Anti-Inflammatory Meroterpenoids of Cordia glazioviana (Boraginaceae)

The phytochemical reinvestigation from the heartwood of the extracts of Cordia glazioviana led to the isolation of four still undescribed hydroquinones derivatives designated as cordiaquinol D (1), cordiaquinol E (2), (10R)-10,11-dihydrofuran-1,4-dihydroxy-globiferin (3) and 2-[(1’E,6’E)-3’,8’-dihydroxy-3’,7’-dimethylocta-1’,6’-dienyl]-benzene-1,4-diol (4), along with the naphthoquinone 6-[(2’R)-2’-hydroxy-3’,6’-dihydro-2H-pyran-5’-yl]-2-methoxy7-methylnaphthalene-1,4-dione (5). Additionally, six previously known compounds were also isolated: rel-1,4-dihydroxy-8α,11α;9α,11α-diepoxy-2-methoxy-8aβ-methyl5,6,7,8,8a,9,10,10a-octahydro-10-antracenone (6), didehydroconicol (7), 1β,6β-dihydroxy-7-epieudesm-3-ene (8), 1β,6β-dihydroxy-7-epi-eudesm-4(15)-ene (9), 10,11-dihydroxybisabolol (10), and hamanasal-A (11). The structures of the new compounds were assigned by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analyses. The relative stereochemistry of 3, 4, and 5 was improved by quantum mechanical calculations. Eight, out of the eleven isolated compounds (2-9), were tested through cellular viability and lipopolysaccharide (LPS)-induced inflammation assays against RAW 264.7 macrophage-like cells. Compounds 3-5 exhibited a stronger effect on LPS-induced NO production (half-maximal inhibitory concentration (IC50) 50.34, 105.83, and 66.73 μM, respectively).


Introduction
Plants of the genus Cordia (Boraginaceae) have been described as a prolific source of bioactive compounds. 1 In fact, several Cordia species (C. dichotoma, C. latifolia, C. verbenacea, C. myxa, C. rothii, C. gharaf, C. obliqua, etc.) have been used in different traditional systems of medicine around the world such as the Ayurveda, Unani, and Siddha 2 due to their ethnopharmacological properties: anti-inflammatory, antimicrobial, anthelmintic, analgesic, and diuretic. 1 Despite the wealthy Brazilian biodiversity, the great traditional knowledge and acceptance of medicinal plants, in contrast to the increasing world demand for phytotherapeutics, the Brazilian herbal medicine market is still very modest. Nevertheless, it is worthwhile to highlight that an anti-inflammatory product incorporative Cordia verbenacea essential oil, is found among the top 20 pharmaceutical drugs marketed in Brazil in 2016. 3 Moreover, recent studies have evidenced the antiinflammatory potential of extracts and pure compounds from other Cordia species. 4 Previous phytochemical studies 5,6 on Cordia genus have reported the isolation of terpenoids, particularly sesquiterpenes and triterpenes, meroterpenoid benzoquinones, and naphthoquinones as well as their respective hydroquinones. Furthermore, the antiinflammatory effect of sesquiterpenes, 7 triterpenes, 8 hydroquinones, 9 and naphthoquinones 10 have been demonstrated.
Cordia glazioviana (Auxemma glazioviana), an endemic Brazilian plant, is largely widespread in the "caatinga" (the characteristic biome of northeastern Brazil). 11 In folk medicine, the water decoction from its barks is indicated to the healing of small cuts and wounds. 6 Previous reports 6,12 on C. glazioviana described the isolation of sesquiterpenes and terpenoids benzoquinones, as well as hydroquinones. Thus, encouraged by the new perspective, we decided to reinvestigate the extracts of C. glazioviana pursuing the isolation of anti-inflammatory natural chemical compounds.

Results and Discussion
Eleven meroterpenoid compounds including sesquiterpenes, hydroquinones, and naphthoquinones, five of which previously unreported (1-5), were isolated from the ethanol (EtOH) extract of the heartwood of C. glazioviana (Figure 1).
Compound 1 had its molecular formula established as C 17 H 18 O 5 by high-resolution electrospray ionization mass spectrometry (HRESIMS) through the deprotonated molecule [M -H]at m/z 301.1076 (calcd. m/z 301.1081). Its infrared (IR) spectrum indicated absorption bands for hydroxy (3405 cm -1 ), carbonyls (1698 and 1630 cm -1 ), and carbon-carbon double bonds (1490 and 1442 cm -1 ). The 1 H NMR (nuclear magnetic resonance) spectrum (Table 1)   . The 13 C NMR spectrum displayed signals for 17 carbon atoms, assigned by heteronuclear single quantum correlation (HSQC) spectra into two methyls (including the methoxyl), two carbons sp 3 hybridized (methylene and methine), two double bonds (terminal and vinyl), an aldehyde carbonyl at d C 195.5 (C-11) and, comparatively, eight non-hydrogenated carbons, including a conjugated ketone carbonyl at d C 202.7 (C-10), Table 1. 1 H and 13 C NMR data analysis were consistent with a 2-methoxy-p-hydroquinone, a vinyl group and an α,β-conjugated propenal moiety. The heteronuclear multiple bond correlation (HMBC) spectrum showed correlations of the methylidene hydrogens at d H 6.38/6.23 (2H-6) with the aldehyde carbonyl at d C 195.5 (C-11) and d C 55.4 (C-10a), and the proton at d H 3.94 (H-10a) with the carbon at d C 202.7 (C-10) supporting the propenal moiety at the alpha position of the ketone carbonyl. The stereocenter C-8a, bearing a methyl and a vinyl group, was supported by the HMBC correlations of the methylidene vinyl protons at d H 4.98/4.97 (2H-7) with the carbon at d C 42.8 (C-8a), and the methyl protons at d H 1.12 (Me-12) with the sp 2 methine carbon at d C 143.3 (C-8). Additional HMBC correlations, as depicted in Figure 2, supported the suggested planar structure. The relative stereochemistry ascribed to the stereocenters C-8a (R*) and C-10a (R*) were determined based on the nuclear Overhauser spectrum (NOESY) correlations between H-10a and the Me-12 ( Figure 3) indicating that both vinyl and propenal moieties are cis-vicinally positioned, what is in agreement with previous compounds isolated from other Cordia species. 5,6 From the above data, the relative configuration of 1, named cordiaquinol D, was established as shown in Figure 3.
Although there is no experimental support for the biosynthesis of the meroterpenoid 1,4-quinones isolated specifically from Cordia species, a reasonable biosynthetic pathway for compounds 1 to 6 was suggested based on previous studies reported to terpenoid quinones. 24 Thus, it seems reasonable that compounds 1-6 could be produced from a C-alkylation of the p-hydroxybenzoic acid with two prenyl unities followed by a sequence of typical reactions of the biogenetic process as intramolecular cyclization, oxidation, hydroxylation, and O-methylation as depicted in Figure 6.
To evaluate the effects of the isolated compounds on the production of sulfated polysaccharides (LPS)-induced oxide nitric (NO) in RAW264.7 cells, the concentrations of NO in the culture medium were measured by the Griess assay. 26 NO levels in the culture supernatants from LPS-stimulated cells were significantly reduced after treatment with the compounds (Table 4). Compounds 3, 4, and 5 were more able to reduce NO production with IC 50 values of 50.34 ± 9.88, 105.83 ± 5.09, and 66.73 ± 10.28 μM, respectively.

Conclusions
Eleven compounds, including four new terpenoid hydroquinones (1-4) and a naphthoquinone (5), were isolated through the reinvestigation of the EtOH extract from the heartwood of C. glazioviana. It is worth highlighting that similar meroterpenoid compounds have been previously isolated from several Cordia species, and they seem to be restricted to woody plants, in particular, in the roots and trunk heartwood. As a significant number of terpenoid quinones and hydroquinones were previously isolated from Cordia species, it seems reasonable to suggest these compounds as possible chemomarkers for the genus. Eight, out of the eleven isolated compounds (2-9), were tested through cellular viability and lipopolysaccharide (LPS)-induced inflammation assays against RAW 264.7 macrophage-like cells, being compound 3 the one that showed the best reduction of the NO synthesis (IC 50 50.34 ± 9.88 μM).

General experimental procedures
Optical rotations were measured on a Jasco P-2000 polarimeter (Tokyo, Japan), operating with a tungsten lamp at a wavelength of 589 nm at 20 °C. Melting points were recorded on a digital Marconi MA-381 (Piracicaba, Brazil) apparatus and were uncorrected. Fourier-transform infrared (FTIR) spectra were obtained on a PerkinElmer Spectrum 100 spectrometer (Waltham, USA), using a universal attenuated total reflectance accessory (UATR). The highresolution mass spectra (HRMS) analysis was acquired on a chromatograph coupled to an ion trap mass spectrometer and time-of-flight (LCMS-IT-TOF, Shimadzu, Kyoto, Japan) system as well as on an Acquity UPLC instrument coupled to a Xevo QToF mass analyzer (Waters, Milford, MA, USA). 1 H and 13 C NMR (1D and 2D) spectra were run on a Bruker Avance DRX-500 spectrometer, using MeOD and C 5 D 5 N  (Cambridge Isotope Laboratories Inc., Tewksbury, USA) as solvents. The high-performance liquid chromatography (HPLC) separations were achieved on a Shimadzu-UFLC semi-preparative HPLC system, equipped with ternary pumps and diode array SPD-M20A UV/VIS detector using a Phenomenex C18 column (Phenomenex, Torrance, USA) (250 × 10 mm, 5 mm) and a mobile phase consisting of water was purified in a Milli-Q system (Millipore, St. Louis, USA) with trifluoroacetic acid (CF 3 CO 2 H, 0.1% v/v) analytical grade was acquired from Vetec (Rio de Janeiro, Brazil) and acetonitrile (MeCN) HPLC grade, were purchased from Tedia (Rio de Janeiro, Brazil), a flow rate of 4.7 or 4.0 mL min -1 , oven temperature of 40 °C, monitored at 210-400 nm. Chromatography columns (CC) were performed using silica gel 60 (70-230 mesh, Vetec, Rio de Janeiro, Brazil), while the analytical thin-layer chromatography (TLC) was carried out on pre-coated TLC silica gel plates (Merck, Frankfurt, Germany) and the spots visualized by spraying with a vanillin/perchloric acid/EtOH (Vetec, Rio de Janeiro, Brazil and Merck, Frankfurt, Germany) solution followed by heating at 100 °C. All PA solvents were purchased from Labsynth (São Paulo, Brazil).

Plant material
Cordia glazioviana was collected in April 2012 at Acarape county, Ceará State, Brazil and was authenticated by Dra Maria Iracema Bezerra Loiola, botanist of Departamento de Biologia, Universidade Federal do Ceará (UFC). A voucher specimen (No. 30824) is deposited at the Herbário Prisco Bezerra-UFC. The collection permit was granted by Biodiversity Authorization and Information, SisGen number A86B918.

Computational details
To establish the relative stereochemistry of compounds 3 and 5, two possible isomers of each one of those compounds (3a/3b and 5a/5b) were drawn and their geometrical structures were optimized by using standard techniques. 27 Optimization calculations were performed by using density functional theory (DFT) 28 method and a functional version of the PW91 exchange in combination with the original PW91 correlation functional and a mixing ratio of exact and DFT exchange of 0.25:0.75, mPW1PW91 29 along with 6-31G(d,p) basis set implemented in Gaussian 16 package. 30 The frequencies of the optimized geometries were calculated to determine whether the resulting geometries were true minima or transition states on the potential energy surface. All optimization calculations were performed in solution by using the polarizable continuum model (PCM) 31 with the integral equation formalism (IEF) 32 using methanol as solvent. The NMR isotropic shielding constants were determined from the optimized geometries of 3a/3b and 5a/5b with mPW1PW91/6-31G(d,p) level of theory based on the GIAO 14 proposal with tetramethylsilane (TMS) as reference implemented in the Gaussian 16. 30 The integral equation formalism and polarizable continuum model (IEF-PCM) solvation method was used with methanol (the solvent used to acquire the 1 H and 13 C NMR spectra) as an implicit solvent to simulate the medium on the chemical shifts of the stereoisomers. A supplemental analysis that correlates NMR chemical shifts and statistical analysis, named DP4+ allows the use of quantum chemical calculated NMR parameters combined with refined statistical data to elucidate the most likely structure among the stereoisomers. 15 Assay for cell viability Cell viability was assessed using the MTT (Sigma-Aldrich, St. Louis, USA) assay as described previously. 25 In brief, RAW 264.7 cells (Merck, Frankfurt, Germany) were seeded into a 96-well plate at a density of 1 × 10 4 cells per well and incubated at 37 °C for 24 h. Compounds at different concentrations (12.5-100 μM) in dimethyl sulfoxide (DMSO, Romil Chemical Ltd., Cambridge, UK) were added to the cell plate for another 24 h, and then MTT (0.5 mg mL -1 ) in phosphate-buffered saline (PBS, Merck, Frankfurt, Germany) was added into each well to form the formazan crystals (3 h). The supernatant was then carefully removed, and 100 μL of DMSO was added into each well to dissolve the MTT formazan crystals and measured at 540 nm using a microplate reader.

Assay for the inhibition of cellular NO production
The nitrite concentration in the medium was measured by the Griess reagent as an indicator of NO production. 26 RAW 264.7 cells were seeded into a 96-well plate at a density of 5 × 10 5 cells per well and incubated at 37 °C for 24 h. After that, the cells were treated with several sample concentrations (6.25-50 μM) or controls (0.01% DMSO or 4 μM dexamethasone) for 2 h and then incubated with 1 μg mL -1 LPS for 24 h. To measure the NO in the culture medium, a total of 100 µL of culture medium from each sample was mixed with the same volume of Griess reagent and incubated at 37 °C for 10 min. The absorbance was measured at 540 nm using a microplate reader.