Phytochemical study of Alzatea verticillata, the sole species belonging to the Alzateaceae family

Alzatea verticillata Ruiz & Pav. (Alzateaceae) is a tropical tree from Central and South America. It is the only living species of Alzatea genus and the Alzateaceae family, all others being extinct. With the aim to investigate the possibility to find unusual natural products, the chemical content of the dichloromethane and methanolic extracts (stems and leaves) of A. verticillata have been investigated. Apolar and polar extracts were purified by semi-preparative HPLC using appropriate stationary phase columns allowing the isolation of 12 compounds: walterolactone B (2) walterolactone A/B β-D-pyranoglucoside (3), gallic acid (4), caffeic acid 4-O-β-D-glucopyranoside (6), walterolactone A/B 6-O-gallate-β-D-glucopyranoside (8), caffeic acid (9), 8-desmethylsideroxylin (11), sideroxylin (12) and 7,7′-bis(3,4-dihydroxyphenyl)-8,8′-cyclobutanedicarboxylic acid (7). Three isolated compounds are natural products described here for the first time: dimethyl-anemonin (1) and two β-truxinic acid derivatives (rel-(7S, 8R, 7′R, 8′S)-7,7′-bis(4-glucosyloxy-3-hydroxyphenyl)-8,8′-cyclobutane dicarboxylic acid (5) and rel-(7S, 8R, 7′R, 8′S)-7,7′-bis(4-glucosyloxy-3-hydroxyphenyl)-8,8′-cyclobutane-9-methyl dicarboxylic acid (10). The structures of the isolated compounds were elucidated by NMR and HRMS. The structure of compound 1 was confirmed by X-ray crystallography. A MS-based metabolite analysis of the A. verticillata extracts revealed additional truxinic acid derivatives that were putatively annotated with the help of feature-based molecular network. The presence of phenolic compounds such as truxinic acid derivatives could explain the traditional use of this plant as these compounds are known to possess anti-inflammatory and anti-nociceptive properties.


Introduction
Alzateaceae (Myrtales) [Q13634079] is a peculiar botanical family of Central and South America, represented by the single species Alzatea verticillata Ruiz & Pavón (Ruiz and Pavón, 1798) [Q131843]. It is a small tree typically found on steep slopes of the low mountain rain forest at an elevation between 1,000 and 2,200 m. Its closest relatives are the South-East Asian Crypteroniaceae and the African Penaeaceae (Graham, 1984;Conti et al., 2002;Schonenberger and Conti, 2003). This led both Rutschmann et al. (Rutschmann et al., 2004;Rutschmann et al., 2007) and Berger et al. (Berger et al., 2016) to suggest that the ancestor of the clade formed by those three families lived on the super-continent Gondwana millions of years ago and that following the Gondwana breakup, the ancestors of the Alzateaceae was isolated on the South American continent (c. 100 mya). Thus, Alzatea has evolved along an independent course over a long period of time acquiring many specialized attributes (e.g., a different chromosome number, n = 14 for A. verticillata subsp. amplifolia versus a base chromosome number of x = 12 for the other families of the order (Almeda, 1997)), ultimately occupying an isolated position in the order Myrtales. This genus is of phytochemical interest because its distant genetical background (and thus its biochemical phenotypes) might have chosen unique evolutionary pathways. Interestingly, the leaves of this plant are used as antipruritic for itching, scaly scalp, shampoo, and for venereal disease, by the Guyana Patamona Indians (Tiwari, 2002). Inspired from the unique diterpenes (ginkgolide derivatives) observed in Ginkgo biloba, which is also the sole species of its family (Forman et al., 2022), we undertook a phytochemical investigation of A. verticillata. To this aim, the chemical profile of the dichloromethane (DCM) and methanolic (MeOH) extracts of A. verticillata stems or leaves were analyzed by untargeted highresolution mass spectrometry (UHPLC-HRMS/MS) and the results interpreted by molecular networking. The main constituents were then isolated by high-resolution semipreparative HPLC.

Results and discussion
To establish a preliminary chemical profile of the specialized metabolites produced by A. verticillata, the DCM and MeOH extracts obtained from the leaves and stems were analysed by HPLC-PDA ( Figure 1) and UHPLC-HRMS/MS (Supplementary Figure S1). Given that the species and genus weren't previously investigated, we have carried out a preliminary isolation and structure elucidation of the main compounds of the extract observed prior to a more advanced annotation procedure of most metabolites detected.
Compound 1 was isolated as an amorphous solid. The HRMS spectrum with an atmospheric pressure chemical ionization source (APCI) showed a protonated molecule at m/z 221.0800 [M + H] + . The 1 H NMR and edited-HSQC spectra of 1 showed a vinylic signal at δ H 5.90 (each, 1H, q, J = 1.5 Hz, H-3, H-3′), a methylene at δ H 2.52 (each, 2H, s, H 2 -6, H 2 -6′) and a methyl at δ H 2.41 (each, 3H, d, J = 1.5 Hz, H 3 -7, H 3 -7′). The HMBC correlations from the methyl to the vinylic C-3 (δ C 117.5), the sp 2 carbon C-4 (δ C 169.6) and the oxygenated quaternary carbon C-5 (δ C 92.4), from H-3 to C-5 and the ester carbonyl C-2 (δ C 172.6) and from H-6 to C-6 (δ C 25.0) and C-4 indicated the presence of a 4-methylfuran-2(5H)-one with extra methylene in C-5, accounting for six carbons. The molecular formula and the fact that the methylene protons correlated in HMBC with their own carbon indicated that 1 should be a symmetric dimer. To confirm its structure and establish its configuration, compound 1 was successfully crystalized in ethyl acetate and the crystals were subjected to X-ray crystallography ( Figure 3). Compound 1 has crystallized in the centrosymmetric space group P-1 and therefore is a S,S and R,R racemic mixture. Dimethyl-anemonin (1) is a dimethylated derivative of the known compound anemonin which was first isolated by Heyer in 1792 (Heyer, 1792). It is well known that, in plant, anemonin is formed by the spontaneous dimerization of protoanemonin, which comes from the enzymatic deglycosylation of raninculin (Pirvu et al., 2022). Suga and Hirata showed that protoanemonin in Ranunculus glaber comes from 2-oxo-glutaric acid (Suga and Hirata, 1982). In our case, we could imagine a methylation of 2-oxo-glutaric acid before cyclisation to obtain methyl-protoanemonin and then the dimethyl-anemonin (1) (Supplementary Figure S2). Moreover, one of the fractions obtained from the purification of the dichloromethane extract of the leaves contained in mixture with 8-desmethylsideroxylin (11), the 5-hydroxy-4,5-dimethylfuran-2(5H)-one and 4,5-dimethylfuran-2(5H)-one which are in line with this biosynthesis proposal (data in Supplementary Figure  S3). This type of dimerisation explains that 1 was, as anemonin, isolated as a racemic mixture. One note that walterolactone B (2) could also be obtain by rearrangement of methyl-protoanemonine.
Compound 10 presented a deprotonated molecule at m/z 697.1991 [M-H]in the ESI-HRMS spectra, suggesting the molecular formula C 31 H 37 O 18 . The 1 H NMR spectra of 10 showed close similarities to that of 5, except for the presence of a methoxy group at δ H 3.72 for 10 and a splitting of the cyclobutane signals for H-7 and H-7′ as well as H-8 and H-8′ protons. In 10, the methylation of only one of the two acid functions results in a loss of symmetry and thus the cyclobutane protons became non-equivalent. The COSY correlations from H-8 at δ H 3.78 to H-7 at δ H 4.19, from H-7 to H-7′ at δ H 4.13 and from H-7′ to H-8′ at δ H 3.75, the HMBC correlations from H-7 and H-7′ to the aromatic carbons C-1/1′(δ C 136.1/136.3), C-2/2′(δ C 116.8/116.9) and C-6/6′(δ C 120.5/120.6), and from H-7, H-8 and the methoxy H-10 (δ H 3.72) to C-9 at δ C 175.2 allowed to identify a truxinic acid derivative (  (Fujiwara et al., 2016). Two different diastereoisomers of this compound could exist depending on the methoxy position in C-9 or C-9′. Indeed, the almost same spectra were obtained from two very close HPLC peaks (10a, 10b) (Figure 1; Supplementary Figure S4) so that it wasn't possible to distinguish these two diastereoisomers. Based on the data described above, compound 10 was thus identified as rel-(7S, 8R, 7′R, 8 ′S)-7,7′-bis(4-glucosyloxy-3-hydroxyphenyl)-8,8′cyclobutane-9-methyl dicarboxylic acid, a new β-truxinic acid derivative.
To investigate the presence of other truxinic acid analogues, a molecular network (Wang et al., 2016) was generated from the DCM and MeOH extracts of leaves and stems of A. verticillata using an UHPLC-HRMS/MS ESI, positive ion mode). A molecular network cluster containing the two isolated truxinic derivatives was observed FIGURE 6 Molecular network cluster containing 5 and 10, as well as other putative truxinic acid derivatives. These data were generated from LC-ESI-HRMS/MS data of the DCM or MeOH extracts of stems or leaves of Alzatea verticillata. The classical molecular networking workflow was used where the node size represents the relative intensity of the precursor ions for each extract.

Frontiers in Natural Products
frontiersin.org 06 as [M+NH 4 ] + adducts ( Figure 6). Interestingly, other non-isolated derivatives were also part of this cluster. An ion at m/z 730.237 suggested a molecular formula differing by an additional CH 2 compared to 10. The examination of the fragmentation spectra allowed to determine that the compound at m/z 730.237 corresponded to a methoxylated derivative of 5 on the free acid function. Further examination of the fragmentation pattern for the remaining ions of this cluster enabled to putatively annotate them. The ion at m/z 568.188 corresponded to the deglucosylated derivative of m/z 730.237. Similarly, the interpretation of the fragmentation spectra of the ion at m/z 554.173 agreed with a deglucosylated derivative of 10. In the case of the latter, the precise location of the deglucosylation on one specific trihydroxybenzen unit could not be established. It is important to note that the methoxylated derivatives such as compound 10 or other annotated derivatives, may be artefacts due to methylation during the extraction process.
Although many compounds belonging to the phenylpropanoid structural class have been isolated, phenylpropanoid dimers that contain a cyclobutane moiety are relatively rare (Yang et al., 2022). These compounds possess strong radical scavenging properties (Shahidi and Chandrasekara, 2010), and they are also involved in the plant tolerance to both biotic and abiotic stresses (Shahidi and Chandrasekara, 2010).
Further insight into the chemical profile of A. verticillata was enabled by annotating the mass spectrometry data by spectral library matching with public libraries. These annotated ions were not isolated and thus most likely corresponded to minor metabolites. The spectral matches obtained were classified with NPClassifier (Supplementary Figure S5) and indicated the presence of multiple derivatives belonging to shikimates and phenylpropanoids (32.3%, n = 19), fatty acids (29.2%, n = 19), terpenoids (21.5%, n = 14), alkaloids (9.23%, n = 6), and polyketides (6.15%, n = 4).

Experimental section
General experimental procedures NMR spectroscopic data were recorded on a 500 MHz Varian (Palo Alto, CA, United States) INOVA NMR spectrometer and on a Bruker Avance III HD 600 MHz NMR spectrometer equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany). Chemical shifts are reported in parts per million (δ) using the residual CD 3 OD signal (δ H 3.31; δ C 49.0) or DMSO-d 6 signal (δ H 2.50; δ C 39.5) as internal standards for 1 H and 13 C NMR, respectively, and coupling constants (J) are reported in Hz. Complete assignments were obtained based on 2D-NMR experiments (COrrelation SpectroscopY (COSY), Nuclear Overhauser Effect SpectroscopY (NOESY), Heteronuclear Single Quantum Correlation (HSQC) and Heteronuclear Multiple Bond Correlation (HMBC)). ESI-HRMS data were obtained on a Micromass LCT Premier time-of-flight mass spectrometer from Waters with an electrospray ionization (ESI) interface (Waters, Milford, MA, United States). HPLC-PDA data were obtained with an Agilent HP 1,260 series system consisting of an autosampler, high-pressure mixing pump and DAD detector (Agilent Technologies, Santa Clara, CA, United States). HPLC-fractionation was performed with an Armen modular spot prep II (Saint-Avé, France) equipped with an UV detector and fraction collector and on a Shimadzu system equipped with a LC-20A module pumps, an SPD-20A UV/VIS, a 7725I Rheodyne ® valve, and an FRC-10A fraction collector (Shimadzu, Kyoto, Japan). The system was controlled by the LabSolutions software, also from Shimadzu.

Plant material
Leaves and stems of A. verticillata were collected at Parque Nacional General de División Omar Torrijos Herrera, in the provincial of Coclé, Panama in July 2013. The species was identified by De Gracia J. and voucher was deposited at the National Herbarium of Panama (FLORPAN, Voucher at PBNB: n°8747).

Extraction
The air-dried plant material (500 g) was pulverized with a Wiley Mill and extracted at room temperature successively with dichloromethane and methanol to give 32 g, and 48.5 g, respectively. The extracts were concentrated under pressure and later lyophilized.

HPLC-PDA analyses
HPLC-PDA analyses were conducted on an Agilent 1,260 system equipped with a photodiode array detector. The separations of the dichloromethane and methanolic extracts of stems and leaves of A. verticillata were performed on a Waters Frontiers in Natural Products frontiersin.org 07 XBridge C 18 column (250 × 4.6 mm i.d., 5 µm) (Waters, Milford, MA, United States) equipped with a Waters C 18 pre-column cartridge holder (20 × 4.6 mm i.d., 5 µm) with MeOH (B) and H 2 O (A), both containing 0.1% formic acid as solvents. The flow rate was set at 1 mL/min, the injection volumes were 10 µL with samples at about 10 mg/mL. The separation for the dichloromethane extracts was performed in gradient mode using the following conditions: 5%-100% B in 60 min, followed by 10 min of washing at 100% B. The separation for the methanolic extracts was also performed in gradient mode using the following conditions: 5% B for 10 min, 5%-17% B in 18 min, 17% B for 30 min, 17%-60% B in 30 min and 60%-100% in 2 min, followed by 10 min washing at 100% B.

UHPLC-ESI-HRMS/MS analyses
The UHPLC-ESI-HRMS/MS analysis was carried out on a Waters Acquity UPLC IClass system interfaced to a Q Exactive Focus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. Chromatographic separation was performed on a column of Waters BEH C 18 50 × 2.1 mm i.d., 1.7 µm, mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), flow rate was 600 μL/min, injection volume was 1 μL, and linear gradient elution from 5% to 100% B in 7 min, followed by isocratic at 100% B for 1 min, and decreased to 5% B at the final step for 2 min. Positive and negative ionization mode were applied in this study. The diisooctyl phthalate C 24 H 38 O 4 [M-H] − ion (m/z 389.2697) was used as an internal lock mass. The optimized HESI-II parameters were set as follows: source voltage, 3.5 kV (pos) or 2.5 kV (neg); sheath gas flow rate (N 2 ), 48 units; auxiliary gas flow rate, 11 units; spare gas flow rate, 2.0; capillary temperature, 300°C (pos), S-Lens RF Level, 55. The mass analyzer was calibrated using a mixture of caffeine, methionine-arginine-phenylalanine-alanineacetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1,621 in an acetonitrile/methanol/water solution containing 1% formic acid by direct injection. The datadependent MS/MS events were performed on the three most intense ions detected in full scan MS (Top3 experiment). The MS/MS isolation window width was 2 Da, and the normalized collision energy (NCE) was set to 35 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35,000 fwhm (at m/z 200) and MS/MS scans at 17,500 fwhm both with a maximum injection time of 50 ms. After being acquired in a MS/MS scan, parent ions were placed in a dynamic exclusion list for 2.0 s.

Mass spectrometry and fragmentation spectra annotation
The UPHLC−HRMS/MS raw data was converted to.mzXML format using the MSConvert software, part of the ProteoWizard package. Molecular networks were generated on the GNPS webplatform using the classical molecular networking workflow (Wang et al., 2016) from the mass spectrometry of the extracts of A. verticillata (DCM or MeOH extracts of stems or leaves). The fragmentation spectra were annotated by spectral library matching using public libraries and reference spectra of the isolated compounds. The interactive view of the entirety of molecular networks generated and the parameters used are available via the job link at https://gnps.ucsd.edu/ProteoSAFe/ status.jsp?task=7e5e5b3e23de4e2395a247a73ff18a85. The reference MS/MS fragmentation spectra (ESI positive ionization mode) of compounds 5-8, 10-12 were added to the GNPS public spectral library (Wang et al., 2016). The NPClassifier taxonomy (Kim et al., 2021) was used to classify the annotated compounds.
Purification of the dichloromethane extract of stems by semi-preparative HPLC-UV The dichloromethane extract of stems was purified using the semi-preparative HPLC-UV equipment and was performed on an Armen modular spot prep II system equipped with an UV detector and fraction collector. The separation was performed with an Interchim Silica HP column (250 × 21 mm i.d., 10 μm; Interchim, Montluçon, France). The flow rate was set at 21 mL/ min. The solvent system used was a mixture of hexane (A) and ethyl acetate (B) in gradient mode: 5%-100% of A in 61.4 min followed by 100% of A for 10 min. The UV absorbance was measured at 210 nm. The sample (90 mg) was injected by loop injection (3 injections of 30 mg in 500 µL of ethyl acetate). After collection, each fraction was evaporated to dryness using a SpeedVac (HT-4X Genevac ® , Stone Ridge, NY, United States). The separation yielded 7 mg of compound 1.
Purification of the dichloromethane extract of the leaves by semi-preparative HPLC-UV For the separation of the dichloromethane extract of the leaves of A. verticillata, the semi-preparative HPLC was performed on a Shimadzu system equipped with a LC-20A module pumps, an SPD-20A UV/VIS, a 7725I Rheodyne ® valve, and an FRC-10A fraction collector. The system was controlled by the LabSolutions software from Shimadzu. The separation was performed as follows: Waters XBridge C 18 column (250 × 19 mm i.d., 5 µm); solvent system MeOH (B) and H 2 O (A), both containing 0.1% formic acid. The separation was performed in gradient mode as follows: 5%-100% B in 60 min. After this the column was washed with 100% of B during 10 min. Flow rate was 17 mL/min. The sample (50 mg of the extract) was injected by dry load using the protocol recently published (Queiroz et al., 2019). The UV absorbance was measured at 210 and 254 nm. The separation yielded compound 6 (1.8 mg), 9 (0.7 mg), 11 (0.6 mg), and 12 (0.4 mg).

Description of the isolated compounds
Dimethyl-anemonin (1) X ray analysis of compound 1 The compound (5 mg) was solubilized in 1 mL of a hexane and ethyl acetate mixture (1:1) in a small glass vial (5 mL). The vial was wrapped with aluminium foil and small holes were made in the cover for slow evaporation of the solvent. The solution was left at room temperature overnight. The next day the crystals appeared in the bottom of the bottle. Summary of crystal data, intensity measurements and structure refinements for 1 were collected in Supplementary Tables S1, S2 (Supplementary information). Supplementary Figures S74 shows the Ortep of compound 1 (Thermal ellipsoids are drawn at 50% probability level). The crystals were mounted on MiTeGen Kapton cryoloops with protection oil. X-ray data collection were performed with a Rigaku Super Nova Dual diffractometer equipped with a CCD Atlas detector (Cu[Kα] radiation). The structures were solved by using direct methods and full-matrix least-square refinements on F 2 were performed with SHELXL-2014 (Sheldrick, 2015). CCDC 1873730 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.