SUPPLEMENTARY Vasorelaxing effect and possible chemical markers of the flowers of the Mexican Crataegus gracilior

The vasorelaxing effect of the methanol extract of the flowers of Crataegus gracilior , a Mexican medicinal plant used to treat some cardiovascular diseases, was assessed, and its possible chemical markers identified. The extract produced a potent vasodilator effect on isolated rat aortic rings (EC 50 = 1.83 ± 1.39 µg/mL; E max = 100 ± 3.4%). Vitexin, the most commonly identified flavonoid in the flowers and used to standardise some Crataegus species, was not found at all in this plant sample. Instead, daucosterol, and corosolic and euscapic acids were purified. The two triterpene acids have been reported to possess beneficial effects on cardiovascular diseases. These results indicate that the vasodilator effect might induce the hypotensive effect claimed by users, and that euscapic and corosolic acids may be the main vasodilator compounds, and can then be employed as the chemical markers towards the future standardisation of the extract.


Vasorelaxant effect assessment
Animals. Six Wistar male rats (250-300 g) were used for the pharmacological study. Rats were provided by the Institute of Neurobiology of the National Autonomous University of Mexico, Campus Juriquilla, Querétaro, Qro. Mexico. All experiments were performed in accordance with the ruling guidelines of the Mexican Official Standard NOM-062- ZOO-1999ZOO- (2017. Rats were housed under 12:12 h light-dark cycle, and tap water and food were provided ad libitum. Determination of the vasorelaxant effect of the methanol extract on Rat Aorta. Rats were killed by decapitation and thoracic aorta was surgically removed and promptly placed in a dish containing ice-cold (4 °C) Krebs-Henseleit medium (containing in mM: 126.8 NaCl, 5.9 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 5.0 D-glucose, 30 NaHCO 3 , 2.5 CaCl 2 ), bubbled with carbogen (95% O 2 and 5% CO 2 ). Then, the luminal space of the vessel was rinsed with cold Krebs-Henseleit solution to avoid clot formation, cleaned from surrounding connective tissue, and sliced into segments (3-4 mm in length). Aortic rings were suspended between two metallic clips passed through the lumen into organ baths chambers containing prewarmed Krebs-Henseleit solution (37 °C) gassed with carbogen. Tissues were allowed to equilibrate for 60 min under a resting tension of 1.5 g. During this period, the organ bath solution was exchanged every 10 min. Then, aortic segments were contracted with KCl solution (100 mM), in order to stimulate the vascular smooth muscle. When a stable contractile tone was reached, Krebs-Henseleit solution was replaced every 10 min to restore the initial resting tension of 1.5 g. For the vasodilator studies, the aortic segments were contracted with L-phenylephrine (Phe, 1 µM). The resulting contraction was defined as 100%, and once the plateau was reached, the test substances were cumulatively added. Acetylcholine (ACh) was used as positive control in a concentration range of 0.1 µg/mL-100 µg/mL. The extract was dissolved in distilled water and tested in a concentration range of 0.1 µg/mL-31.6 g/mL. Changes in tension caused by the tested concentrations were detected by Grass FT03 force transducers coupled to a Grass 7D Polygraph. These changes were expressed as percentages of relaxation based on the contraction generated by adding Phe (Ibarra-Alvarado et al. 2010).
Statistical Analysis. The vasodilator effect of each concentration of the methanol extract and acetylcholine was performed using aortas from six different rats (experiments in duplicate, n = 12). Vasorelaxant percentage values were expressed as the mean ± standard error of the mean (SEM). The resulting data obtained from each evaluation were plotted and analysed with the statistic software Prism 7.02 (GraphPad, San Diego, CA, USA).

Total phenolics determination
Total polyphenols were determined according to the described Folin-Ciocalteu method (Dewanto et al. 2002). First, 0.5 mg of gallic acid (Sigma-Aldrich  ), used as standard, was dissolved in 1 mL of pure water. For the calibration curve construction, different concentrations ranging from 0.005 to 0.1 mg/mL of the stock solution were added to methanol (80%) until 1 mL was reached. For total phenolic determination, 300 L of each stock solution or test sample (0.5 mg dry extract in 1 ml ethanol) were transferred to a 25 mL volumetric flask containing 1.5 mL of Folin-Ciocalteu reagent (1:10). After one-hour incubation, 1.2 mL of aqueous Na 2 CO 3 (7.5%) were added. Then water was transferred to the flask until 25 mL were reached. Subsequently, after an incubation period of 90 minutes, the samples were scanned in a UV-Vis spectrophotometer (Benchmark TM Plus Microplate Reader Spectrophotometer, Bio Rad, Hercules, CA, USA) at 765 nm.

Total flavonoids determination
Total flavonoids were determined according to the method described by Zhishen (1999) with slight modification. The standard stock solution was prepared by dissolving 0.06 mg of (+)catechin (Sigma-Aldrich  ) in 1 mL of water. For the calibration curve construction, different concentrations ranging from 0.02 to 0.2 mg/mL of the stock solution were added to methanol (80%) until 1 mL was reached. Additionally, aqueous solutions of NaNO 2 (5%), AlCl 3 (3%) and NaOH (1M) were prepared. Subsequently, to prepare the test solution, in a 10 mL volumetric flask, the following solutions were purred: 4 mL of water, 1 mL of the stock solution or the methanol extract (10 min sonicated 0.5 mg dry extract in 1 ml water), and 300 L of NaNO 2 . After 5 minutes, 300 L of AlCl 3 (10% aqueous) were added to the solution. Six minutes later, 2 mL of the NaOH solution (5%) and water were added until 10 mL were reached. The solutions were stirred and scanned at 510 nm.

Fractionation and purification of Compounds 1, 2 and 3
The methanol extract (7.3 g) was carefully fractionated in an open CC (silica gel Kiesegel 60 Merck, 70-230 mesh ASTM, particle size 0.063-0.200 mm) which was first eluted with hexane to remove the remaining fats, followed by progressive mixtures of dichloromethane, acetone and methanol in an increasing stepwise. In total, 237 fractions were collected, monitored by thin layer chromatography (TLC) and most of them pooled together into 9 fractions according to their similarities. No further chemical or pharmacological study was carried on the oily pooled fractions 1-40 (297.8 mg). Fraction 108 (12.2 mg) eluted with pure CH 2 Cl 2 and isolated as a clean white powder was left alone and labelled as compound components, which were purified after multiple injections at the analytical scale, provided that a semi-preparative scaling up attempt failed to achieve good resolution. Four peaks were collected, dried under reduced pressure, and their NMR data recorded: peaks 5 (30 mg, Rt 7.55 min), 6 (5.0 mg, Rt 8.36 min), 7 (4.8 mg, Rt 12.61 min), and 8 (4.6 mg, Rt 13.42 min). Only peaks 5 (compound 2) and 8 (compound 3) produced spectra suitable for structure elucidation. The NMR of the two remaining peaks revealed that they were triterpenes, but still had considerable impurities. Owing to their low quantities available, no further purification was undertaken.