Supplementary Material Characterization of polyphenols in the methanol leaf extract of Bathysa gymnocarpa (Rubiaceae) by HPLC-DAD-ESI-MS/MS

: The species Bathysa gymnocarpa K.Schum is a tree belonging to the Rubiaceae family, endemic in Brazil. So far, there are reports neither of phytochemical work nor of biological evaluation of it. The analysis by High Performance Liquid Chromatography coupled to a Diode Array Detector and a tandem Mass Spectrometer with an Electrospray Ionization source (HPLC‐DAD‐ESI‐MS/MS) of its crude extract allowed to characterize in a complex mixture, without isolation, fourteen compounds, being two as cinnamic acid derivatives, and the others as mono-, di-and triglycosilated derivatives of the flavonols quercetin and kaempferol. These compounds are reported for the first time in Bathysa spp.


HPLC-DAD-MS-MS analysis
The HPLC-DAD-MS/MS analysis was carried out in an Alliance 2695 (Waters Cop., Milford, USA) equipment with a quaternary pump, autosampler, degasser, column oven, diode array detector (DAD) Waters 2996 and equipped with a Phenomenex Luna C18(2) column (Phenomenex, Torrance, USA) (3 µm, 150 x 4.6 mm i.d.) with a Waters Nova-Pack C18 guard column (4 µm, 10 x 3.9 mm i.d).A previously reported gradient program was employed : acetic acid (AcOH)-H2O (0.5:99.5 v/v) (phase A) and MeOH (phase B).The applied elution conditions were: 0-2 min, 0% B isocratic; 2-6 min, linear gradient from 0% to 15% B; 6-12 min, 15% B isocratic; 12-17 min, linear gradient from 15% to 20% B; 17-35 min, 20% B isocratic; 35-90 min, linear gradient from 20% to 35% B; 90-136 min, 35% B isocratic, and finally, washing and reconditioning of the column was done.The diode array detector was set at an acquisition range of 250-600 nm and phenolic compound monitoring was performed at 280, 320 and 370 nm.The injection volume was 50 µL.The online MS were obtained on a Micromass (Waters Cop.) Quattro micro triple quadrupole mass spectrometer coupled to the exit of the diode array detector and equipped with a Z-spray Electrospray Ionization (ESI) source.A flow of 70 µL min -1 from the DAD eluent was directed to the ESI interface using a flow-splitter.Nitrogen was used as desolvation gas, at 300ºC and a flow rate of 450 L h -1 , and no cone gas was used.A potential of 3.2 kV was used on the capillary for positive ion mode and 2.6 kV for negative ion mode.The source block temperature was held at 120ºC.Full scan mass spectra (MS), within the m/z range 50-1000, were performed in the positive mode at different cone voltages (15, 30 and 45 V) and in the negative mode at -30 V. MS/MS product ion spectra in positive and negative modes were recorded using argon as collision gas at 1.5.10 - 3 mbar and under different collision energies in the range 10-40 eV and -30 V (for negative) and optimized (for positive polarity) cone voltages.The optimum cone voltages were those which produced the maximum intensity for protonated molecule [M+H] + and protonated aglycone ion [Y0] + in the previous MS experiments.Phenolic standards were supplied as previously described (Wolff et al. 2019).All stock standard solutions (in concentrations ranging from 300 to 2700 μg mL -1 , depending on each phenolic compound, were prepared in MeOH.
All were stored at 4 ºC in darkness.

Plant material
The species Bathysa gymnocarpa was collected in January 2018 at the Parque Nacional da Tijuca, Rio de Janeiro, RJ, Brazil.Voucher specimen was deposited at the Herbarium of the Botanic Garden of Rio de Janeiro, RJ, Brazil, under number RB 791658.The collection had the previous permission from SISBIO-ICMBio-MMA-Brazil under number 56756-1.Legal access of the Brazilian genetic heritage component is registered in the SisGen platform under number A830934.

Extraction and TLC polyphenolic prospection
B. gymnocarpa leaves were dried at 40°C for 24 h and milled.A MeOH extract was prepared from 42.0 g of the resulted leaves (12 cycles x 150 mL) with ultrasonic assistance (15 min each cycle) at room temperature.The solvent was evaporated under low pressure, yielding 5.5 g of dried crude extract.TLC analyses were performed in pre-coated silica gel 60 F254 plates (Merck, Frankfurt, Germany) using ethyl acetate (EtOAc)/formic acid (HCOOH)/AcOH/H2O 100:11:11:27 (v/v/v/v) as mobile phase.The spots were visualized under UV-irradiation at 254 and 365 nm, and then sprayed with NP reagent (diphenylboric acid β-aminoethyl ester) followed by irradiation under 365 nm, and with anisaldehyde/sulfuric acid (H2SO4)/heated at 100 o C.

Total Phenolic Content
The total phenolic content was determined by the Folin-Ciocalteu method as previously described (Santos et al. 2018).Briefly, in 96-well microplates, 20 μL of methanolic solution of the extract (1 mg mL -1 ) or standard solutions or solvent (blank) were added and mixed with 0.2 N Folin-Ciocalteu solution (100 µL).After 5 min of incubation, 7.5%, w/v Na2CO3 water solution (80 µL) was added, allowed to stand for 2 h in dark ambient and then the absorbance at 760 nm was measured in a SpectraMax Plus 384 microplate reader (Molecular Devices).The difference between the absorbances of the extract or standard solutions and blank was determined.The standard curve of rutin was prepared with five-points (triplicate) ranging from 20.00 -100.0 µg mL -1 (y = 0.0039x + 0.0246, R = 0.9944).The concentration of total phenolic compounds of the extract was performed by comparing with the rutin standard curve and the results were expressed as milligram of rutin equivalents (RuE) per g of the extract.The results were presented as the means of three replications ± standard deviation (SD).

Total Flavonoid Content
The measurement of flavonol and flavone total content was performed as previously reported (Santos, et al. 2018).Briefly, in 96-well microplates, 20 μL of methanolic solution of the extract (1 mg mL -1 ) or standard solutions or solvent (blank), 20 μL of 1 M sodium acetate water solution (pH 8) and 180 μL of distillated water were previously mixed, followed by the addition of 20 μL of 5% AlCl3 methanolic solution.After 30 min of incubation, the absorbance was measured at 430 nm in a SpectraMax® M2e microplate reader (Molecular Devices).The standard curve of rutin was built with five-points (triplicate) ranging from 10.00 -70.0 µg mL - 1 (y = 0.002x -0.0043, R = 0.9940).The concentration of total phenolic compounds of the extract was performed by comparing with the rutin standard curve and the results were expressed as milligram of rutin (RuE) per gram of the extract.The results were presented as the means of three replications ± standard deviation (SD).

Results
Figure S1.HPLC-DAD chromatograms of the methanol leaf extract of Bathysa gymnocarpa at 280 nm and 370 nm.

Table S1 .
HPLC-DAD-MS/MS data of the identified cinnamic acid derivatives from Bathysa gymnocarpa leaves.

Table 2S .
HPLC-DAD-MS/MS data of the identified flavonoids from Bathysa gymnocarpa leaves.