[0032] Example 1 Compound 1 (C 15 H 19 Preparation of NO4 1. Liquid fermentation culture: The *Auricularia auricula-judae* strain with preservation number GDMCC NO: 62288 was inoculated onto Petri dishes containing potato dextrose agar (PDA) solid medium and cultured at 28℃ for 7 days. After mycelial germination and growth, mycelial blocks were selected from the edges of well-grown colonies and cut using a 0.5 cm pore size sterile punch. These blocks were then transferred to Erlenmeyer flasks containing 100 mL of potato dextrose liquid medium and placed in a constant-temperature shaker at 28℃ and 220 rpm for 15 days to obtain the liquid fermentation seed culture. Subsequently, the liquid fermentation seed culture was inoculated at a rate of 2.5% (v / v) into Erlenmeyer flasks containing 200 mL of potato dextrose liquid medium, for a total culture volume of 15 L. The mixture was then cultured at 28℃ and 220 rpm for 15 days to obtain the fermentation broth.

[0033] 2. Organic extraction: The fermentation broth was concentrated under reduced pressure to 1 / 5 of its original volume, and extracted three times with an equal volume of ethyl acetate. The ethyl acetate extract phases were combined, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 1 g of crude extract.

[0034] 3. Separation and purification: (1) Reversed-phase silica gel column chromatography: Dissolve 1 g of crude extract in 1 mL of methanol and load it onto a 150 g reversed-phase silica gel column. Elute with a gradient of 1.5 L of methanol-water mixture, gradually increasing the methanol volume percentage from 30% to 100% (30%, 50%, 70%, 90%, 100%). Collect the eluent in separate bottles. Take 3 mL from each bottle, concentrate it, and perform thin-layer chromatography. Combine similar components and obtain the component containing the target analyte (88.8 mg) from the 50% methanol-water elution gradient.

[0035] (2) Dextran gel column chromatography: Take the above components and load them onto a Sephadex LH20 dextran gel column (column size 26×1000 mm, packing volume 500 mL). Use methanol as the eluent and control the flow rate at 15-20 s / drop. Collect 5 mL from each tube. Take samples during the elution process for thin-layer chromatography detection. Combine similar components. The component containing the target analyte (17.7 mg) is obtained from the 8th component after combination (elution volume is about 1000~1200 mL).

[0036] (3) Normal phase silica gel column chromatography: Take the above components, load them onto a 0.7 g normal phase silica gel column, and elute with a dichloromethane-methanol mixture (volume ratios of 600:1, 500:1, 400:1 and 300:1). Collect the eluents under the dichloromethane-methanol elution gradient with a volume ratio of 400:1, combine them, and concentrate them to dryness under reduced pressure at 40 °C to obtain 12 mg of pure compound 1.

[0037] 4. Structural identification: Nuclear magnetic resonance spectroscopy was performed on compound 1, and the data are as follows: 1 H NMR [600 MHz, Methanol-d4]: δ 8.12 (d, J = 6.1 Hz, 1H, H4), 8.08(s,1H, H2), 7.81 (d, J = 7.4 Hz, 1H, H7), 7.21-7.14 (m, 2H, H-5, H6), 4.45 (dd, J = 8.2, 2.9 Hz, 1H, H2'), 3.87 (d, J = 1.8 Hz, 3H, H9), 3.44-3.32 (m, 2H, H3'), 1.76 (m, 6H, H4', H5').

[0038] 13 C NMR (151 MHz, Methanol-d4): δ 167.51 (C8), 137.24 (C7a), 134.82(C2), 129.61 (C4a), 123.18 (C4), 122.57 (C6), 122.54 (C5), 115.55 (C7),106.69 (C3), 76.07 (C2'), 63.90 (C3'), 63.59 (C1'), 51.38 (C9), 25.06 (C4'), 24.13 (C5').

[0039] Spectroscopic data analysis confirmed that the obtained compound was an N1-reverse isopentenylindole alkaloid with the molecular formula C2. 15 H 19 NO4, structure as follows: .

[0040] Example 2 Compound 2 (C 14 H 15 Preparation of NO2 1. Liquid fermentation culture: Same as step 1 in Example 1, to obtain 15L of purple fungus fermentation broth.

[0041] 2. Organic extraction: Same as step 2 in Example 1, to obtain 1g of crude extract.

[0042] 3. Separation and purification: (1) Reversed-phase silica gel column chromatography: Same as step 3 (1) in Example 1, similar components were combined and the fraction containing the target analyte (56.7 mg) was obtained from the elution gradient of 70% methanol aqueous solution.

[0043] (2) Dextran gel column chromatography: Take the above components and load them onto a Sephadex LH20 dextran gel column. Use methanol as the eluent and control the flow rate at 15-20 s / drop. Collect 5 mL from each tube. Take samples during the elution process for thin-layer chromatography detection. Combine similar components. The pure product of target compound 2 (3.2 mg) is obtained from the 7th component after combination (elution volume is about 800-1000 mL).

[0044] 4. Structural identification: The obtained pure sample was subjected to nuclear magnetic resonance spectroscopy, and the data are as follows: 1 H NMR [600 MHz, Methanol-d4]: δ 8.11 (d, J = 6.1 Hz, 1H, H4), 8.10(s,1H, H2), 7.57 (d, J = 7.4 Hz, 1H, H7), 7.16 (ddd, J = 7.5, 5.2, 1.7 Hz, 2H,H5, H6), 6.18 (dd, J = 8.2, 2.9 Hz, 1H, H2'), 5.27 (d, J = 10.7 Hz, 1H,H3a'), 5.17 (d, J = 17.5 Hz, 1H, H3b'), 1.80 (m, 6H, H4', H5').

[0045] 13 C NMR (151 MHz, Methanol-d4): δ 168.96 (C8), 144.48 (C7), 137.38(C7a), 133.79 (C2), 129.57 (C4a), 122.82 (C6), 122.47 (C4, C5), 115.77 (C3'), 114.84 (C2'), 107.43 (C3), 61.40 (C1'), 28.20 (C4', C5').

[0046] Spectroscopic data analysis confirmed that the obtained compound was an N1-reverse isopentenylindole alkaloid with the molecular formula C2. 14 H 15 NO2 has the following structure: .

[0047] Example 3 Cytotoxicity of Compound 1 and Compound 2 The cytotoxicity of compounds 1 and 2 on mouse monocyte-macrophage RAW264.7 cells was detected using the CCK-8 assay. The experiment included a normal control group, a 10% absolute ethanol (EtOH) positive toxicity control group, a 0.25% DMSO blank control group, and groups containing different concentrations (1, 10, 50, 100, 200 μM) of compounds 1 and 2. Each group was in duplicate. After culturing for 24 h, 10% CCK-8 solution was added for an additional 50 min of incubation, followed by measurement of OD. 450 Values ​​are used to calculate cell viability.

[0048] The results are as follows Figures 5-6 As shown, compared with the normal control group, compound 1 showed no statistically significant difference in RAW264.7 cell viability within the concentration range of 1-200 μM (P>0.05); compound 2 also showed no significant change in RAW264.7 cell viability within the same concentration range (P>0.05). Cell viability was significantly reduced in the 10% anhydrous ethanol group (P<0.001), verifying the reliability of the experimental system. These results indicate that compounds 1 and 2 have no significant toxicity to RAW264.7 cells within the concentration range of 1-200 μM, exhibiting good cell safety and suitable for subsequent evaluation of anti-inflammatory activity.

[0049] Example 4: Anti-inflammatory activity of compounds 1 and 2 An inflammation model was established in RAW264.7 cells induced by lipopolysaccharide (LPS). The anti-inflammatory activity of two derivatives was evaluated by detecting the nitric oxide (NO) content in the cell supernatant. The experiment included a normal control group (no treatment), an LPS model group (400 ng / mL), a dexamethasone (DXM, 30 mM) group, and groups of compounds 1 and 2 at different concentrations (1, 10, 50, 100, 200 μM, DMSO system).

[0050] The results are as follows Figures 7-8 As shown: Compared with the normal control group, the NO content in the LPS model group was significantly increased (P<0.001), indicating that the inflammation model was successfully constructed; compared with the LPS model group, compound 1 significantly reduced NO production at concentrations of 100 μM and 200 μM (P<0.01), showing a significant anti-inflammatory effect; compound 2 significantly inhibited NO release at concentrations of 50 μM and 100 μM (P<0.05), and the difference was extremely significant at a concentration of 200 μM (P<0.001), showing that the anti-inflammatory activity increased in a concentration-dependent manner.

[0051] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing N1-reverse isopentenylindole alkaloids, characterized in that: Using *Purple Ear* strain with accession number GDMCCNO: 62288 as raw material, two N1-reverse isopentenyl indole alkaloids were obtained through liquid fermentation, organic extraction, and separation and purification. These N1-reverse isopentenyl indole alkaloids are both N1-substituted indole-3-carboxylic acid alkaloids, including compound 1 and compound 2, wherein compound 1 has the molecular formula C1. 15 H 19 NO4, compound 2 has the molecular formula C 14 H 15 NO2 has the following structural formulas: or .

2. The method according to claim 1, characterized in that: Specifically, the following steps are included: (1) Liquid fermentation: The purple ear fungus strain was activated to obtain liquid fermentation seed liquid, and then the liquid fermentation seed liquid was inoculated into liquid fermentation medium and cultured in a shaker at a constant temperature to obtain fermentation broth; (2) Organic extraction: The fermentation broth is concentrated to 1 / 4 to 1 / 6 of its original volume, extracted with an equal volume of organic solvent, the organic phase is collected, and after dehydration and concentration, the crude organic extract is obtained. (3) Separation and purification: After dissolving the crude organic extract, reversed-phase silica gel column chromatography, Sephadex LH20 dextran gel column chromatography, or normal-phase silica gel column chromatography were performed sequentially to obtain pure products of compound 1 and compound 2, respectively.

3. The method according to claim 2, characterized in that: The liquid fermentation medium mentioned in step (1) is potato glucose liquid medium, and the culture conditions are 25-28℃, shaker 160-280 rpm, and constant temperature culture for 15-20 days.

4. The method according to claim 2, characterized in that: The activation described in step (1) involves inoculating the *Auricularia auricula-judae* strain onto potato dextrose agar slant medium and culturing it at 25-30℃ for 5-9 days. Subsequently, the bacterial blocks are cut and transferred into potato liquid medium and cultured continuously at 25-30℃ and 200-250 rpm for 12-20 days to obtain the liquid fermentation seed liquid. The inoculation amount is 1-10% of the culture medium volume.

5. The method according to claim 2, characterized in that: The organic solvent in step (2) is ethyl acetate or ethyl butyrate.

6. The method according to claim 2, characterized in that: The reversed-phase silica gel column chromatography conditions in step (3) are as follows: gradient elution is performed using organic solvent-water of different concentrations as eluents, wherein the organic solvent is methanol, acetone or acetonitrile, with a volume ratio of 30-100%, and finally detected by thin-layer chromatography. The developing solvent is a mixture of chloroform and methanol in a volume ratio of 10:1, and the colorimetric reagent includes iodine or 10 wt% sulfuric acid ethanol solution. The eluents under the gradient are combined with 50-70% organic solvent-water mixture. When the volume ratio of organic solvent is 50%, the eluent is collected as the component containing compound 1, and when the volume ratio of organic solvent is 70%, the eluent is collected as the component containing compound 2.

7. The method according to claim 2, characterized in that: In step (3), the Sephadex LH20 dextran gel column chromatography conditions are as follows: methanol or acetone is used as the eluent, the flow rate is 15-20 s / drop, and 5 mL is collected from each tube; the fractions with an elution volume of 800-1200 mL are combined, of which the elution volume of 1000-1200 mL is the fraction containing compound 1, and the elution volume of 800-1000 mL is the pure product of compound 2.

8. The method according to claim 2, characterized in that: In step (3), the normal phase silica gel column chromatography conditions are as follows: using dichloromethane-methanol with a volume ratio of 600:1 to 300:1 as the eluent, combining similar components, and concentrating under reduced pressure to dryness to obtain the pure compound; wherein the eluents under the dichloromethane-methanol elution gradient with a volume ratio of 400:1 are combined and concentrated under reduced pressure to dryness to obtain the pure compound 1.

9. The application of N1-reverse isopentenyl indole alkaloids in the preparation of anti-inflammatory drugs or anti-inflammatory functional foods, characterized in that: The N1-reverse isopentenylindole alkaloid is prepared by the method described in any one of claims 1 to 9, and its structural formula is as follows: or .

10. The application according to claim 9, characterized in that: The anti-inflammatory drugs or anti-inflammatory functional foods also include pharmaceutically or food-grade excipients.

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