Pasuchaca (Geranium dielsiaum Knuth): A New Source of Astilbin with Antiglycation Activity

Abstract

Pasuchaca (Geranium dielsianum Knuth), a traditional Peruvian medicinal plant from the Geraniaceae family used for diabetes management, was investigated for its antiglycative properties. This study aimed to screen, isolate, and identify the active antiglycative compounds from its aerial parts. By coupling a methylglyoxal (MGO)-HPLC screening assay with high-speed counter-current chromatography (HSCCC), seven dihydroflavonol derivatives were separated and identified from the 80% methanol extract. The compounds were identified as 2,3-dihydromyricetin 3-O-α-rhamnopyranoside (1), (+)-taxifolin 3-O-β-D-xylopyranoside (2), astilbin (6), isoastilbin (8), 3″-acetyl astilbin (9), and 2″-acetyl astilbin (11). Astilbin was identified as the major constituent, with remarkably high contents of 252.41 mg/g in the 80% methanol extract and 541.04 mg/g in the partitioned upper layer fraction. Astilbin demonstrated potent antiglycation activity across all stages of protein glycation (early, middle, late, and whole stages), significantly surpassing the positive control aminoguanidine. Furthermore, the formation of MGO-astilbin adducts was confirmed by LC-ESI-MS, validating its role as an effective MGO scavenger. This report is the first to isolate these phytochemicals from Pasuchaca. The findings establish astilbin as the key antiglycative component of Pasuchaca, substantiating its traditional use and highlighting its potential as a source of functional food ingredients or natural therapeutics for mitigating glycative stress.

Keywords: Geranium dielsiaum Knuth; HSCCC; MGO-HPLC; Pasuchaca; antiglycative activity; astilbin.

Figure 1

Antiglycation activities of the extracts of Pasuchaca against the formation of fructosamine-mediated early-stage AGEs (A), BSA-MGO mediated middle-stage AGEs (B), BSA-fructose mediated whole-stage AGEs (C), and G.K. peptide-ribose mediated late-stage AGEs (D). Bars marked with different letters were significantly different from each other (p < 0.05). Aminoguanidine hydrochloride (AG) was used as a positive control.

Figure 2

Screening of methylglyoxal (MGO) scavengers in the 80% MeOH extract of Pasuchaca by MGO-HPLC assay. (A) Incubation of the sample in water (black line) and 0.1 M PBS at pH 7.4 (pink line) for 1 h. (B) Incubation of the sample with MGO (blue line) or without MGO (pink line) in 0.1 M PBS at pH 7.4 for 1 h. Compounds 1, 2, 6, 8, 9, 10, and 11 were screened as potential MGO scavengers.

Figure 3

Partition of the 80% MeOH extract of Pasuchaca and antiglycative activity of the partitions. (A) HPLC profiles of the partitioned upper layer (PU) and lower layer (PL) of the 80% MeOH extract. Antiglycative activity of the 80% MeOH extract and its PU and PL against the formations of fructosamine-mediated early-stage AGEs (B), MGO-BSA mediated middle-stage AGEs (C), BSA-fructose mediated whole-stage AGEs (D), and G.K. peptide-ribose mediated late-stage AGEs (E). Bars marked with different letters were significantly different from each other (p < 0.05). ns stands for not significant (p > 0.05).

Figure 4

HPLC and pre-HPLC chromatograms of the precipitation and separation process of the 80% MeOH extract. (A) The supernatant (PUS) and precipitate (PUP) of the partitioned upper layer (PU) of the 80% MeOH extract and the purified compound 6 by pre-HPLC; (B) separation of compound 6 by Pre-HPLC; (C) fractionation of the target compounds by sephadex LH-20 column chromatography (SCC).

Figure 5

Separation of the target compounds from the Sephadex LH-20 column chromatography (SCC) fractions of Pasuchaca by conventional HSCCC and recycling HSCCC. (A) Separation of compounds 1, 6, and 8 from SCC fractions 135–150 by HSCCC and recycling HSCCC (171–540 min, marked by a green box) using the solvent system (n-hexane/EtOAc 1:5, v/v)/(MeOH/water 1:5, v/v) (1:1, v/v), whereas the chromatographic peak positions of compounds 6 and 8 in recycling HSCCC were marked by red boxes; (B) Separation of compounds 2 and 8 from SCC fractions 123–127 by conventional HSCCC using the solvent system (EtOAc/(MeOH/Water 1:5, v/v) (1:1, v/v); (C) Separation of the mixture of 9 and 11 from SCC fractions 173–206 by conventional HSCCC using the solvent system (n-hexane/EtOAc 1.5:5, v/v)/(MeOH/water 1:5, v/v) (1:1, v/v); (D) Separation of compounds 9 and 11 from the mixture of 9 and 11 by recycling HSCC using the solvent system (n-hexane/EtOAc 1.5:5, v/v)/(MeOH/water 1:5, v/v) (1:1, v/v), whereas the chromatographic peak positions of compounds 9 and 11 in recycling HSCCC were marked by green and red boxes, respectively.

Figure 6

Identification of the separated compounds in Pasuchaca.

Figure 7

Antiglycative activity of astilbin separated from Pasuchaca. Inhibitory activities of astilbin against the formation of fructosamine-mediated early-stage AGEs (A), BSA-MGO mediated middle-stage AGEs (B), BSA-fructose mediated whole-stage AGEs (C), and G.K. peptide-ribose mediated late stage AGEs (D). Bars marked with different letters were significantly different from each other (p < 0.05). Aminoguanidine hydrochloride was used as a positive control.

Figure 8

Methylglyoxal scavenging activity of compounds 1, 2, 6, 8, 9, and 11 from Pasuchaca. The compounds are 2,3-dihydromyricetin 3-O-α-rhamnopyranoside (Com.1), (+)-taxifolin 3-O-β-D-xylopyranoside (Com.2), astilbin (Com.6), isoastilbin (Com.8), 3″-acetyl astilbin (Com.9), and 2″-acetyl astilbin (Com.11). Aminoguanidine hydrochloride (AG) was used as a positive control. The methylglyoxal (MGO) scavenging activity of astilbin (Com.6) was not measurable since the HPLC peaks of 2-methyl quinoxaline and the produced MGO-astilbin adducts overlapped. Different letters indicate significant differences in the compounds’ activity in scavenging methylglyoxal at the same concentration (p < 0.05).

Figure 9

Identification of methyl glyoxal (MGO)-astilbin adducts by LC-ESI-MS. (A) Chromatograms of samples after incubating astilbin with different concentrations of MGO for 1 h at 37 °C; (B) Identification of compounds a–f by LC-MS; (C) The structures of compounds a–f.