Intra- and Interspecies Differences of Two Cecropia Species from Tabasco, Mexico, Determined through the Metabolic Analysis and 1H-NMR-Based Fingerprinting of Hydroalcoholic Extracts

Abstract

The genus Cecropia is used in the traditional medicine of Tabasco, Mexico, in diabetes and hypertension treatments, mainly without distinction of the species. This contribution aimed to carry out the metabolic analysis and Proton Nuclear Magnetic Resonance (¹H-NMR) spectroscopy-based fingerprinting of the hydroalcoholic leaf extracts of Cecropia peltata (Cp) and Cecropia obtusifolia (Co) collected in five sub-regions of the State of Tabasco (Cp1, "Centro"; Cp2, "Chontalpa"; Cp3, "Pantanos"; Cp4, "Ríos" and Co5, "Sierra"). Firstly, the extracts were evaluated for their Total Phenol Content (TPC) and Total Flavonoid Content (TFC) by spectrophotometric methods. In addition, metabolic analysis was performed using High-Performance Liquid Chromatography with Diode-Array Detection HPLC-DAD, which allowed the quantification of the chemical markers: chlorogenic acid, isoorientin, and orientin, as well as a vitexin analog. Finally, metabolomic analysis was carried out based on the ¹H-NMR spectra. The Cp4 extract (C. peltata from the "Ríos" sub-region) presented the highest values of TPC (155 ± 9.1 mg GAE/g E) and TFC (724 ± 22.2 mg RE/g E). The metabolic analysis was similar among the five samples; the highest concentrations of the four chemical markers were found in Cp3 (C. peltata from the "Pantanos" sub-region) for chlorogenic acid (39.8 ± 2.3 mg/g) and isoorientin (51.5 ± 2.9 mg/g), in Cp4 for orientin (49.9 ± 0.6 mg/g), and in Cp2 (C. peltata from the "Chontalpa" sub-region) for the vitexin analog (6.2 ± 0.2 mg/g). The metabolic analysis and the ¹H-NMR fingerprint analysis showed intraspecies differences among the C. peltata samples and interspecies between C. peltata and C. obtusifolia, which were attributed to variations in the metabolite groups as well as in the proportion of sugars such as glucose and xylose.

Keywords: Cecropia obtusifolia; Cecropia peltata; chemical markers; metabolomic profile.

Figure 1

Comparison of TPC (A) and TFC (B) in the hydroalcoholic extracts of C. peltata (Cp1–Cp4) and C. obtusifolia (Co5). TPC: Total phenol content; mg GAE/g E: Gallic acid equivalents per gram of extract. TFC: Total flavonoid content; mg RE/g E: rutin equivalents per gram of extract. The data represent the mean ± standard error (n = 3) of each extract. Statistical significance was determined using analysis of variance (ANOVA) followed by a post hoc Tukey test. Bars with the same letter (a–d) represent no significant difference at p < 0.05.

Figure 2

HPLC chromatograms of the hydroalcoholic extracts of C. peltata (Cp1–Cp4) and C. obtusifolia (Co5). The peaks are numbered in ascending order according to their retention times (λ = 330 nm). 1, chlorogenic acid; 2, isoorientin; 3, orientin; 4, vitexin analog; 5 cathequin analog.

Figure 3

Concentration (mg/g) of chlorogenic acid, isoorientin, orientin, and the vitexin analog in the hydroalcoholic extracts of C. peltata (Cp1–Cp4) and C. obtusifolia (Co5). The data represent the mean ± standard error (n = 3) for each extract. Statistical significance was determined using analysis of variance (ANOVA) followed by a post hoc Tukey test. Bars with the same letter (a–l) represent no significant difference at p < 0.05.

Figure 4

¹H-NMR profiles (D₂O, 600 MHz; region δ 8.00 to 0.00 ppm) of the hydroalcoholic extracts of C. peltata (Cp1–Cp4) and C. obtusifolia (Co5); a–c corresponds to triplicates of each sample. Inserted spectra show the region from δ 3.40 to δ 4.20 ppm with the characteristic signals of sugars.

Figure 5

Principal component analysis score plot (PC1 vs. PC2, 65.2% explained variance); (A) ¹H-NMR profiles of the hydroalcoholic extracts of C. peltata (Cp1–Cp4) and C. obtusifolia (Co5); a–c correspond to triplicates of each sample. (B) Variable Importance in Projection (VIP) of the defined metabolites of the ¹H-NMR profiles of the hydroalcoholic extracts from Cecropia species.

Figure 6

Collection points in Tabasco, Mexico, of leaves of C. peltata (Cp1–Cp4) and C. obtusifolia (Co5); map was made using QGIS^® v. 3.30.3 (Beaverton, OR, USA).