Method for comprehensive extraction of eicosanoid containing EPA and ARA from phaeodactylum tricornutum and application

Abstract

The application provides a method and application for comprehensively extracting eicosanoid containing EPA and ARA from Phaeodactylum tricornutum, establishes a complete process chain of "algal culture-algal mud acquisition-ASE rapid extraction of microalgal oil-SPE enrichment-semi-preparation HPLC purification", the composition ratio of the anti-inflammatory mediators is clear, and the structure is identified by high performance liquid chromatography; the existing literatures and patents do not disclose that the algal strain can simultaneously enrich the combination of the above-mentioned various anti-inflammatory mediators. And the raw material of Phaeodactylum tricornutum is derived from marine diatoms which can be cultured on a large scale, there is no risk of pesticide residue of terrestrial plants, and there is no organic chlorine solvent in the production process, which meets the requirements of FDA / EFSA on natural marine source functional ingredients; can be simultaneously used for anti-inflammatory prescription drugs, postoperative inflammation relief food and soothing and repairing cosmetics, and has a wide market adaptation.

Description

Technical Field

[0001] This invention belongs to the technical field of microalgae cultivation and extraction of active substances, and in particular relates to a method and application for the comprehensive extraction of eicosanoic acid containing EPA and ARA from *Phaeodactylum tricornutum*. Background Technology

[0002] Inflammation is a complex defense response of the body to external or endogenous stimuli. However, excessive or persistent inflammation can lead to various chronic diseases such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, and neurodegenerative diseases. While commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids can rapidly relieve symptoms, they are accompanied by side effects such as gastrointestinal damage, immunosuppression, and metabolic disorders. Therefore, developing naturally derived, highly effective, low-toxicity, and novel anti-inflammatory active molecules has become an important direction in the pharmaceutical and functional food fields.

[0003] Lipooxygenates (LX) are tetraene eicosanoic acid derivatives generated from arachidonic acid through several pathways involving the combined action of various lipoxygenases. Two major lipoxygenate structural types have been identified: LXA4 and LXB4.

[0004] A key feature distinguishing lipoxygenin (LX) from most other inflammatory mediators (or modulators) is its potent anti-inflammatory activity. The most well-studied in vitro and in vivo activities of lipoxygenin LXA4 to date include: (a) inhibition of neutrophil chemotaxis, adhesion, and transmembrane migration; (b) inhibition of neutrophil activation (including NF-κB activation, superoxide production, and elastase secretion); (c) inhibition of IL-8 production by epithelial cells and leukocytes; (d) upregulation of bactericidal permeability and increased protein expression in epithelial cells; (e) upregulation of monocyte chemotaxis; and (f) upregulation of monocyte phagocytosis of apoptotic neutrophils. In various in vivo models, lipoxygenin has been shown to prevent neutrophil-mediated damage and promote neutrophil-mediated inflammatory resolution. The synthetic pathway of lipoxygenin LXA4 is disclosed in WO2000055109A1, and the synthetic pathway of phenyllipoxygenin analogues is disclosed in US7683193B2.

[0005] No existing technology has disclosed the extraction of active substances from algae that belong to the arachidonic acid (AA) bioactive metabolite group and have a clear chemical structure, a clear mechanism of action, and significant in vitro and in vivo anti-inflammatory effects. Summary of the Invention

[0006] This invention isolates, purifies, and identifies a variety of active substances with significant anti-inflammatory activity from *Phaeodactylum tricornutum*, providing a new method for obtaining extracts containing lipoxygen components. These extracts can be used to prepare anti-inflammatory drugs, functional foods, or cosmetics, solving the problem of high costs associated with the current chemical synthesis of lipoxygen.

[0007] The technical solution of this invention is: a method for comprehensively extracting eicosanoic acid containing EPA and ARA from *Phaeodactylum tricornutum*, comprising the following steps:

[0008] a. Centrifuge the culture medium of *Phaeodactylum tricornutum*, collect the algal sludge, freeze-dry the algal sludge, and grind it into algal powder.

[0009] b. Mix the algae powder and diatomaceous earth and fill them into an extraction tank that has been lined with an organic filter membrane. Add the solvent system to the extraction tank, heat the solvent system in the extraction tank and apply pressure to accelerate the penetration and dissolution of the solvent system into the filler. Recover the extract and repeat the extraction several times.

[0010] c. After extraction, add water to the extract, collect the lower organic phase, rotary evaporate the organic phase, resuspend it in methanol, add double-distilled water to make the methanol volume concentration 10% to obtain the sample solution, and acidify the sample solution to pH=3.5 with hydrochloric acid.

[0011] d. The sample solution was washed with an SPE extraction column to neutralize the acid and obtain an eluent. The eluent in the extraction column was then washed with methyl formate, and the solvent was evaporated under a nitrogen stream to concentrate the lipids, yielding an eicosanoic acid precursor containing LXB4, 15-epi-LXA4, 18-HEPE, RvE1, RvE2, RvE3, and RvE4.

[0012] Furthermore, the *Phaeodactylum tricornutum* was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, with the species number FACHB-843.

[0013] Furthermore, in step b, the solvent system is methanol and chloroform in a volume ratio of 2:1.

[0014] Furthermore, after adding water to the extract in step c, the solvent system becomes a solvent system of methanol, chloroform and water with a volume ratio of 2:1:2.

[0015] Furthermore, the washing of the sample solution with the SPE extraction column in step d includes: activating the column with methanol and double-distilled water at a volume ratio of 2:3, adding the sample solution, and then rapidly washing with double-distilled water to neutralize the acid.

[0016] The eicosanoic acid precursors are used to prepare anti-inflammatory drugs, functional foods, or cosmetics.

[0017] The eicosanoic acid precursor is used to separate arachidonic acid.

[0018] The culture medium of *Phaeodactylum triangularis* is obtained through the following steps:

[0019] (1) Culture of *Phaeodactylum tricornutum*: *Phaeodactylum tricornutum* was inoculated at a certain density on f / 2 medium and cultured under light / dark cycle conditions with aeration until the cell density reached the peak before the plateau phase and the optical density OD reached the peak value. 680 When nm no longer increases significantly, the culture is terminated.

[0020] Furthermore, the f / 2 culture medium in step (1) has a salinity of 32‰, a pH of 8.0±0.2, and the culture conditions are a light intensity of 40 μmol / (m²). 2 The culture conditions were as follows: light-dark ratio of 12 h:12 h, culture temperature of 23℃, single-use aerated culture flasks, and inoculation density of 5 × 10⁻⁶. 5 cells mL -1 The culture flasks were shaken three times daily, and cultured until day 7, 168 hours post-inoculation, at which point the cell density reached its peak before the plateau phase, at 2.8–3.2 × 10⁻⁶. 7 cells mL -1 Optical density OD 680 Once the nm value no longer increases significantly, it is considered the end of the culture, and the next step of algal collection should be carried out immediately.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The raw material for *Phaeodactylum tricornutum* comes from marine diatoms that can be cultivated on a large scale. It has no risk of pesticide residues in terrestrial plants, and the production process is free of organochlorine solvents. It meets the FDA / EFSA "GRAS" requirements for natural marine-derived functional ingredients. It can be used in anti-inflammatory prescription drugs, postoperative inflammation relief foods, and soothing and repairing cosmetics, and has a wide market applicability. Attached Figure Description

[0023] Figure 1 The secondary mass spectrum of LXB4 in the AA bioactive metabolome of *Phaeodactylum tricornutum* oil is shown.

[0024] Figure 2 The secondary mass spectrum of 15-epi-LXA4 of the AA bioactive metabolome in brown finger algae oil;

[0025] Figure 3 The image shows the 18-HEPE secondary mass spectrum of the EPA bioactive metabolome in brown finger algae oil.

[0026] Figure 4 The image shows the secondary mass spectrum of RvE1 in the bioactive metabolome of EPA in brown finger algae oil.

[0027] Figure 5 The image shows the secondary mass spectrum of RvE2 in the bioactive metabolome of EPA in *Phaeodactylum tricornutum* oil.

[0028] Figure 6 The image shows the secondary mass spectrum of RvE3 in the bioactive metabolome of EPA in brown finger algae oil.

[0029] Figure 7 This is a secondary mass spectrum of 5S,15S-diHEPE (RvE4) of the EPA bioactive metabolome in brown finger algae oil. Detailed Implementation

[0030] The present invention will be further illustrated below through specific embodiments. The following embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the following embodiments.

[0031] The *Phaeodactylum tricornutum* used in the following examples was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, species number FACHB-843.

[0032] Example 1

[0033] 1. Cultivation of *Phaeodactylum tricornutum*

[0034] The algae species *Phaeodactylum tricornutum* (FACHB-843) were cultured on f / 2 medium at a salinity of 32‰ and a pH of 8.0±0.2. The culture conditions included a light intensity of 40 μmol / (m²). 2 •s), light mode: dark cycle (12 h: 12 h), culture temperature: 23 ℃, disposable aerated culture flasks (5 L), inoculation density of *Phaeodactylum tricornutum*: 5 × 10⁻⁶ 5 cells mL -1 Shake the culture flask three times a day.

[0035] By day 7 (168 h post-inoculation), the cell density reached its pre-plateau peak of 2.8–3.2 × 10⁻⁶. 7 cellsmL -1 Optical density OD 680 Once the nm value no longer increases significantly, it is considered the end of the culture, and the next step of algal collection should be carried out immediately.

[0036] 2. Collection of cultures of *Phaeodactylum tricornutum* species

[0037] The culture of *Phaeodactylum tricornutum* was centrifuged at 5000 rpm for 10 min in an ultracentrifuge. After removing the supernatant, the algal sludge was frozen at -80 ℃.

[0038] In step 1 above, the culture medium (f / 2) for *Phaeodactylum tricornutum* is shown in Table 1.

[0039] Table 1. Components of f / 2 Culture Medium

[0040]

[0041] Preparation of trace element solutions: 4.4 g / L Na2EDTA·2H2O, 3.16 g / L FeCl3·6H2O, 0.012 g / L CoSO4·7H2O, 0.021 g / L ZnSO4·7H2O, 0.18 g / L MnCl2·4H2O, 0.007 g / L CuSO4·5H2O, and 0.007 g / L Na2MoO4·2H2O.

[0042] Vitamin solution preparation: 100 mg / L vitamin B1, 0.5 mg / L biotin, 0.5 mg / L vitamin B1 12 Dissolve in 1 L of distilled water, filter and sterilize, then dispense and store in the dark at 4 ℃.

[0043] Example 2

[0044] 1. Freeze-drying and extraction of microalgae oil

[0045] The algal mud obtained in Example 1 above, which was frozen at -80 ℃, was dried in a freeze dryer, ground into algal powder in a mortar, sealed, and stored at -20 ℃.

[0046] Microalgae oil extraction was performed using an ASE350 rapid solvent extractor. An organic filter membrane, resistant to chloroform and methanol, and resistant to high temperature and pressure, was pre-placed at the bottom of the ASE extraction cell. A polytetrafluoroethylene (PTFE) membrane was selected as an example. 200 mg of algal powder was mixed with diatomaceous earth and filled into the extraction cell, gently compacted to avoid channel flow. A methanol-chloroform mixture (2:1 volume ratio) was added to the extraction cell as the solvent. Under controlled high temperature (100 °C) and high pressure (typically 10–15 MPa) conditions, the ASE350 extractor accelerated the penetration and dissolution of the solid or semi-solid sample, achieving rapid and high recovery extraction. For the extraction of freeze-dried algal biomass, the extraction temperature was 100 °C, the static time was 15 min, and four static cycles were performed.

[0047] After extraction, water was added to the extraction solution to make the solvent system methanol:chloroform:water (2:1:2). The lower organic phase was collected, and after rotary evaporation at 37 °C, the extract was resuspended in methanol. Double-distilled water was added to the extract dissolved in methanol to make the methanol volume concentration 10%, and the sample solution was acidified to pH=3.5 with hydrochloric acid.

[0048] 2. Solid-phase extraction purification of algal oil

[0049] The sample obtained in the above steps was purified using an SPE extraction column. The column was first activated with 6 mL of methanol and 9 mL of double-distilled water. After adding the sample, it was rapidly washed with 4 mL of double-distilled water to neutralize the acid. The product was then eluted with 9 mL of methyl formate and concentrated under a nitrogen (N2) stream at 37 °C. After complete solvent evaporation, 300–500 μL of methanol:water (1:1) was added. The sample was centrifuged at 2000 × g for 2 min. The supernatant was collected, filtered through a 0.22 µm filter membrane, and the filtrate was transferred to a sample vial for LC-MS / MS analysis.

[0050] 3. LC-MS / MS analysis

[0051] The processed samples were analyzed by LC-MS / MS using an ultra-high resolution liquid chromatography-mass spectrometry (HPLC) system. HPLC conditions: C18 column (2.1*100 mm, 1.7 μm); column temperature 30 ℃; mobile phase: methanol (A) - 0.1% acetic acid solution (B); gradient elution program: 0.0–10.0 min, 0%–100% A, 10.0–16.0 min 100% A, 16.0–16.1 min, 100%–0% A; flow rate: 0.3 mL / min. -1 Instrument settings: Ion source: electrospray ionization; negative ions (V): 3000; sheath gas: 30; auxiliary gas: 8; purge gas: 0; temperature of ion transfer tube (°C): 320; temperature of evaporator (°C): 150; MS mode: negative electrode; resolution: 120,000.

[0052] Test results:

[0053] Using Xcalibur® software (Thermo Fisher Scientific), after acquiring raw mass spectrometry data by LC-MS / MS, the measured first-order precise mass (m / z) and second-order fragment ion spectra were compared with data in publicly available spectral libraries (or literature). Through qualitative analysis by LC-MS / MS, it was preliminarily determined that the oil of *Phaeodactylum tricornutum* contains 18-HEPE, LXB4, 15-epi-LXA4, and RvE1-4.

[0054] Among them, the secondary mass spectra of LXB4 and 15-epi-LXA4 of the arachidonic acid (AA) bioactive metabolome in *Phaeodactylum tricornutum* oil were obtained from... Figure 1 and Figure 2 As shown.

[0055] The diagnostic ion and fragmentation patterns of LXB4 and 15-epi-LXA4 are as follows: LXB4 (351=MH; 333=MH-H2O; 315=MH-2H2O; 307=MH-CO2; 289=MH-CO2-H2O; 271=MH-CO2-2H2O; 261=279-H2O; 233=251-H2O; 217=235-H2O) ;207=251-CO2; 203=221-H2O; 189=251-CO2-H2O; 177=221-CO2); 15-epi-LXA4 (351= MH; 333=MH-H2O; 315=MH-2H2O; 271=MH-CO2-2H2O; 217=235-H2O; 189=251-CO2-H2O).

[0056] The secondary mass spectra of 18-HEPE and RvE1-4 of the bioactive metabolome of eicosapentaenoic acid (EPA) in *Phaeodactylum tricornutum* oil are obtained by... Figure 3 and Figures 4 to 7 As shown.

[0057] The diagnostic ion and fragmentation patterns of 18-HEPE and RvE1-4 are as follows: 18-HEPE (317=MH, 299=MH-H2O, 273=MH-CO2, 255=MH-H2O-CO2, 243=287-CO2, 215=259-CO2); RvE1 (349=MH, 287=MH-H2O-CO2, 269=MH-2H2O-CO2, 205=223-H2O, 161=223-CO2-H2O, 151=195-CO2); RvE2 (333=MH, 315=MH-H2O, 297=MH-2H2O, 271=MH-CO2-H2O, 253=MH-CO2- 2H2O, 213=275-CO2-H2O, 199=217-H2O); RvE3 (333=MH, 315=MH-H2O, 297=MH-2H2O, 289=MH-CO2, 271=MH-CO2-H2O, 253=MH-CO2-2H2O, 213=275-CO2-H2 O, 201=245-CO2); RvE4 (333=MH, 315=MH-H2O, 289=MH-CO2, 271=MH-CO2-H2O, 253=MH-CO2-2H2O, 201=263-CO2-H2O, 199=217-H2O, 173=235-CO2-H2O).

[0058] Lipooxygenates (LXs) are derivatives of arachidonic acid (AA) and include four different structures: lipoxygenate A4 (LXA4), lipoxygenate B4 (LXB4), and aspirin-induced 15-epi-LXA4 and 15-epi-LXB4. Due to their unique bioactivity in reducing inflammation in vivo, LXs show great potential in neuroprotection and anti-inflammatory treatment of COVID-19 and related diseases. In this embodiment, two bioactive substances belonging to the arachidonic acid (AA) bioactive metabolome, namely LXB4 and 15-epi-LXA4, were extracted from *Phaeodactylum tricornutum* oil, which have not been previously reported.

[0059] 18-HEPE and regressors (RvE1-4) are derivatives of eicosapentaenoic acid (EPA) and possess anti-inflammatory and pro-inflammatory-resolving functions. RvE1 has been extensively studied and has been shown in vitro to inhibit leukocyte infiltration, downregulate the expression of pro-inflammatory mediators, and promote tissue regeneration. In this embodiment, five active substances belonging to the eicosapentaenoic acid (EPA) bioactive metabolome—namely 18-HEPE and RvE1, RvE2, RvE3, and RvE4—were extracted from *Phaeodactylum tricornutum* oil.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the comprehensive extraction of eicosanoic acid-like substances containing EPA and ARA from *Phaeodactylum tricornutum*, characterized in that, The steps are as follows: a. Centrifuge the culture medium of *Phaeodactylum tricornutum*, collect the algal sludge, freeze-dry the algal sludge, and grind it into algal powder. b. Mix the algae powder and diatomaceous earth and fill them into an extraction tank that has been lined with an organic filter membrane. Add the solvent system to the extraction tank, heat the solvent system in the extraction tank and apply pressure to accelerate the penetration and dissolution of the solvent system into the filler. Recover the extract and repeat the extraction several times. c. After extraction, add water to the extract, collect the lower organic phase, rotary evaporate the organic phase, resuspend it in methanol, add double-distilled water to make the methanol volume concentration 10% to obtain the sample solution, and acidify the sample solution to pH=3.5 with hydrochloric acid. d. The sample solution was washed with an SPE extraction column to neutralize the acid and obtain an eluent. The product was then eluted with methyl formate, and the solvent was evaporated under a nitrogen stream to concentrate the lipids, yielding an eicosanoic acid precursor containing LXB4, 15-epi-LXA4, 18-HEPE, RvE1, RvE2, RvE3, and RvE4. In step b, the solvent system is methanol and chloroform in a volume ratio of 2:1; the heating temperature of the solvent system in the extraction tank in step b is 100℃, and the pressure is set to 10-15 MPa. The step d of washing the sample solution with the SPE extraction column to neutralize the acid and obtain the eluent includes: activating the column with methanol and double-distilled water in a volume ratio of 2:3, adding the sample solution, and then rapidly washing with double-distilled water to neutralize the acid. The steps for obtaining the *Phaeodactylum tricornutum* culture medium are as follows: *Phaeodactylum tricornutum* is cultured on f / 2 medium at a certain density, and cultured under light / dark cycling conditions with aeration until the cell density reaches its peak before the plateau phase and the optical density OD... 680 When nm no longer increases significantly, the culture is terminated.

2. The method according to claim 1, characterized in that, After adding water to the extract in step c, the solvent system becomes a solvent system of methanol, chloroform and water with a volume ratio of 2:1:2.

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