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From Vietnamese plants to a biflavonoid that relieves inflammation by triggering the lipid mediator class switch to resolution

Chronic inflammation results from excessive pro-inflammatory signaling and the failure to resolve the inflammatory reaction. Lipid mediators orchestrate both the initiation and resolution of inflammation. Switching from pro-inflammatory to pro-resolving lipid mediator biosynthesis is considered as efficient strategy to relieve chronic inflammation, though drug candidates exhibiting such features are unknown. Starting from a library of Vietnamese medical plant extracts, we identified isomers of the biflavanoid 8-methylsocotrin-4′-ol from Dracaena cambodiana, which limit inflammation by targeting 5-lipoxygenase and switching the lipid mediator profile from leukotrienes to specialized pro-resolving mediators (SPM). Elucidation of the absolute configurations of 8-methylsocotrin-4′-ol revealed the 2S,γS-isomer being most active, and molecular docking studies suggest that the compound binds to an allosteric site between the 5-lipoxygenase subdomains. We identified additional subordinate targets within lipid mediator biosynthesis, including microsomal prostaglandin E2 synthase-1. Leukotriene production is efficiently suppressed in activated human neutrophils, macrophages, and blood, while the induction of SPM biosynthesis is restricted to M2 macrophages. The shift from leukotrienes to SPM was also evident in mouse peritonitis in vivo and accompanied by a substantial decrease in immune cell infiltration. In summary, we disclose a promising drug candidate that combines potent 5-lipoxygenase inhibition with the favorable reprogramming of lipid mediator profiles.


Introduction
Inflammation is a self-defensive process of the host microcirculation against external stimuli like pathogen infection and injury 1 . Both initiation and termination of inflammation are regulated through a network of oxygenated lipid mediators, which are produced from polyunsaturated fatty acids by a limited set of biosynthetic enzymes that are differentially expressed in innate immune cells and are spatially and temporally controlled 2 . Inflammation is initiated and maintained by the production of pro-inflammatory mediators, among which leukotrienes (LTs) and prostaglandins (PGs) are dominating 3 . The physiological inflammatory response is actively terminated in the resolution phase, a homeostatic process that is governed by the biosynthesis of specialized pro-resolving mediators (SPM) 4,5 . Impaired resolution together with the excessive production of proinflammatory mediators and the failure to dampen their signaling allow acute inflammation to turn into a chronic state, which causes permanent tissue damage and is closely related to chronic inflammatory pathologies, such as osteoarthritis, atherosclerosis, asthma, and cancer 6 . Classical strategies to treat inflammation mainly focus on suppressing pro-inflammatory signaling, while novel approaches aim at promoting resolution programs.
LTs and PGs are produced by lipoxygenases (LOXs) and cyclooxygenases (COXs) in concert with further redox-and nonredox-active enzymes 7 , whose inhibition relieves inflammationrelated diseases like asthma, allergy, and osteoarthritis 8,9 . However, current anti-inflammatory drugs targeting COX and 5-LOX are associated with gastrointestinal, renal, and cardiovascular side effects 10e12 and were proposed to impair resolution 13 . Additional targets within eicosanoid biosynthesis, i.e., microsomal prostaglandin E 2 synthase 1 (mPGES-1), leukotriene C 4 synthase (LTC 4 S), and soluble epoxide hydrolase (sEH), were reported 12,14,15 , and polypharmacological strategies were explored 16,17 , including dual inhibition of LT biosynthesis and mPGES-1 18 . While some of these approaches favorably modulate pro-inflammatory lipid mediator signaling, strategies that alter lipid mediator networks from a pro-inflammatory to a pro-resolving state have not yet been realized, despite their proclaimed high therapeutic potential 19,20 .
SPM biosynthesis essentially depends on the activity of 12-LOX and 15-LOX, which are abundantly expressed in antiinflammatory (M2-like) macrophage subsets and other immune cells 21,22 . 5-LOX also participates in SPM biosynthesis but its role is diffuse. Increasing evidence shows that SPM inhibit neutrophil trafficking, scavenge pro-inflammatory cytokines, stimulate efferocytosis and bacterial clearance, promote tissue regeneration, and protect from oxidative stress 4,5 .
To identify small molecules that induce a lipid mediator class switch from inflammation to resolution, we started from an inhouse library of Vietnamese medical plants to access structurally diverse pharmacophores 23,24 . Detailed investigations disclose the isomers of 8-methylsocotrin-4 0 -ol (2) that limit inflammation by suppressing pro-inflammatory lipid mediator formation and inducing SPM biosynthesis, with the 2S,gS-isomer being most active. Compound 2 inhibits LT formation across human innate immune cells while specifically triggering SPM production in macrophages of the M2 phenotype. The lipid mediator class switch by compound 2 was confirmed in vivo using an experimental mouse model of zymosan-induced peritonitis.

Materials
Solvents used for extract fractionation and compound isolation were purchased from VWR International (Darmstadt, Germany). Solvents for high performance liquid chromatography (HPLC) were obtained from Merck (Darmstadt, Germany). Ultrapure water was produced by a Sartorius Arium 611 UV water purification system (Göttingen, Germany). Deuterated and nondeuterated lipid mediator standards for ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were purchased from Cayman Chemicals (Ann Arbor, MI, USA).
Zymosan, indomethacin, staurosporine, and all other reagents were purchased from Merck (Darmstadt, Germany) unless indicated otherwise.

Plant material
The red wood of D. cambodiana was collected in Tinh Bien (An Giang, Vietnam) in March 2014, identified by Prof. Dr. Tran Hung (Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy of HoChiMinh city, Vietnam) and airdried. A voucher specimen (AG3032014) is stored at the Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy of HoChiMinh City, Vietnam.

Computational methods for ECD calculation
3D structures of 1 and 2 were drawn and subjected to a conformational search by using OPLS-3 force field in Macro-Model v.9 (Schrödinger Ltd., New York, NY, USA) in water. The number of steps was set high enough to include all low energy conformers. Conformers occurring in the energy window of 10 kcal/mol from the global minima were subjected to geometrical minimization and optimization in the DFT/B3LYP/6-31þG(d,p) basis set and level of theory and using the conductor-like polarizable continuum model (CPCM) in ethanol, using Gaussian 16 v. A.03 software (Gaussian, Inc., Wallingford, CT, USA) (Supporting Information Fig. S1). Frequency calculations were performed at the same level and no imaginary frequencies were observed. Calculation of the excitation energy (nm), rotatory strength dipole velocity (Rvel) and dipole length (Rlen) were performed by using time-dependent density-functional theory (TD-DFT) at PBEPBE/def2-SV/6-31þG(d,p)/ CPCM level in ethanol. ECD curves were simulated on the basis of rotatory strengths using SpecDis v1.7 with half-band of 0.2 eV and UV shift of 10e30 nm.
Pdb entry 6ncf 25 , stable 5-LOX, co-crystalized with 3-Oacetyl-11-keto-b-boswellic acid (AKBA) at the allosteric binding site was used as a target structure. The protein was energetically minimized in Discovery Studio (version 2018; Biovia, San Diego, CA, USA). Hydrogens were added with GOLD, using default settings. The binding site was defined on the B chain in a 9 Å radius at the site of the co-crystalized ligand. Protein-ligand interactions of the docking poses were analyzed using LigandScout 4.2 (www.inteligand.com/ligandscout). A re-docking of AKBA was conducted to optimize the docking settings. The best ranked pose with the final docking settings had an RMSD of 1.061 compared to the co-crystallized ligand.

Isolation of primary cells from human blood
Human blood (3.13% sodium citrate) and human leukocyte concentrates from freshly withdrawn venous blood (16 I.E. heparin/ mL blood) were provided by the Institute for Transfusion Medicine of the University Hospital Jena (Germany) with informed consent of the volunteers 26 . The selected fasted (12 h) healthy male and female blood donors (18e65 years) were registered and regularly donated blood every 8e12 weeks. They were physically inspected by a clinician and free of obvious infections and inflammatory or allergic disorders and neither had taken antibiotics nor anti-inflammatory drugs within 10 days before blood collection.
Human polymorphonuclear leukocytes (PMNL), peripheral blood mononuclear cells (PBMC) and platelets were freshly isolated from leukocyte concentrates as described 26 . Briefly, PMNL, PBMC and platelets were separated by dextran sedimentation, followed by density gradient centrifugation using lymphocyte separation medium (LSM 1077, GE Healthcare, Freiburg, Germany). Platelets and PBMC were collected from the supernatant and the intermediate layer, respectively, whereas PMNL were obtained from the pelleted cells after removal of erythrocytes by hypotonic lysis. To enrich monocytes, PBMC were seeded in RPMI 1640 medium plus 5% heat-inactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (GE Healthcare) in culture flasks (Greiner Bio-one, Frickenhausen, Germany) and incubated for 1.5 h at 37 C and 5% CO 2 . Adherent monocytes were recovered, and their purity (>85%) was defined by flow cytometry 26 . Experimental procedures on human blood and blood cells were approved by the ethical commission of the University Hospital Jena.
To investigate whether compounds 2 and 5 reversibly inhibit 5-LOX activity, the purified enzyme was pre-incubated with the test compounds at 30 or 3 mmol/L for 15 min and then 10-fold diluted to a final compound concentration of 3 or 0.3 mmol/L, respectively. Arachidonic acid (20 mmol/L) and CaCl 2 (2 mmol/L) were added and after 10 min at 37 C 5-LOX products were extracted and analyzed as described above.
Supernatants of treated cells (PMNL or M1/M2 macrophages in 1 mL PBS pH 7.4 plus 1 mmol/L CaCl 2 ), blood (1 mL) or exudates from murine peritonitis (1 mL) were mixed with icecold methanol (2 mL) supplemented with deuterium-labeled internal standards (10 mL; 200 nmol/L d 8 -5S-HETE, d 4 -LTB 4 , d 5 -LXA 4 , d 5 -RvD2, d 4 -PGE 2 , and 10 mmol/L d 8 -arachidonic acid). Lipid mediators were extracted by SPE on Sep-Pak C18 35 cc Vac Cartridges (Waters) 22 . In brief, samples were placed at À20 C for at least 45 min to allow protein precipitation. After centrifugation (1200Âg, 4 C, 10 min), 8 mL acidified H 2 O (pH Z 3.5) was added to the supernatants, and the samples were loaded onto equilibrated RP-columns. After extensive washing with H 2 O and n-hexane, lipid mediators were eluted with methyl formate. Eluates were brought to dryness (TurboVap LV, Biotage, Uppsala, Sweden) and dissolved in methanol/water (50/50) for UPLC-MS/MS analysis. Lipid mediators were analyzed by UPLC-MS/MS using an Acquity UPLC system (Waters) coupled to a QTRAP 5500 mass spectrometer (Sciex, Darmstadt, Germany), which was equipped with a Turbo V™ electrospray ionization source 22 . Analytes were separated on an Acquity UPLC BEH C18 column (1.7 mm, 2.1 mm Â 100 mm; Waters) at 50 C with a flow rate of 0.3 mL/min. The mobile phase consisting of methanol, water, and acetic acid (42:58:0.01) was ramped to 86:14:0.01 over 12.5 min and then switched to 98:2:0.01 for isocratic elution (3 min). Diagnostic ions were detected in the negative ionization mode by scheduled multiple reaction monitoring using optimized MS parameters for each lipid mediator 22 . Retention times were confirmed using external standards (Cayman Chemicals). Automatic peak integration was performed with Analyst 1.6 software (Sciex) using IntelliQuan default settings. Lipid mediator amounts were calculated from signal intensities normalized to internal standards using external calibration curves.

Determination of cell viability
Mitochondrial dehydrogenase activity was measured by MTT assay to assess cell viability 27 . Briefly, monocytes were preincubated with vehicle (DMSO), test compounds or staurosporine (1 mmol/L, cytotoxic reference) for 15 min and incubated for 24 h at 37 C and 5% CO 2 . 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was added, and cells were incubated for 2e4 h. After cells were lysed and the formazan crystals solubilized with SDS (10% in 20 mmol/L HCl), the absorption was read at 570 nm using a Multiskan Spectrum Microplate Reader (Thermo Fisher Scientific).

Determination of COX-1 and COX-2 activity
For the determination of cell-free COX activity, isolated ovine COX-1 (50 units, Cayman Chemicals) or human recombinant COX-2 (20 units, Cayman Chemicals) were pre-treated with vehicle (DMSO) or test compounds in 100 mmol/L Tris pH 8 (supplemented with 5 mmol/L glutathione, 5 mmol/L hemoglobin and 100 mmol/L EDTA) for 5 min at 4 C. The mixture was prewarmed for 1 min at 37 C, and arachidonic acid was added (5 mmol/L for COX-1 and 2 mmol/L for COX-2). The reaction was stopped by addition of an equal volume of ice-cold methanol, and COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) was extracted by SPE using Sep-Pak C18 35 cc Vac Cartridges (Waters) and analyzed by RP-UV-HPLC on a Nova-Pak C18 Radial-Pak Column as described for 5-LOX products. The COX-1/2 inhibitor indomethacin (10 mmol/L) was used as control.
For the determination of cellular COX-1 activity, freshly isolated human platelets (1 Â 10 8 ) were pre-treated with vehicle (DMSO), test compounds or indomethacin (10 mmol/L) for 5 min at room temperature, and arachidonic acid (5 mmol/L) was added. After 5 min at 37 C, the reaction was stopped by addition of an equal volume of ice-cold methanol, and formed 12-HHT was extracted and analyzed as described above.

Determination of LTC 4 S activity
Human embryonic kidney (HEK)293 cells (ATCC, Manassas, VA, USA) were cultivated in DMEM/high glucose (4.5 g/L; GE Healthcare) supplemented with heat-inactivated FCS (10%) and penicillin/streptomycin at 37 C and 5% CO 2 . Confluent cells were reseeded at 1 Â 10 4 /cm 2 after being detached with trypsin/ EDTA (GE Healthcare). Microsomal membranes (2.5 mg total protein) from HEK293 cells that stably express LTC 4 S were pretreated with vehicle (DMSO) or test compounds in potassium phosphate buffer (0.1 mol/L, pH 7.4, containing 5 mmol/L glutathione) for 10 min 28 . The reaction was started by addition of LTA 4 methyl ester (1 mmol/L, Cayman Chemicals) and stopped after 10 min at 4 C by addition of an equal volume of ice-cold methanol. LTC 4 -d 5 methyl ester (5 ng, Cayman Chemicals) was used as internal standard. Formed LTC 4 was extracted by SPE and analyzed by UPLC-MS/MS 28 . MK886 (10 mmol/L, Cayman Chemicals) was used as reference for LTC 4 S inhibition.

Zymosan-induced peritonitis in mice
Male CD-1 mice (33e39 g, 6e8 weeks, Charles River Laboratories, Calco, Italy) were housed at 21 AE 2 C, provided with standard rodent chow and water and subjected to a 12 h light/12 h dark cycle. Mice were randomly assigned for the experiments and allowed to acclimate for four days. The experimental procedures comply with the ARRIVE guidelines and were approved by the Italian Ministry (744/2019) and conducted according to European and Italian regulations for animal protection (directives 2010/63/ EU and DL 26/2014).
Mice (n Z 6 per experimental group) received vehicle (0.5 mL, 2% DMSO in saline), compound 2 (10 mg/kg) or MK886 (3 mg/kg) by intraperitoneal injection (i.p.) 30 min prior to peritonitis induction by zymosan (0.5 mL, 2 mg/mL suspension in 0.9% saline, i.p.) 30 . Mice were euthanized 120 min post zymosan injection with CO 2 , followed by a peritoneal lavage with 3 mL of cold PBS pH 7.4. Exudates were collected from the peritoneal cavity, and cells were immediately counted by vital trypan blue staining. After centrifugation (18,000Âg, 5 min, 4 C), supernatants were collected, and lipid mediators were extracted by SPE and analyzed by UPLC-MS/ MS. LTC 4 levels in the exudates were analyzed by enzyme immunoassays (Enzo Life Sciences, Lörrach, Germany) according to manufacturer's instructions. To measure myeloperoxidase activity, cell pellets were sonicated in PBS buffer (50 mmol/L, pH 6) with 0.5% hexadecyltrimethylammonium bromide, freeze-thawed three times, and centrifuged (32,000Âg, 20 min, 4 C). Cell supernatants were collected, and the reaction was initiated by addition of odianisidine (0.167 mg/mL) and 0.0005% hydrogen peroxide. Changes in absorbance were time-dependently monitored using an iMark microplate Reader (Bio-Rad, Segrate, Milan, Italy). Myeloperoxidase activity was determined from the calibration curve using human neutrophil myeloperoxidase as reference standard and is given in U/mL 31 .

Statistical analysis
Data are presented as mean AE SEM of n observations, where n represents the number of independent experiments (including independent immune cell preparations) or the number of animals per group, as indicated. GraphPad Prism 8 software (San Diego, CA, USA) was used for statistical calculations. Normal distribution of the data was addressed by KolmogoroveSmirnov tests. Samples were blinded for screening of plant extracts, fractions and natural products and for counting infiltrated cells in the mouse peritoneum. Data were log-transformed for statistical analysis where indicated. Outliers were determined using a Grubb's test. For multiple comparison, ordinary or repeated-measures one-way ANOVA with Tukey or Dunnett's post hoc tests or ordinary twoway ANOVA with Tukey post hoc tests were applied. For comparison of two groups, paired t-tests were used. The criterion for statistical significance was * P < 0.05. IC 50 values were calculated by non-linear regression using GraphPad Prism 8.

Targeting LOX isoenzymes by 8-methylsocotrin-4 0 -ol from Vietnamese plant extracts
To identify novel natural product-derived pharmacological agents that induce a lipid mediator class switch from inflammation to resolution, we evaluated an in-house library of 94 extracts from 31 Vietnamese medicinal plants in activated PMNL (Fig. 1A). Our aim was to identify extracts, which potently suppress 5-LOXdependent LT biosynthesis (IC 50 10 mg/mL) while enhancing 15-LOX (residual activity ! 100%) and optionally 12-LOX product formation (Supporting Information Table S1 32,33 ). The methanolic red wood extract of Dracaena cambodiana Pierre ex Gagnep. (Asparagaceae) showed the most favorable pharmacological profile. It inhibited the formation of LTB 4 and other 5-LOX products with an IC 50 of 0.4 mg/mL (Fig. 1B), increased the biosynthesis of the 15-LOX metabolite 15-HETE (Fig. 1C), and marginally decreased 12-HETE formation, the major 12-LOX metabolite of platelets in the PMNL fraction (Fig. 1D).
Aiming at the identification of the active principle(s) responsible for this favorable pharmacological effect, we prepared an ethanolic extract from the D. cambodiana red wood material and fractionated it by liquideliquid extraction with dichloromethane, ethyl acetate, and n-butanol to afford fractions of different polarity. Biological testing confirmed the potent bioactivity of the ethanolic extract on 5-LOX product formation in PMNL. Evaluation of the subfractions revealed strong 5-LOX-inhibitory activity of the dichloromethane and the ethyl acetate subfraction, while the n-butanol fraction turned out to be inactive (Fig. 1E). Therefore, the ethyl acetate subfraction, which showed a strong 5-LOXinhibitory potential, was selected for further bioactivity-guided fractionation. Separation by silica gel column chromatography yielded 20 subfractions, which were analyzed for their 5-LOX-inhibitory activity in PMNL. At 3 mg/mL, subfractions 5e18 showed a statistically significant 5-LOX inhibition, with subfractions 11 and 12 being most active ( Fig. 1F and G). By using different chromatographic techniques, 18 (poly)phenolic compounds were isolated from the bioactive subfractions (Table 1, Fig. 1F), among them three new natural products (3e5) and another three (9, 10, 16) that have not yet been reported in Dracaena species (Supporting Information Section S5). Details on the elucidation of the chemical structures and the obtained physical and spectral data of the new compounds 3e5 are given in Sections S2e4. Due to the observed small optical rotation values of all chiral compounds (1e11), with the exception of compound 10, we assumed the presence of racemic/scalemic mixtures of the respective compounds. This assumption could be verified by chiral HPLC analysis, which revealed for all chiral compounds, except for 10, at least two peaks (Section S1).
All seven dimeric compounds 1e7 directly inhibited human recombinant 5-LOX in a cell-free assay (IC 50 Z 0.3e1.1 mmol/L) ( Table 1). Neither cytotoxic (Fig. 2C) nor radical scavenging activities (Fig. 2D) were observed at relevant concentrations. The two compound series, represented by compound 2 and 5, inhibited 5-LOX independently of the substrate concentration (Fig. 2E) in a reversible manner (Fig. 2F). Together, we identified a group of potent 5-LOX inhibitors from a library of medicinal plant extracts, which shift the balance from 5-LOX to 15-LOX product formation in PMNL.
Molecular docking studies on the allosteric binding site of 5-LOX suggest that the most active stereoisomer 2b forms hydrogen bonds with Lys140, Glu136, and Arg101 (Fig. 3C, Supporting Information Table S2). While the first two bonds are also formed by 1a and 1b, 2a is turned around in the binding site, due to steric hindrance, and forms other interactions, which could explain the loss of activity (Fig. 3D).
Due to the poor accessibility of the purified stereoisomers, we selected the racemic/scalemic mixtures 2 and 5 (as representatives of the two series of dimeric compounds) for further pharmacological evaluations. Note that lower effective concentrations would be expected for the purified isomer 2b.

Subordinate modulation of lipid mediator biosynthesis
Multiple enzymes besides LOXs cooperate in accomplishing the complex lipid mediator networks, thereby regulating initiation, maintenance, and resolution of inflammation 2,20 . Compounds 2 and 5 substantially inhibited mPGES-1 in a cell-free assay (IC 50 w 1 mmol/L) (Fig. 4A) and to some extend weakly COX-1, both as purified enzyme (IC 50 > 10 mmol/L) (Fig. 4B) and in activated human platelets (IC 50 > 1 mmol/L) (Fig. 4C). sEH activity was slightly suppressed at 10 mmol/L, exclusively by compound 5 (Fig. 4D), and both 2 and 5 showed a minor inhibitory effect on LTC 4 S by trend, again only at 10 mmol/L (Fig. 4E). Neither of the two compounds affected COX-2 activity (Fig. 4B). Together, in addition to prominent 5-LOX inhibition and the efficient increase of 15-LOX product formation in PMNL, we identified mPGES-1 and COX-1 as subordinated targets of 2 and 5.

Efficient inhibition of 5-LOX in blood and in activated PMNL
To investigate the effect of compounds 2 and 5 on more complex lipid mediator profiles, we performed metabololipidomics studies on human blood, which contains PMNL and monocytes as major cells contributing to large lipid mediator networks 34 . When lipid mediator production was initiated by A23187, compound 2 efficiently suppressed the biosynthesis of LTs (LTB 4 , LTB 4 isomers, 20-OH-LTB 4 ) and other 5-LOX products (5-HETE, 5,6-diHETE, 5-HEPE) (Fig. 5A and Supporting Information Table S3). The formation of the 15-LOX product 15-HETE and to a minor extend the 12-LOX-derived 12-HETE, 12-HEPE, and 14-HDHA was moderately elevated, with strong inter-individual differences, whereas the generation of COX-derived prostanoids (PGE 2 , PGD 2 , thromboxane (TXB 2 )), and the release of fatty acid substrates (arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid) was not influenced. Comparable effects on 5-LOX production were observed when pathogenic E. coli was used as physiological stimulus ( Fig. 5B and Supporting Information Table S4). Compound 5, on the other hand, failed to suppress the formation of 5-LOX products in human blood, even at the highest concentration tested (30 mmol/L), potentially due to unfavorable plasma protein binding. PMNL, which are the major 5-LOX-expressing cells in blood, predominantly produce LTs and other pro-inflammatory lipid mediators 2 . Compound 2 potently suppressed the biosynthesis of 5-LOX products in activated PMNL and moderately elevated the formation of the 15-LOX products 15-HETE and 15-HEPE at concentrations in the range of 0.3 and 3 mmol/L (Fig. 6). SPM biosynthesis was instead not upregulated. Compound 2 equally inhibited the formation of resolvins, lipoxins, and 5-LOX products. Comparable changes in the lipid mediator profile were evident for the 5-LOX reference inhibitor zileuton, which underlines a role for 5-LOX in SPM biosynthesis. It should be noted that PMNL dominate during the initial phase of inflammation while SPM are mainly produced by cells related to the resolution phase, i.e., macrophages 2 .

Induction of SPM biosynthesis in M2 macrophages
Macrophages with M2-like phenotype strongly express 15-LOX-1 and are the major innate immune cells responsible for SPM production at sites of inflammation during resolution 21,22,35 . The M1 subtype, on the other hand, excels in generating pro-inflammatory lipid mediators, including PGs and LTs, and is associated with acute and unresolved inflammation 21,22,34 . To obtain M1 and M2 subsets, we differentiated human primary monocytes into macrophages and subsequently induced their polarization. By monitoring the expression of typical M1 (CD54 and CD80) and M2 surface markers (CD163 and CD206), we could exclude that compound 2 affects macrophage polarization (Supporting Information Fig. S3). We then investigated the two subsets for the effect of compound 2 on the lipid mediator network and confirmed that the formation of LTs and other 5-LOX products is strongly attenuated, regardless of whether cells were stimulated with E. coli (Fig. 7 and Supporting Information Table S5) or S. aureus (Supporting Information Table  S6). Most remarkably, M2-like macrophages produced substantial amounts of SPM, including resolvins (RvE3, RvD5), protectins (PD1, AT-PD1, PDX), and maresins (MaR1), as well as monohydroxylated precursors derived from 12-and 15-LOX, when treated with compound 2. In M1 macrophages, compound 2 moderately increased the biosynthesis of 15-LOX products (i.e., 15-HETE, 15-HEPE, 17-HDHA) without substantially influencing the minute SPM generation (Fig. 7 and Table S5). Neither protein expression of 15-LOX-1, 15-LOX-2 nor 5-LOX were affected by compound 2 in M2 macrophages within 24 h (Supporting Information Fig. S4). Together, compound 2 efficiently suppresses pro-inflammatory LT biosynthesis throughout innate immune cells and selectively stimulates M2 macrophages to generate SPM.

Lipid mediator class switch to relieve inflammation in murine peritonitis
Whether the lipid mediator class switch from LTs to SPM, which we observed in vitro, also occurs at inflammatory sites in vivo, was  1a, 1b, 2a, and 2b on cell-free 5-LOX and 5-LOX product formation in PMNL that were treated with A23187 alone or in combination with arachidonic acid. 5-LOX products were analyzed by RP-UV-HPLC. Results are given as means þ SEM, percentage of vehicle control, n Z 3. Data were log-transformed for statistical analysis. ***P < 0.001, **P < 0.01; ordinary two-way ANOVA. (C, D) Docking poses of compound 2b (C) or compounds 1a and 1b (green), 2b (light green), and 2a (red) (D) at an allosteric binding site at 5-LOX, suggesting key interactions with Lys140, Glu136 and Arg101. Compound 2a is turned within the binding pocket, which might explain the weaker activity (D). investigated for zymosan-induced mouse peritonitisda selfresolving model of acute inflammation that is initiated by LTs (Fig. 8A) 36 . Mice were sacrificed at 2 h after zymosan administration to allow the simultaneous analysis of LTs and SPM. Intraperitoneal administration of compound 2 prior to zymosan effectively reduced 5-LOX product levels, including LTs, in peritoneal exudates but neither substantially altered the release of PGs nor of polyunsaturated fatty acid substrates (Fig. 8B). Resolvin (RvE3 and RvD5) concentrations decreased, though this 2-to 3-fold drop was overcompensated by the strong increase of 12-LOX-and 15-LOXderived SPM precursors and the 2.5-to 15-fold upregulation of protectins (PD1 and PDX) and MaR1 (Fig. 8B). MK886, an inhibitor of 5-LOX-activating protein (FLAP), had a similar but less prominent effect. The vehicle control (0.5 mL, 2% DMSO in saline) did not substantially affect the inflammatory reaction in the peritoneal cavity within 30 min to 4 h after zymosan injection, as suggested from the barely altered cell infiltration, myeloperoxidase activity (as marker for infiltrated neutrophils), and peritoneal LTC 4 levels (Supporting Information Fig. S5). Together, compound 2 shifts the lipid mediator profile in experimental peritonitis from a pro-inflammatory to a pro-resolving signature, thereby attenuating inflammation, as indicated by the drop of immune cell infiltration into the peritoneal cavity (Fig. 8C).

Discussion
Chronic inflammation and inflammation-related diseases are accomplished by pro-inflammatory signaling, which essentially orchestrates the acute phase of inflammation but becomes pathological when being persistent 6 . Current pharmacological strategies to combat inflammatory disorders, i.e., NSAIDs, focus on the proinflammatory branch of inflammation, but are confronted with major challenges, including i) lack of selectivity and efficacy, ii) undesired gastrointestinal, renal, and cardiovascular safety issues, and iii) impaired resolution 10,11,13,14 . By identifying a unique natural product that induces the lipid mediator class switch from pro-inflammatory LTs to SPM, our study gives access to a novel anti-inflammatory strategy that, in addition to suppressing LTdriven inflammation, may actively promote resolution and the return to homeostasis.
We took advantage of a comprehensive library of 94 Vietnamese medical plant extracts (31 different plants), from which we identified two structural different series of dimeric phenolic compounds that i) are present in D. cambodiana, ii) potently inhibit 5-LOX, and iii) trigger 15-LOX product formation. D. cambodiana is an important source for the preparation of 'Dragon's Blood', a dark red plant resin, which is used in many cultures as antimicrobial, antiviral, antitumor, and anti-inflammatory remedy 37,38 . The promising anti-inflammatory and analgesic activities of 'Dragon's Blood' were confirmed in pre-clinical and clinical studies 39e46 . Li and co-workers showed reduction of chronic inflammatory and neuropathic pain in rats, which they ascribed to an interference with Ca 2þ signaling, COX-2 expression, and substance P release 46 by components of 'Dragon's Blood' that relieve central pain 39,40,46 . The impact on lipid mediators was not evaluated, despite the central role of PGs and related mediators in pain sensitization 47 . 'Dragon's Blood' further exhibits protective effects against radiation-induced oxidative stress, brain injury and myelosuppression in rodents 41e44 , and as a cream, improved wound healing in a randomized, double-blind and placebo-controlled trial on 60 patients with removed skin tags 45 . We speculate that the bioflavonoids identified in this study might contribute to these activities by increasing the biosynthesis of SPM that actively terminate neutrophil transmigration, suppress oxidative stress, stimulate phagocytosis and promote tissue regeneration 4,48,49 including dermal tissue repair 50e52 . For the natural products 1, 2, and 6, which are representatives of the two identified series of dimeric phenols, anti-Helicobacter pylori, anti-thrombin, and anti-diabetic activities have been reported in vitro 53,54 . However, their anti-inflammatory potential remained elusive. Our findings suggest that especially the biflavonoid isomer 2b contributes to the anti-inflammatory activity of D. cambodiana by suppressing the biosynthesis of proinflammatory 5-LOX-derived LTs and causing a lipid mediator class switch towards SPM that promote resolution. Flavonoids are excellent scaffolds for 5-LOX inhibition 55,56 , but neither were adducts of them reported (as realized in the two biflavonoid series 1e7 of this study) nor effects on SPM biosynthesis addressed. Strong differences between the two biflavonoid series seem to exist for plasma protein binding. While the IC 50 value of compound 2 for 5-LOX inhibition was only slightly shifted to higher values in human blood as compared to PMNL, compound 5, which is comparably effective to 2 in PMNL, failed to inhibit 5-LOX. Our finding is in agreement with a previous study, showing that compound 6, which differs from 5 only in its substitution pattern, is prone to human serum albumin binding 57 .
5-LOX inhibitors can target the active site iron (redox-active compounds and iron chelators) or compete with arachidonic acid or fatty acid peroxides for binding to 5-LOX (non-redox type inhibitors) 9,58e60 . Higher efficacy under clinical settings is expected for allosteric inhibitors that interact with the ATP or membrane binding site 61 . Recently, we identified a series of allosteric 5-LOX inhibitors 26 and proposed that other reported inhibitors actually bind to this site. We confirmed this theory by co-crystalizing 5-LOX with the pentacyclic triterpene acid AKBA 25 . Since compound 2 inhibits 5-LOX like AKBA in a substrate concentration-independent manner and exhibits poor radical scavenging activity, we explored the structureeactivity relationship at this allosteric binding site. The scoring function did not directly correlate with the 5-LOX-inhibitory activity of compounds 1e18, but the analysis of the docking simulation suggests three key interaction partners on the protein side, Lys140, Glu134, and Glu136, which form hydrogen bonds to those compounds that display IC 50 values in the low or even submicromolar range (Table S2).
While strong 5-LOX expression in PMNL and the proinflammatory macrophage subtype M1 enables efficient LT biosynthesis 22,62,63 , macrophages of the M2 phenotype instead express substantial 15-LOX-1, generate high amounts of SPM 4,21,22 , and promote the resolution phase of inflammation as well as tissue repair 64 . Compound 2 inhibits LT biosynthesis throughout the cellular systems investigated in this study, i.e., PMNL, the macrophage subtypes M1 and M2, and blood, and slightly enhanced the formation of 15-LOX products (e.g., 15-HETE, 15-HEPE) in most settings. However, a strong increase of both 12-and 15-LOX products was mainly evident for M2 macrophages. Accordingly, the formation of distinct SPM and their precursors was exclusively elevated in this macrophage subtype.
Substrate redirection of polyunsaturated fatty acids might partially explain the moderately increased biosynthesis of 15-LOX products throughout innate immune cells and blood. In support of this hypothesis, compound 2 also increased the production of PGE 2 and other prostanoids in vitro and in vivo. However, this effect is weak, and we hypothesize that the shunting of arachidonic acid towards prostanoid biosynthesis is buffered by the inhibition of the low-affinity targets COX-1 and mPGES-1. Compound 2 (COX-1: IC 50 Z 6.8 mmol/L; mPGES-1: IC 50 Z 1.6 mmol/L) inhibits COX-1 less potent than indomethacin (IC 50 Z 0.02 mmol/L) 65 but is comparably efficient in inhibiting mPGES-1 as the control inhibitor MK886 (IC 50 Z 1.6 mmol/L) 66 . Accordingly, PGE 2 formation slightly increased at low concentrations ( 3 mmol/L) of compound 2 in PMNL and again decreased at effective concentrations to inhibit COX-1 and mPGES-1 (10 mmol/L). Depending on the spatial and temporal distribution, PGE 2 and other prostanoids display pro-inflammatory or anti-inflammatory activities during the course of inflammation 3 . Helminth larval products have been proposed to exploit this bias by lowering LT and enhancing PG production 67 . However, the balance between pro-and anti-inflammatory PG activities is sensitive, and we therefore consider the switch from proinflammatory LT to pro-resolving SPM as more robust and safer for pharmacotherapy.
Pharmacological strategies that trigger a lipid mediator class switch from inflammation to resolution are still rare. Endogenous metabolites such as PGE 2 68 and oxidized phosphatidylcholine 69 inhibit LT formation while enhancing the biosynthesis of lipoxins, Line charts present data in pg/5 Â 10 6 cells for selected lipid mediators as means þ SEM, n Z 3. Data were log-transformed for statistical analysis. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vehicle control; repeated measures one-way ANOVA plus Tukey post hoc tests. a class of 5-LOX-derived SPM 70 , though they do qualify for therapeutic application. The allosteric 5-LOX inhibitor AKBA (IC 50 Z 3 mmol/L 71 ) increases the formation of 12-LOX (270%, at 10 mmol/L) and less 15-LOX metabolites (210%, at 10 mmol/L) in human neutrophils 25 with unknown consequences on SPM production. Endogenous long-chain vitamin E metabolites excel in 5-LOX inhibition (IC 50 Z 0.035e0.075 mmol/L) but the increase in SPM is mainly restricted to systemic RvE3 levels during the resolution of murine peritonitis 26 . Other small molecules that trigger SPM formation in human neutrophils or macrophages include benzenesulfonamide-derivatives (110%e140% at 10 mmol/L) 72 , ginkgolic acid (110%e240% at 10 mmol/L) 73 , benzoxanthene lignans (130%e170% at 10 mmol/L) 74 , and oxymetazoline (130% at 400 mmol/L) 75 . While oxymetazoline failed to inhibit 5-LOX product formation (at 1000 mmol/L), the other compounds inhibited 5-LOX with IC 50 values of 1.2e2.3 mmol/L, being considerably less potent than compound 2 (IC 50 Z 0.3 mmol/L), except for ginkgolic acid (IC 50 Z 0.2 mmol/L). Evidence for a lipid mediator class switch in vivo was limited to the benzoxanthene ligans 1 and 2, which were less effective in raising SPM (precursor) levels in Figure 7 Modulation of lipid mediator profiles in macrophages by compound 2. Lipid mediator profiles of human macrophages of the M1 and M2 phenotype stimulated with pathogenic E. coli as determined by UPLC-MS/MS. (A) Heatmaps show mean percentage changes upon treatment with compound 2 relative to the vehicle control. HEPE, hydroxy-eicosapentaenoic acid; (di)HETE, (di)hydroxy-eicosatetraenoic acid; HDHA, hydroxy-docosahexaenoic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. Line charts present data in pg/ 2 Â 10 6 cells for selected lipid mediators as single data with values from the same independent experiment connected by lines, n Z 3. Data were log-transformed for statistical analysis. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vehicle control; repeated measures one-way ANOVA plus Tukey post hoc tests. (B) Quantitative illustration of the lipid mediator network in M2 macrophages treated with compound 2 (3 mmol/L) and stimulated with pathogenic E. coli as compared to vehicle control. The node size represents the mean concentration in pg and the color intensity denotes the fold increase for each lipid mediator; n Z 3. HpEPE, hydroperoxy-eicosapentaenoic acid; HpETE, hydroperoxy-eicosatetraenoic acid; HpDHA, hydroperoxy-docosahexaenoic acid.
inflamed peritoneal exudates than compound 2. Additional mechanisms besides substrate redirection are needed to explain the extraordinarily strong increase in 12-LOX and 15-LOX metabolites as well as SPM in activated M2 subsets both in vitro and in the inflamed peritoneal cavity in vivo. On the one hand, binding of AKBA to the allosteric site that is also addressed by compound 2 changes the regioselectivity of 5-LOX from C5-to C12hydroperoxidation of arachidonic acid 25 . It is therefore tempting to speculate that compound 2 exploits a similar mechanism to reprogram the 5-LOX enzyme, thereby triggering the generation of 15-LOX products in innate immune cells. On the other hand, compound 2 might indirectly act on signaling pathways that are specific for M2 macrophages. Along these lines, we have recently shown that 5-LOX inhibition by zileuton neither enhanced the formation of SPM (precursors) in M2 macrophages 76 nor in mouse peritonitis 26 . FLAP inhibition by MK886, however, manipulated the lipid mediator profile similar to compound 2 (Fig. 8B), although upregulation of SPM (precursor) was less pronounced. This finding is surprising because substantial FLAP inhibition can be excluded for compound 2, which suppressed cellular 5-LOX product formation in presence of excess substrate, thereby circumventing the need for FLAP in LT biosynthesis 77 . How macrophages switch between LT and SPM biosynthesis was recently ascribed to HMGB1-C1q complexes that crosslink receptor for advanced glycation end product (RAGE) and leukocyte-associated immunoglobulin-like receptor (LAIR)-1 78 . Whether compound 2 targets this pathway besides direct inhibition of 5-LOX needs further investigation. However, such a mechanism would explain why SPM upregulation is limited to macrophages, while 5-LOX inhibition occurs also in other immune cells.
The compound 2-induced switch from LT to SPM biosynthesis is not equally efficient for all SPM. In zymosan-induced mouse peritonitis, which is driven by infiltrated PMNL and peritoneal macrophages 36 , compound 2 lowered LT and elevated protectin (PD1, PDX) and MaR1 levels, whereas resolvin concentration is given in pg/mL exudate as means AE SEM, n Z 6 mice. Data were log-transformed for statistical analysis. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vehicle control; ordinary one-way ANOVA plus Dunnett's post hoc tests. The heatmap shows percentage changes upon administration of compound 2 relative to the vehicle control. HEPE, hydroxy-eicosapentaenoic acid; (di)HETE, (di)hydroxy-eicosatetraenoic acid; HDHA, hydroxy-docosahexaenoic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. (C) Cell infiltration into the peritoneal cavity. Data are shown as mean plus single data points, n Z 6 mice. **P < 0.01, *P < 0.05 compared to vehicle control; ordinary one-way ANOVA plus Dunnett's post hoc tests.
(RvD5, RvD3) concentrations even decreased; an effect that was overcompensated by the strong increase of PDX. It should be noted that mouse 12/15-LOX differs in regiospecificity to the human orthologues 15-LOX-1 and 15-LOX-2 79 . In line with these findings, 5-LOX has been reported to participate in resolvin biosynthesis 80 . On the other hand, compound 2 induces the production of resolvins along with protectins and maresins in human M2 macrophages in vitro. This apparent discrepancy likely depends on our focus on the early phase of peritonitis, for which PMNL are the dominating immune cells in the peritoneal cavity 36 . Compound 2 decreases resolvin biosynthesis in PMNL, and although they do not substantially contribute to SPM formation during resolution, they seem to be the major cell type that generates the low resolvin levels at 2 h after administration of the inflammatory stimulus. When PMNL are replaced by infiltrated macrophages as major innate immune cells in the peritoneal cavity during the late acute and resolution phase of inflammation 4 , we rather expect compound 2 to increase resolvin levels along with other SPM.

Conclusions
Promoting signaling pathways that resolve inflammation is considered as promising complementary or even superior therapeutic approach to treat chronic inflammatory diseases over simply suppressing pro-inflammatory signals 19 . Starting from the investigation of an extract library of Vietnamese medicinal plants, bioactivity-guided extract fractionation led to the identification of the potent biflavonoid 2 as promising drug candidate that relieves inflammation by inducing the lipid mediator class switch from inflammation to resolution in anti-inflammatory macrophage subsets. Our study provides a molecular basis for the antiinflammatory, wound-healing, and protective activities of D. cambodiana and gives access to a unique pharmacological tool that might open the door to a more efficient and safe strategy to fight chronic inflammation.