Anti-biofilm and anti-inflammatory active diterpene isolated from the fruit of Xylopia benthamii R.E.Fr.

Abstract Xylopia benthamii (Annonaceae) is a plant with limited phytochemical and pharmacological evidence. Thus, using LC-MS/MS, we performed exploratory analyses of the fruit extract of X. benthamii, resulting in the tentative identification of alkaloids (1–7) and diterpenes (8–13). Through the application of chromatography techniques with the extract of X. benthamii, two kaurane diterpenes were isolated, xylopinic acid (9) and ent-15-oxo-kaur-16-en-19-oic acid (11). Their structures were established using spectroscopy (NMR 1D/2D) and mass spectrometry. The isolated compounds were submitted to anti-biofilm analysis against Acinetobacter baumannii, anti-neuroinflammatory and cytotoxic activity in BV-2 cells. Compound 11 (201.75 µM) inhibited 35% of bacterial biofilm formation and high anti-inflammatory activity in BV-2 (IC50 = 0.78 µM). In conclusion, the results demonstrated that compound 11 was characterized for the first time with pharmacological potential in the development of new alternatives for studies with neuroinflammatory diseases. Graphical Abstract


Introduction
In terms of biodiversity, the Amazon is one of the richest ecosystems on the planet; however, this biome has been under constant pressures that threaten its functioning and diversity, especially in regards to its plant species (Hofhansl et al. 2020;Caetano 2021).The flora of the Amazon possesses a promising natural reservoir of bioactive substances distributed in certain botanical families, with Annonaceae being one of the most studied and most promising in relation to species diversity (Cascaes et al. 2021).
The Annonaceae family is distributed almost exclusively in tropical regions (Heywood 1985), and comprises around 2,500 known species distributed in 130 genera.The family is extremely diverse, with 40 genera in the neotropical region, and is composed of approximately 900 species distributed throughout the Amazon and Guianas (Smith et al. 2004).
The species X. benthamii is of Amazonian origin (Jurgens et al. 2000) that is little known chemically and pharmacologically.Published studies on this species have already reported the isolation of benzenoids, terpenes (Jurgens et al. 2000;Jagessar and Maxwell 2012), aporphine alkaloids and phenolic amides (feruloylamides) (Pimenta and Mendonça 2012).
Considering the potential of the bioactive compounds of the species of the genus Xylopia, as well as the scarcity of chemical and biological studies of X. benthamii, the present work exposes the chemical profile through LC-MS/MS analysis and reports the isolation and structural characterization of two diterpenes in the species (Figure 1) .In addition, these compounds and the crude extract were evaluated for their anti-inflammatory and cytotoxic activity in BV-2 lineage microglial cells (Figure 1).

Anti-biofilm activity
The minimum inhibitory concentration of biofilm (MICB) was evaluated by the inhibition of biofilm formation in bacterial cultures of A. baumannii that were treated with increasing concentrations of the ent- .When treated with ent-15-oxo-kaur-16-en-19-oic acid, the MICB of A. baumannii was of 201.75 μM, with an inhibition of biofilm formation of 35% (Figure 2A).The positive control ciprofloxacin reduced A. baumannii biofilm formation by 80% at concentrations of 96.5 μM (Figure 2B), which indicates that ent-15-oxo-kaur-16-en-19-oic acid has a low anti-biofilm activity.
Biofilm is a complex matrix of microorganisms in which cells unite and attach to the biotic or abiotic surface (Bazargani and Rohloff 2016).Bacteria within biofilms are more resistant to antibiotics and chemicals than planktonic cells in suspension (Alam et al. 2020), and chemical agents that penetrate the biofilm matrix are less effective because most chemicals are only active against loose microorganisms.The ent-15-oxo-kaur-16-en-19-oic acid inhibited biofilm formation by only 35%.Previous studies have shown evidence that medicinal plants are promising in the combat against different types of infectious diseases caused by numerous microbial species (Famuyide et al. 2019;Manandhar et al. 2019).Previous studies report the antibacterial activity of kaurane diterpenes against Gram-positive bacteria (1.56 − 10 µg.mL −1 ) (Ambrósio et al. 2008;Fonseca et al. 2013;Barbosa et al. 2019) and Gram-negative (0.10 − 3.12 µg.mL −1 ) (Ren et al. 2015;Yang et al. 2016).Ambrósio et al. (2008) suggest that the presence of the carboxy group (C-18) makes an important contribution to the antibacterial activity.This fact was sustained in the work of Çiçek et al. (2020), noting that the presence of these groups in the kaurane structures had an influence on the antibacterial action.Although the mechanism of action of this class of substance on the biofilm is not well understood, it is reported that may occurs through disassembly and weakening of the bacterial membrane (Kamaruzzaman et al. 2018).

Anti-neuroinflammatory and cytotoxic activity in BV-2 cells
The cell viability of the X. benthamii extract and the isolated compound ent-15-oxo-kaur-16-en-19-oic acid against BV-2 cells was evaluated after 24 h of incubation using the MTT method (Figure 3A).The results showed that the X. benthamii extract showed low cytotoxic activity against BV-2 cells, with a reduction of 24% at 32 µg.mL −1 .In contrast, the X. benthamii extract showed anti-inflammatory activity in BV-2 microglial cells when they were stimulated with LPS (Figure 3B).The X. benthamii extract inhibited the inflammation induced by LPS by 100% at 32 µg.mL −1 and presented an IC 50 of 0.47 ± 0.37 µg.mL −1 .Similarly, to the extract, the compound ent-15-oxo-kaur-16-en-19-oic acid showed low cytotoxic activity (Figure 3C). on the other hand, the isolated compound displayed a more potent anti-inflammatory activity in BV-2 stimulated cells with a IC 50 at 0.78 µM (Figure 3D).
The triggering of neurodegenerative diseases such as Alzheimer's, Parkinson's and multiple sclerosis are caused by inflammatory processes involving cells of innate and adaptive immunity.The initiation, as well as the amplification of the neuro-inflammatory process, involves the presence of brain-resident microglial cells that are recruited to the site of inflammation during injury, and which in turn recruit other cellular elements through the release of chemokines and cytokines, thus contributing to the progression of these diseases (De Caris et al. 2020).
Microglia, the key cells involved in the innate immune response that defends against central nervous system (CNS) insults, play essential roles in maintaining homeostasis and responding to conditions of neuroinflammation (Boleti et al. 2020).In response to lipopolysaccharide (LPS) stimulation, microglia adopt the classic M1 phenotype and exert a pro-inflammatory role through the secretion of TNF-α, IL-1β, IL-6 and RoS/RNS (Khalil et al. 2021).It was shown that the extract of X. benthamii reduced nitric oxide production in a concentration-dependent manner and maintained the viability of BV-2 microglial cells, which indicated absence of cytotoxicity.Similar results were also obtained with Moringa oleifera leaf extract (Sivaprakasam et al. 2019).Kim et al. (2021) also reported that the extract of Fructus ligustri lucidi was not toxic to BV2 cells up to a concentration of 500 µg.mL −1 and inhibited the production of No in a concentration-dependent manner (Kim et al. 2021).
Some studies have reported the anti-neuroinflammatory activities of diterpenes from plants (Gan et al. 2015;Kim et al. 2016;Wei et al. 2017).However, there are still no reports on the anti-neuroinflammatory effects of the two ent-kaurenoic diterpenes isolated from X. benthamii.In this study, we therefore evaluated the anti-neuroinflammatory activities of two isolated ent-15-oxo-kaur-16-en-19-oic acid compounds using BV-2 cells activated with LPS.our results show that the ent-15-oxo-kaur-16-en-19-oic acid compound present in X. benthamii extract has anti-inflammatory activity in BV-2 microglial cells.These results with ent-15-oxo-kaur-16-en-19-oic acid compound were superior when compared to other ent-kauranes, such as 2β,15α-dihydroxy-ent-kaur-16-ene and pterokaurane, isolated from Pteris multifida roots with IC 50 values of 13.9 µM and 10.8 µM, respectively (Kim et al. 2016).Moreover, Lee et al. (2014) studied kauranes isolated from Siegesbeckia pubescens (Asteraceae) on their significant inhibitory activity against induced No production.The authors suggested that the tetracyclic core kauran-19-oic acids are essential to promote inhibitory activity on No production in LPS-stimulated BV2 microglial cells.Regarding our compounds, we observe that the same moiety is encountered for ent-15-oxo-kaur-16-en-19-oic acid.Beyond this, an interesting feature of our compound is the presence of an α,β-unsaturated ketone at the cyclopentane ring, which may lead to a more reactive compound toward key proteins, thus giving more potency to ent-15-oxo-kaur-16-en-19-oic acid.To the best of our knowledge, this is the first report of two diterpenes from X. benthammi with anti-neuroinflammatory effects in BV-2 microglial cells.

Plant material
The fruit of X. benthamii (SISGEN Register AEDBD42) was obtained in the city of Manaus, Amazonas state, in the botanical garden located in the Adolpho Ducke Forest Reserve (coordinates 3° 00′ 18.3" S, 59° 56′ 26.5" W).The species was identified and deposited in the herbarium of the Instituto Nacional de Pesquisas da Amazônia (INPA) under the registration number #281751.

Processing and extraction
The dried and pulverized fruit powder of X. benthamii (75.91 g) was subjected to extraction by cold maceration with 95% ethanol at 25 °C.Every 72 h, the solvent was renewed, and six extractions were performed.Later, the extractions were combined, the resulting extract filtered and concentrated in a rotary evaporator under reduced pressure at 50 °C to give the crude ethanolic extract, called EEBXB (11.08 g).

Fractionation, isolation and characterization
A portion (2 g) of the EEBXB was subjected to open column chromatography containing 40 g of C18 (reverse phase) (200-400 mesh, 60 Å).Initially, it was eluted with only a gradient consisting of MeoH/H 2 o (7:3), followed by MeoH (100%), resulting in two fractions: F1 (400 mg) and F2 (300 mg).For the isolation of the substances of interest, a chromatograph (LC-6AD, Shimadzu®) equipped with uV/VIS detector (SPD-20A) was used, and measured the absorbance of the sample at 240 and 260 nm.An aliquot of fraction F2 (100 mg) was solubilized in 400 µL DMSo and injected twice (200 µL) in a Phenomenex Luna C18 column (5 µm, 250 mm x 15 mm id) (Torrance, CA, uSA).Milli-Q Water (A) and MeoH (B) were used as the mobile phases.For elution, milli-Q Water (A) and MeoH (B) were used in gradient mode from 60 to 100% in 30 min, with another 10 min at 100% (6 mL.min −1 ), which resulted in the isolation of substances 9 and 11.

Chemical analyses
The extract was solubilized in 1 mL of HPLC grade MeoH, centrifuged at 13,000 rpm for 10 min, and then the supernatant was separated.The supernatant was subjected to LC-MS analysis using a chromatograph (Nexera X2, Shimadzu, Japan) with a diode array detector (DAD)-SPD M20A coupled to a spectrometer with a Q-ToF analyzer, (MicroToF-QII, Bruker Daltonics, uSA), and equipped with electrospray source (ESI) in positive mode.The operating parameters of the equipment were as follows: capillary 4,500 V, nebulizer gas (nitrogen) 4 bar, drying gas (nitrogen) 9.0 L.min −1 , source temperature 200 °C and mass range of m/z 50-1,200.For internal calibration of the system, a solution of 10 nM sodium formate in isopropanol/water (1:1 v/v) was used.Chromatographic separation was performed on a Kinetex C18 analytical column (100 × 2.1 mm, 2.6 µm) (Phenomenex, uSA) maintained at 50 °C, with a flow rate of 0.35 mL.min −1 .De-ionized water (A) and acetonitrile + formic acid (20 mM) (B) (HPLC grade) were used for elution.Initially, 0-2 min of isocratic elution was applied in 15% of (B), followed by 2-12 min of elution from 15 to 95% of (B) and again 12-17 min of isocratic elution at 95% of B (5 µL) of sample were injected in each analysis.
For the characterization of the isolated constituents, 1D and 2D nuclear magnetic resonance (NMR) analyses were performed.The spectra were acquired at 293 K in CDCl 3 in a spectrometer (Bruker Avance III HD NMR) operating at 11.75 Tesla, and the 1 H and 13 C nuclei were observed at 500 and 125 MHz, respectively.All 1 H and 13 C (δ) NMR chemical shifts are given in ppm relative to the tetramethylsilane signal (0.00 ppm) as the internal reference and coupling constants (J) have been assigned in Hz.

Anti-biofilm activities
The minimum inhibitory concentration of biofilm (MICB) was obtained by inhibiting biofilm formation using Basal Medium 2 (BM2) bacterial cultures of A. baumannii (clinical isolate-003326263), which were cultured for 18 h in Mueller-Hinton broth (MHB) and were diluted 1:1:100 (v/v) in BM2.The bacterial suspension was plated in 96-well round-bottomed plates containing the samples in serial dilutions, ranging from 8 to 256 μg.mL −1 (final volume: 100 µL).The microplates were then incubated for 24 h at 37 °C.The negative growth control contained only bacterial cells, while ciprofloxacin was used as a positive control at the same concentrations as the samples.The growth of planktonic cells was evaluated using absorbance at 600 nm.
To assess biofilm formation, the medium was removed from the microplates and the wells were washed twice with deionized water.The adherent cells were stained with 0.01% crystal violet for 20 min, after which the microplate wells were washed twice with deionized water, air dried and the crystal violet adhered to the cells was solubilized with 110 µL of ethanol at 60%.Biofilm formation was measured using absorbance at 595 nm.All absorbance readings were performed using a microplate reader (Bio-TekInstruments, Inc., united States).All experiments were performed in biological and technical triplicate.

Cell cultures
In this study, we used microglial cells (BV-2) acquired from the Rio de Janeiro Cell Bank.The BV-2 cell lines were stored in liquid nitrogen at a temperature of approximately −196 °C at the universidade Católica Dom Bosco (uCDB, Brazil).The BV-2 cells were cultured in the Immunology Laboratory (uCDB) in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 u.mL −1 penicillin and 100 µg.mL −1 streptomycin (Gibco, Brazil) at 37 °C in an incubator at 5% Co 2.

Cell viability test using MTT methodology
To verify whether the extracts and compounds are cytotoxic, the viability of BV-2 cells was evaluated according to a method adapted from Mosmann (1983), based on the enzymatic reduction of 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) to formazan crystals.BV-2 cells were plated at 5 x 10 5 cells.mL−1 in 96-well microplates and treated with 100 µL of different molar concentrations of the X. benthamii extract (0.5 to 32 μg.mL −1 ) for 24 h.The culture medium was used as a negative control.After the incubation period, the supernatant was removed and 100 μL of MTT solution (1 mg.mL −1 diluted in culture medium) was added to the cells.After 4 h of incubation, the formazan crystals were resuspended with 100 μL of dimethyl sulfoxide (DMSo) and read at 570 nm on the microplate reader (MultiSkan Go, Thermo scientific) (Mosmann 1983).Three independent experiments were performed in triplicate, and cell viability was calculated from the following formula:

Inhibition of microglial activation by LPS
BV-2 microglial strains were plated at a density of 5 x 10 5 cells.mL−1 in 96-well plates followed by adhesion for 24 h at 37 °C in a 5% Co 2 atmosphere.After adhesion, the medium was removed, and cells were stimulated with lipopolysaccharide -LPS (final concentration 1 µg.mL −1 ) together with X. benthamii extract and treated at various concentrations (0.5 to 32 μg.mL −1 ) in a final volume of 100 μL.well −1 of RPMI culture medium supplemented with 1% FBS.For the control experiment, cells were cultured in culture medium and the medium with LPS.Then, the cells were incubated for another 24 h at 37 °C, 5% Co 2 , and the cell supernatant was collected for No analysis and with adhered cells.The cell viability assay was performed using the MTT method.The production of nitric oxide was measured by the dosage of its most stable degradation product, nitrite, using Griess' reagent (Green et al. 1982).For the determination of No production, 100 μL of cell supernatant was subjected to the reaction with an equal volume of Griess' reagent.For the preparation of this reagent, solutions of N-(1-naphthyl) ethylenediamine dihydrochloride (0.1%) dissolved in water and 1% sulfanilamide dissolved in H 3 Po 4 (5%) were used.Just before use, the solutions were added in a 1:1 ratio, thus forming the Griess' reagent proper.After the 10 min incubation period, the samples were read in a microplate reader at 540 nm.The calculation of nitrite concentrations was performed based on standard curves using different concentrations of nitrite (3.12 to 200 μM).

Statistical analysis
The results were expressed as mean ± standard error of the mean (SEM).Analysis of variance (ANoVA) was used to compare the results, followed by a multiple comparison test (Dunnett's test).Data were considered significant when p < 0.0001.Statistical analyses were performed using the statistical program GraphPad Prism 8.0.

Conclusions
Through exploratory LC-MS/MS analysis of the fruit of X. benthamii, thirteen known compounds were identified, including seven alkaloids and five terpenes.Compounds xylopinic acid (9) and ent- 15-oxo-kaur-16-en-19-oic acid (11) were isolated, with 11 being biologically active on LPS-induced anti-inflammatory activity (IC 50 = 0.78 µM).Additionally, the crude extract of X. benthamii showed promising anti-inflammatory activity on LPS (IC 50 = 0.47 µg.mL −1 ) and low cytotoxic activity against BV-2 cells, with a reduction of 24% at 32 µg.mL −1 .This is the first report of substance 11 in this species with antineuro-inflammatory activity in cells (BV-2).Therefore, the species proved to be a source of substances with pharmacological properties that encourage new studies in the search for bioactive metabolites.Moreover, future studies on in vivo activities and the safety of 11 should be addressed in order to develop this diterpene as a drug candidate to treat inflammation-mediated neurodegenerative disorders and of as an antibacterial agent.