A New Biflavonoid from Schinopsis brasiliensis ( Anacardiaceae )

Phytochemical investigation of the MeOH extract of Schinopsis brasiliensis Engl. stems led to the isolation of the new biflavanone (2R*,3R*,2’’R*,3’’R*)-7-hydroxy-4’-methoxy-flavanone(3→3’’)-3’’’,7’’-dihydroxy-4’’’-methoxy-flavanone and 4,2’,4’-trihydroxychalcone-(3→O→4’’)2’’’,4’’’-dihydroxychalcone, whereas methyl gallate, gallic acid, (6R,9R)-megastigma-4-en-3-one9-O-β-glucopyranoside, quercetin-3-O-β-D-xylopyranoside and tricetin-3’-O-β-D-glucopyranoside were isolated from the MeOH extract of leaves. Their chemical structures were elucidated using spectroscopic methods and comparison with the literature data. Both biflavonoids showed weak inhibition activities of acetyl and butyrylcholinesterase enzymes when compared with eserine.


Introduction
The genus Schinopsis (Anacardiaceae) is composed of fourteen species, and their woods are mainly used in the leather tanning and wood industries due their resistance to degradation by humidity, attack by insects and ultraviolet radiation. 1Schinopsis brasiliensis Engl. is a large tree that is popularly known in the Brazilian northeastern region as "baraúna".The local population employs its leaves and bark in the treatment of inflammation, sexual impotence, diarrhea, and cattle intestinal worms. 2 Unfortunately, it is presently considered an endangered species due to the extensive use of its wood in house construction.

Experimental
General 1 H NMR, NOESY (nuclear Overhauser effect spectroscopy) and HMBC (heteronuclear multiple bond correlation) spectra were obtained using a Bruker AC-500 instrument operating at 500 MHz for 1 H and 125 MHz for 13 C. 13 C (PND and DEPT) NMR spectra were obtained on a Varian Gemini 2000 instrument operating at 300 MHz for 1 H and 75 MHz for 13 C.In all spectra were employed CO(CD 3 ) 2 , C 5 D 5 N, and CD 3 OD as the solvent and reference.High-resolution electrospray ionisation mass spectrometry (HRESIMS) spectra were recorded in negative mode using a micrOTOF system from Bruker Daltonics.Optical rotation was performed using a microcell (Perkin-Elmer, model 343 polarimeter).The inhibition of AChE was measured using a microplate reader (Biotek, model EL 800).Column chromatography (CC) was conducted on silica gel 60 (Acros) or Sephadex LH-20 (Sigma).The fractions were monitored using TLC on silica gel, and the spots were visualized with iodine fumes and UV light (254/366 nm).All reagents and enzymes were purchased from Sigma.

Plant material
The stems and leaves of S. brasiliensis were collected in the surroundings of Valente-BA, Brazil.The plant was identified by Prof Maria L. S. Guedes, and a voucher was deposited at the Herbarium Alexandre Leal Costa at the Universidade Federal da Bahia (UFBA) under number 038056.

Extraction and isolation
The stems (2 kg) and leaves (579 g) were separated, dried and powdered.Then, they were exhaustively extracted with MeOH at room temperature, furnishing crude extracts (56.3 and 79.2 g, respectively).
The crude methanolic extract of the leaves (79.2 g) was dissolved in MeOH:H 2 O (7:3) and subjected to liquid-liquid extraction with CH 2 Cl 2 , yielding 6.71 g of CH 2 Cl 2 soluble compounds.The CH 2 Cl 2 fraction was subjected to CC on silica gel using CHCl 3 and mixtures of

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition tests
The compounds and CHCl 3 soluble fraction of the MeOH extract of stems of S. brasiliensis were evaluated using bioautographic TLC test for acetylcholinesterase activity, which showed positive spots using a methodology based in the colorimetry, described by Marston et al. 5 The in vitro quantification of AChE and BuChE inhibition by compounds 1 and 2 were determined by spectrophotometry using a colorimetric method adapted from Ellman et al. 6 Briefly, 15 μL of acetylcholine iodide or butylcholine and 62 μL of DTNB (5,5'-dithiobis-(2-nitrobenzoic acid) (3 mmol L -1 ) were incubated with 5 μL of pure compound (500-4 μmol L -1 ), eserine (positive control), or buffer for 15 min in a 96-well microplate.The reaction was then started by adding 12 μL of enzyme in buffer (0.22 U mL -1 ).The change in absorbance was recorded at 405 nm using the microplate reader.DTNB, AChE or BuChE and the substrate were dissolved in 0.1 mol L -1 sodium phosphate buffer (pH 7.4).All samples were analyzed in triplicate.

Results and Discussion
The active chloroform soluble fraction from the MeOH crude extract partition of S. brasiliensis stems, was submitted to various chromatographic procedures in silica gel and Sephadex LH-20 to afford compounds 1 and 2, and their structures were elucidated by HRMS (high resolution mass spectrometry) and mono-and bi-dimensional NMR experiments.The molecular formula of 1 (C 32 H 26 O 9 ) was obtained from the peak [M -H] − at m/z 553.1494 observed in the negative HRESIMS (calc.553.1499).The ¹³C NMR and DEPT (distortionless enhancement by polarization transfer) spectra indicated the presence of two methoxyl groups, thirteen aromatic methine carbons, six oxygenated aromatic carbons, two methine sp 3 carbons and two similar acyl groups (d 191.6 and 191.5).These data were consistent with the biflavanone skeletal units, which are linked by the C-ring flavonoid.The 1   The assignment of all of the hydrogenated carbons was made possible by observing the heteronuclear correlations plotted in the HMQC (heteronuclear multiple quantum correlation) experiment.The location of each substituent in the flavonoid skeleton was assigned by correlations observed in the HMBC spectrum and corroborated by the NOESY experiment.The correlations of the methoxy hydrogens at d H 3.84 and C-4', besides the peaks at d H 5.98 (H-2) and d H 6.94 (H-3'/H-5'), and C-1' (d C 130.1) permitted the identification of the B-ring of unit I as a 1,4-disubstituted aromatic ring and unequivocal assignment of all of the ¹H and ¹³C resonances.The correlations observed in the NOESY spectrum between the methoxyl hydrogens, H-3'/H-5', H-2'/-6' and H-2 corroborated the above proposition.The long range correlations also allowed to confirm that the C-4''' and C-3''' of the B-ring of unit II bore a methoxyl and hydroxyl group, respectively.These findings were possible mainly due to the two key correlations observed in the HMBC spectra.Firstly, the correlation of methoxyl hydrogens at d H 3.84 and the peak at d C 161.2 permitted to assign C-4' of unit I.The singlet at d H 3.89 (4'''-OCH 3 ), the peaks centered near d H 6.63 (H-6'''/H-2'''), the doublet at d H 6.95 (H-5''') correlating with the carbon at d C 149.2 (C-4''') corroborated with the proposed substitution on unit II.The correct position of the methoxyl group bearing the C-4''' was unequivocally attributed by the NOESY interaction of its hydrogens and H-5''' displayed as a doublet.The correlations observed in the NOESY and HMBC spectra also corroborated the unusual C-3→C-3" connection between the biflavanone moieties (Figure 2), permitting the identification of 1 as new (2R*,3R*,2''R*,3''R*)-7-hydroxy-4'-methoxy-flavanone-(3→3'')-3''',7''-dihydroxy-4'''-methoxy-flavanone.
The structure of 2 (Figure 1) was established by HRESIMS, 1D and 1 H NMR spectral analyses, as well as by comparison with literature data. 7This finding is the second report of 4,2',4'-trihydroxychalcone-(3→O→4")-2"',4"',-dihydroxychalcone (2) as a natural product.This compound was isolated for first time from Luxemburgia octandra St. Hil (Ochnaceae), and it was previously named luxenchalcone. 7[10] Both extracts showed acetylcholinesterase-inhibiting activities employing Marston's TLC colorimetric method based on the hydrolysis of naftil acetate. 5However, when the isolates were submitted to the Elmann's test employing acetyl and butyrylcholinesterase, only compounds 1 and 2 showed weak inhibition of these enzymes when compared with serine (
3 and 85.0.These resonances and the coupling constants observed in the ¹H NMR spectrum suggested the presence of two cyclic systems bonded by the two C-rings of the biflavanone units.The coupling constants displayed by H-2 and H-2'' indicated that these hydrogens are in the pseudo-axial position with the vicinal hydrogens H-3 and H-3'', respectively.This finding and the similarities of the ¹³C chemical shifts of C-2/C-2'' and C-3/C-3'' indicated that both units show the same relative configurations.The 1 H NMR and the ¹H-¹H COSY (correlation spectroscopy) spectra permitted the assignment of the four aromatic rings with two AMX, one ABX, and one AA'BB' set of hydrogen coupling systems.The substitution pattern of the A-rings of the two units of compound 1 was shown by the peaks, integrating for 2 H each, displayed as doublets at d H 6.32 (2.3 Hz) and 7.71 (8.7 Hz) and a double doublet at d H 6.58 (2.3 and 8.7 Hz).These signals indicated that the A-rings of the biflavanone moieties are substituted in C-7 and C-7'' by the hydroxyl groups.The B-rings of this compound were identified as being 1,4-disubstituted and 1,3,4-trisubstituted aromatic rings based on the ¹H and ¹³C NMR data.The doublets at d H 7.07 and 6.94 (2H each) and the methines at d C 130.0 and 114.8 were indicative of a 1,4-disubstituted aromatic system.The set of signals in the range of d H 6.62-6.95, in addition to the characteristic resonances observed in the ¹³C NMR (d C 131.0, 115.1, 147.7, 149.2, 112.2 and 120.2), indicated the presence of a 1,3,4-trisubstituted aromatic ring with oxygenated substitution in positions 3 and 4.

Table 2 )
. Despite the isolated biflavonoids present weak inhibitory activities, there are few examples in literature of this class of compounds showing AChE or BuChE activities. 11

Table 2 .
IC 50 values of acetyl and butyrilcholinesterase inhibition by compounds 1 and 2