Investigation of Antioxidant and Elastase Inhibitory Activities of Geum urbanum Aerial Parts,Chemical Characterization of Extracts Guided by Chemical and Biological Assays

Abstract

This study presents the bioguided chemical investigation of the 80% aqueous methanol extract of Geum urbanum aerial parts. Liquid–liquid partitioning of this extract in solvents of increasing polarity combined with biological screening showed that the ethyl acetate (EtOAc) soluble fraction was the most active part of the extract. This fraction was chemically profiled by a ¹³C nuclear magnetic resonance (NMR)-based dereplication method, resulting in the identification of 14 compounds. The dereplication process was followed by the purification of unknown and minor compounds of the EtOAc fraction. A new glycosylated phenol, namely, 3-(3,4-dihydroxyphenyl)propyl-α-l-rhamnopyranoside, together with 6 known compounds were isolated. Their structures were elucidated by spectroscopic methods including NMR and high-resolution electrospray ionization mass spectrometry. The antioxidant activity of fractions and isolated compounds were evaluated by 2,2,1-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging, and by cupric ion reducing antioxidant capacity assays. In parallel, their enzyme inhibitory property against human neutrophil elastase was assessed. Four subfractions, essentially containing polyphenols and triterpenes, exhibited a significant elastase inhibitory activity and an ellagitannin showed a very high radical scavenging activity.

Keywords

ellagitanins phenols NMR dereplication antioxidant elastase

Skin aging is a complex biological process mainly characterized by wrinkle formation, uneven pigmentation, darkening, thinning, and roughening of the skin. This could be caused by intrinsic (genetically determined) or extrinsic (mediated by exposure to ultraviolet [UV] radiation and toxic compounds) mechanisms. One of the underlying mechanisms of these processes is excessive oxidative stress, which is probably the single most harmful contributor to skin aging, leading to loss of cells and extracellular matrix degradation as the most prominent features of chronologically aged skin. Oxidative stress results from an imbalance between reactive oxygen species production and antioxidative defense.¹ Prevention of these dynamic processes is a major issue for dermocosmetics and substantial efforts are being made to discover new protective ingredients.^2,3 Plant extracts can contain a high concentration of phenolic compounds, carotenoids, terpenoids, etc., able to inhibit free radicals and therefore to slow down skin aging.^4,5 The antioxidant potency of phenolic compounds (phenolic acids, flavonoids, anthocyanidins, tannins) can be easily determined by chemical tests such as 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) assay, the ferric reducing/antioxidant power (FRAP) assay, the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the cupric ion reducing antioxidant capacity (CUPRAC) assay.⁴ Phenolics have also been reported as effective compounds in matrix remodeling activation through inhibition of elastases,⁶ a class of proteases highly involved in aging.

In the continuation of our search for local plants with antiaging properties,^7,8 we were interested in Geum urbanum L., a perennial herbaceous plant belonging to the Rosaceae family. The genus Geum encompasses about 70 species distributed in temperate regions all over the world. Geum urbanum is native to humid woods of Europe and Asia⁹ and is a common species in France, especially in cool places such as hedges and undergrowth.¹⁰ Infusions from the roots and rhizomes of G. urbanum have been traditionally used in folk medicine in the treatment of acute diarrhea and as astringent agent for inflammations of the mucosa and gums and treatment of hemorrhoids.^9,11 Previous works have shown that plants species belonging to the Geum genus produce ellagitannins, gallotannins, flavonoids, terpenoids, and phenylpropanoids.^12
-14 Ellagitannins (tellimagrandin, stachyurin, casuarynin, gemin A, and ellagic acid derivatives), procyanidins (procyanidin B₃, procyanidin C₂, and catechin derivatives), gallic acid and its derivatives, steroids, and triterpenoids were isolated from the roots of G. urbanum.^11,15

-18 To our knowledge, the antioxidant and antielastase potency of G. urbanum aerial parts and their relation to chemical composition have not been reported. Recently, the radical scavenging activity of extracts and fractions obtained from aerial and underground parts of G. urbanum were investigated and the ethyl acetate (EtOAc) fractions from roots and aerial parts were found to be the most active, but only the EtOAc extract of the roots was investigated.¹⁵

Thus, the aim of the present study was to investigate the antioxidant and elastase inhibitory activities of extracts, fractions and isolated compounds obtained from the aerial parts of G. urbanum. A ¹³C nuclear magnetic resonance (NMR)-based dereplication methodology combined with a bioguided fractionation and purification procedure was used to identify some of its metabolites.

Results and Discussion

An 80% aqueous methanol extract (hereafter referred to as GUM) was obtained from G. urbanum aerial parts. Its water (H₂O) solution was fractionated by extraction with a series of solvents of increasing polarity, resulting in a dichloromethane fraction (DCMF), EtOAc fraction (EAF), and n-butanol fraction (n-BF). The free radical scavenging activities of GUM and obtained fractions were determined by the DPPH and hydroxyl radical assays whereas their cupric reducing capacity was evaluated by the CUPRAC assay. In addition, their ability to inhibit human neutrophil elastase activity was tested. The results are shown in Table 1. As previously described for roots and aerial parts of G. urbanum,¹⁵ the EAF had the best antioxidant activities. n-BF exhibited the best DPPH scavenging activity (half-maximal inhibitory concentration [IC₅₀] <12.5 µg/mL) followed by EAF and GUM (IC₅₀ 14.7 µg/mL and 21.8 µg/mL, respectively). The hydroxyl radicals scavenging activity was better for EAF and GUM (IC₅₀ 142.7 µg/mL and 174.5 µg/mL, respectively). For all extracts, a substantial cupric ion reducing capacity was observed (IC₅₀ <2.0 µg/mL), but lower than the one of trolox, used as a positive standard. In addition, a very good elastase inhibitory activity was observed for DCMF and n-BF (IC₅₀ 17.6 µg/mL and 14.3 µg/mL, respectively), but EAF showed the best activity (IC₅₀ 6.0 µg/mL), identical to quercetin used as a positive standard. The EAF having interesting biological activities, in both antioxidant and anti-elastase assays, was chemically investigated through a bioassay-guided isolation strategy in order to tentatively determine the active constituents.

Table 1

Antioxidant and Elastase Inhibitory Activities of Extracts, Fractions and Compounds Isolated From Geum urbanum.

	DPPH radical scavenging activity		OH radical scavenging activityIC₅₀ (µg/mL)	Power cupric ion reducing (CUPRAC)IC₅₀ (µg/mL)	Human neutrophil elastase inhibitionIC₅₀ (µg/mL)
	IC₅₀ (µg/mL)	IC₅₀ (µM)	OH radical scavenging activityIC₅₀ (µg/mL)	Power cupric ion reducing (CUPRAC)IC₅₀ (µg/mL)	Human neutrophil elastase inhibitionIC₅₀ (µg/mL)
GUM	21.8 ± 1.3		174.5 ± 1.8	1.5 ± 0.1	110.4 ± 1.1
DCMF	(42%)^a		188.3 ± 3.5	1.8 ± 0	17.6 ± 1.1
EAF	14.7 ± 0.8		142.7 ± 5.7	0.2 ± 0	6.0 ± 1.1
n-BF	<12.5		263.0 ± 3.3	0.5 ± 0	14.3 ± 1.1
EAF₁	13.8 ± 0.1		365.0 ± 3.1	4.8 ± 0.3	2.1 ± 1.2
EAF₂	8.9 ± 0.6		199.2 ± 3.8	2.9 ± 0.1	2.9 ± 1.1
EAF₃	11.5 ± 0		309.0 ± 4.8	2.3 ± 0	5.1 ± 1.1
EAF₄	3.4 ± 0.1		139.0 ± 4.6	3.0 ± 0	8.0 ± 1.1
EAF₅	3.5 ± 0.1		129.5 ± 3.3	1.8 ± 0	13.1 ± 1.1
EAF₆	3.0 ± 0.1		131.5 ± 0	1.2 ± 0	11.9 ± 1.1
EAF₇					12.2 ± 1.2
2	22.6 ± 2.2	77.9 ± 7.6
3	28.5 ± 0.8¹⁹	133.1 ± 3.7¹⁹
4	47.8 ± 3.1²⁰	50.9 ± 3.3²⁰
5	20.1 ± 0.4	44.9 ± 0.9
6	18.5 ± 0.2	36.5 ± 0.4
7	5.0 ± 0.3	2.7 ± 0.2
Quercetin^b	5.4 ± 0.2	17.9 ± 0.7	52.2 ± 4.8	13.6 ± 1.3	6.0 ± 0²¹
Ascorbic acid^b	2.3 ± 0.4	13.1 ± 2.3	229.2 ± 2.3	13.3 ± 0.5
Trolox^b				5.4 ± 0.3

CUPRAC, cupric ion reducing antioxidant capacity; IC₅₀, half-maximal inhibitory concentration; DCMF, dichloromethane fraction; EAF, ethyl acetate fraction; n-BF, n-butanol fraction.

^a% Inhibition at 200 µg/mL

^bUsed as positive control.

The major compounds of EAF were identified using a dereplication method²² combining centrifugal partition chromatography (CPC), NMR analysis, clustering of NMR peak emergence profiles, and database search, without purification of individual components. The CPC fractionation of EAF was performed with the biphasic solvent system methyl tert-butyl ether (MtBE)/acetonitrile (CH₃CN)/H₂O (3/3/4, v/v/v) to afford 11 subfractions (EAF_1–11). After ¹³C NMR analyses of EAF_1–11, all spectra of the fraction series were processed and submitted to hierarchical clustering analysis (HCA) for the recognition of the similarity between emergence profiles of ¹³C NMR peaks throughout the fractionation process. In this way, ¹³C NMR signals belonging to the same compounds were grouped to build “chemical shift clusters” represented in the heat map drawn in Figure 1. As a result, 13 major chemical shift clusters corresponding to the major metabolites of the EAF (Figure 1), colored in yellow, were revealed by the heat map. An in-house database containing predicted chemical shift values of natural metabolites was used to identify the metabolites from the chemical shifts within clusters. Cluster 1 in fraction EAF₁₀ was assigned to saccharose and β-fructose. The identification of saccharose and β-fructose was easily confirmed by checking heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), and correlation spectroscopy (COSY) data of fraction EAF₁₀ and by comparison with literature data.²³ Similarly, clusters 2–11 were identified as ellagic acid and gallic acid²⁴ (clusters 2, 2′, and 2″; fraction EAF₆), a mixture of 6-O-E-caffeoyl-β-d-glucopyranoside²⁵ and malic acid²³ (clusters 3 and 7; fractions EAF_2–8), succinic acid²⁶ (cluster 4; fractions EAF_3–4), dotorioside II²⁷ (cluster 5; fractions EAF_3–4), tormentic acid²⁸ (cluster 6; fraction EAF₁), caffeic acid²⁹ (cluster 7, fraction EAF₇), ellagic acid 4-O-pentoside (cluster 8; fractions EAF_3–5), (+)-catechin (2)³⁰ (cluster 10; fractions EAF_2–3), and 3,3′-di-O-methylellagic acid³¹ (cluster 11; fraction EAF₃). For the fractions EAF_4–5, the database proposed a 3-(3,4-dihydroxyphenyl)propyl-glycoside (1), which could not be identified unambiguously (cluster 9).

Figure 1

¹³C nuclear magnetic resonance (NMR) chemical shift clusters obtained by applying hierarchical clustering analysis on ethyl acetate fraction on centrifugal partition chromatographyfractions from Geum urbanum.

Fractions EAF_1–7 containing the phenolic compounds identified by dereplication were further screened for their antioxidant and elastase inhibitory activities. As shown in Table 1, all tested fractions presented very good antioxidant activities, revealed by DPPH and CUPRAC assays. EAF_1–7 showed also a significant elastase inhibitory activity, in particular EAF_1–4 (IC₅₀ 2.1 µg/mL, 2.9 µg/mL, 5.1 µg/mL, and 8.0 µg/mL, respectively). The most active fractions EAF_2–4 and EAF₇ were further purified using semipreparative high-performance liquid chromatography (HPLC), to identify the compounds responsible for the activities, and afford an undescribed glycosylated phenol (1) and 6 known compounds (2–7). The known compounds were identified as (+) catechin (2),³⁰ 1-(4-hydroxy-3-methoxyphenyl)-1,2,3-propanetriol (guaiacylglycerol) (3),³² tellimagrandin II (4),³³ 3-O-methylellagic acid-4′-O-β-d-xylopyranoside (5),³⁴ 2,3-dihydro-3-hydroxymethyl-7-methoxy-2-(4′-hydroxyphenyl-3′-methoxy)-5-benzofuranpropanol-4′-O-α-l-rhamnopyranoside (icariside E4) (6),³⁵ and gemin A (7)³⁶ (Figure 2) by comparison of physical data with literature values and from spectroscopic evidence.

Figure 2

Chemical structure of compound 1 isolated from Geum urbanum aerial parts.

Compound 1 was obtained in a yellowish-brown solid mixture with compound 3 (1:1 based in ¹H NMR data). The spectral analysis of this mixture showed for compound 1 a molecular formula C₁₅H₂₂O₇, deduced from its negative ionization high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis (m/z 313.1284, [M − H]⁻). The ¹H and ¹³C NMR spectra of 1 presented resonances corresponding to aromatic and glycosidic protons and carbons. The ¹H NMR spectrum of 1 showed 3 signals accounting for 1 proton each at δ _H 6.65 (d, J = 2.0 Hz), 6.77 (d, J = 8.2 Hz), and 6.59 (dd, J = 8.2, 2.0 Hz), due to H-2′, H-5′, and H-6′ in a tri-substituted aromatic ring. These protons were correlated in the HSQC spectrum with their aromatic carbon atoms at δ _C 116.4, 116.3, and 121.0, respectively. In addition, 6 protons at δ _H 3.28 (1H, m)/3.57 (1H, m), 1.76 (2H, quin, J = 6.9 Hz), and 2.50 (1H, q, J = 7.3 Hz)/2.52 (1H, q, J = 7.2 Hz), due, respectively, to the protons H-1, H-2, and H-3 of an alkyl chain correlated in the HSQC spectrum to their alkyl carbon atoms at δ _C 66.2, 31.1, and 31.1, respectively. Complete assignment of the remaining resonances of the aglycone in the ¹³C NMR spectrum of 1 was achieved by analysis of the HMBC data which confirmed the presence of a diphenol linked to an alkyl chain. The linkage site of the alkyl chain was determined by analysis of the HMBC spectrum. The correlation between H-3 (δ _H 2.50/2.52) and C-1′ of the aromatic ring (δ _C 134.7) indicated the position of alkyl chain on the diphenol. A full list of the corresponding assignments is given in the experimental section. Furthermore, an anomeric proton resonance corresponding to O-linked sugar was observed in the ¹H NMR spectrum at δ _H 4.53 (d, J = 1.9 Hz, H-1″) and the signal of his corresponding ¹³C NMR carbon was at δ _C 100.3 (C-1″). A methyl signal at δ _H 1.14 (d, J = 6.2 Hz, H-6″) and δ _C 16.6 (C-6″) indicated a 6-deoxy-hexose. Analysis of COSY correlations allowed us to assign complete spin systems of an α-l-rhamnopyranose³⁷ (see experimental). The α-configuration of rhamnose deduced from the small ³ J _H-1,H-2 value, was confirmed by the chemical shift of C-5 (δ _C 68.4).³⁸ The HMBC correlation between H-1″ (δ _H 4.53) and C-1 of the aglycone (δ _C 66.2) indicated the position of the rhamnose moiety on the alkyl chain. Therefore, the structure of 1 was elucidated as 3-(3,4-dihydroxyphenyl)propyl-α-l-rhamnopyranoside, as shown in Figure 2.

Isolated compounds 2, 5, 6, and 7 were evaluated only for their antioxidant activity using the DPPH scavenging assay due to their low available mass. Compounds 1 and 3 were isolated in a mixture, so their activities could not be evaluated. The biological activity of compounds 3 and 4 was discussed based on literature data. As summarized in Table 1, compounds 2, 5, 6, and 7 exhibited a very good antioxidant activity (IC₅₀ <23.0 µM), with compound 7 achieving noticeably at best (IC₅₀ 2.7 µM). According to literature, compounds 3 ¹⁹ and 4 ²⁰ showed a moderate DPPH scavenging activity (3: IC₅₀ 133.2 µM, 4: IC₅₀ 50.9 µM), while catechin (2) showed good antioxidant activities on DPPH (IC₅₀ 18.3 µM), FRAP (IC₅₀ 13.4 µM), and ABTS (IC₅₀ 3.8 µM) assays.³⁹ The antioxidant activities of caffeic acid (cluster 7), gallic acid (cluster 2), and ellagic acid are well known with IC₅₀ 58.8, 10.6, and 3.9 µM on DPPH assay, respectively.^40
-42 The radical scavenging activity on DPPH of dotorioside II (cluster 5) and tormentic acid (cluster 6) were also previously evaluated and showed to be very low.^43,44 The very high activity of compound 7 is probably due to the presence of several gallic acid units containing numerous aromatic hydroxyl groups known to be responsible for antioxidant activity.^41,45 Indeed, the phenols/polyphenols have the capacity to lose one or more H⁺ ions and thus to trap the free radicals in the ortho and para position of the donor hydroxyl group. In the case of a polyphenol such as catechol, the 2 hydroxyl groups provide 2 ortho sites and 2 para sites, which allows efficient trapping of free radicals. The antioxidant capacity of compound 7 being 17 times higher than that of compound 4, we can suggest that the more gallic acid units in ellagic tannins, the higher its activity.

Conclusions

The present study reports for the first time the isolation and identification of 19 secondary metabolites including ellagic tannins, ellagic acid derivatives, phenolic acids, glycosides derivatives, and triterpenes from aerial parts of G. urbanum. Our studies demonstrated that EAFs were characterized by the highest antielastase and antiradical activities among all prepared fractions. For this reason, we isolated individual constituents of the EAF fraction and evaluated their contribution to the observed activities. We isolated and identified a new glycosylated phenol, together with 6 compounds.

From a chemotaxonomic viewpoint, catechin (2) was isolated earlier in G. urbanum ¹⁵ and Geum iranicum.⁴⁶ Gemin A (7) was previously reported from Geum japonicum,³⁶ Geum aleppicum Jacq. and Geum calthifolium var. nipponicum.⁴⁷ Among the compounds elucidated by dereplication, 6-O-E-caffeoyl-β-d-glucopyranose, succinic acid, and 3-(3,4-dihydroxyphenyl)propyl-glycoside are reported here for the first time in the genus Geum. Eight compounds were previously isolated from G. urbanum roots: tormentic acid,¹⁵ ellagic acid pentoside,¹⁶ ellagic acid, gallic acid, caffeic acid, (+)-catechin,^15,17 malic acid,¹⁸ and dotorioside II.¹¹ The 3 other compounds were already known in the genus Geum.^31,46,48 Two compounds previously isolated in G. urbanum roots,^15,16 the ellagic tannins 4 and 7, could be considered as chemotaxonomic markers of this species.

Regarding biological activities, 4 subfractions, essentially containing polyphenols, exhibited a significant elastase inhibitory activity and, among the identified compounds, phenolics 2, 5, 6, and 7 showed high antioxidant property. These tests have highlighted the antioxidant activity of polyphenols and more particularly catechin (2) and gemin A (7). The EAF and the polyphenols identified from G. urbanum are promising compounds for applications in the dermocosmetic field due to their very interesting antioxidant and antielastase activities.

Experimental

General Experimental Procedures

The 1-dimensional (1D) and 2D-NMR spectra were recorded at 298 K in methanol-d ₄ or dimethyl sulfoxide (DMSO-d ₆) on a Bruker AvanceIII-600 spectrometer (Karlsruhe, Germany) (¹H at 600 MHz and ¹³C at 151 MHz) equipped with a 5 mm TCI cryoprobe. The Bruker TopSpin 3.6 software was used for NMR data acquisition and processing. HR-ESI-MS spectra were recorded on a Micromass Q-TOF micro instrument (Manchester, UK). Mass spectra were recorded in the negative ionization mode in the m/z range 100-2000, with a mass resolution of 20 000 and an acceleration voltage of 0.7 kV. Semipreparative HPLC purification was carried out on a Dionex apparatus equipped with an ASI-100 automated sample injector, an STH 585 column oven, a P580 pump, and a UVD 340S diode array detector, all driven by the Chromeleon software version 6.8. Analytical HPLC experiments were performed using a Thermofisher Ultimate 3000 (Thermo Fischer Scientific, Villebon-sur-Yvette, France), equipped with a 4-way pump LPG 3400 SD, an automatic injector WPS 3000 SL, a UV/visible diode array detector 3000, and the Chromeleon software version 6.8. A prepacked C₁₈ column (Interchim, 250 × 10 mm, 5 μm) was employed for semipreparative HPLC. The mobile phase was composed of H₂O acidified with trifluoroacetic acid (TFA; 0.025%) and CH₃CN with a flow rate of 5 mL/min, and the chromatograms were monitored at 205, 254, 300, and 360 nm. A prepacked C₁₈ column Uptisphere Strategy C₁₈ (Interchim, 250 × 4.6 mm, 5 μm) was used for analytical HPLC and the mobile phase contained H₂O with TFA (0.025% v/v) and CH₃CN. A gradient elution method was applied from 5% to 80% of CH₃CN in 30 minutes with a flow rate of 1 mL/min, and the chromatograms were monitored at 205, 254, 300, and 360 nm. Thin-layer chromatography (TLC) was carried out on silica gel 60 F₂₅₄ precoated aluminum plates (0.2 mm, Merck), using CHCl₃/MeOH/H₂O (14/6/1, v/v/v) as mobile phase. The compounds were visualized under UV light (254 and 366 nm) after high-performance TLC (HPTLC, CAMAG TLC Visualizer 2) and sprayed with 50% sulfuric acid followed by heating. A FLUOstar Omega spectrophotometer (BMG LABTECH) was used for absorbance measurement in antioxidant assays. An Infinite F200 PRO spectrofluorimeter (Tecan, Lyon, France) was used for measuring fluorescence during antielastase assays.

Plant Material

The aerial parts of G. urbanum L. (Rosaceae) were collected in Châlons-sur-Vesle (Northeastern part of France: 49°2167′N, 4°05′E) in July 2016, authenticated by Dr Abdulmagid Alabdul Magid and dried at room temperature. A voucher specimen (MA-GU-2016-07) was deposited at the Herbarium of the Botanic Laboratory-Faculty of Pharmacy, University of Reims Champagne-Ardenne.

Extraction and Isolation

The dried and powdered G. urbanum aerial parts (300 g) were macerated in MeOH/H₂O (4/1, v/v, 3 × 3 L, 24 hours) at room temperature. The macerate was concentrated to about 1 L at 40°C under vacuum. An aliquot of the aqueous solution (100 mL) was evaporated to dryness to obtain the GUM extract (2.6 g). The aqueous solution (1 L) of GUM was successively extracted with dichloromethane (3 × 750 mL), EtOAc (3 × 750 mL), and n-butanol (3 × 750 mL), then dried under reduced pressure to yield DCMF (6.1 g), EAF (6.2 g), and n-BF (24.2 g) extracts, respectively, and a water-soluble part (29.2 g).

Centrifugal Partition Chromatography

CPC fractionation was carried out on a lab-scale FCPE300 column of 303 mL inner volume (Rousselet Robatel Kromaton, Annonay, France) made of 7 circular partition disks and engraved with a total of 231 partition twin cells (≈1 mL per twin cell). The liquid phases were pumped by a KNAUER Preparative 1800 V7115 pump (Berlin, Germany). The column was coupled online with a UVD 170 s detector set at 210, 254, 280, and 366 nm (Dionex, Sunnivale, CA, USA). Fractions of 20 mL were collected by a Pharmacia Superfrac collector (Uppsala, Sweden). The solvent system was MtBE/CH₃CN/H₂O (3/3/4, v/v/v). The column rotation speed was set at 1200 rpm and the flow rate at 20 mL/min. EAF (1.5 g injected mass) was dissolved in 15 mL of a mixture of both lower phase (10 mL) and upper phase (5 mL). The upper phase of the biphasic solvent system was pumped for 85 minutes in the ascending mode. The stationary phase was then extruded by pumping the organic phase in the descending mode at 20 mL/min. Fractions of 20 mL were collected over the whole process. All fractions were analyzed by TLC and HPLC and then pooled to yield fractions EAF_1–11.

NMR Analysis and Dereplication of the Metabolites

The structures and names of metabolites already reported in the genus Geum (n = 50) were collected from articles available in the scientific literature. The predicted ¹³C NMR chemical shifts of each one were then stored into a local database already comprising 3038 structures of natural compounds (NMR Workbook Suite 2012, ACD/Labs, Ontario, Canada). In the second step, all CPC fractions were dried under vacuum and an aliquot (up to 20 mg when possible) was dissolved in 600 µL of DMSO-d ₆ and analyzed by ¹³C NMR. A standard zgpg pulse sequence was used with an acquisition time of 0.9 seconds and a relaxation delay of 3 seconds. For each sample, 1024 scans were added to obtain a satisfactory signal-to-noise ratio. The spectral width was 240 ppm and the receiver gain was set to the highest possible value. Spectra were then manually phased, baseline corrected, and calibrated on the central resonance of DMSO-d ₆ (δ 39.8 ppm). The last step consisted of the binning of all ¹³C NMR signals followed by the visualization of the whole dataset as a heat map. For this purpose, the absolute intensities of all ¹³C NMR signals detected in the spectra of the fraction series were automatically collected and each resulting peak list was stored as a text file. The binning step was performed by a locally developed computer script written in Python language. Its principle was to divide the ¹³C spectral domain (from 0 to 240 ppm) into chemical shift intervals (or bins) of identical width (Δδ = 0.2 ppm) and to associate the absolute intensity of each peak to the corresponding bin. The resulting table was imported into the PermutMatrix version 1.9.3 software (LIRMM, Montpellier, France) and submitted to HCA for data visualization. The chemical shift clusters regrouped with the HCA were then submitted to the database search for compound identification. Additional 1D and 2D NMR experiments (¹H NMR, HSQC, HMBC, and ¹H-¹H-COSY) were recorded and analyzed in order to confirm the structures of the identified compounds.

HPLC Purification of EAF

Fraction EAF₂ was subjected to semipreparative HPLC using the gradient system (15%–40% CH₃CN, 30 minutes) to yield compounds 2 (t_R = 5.9 minutes, 3.8 mg), 4 (t_R = 9.5 minutes, 4.3 mg), and the mixture of 1 and 3 (t_R = 20.2 minutes, 5.1 mg). Fractions EAF_3–4 were purified by semipreparative HPLC with gradient system (10%–40% CH₃CN, 30 minutes) to yield compounds 1 and 3 in mixture (t_R = 12.6 minutes, 2.7 mg), 5 (t_R = 13.4 minutes, 3.8 mg), and 6 (t_R = 13.8 minutes, 3.7 mg). Fraction EAF₇ was purified by semipreparative HPLC with gradient system (10%–25% CH₃CN, 20 minutes) to yield compound 7 (t_R = 11.2 minutes, 4 mg).

3-(3,4-Dihydroxyphenyl)propyl-α-L-Rhamnopyranoside (1)

Yellowish-brown solid; ¹H NMR (600 MHz, CD₃OD, supplemental data, supplemental figure S1) 3.28 (1H, m, H-1a), 3.57 (1H, m, H-1b), 1.76 (1H, quin, J = 6.9 Hz, H-2a), 2.50 (1H, qd, J = 7.3 Hz, H-3a), 2.52 (1H, qd, J = 7.2 Hz, H-3b), 6.65 (1H, d, J = 2.0 Hz, H-2′), 6.77 (1H, d, J = 8.2 Hz, H-5′), 6.59 (1H, dd, J = 8.2,2.0 Hz, H-6′), 4.53 (1H, d, J = 1.9 Hz, H-1″), 3.69 (1H, dd, J = 3.4,1.9 Hz, H-2″), 3.55 (1H, dd, J = 9.4,3.4 Hz, H-3″), 3.26 (1H, t, J = 9.4 Hz, H-4″), 3.48 (1H, m, H-5″), 1.14 (3H, d, J = 6.2 Hz, H-6″) ; ¹³C NMR (125 MHz, CD₃OD, supplemental data, supplemental figure S2) 66.2 (C-1), 31.1 (2), 31.2 (3), 134.7 (1′), 116.4 (C-2′), 143.7 (C-3′), 141.6 (C-4′), 116.3 (C-5′), 121.0 (C-6′), 100.3 (C-1″),70.9 (C-2″), 71.0 (C-3″), 72.6 (C-4″), 68.4 (C-5″), 16.6 (C-6″) ; HR-ESI-MS (negative-ion mode) m/z: 313.1284 [M − H]⁻ (calculated for C₁₅H₂₁O₇, 313.1287).

DPPH Radical Scavenging Activity

Extracts, fractions, and compounds 2, 5, 6, and 7 were evaluated for their DPPH radical scavenging activity according to a recently published procedure.⁸ Samples were prepared at concentrations of 200, 100, 50, 25, 12.5, and 6.25 µg/mL. Ascorbic acid and quercetin were used as positive controls. All the tests were conducted in triplicate for each concentration.

Hydroxyl Radical Scavenging Activity

Extracts and fractions were evaluated for their hydroxyl radical scavenging activity at various concentrations, ranging from 1330 to 41.56 µg/mL, according to a recently published procedure.⁸

Power Cupric Ion Reducing (CUPRAC) Assay

Extracts and fractions were tested for their cupric ion reducing ability. The cupric ion reducing activity (CUPRAC) was determined according to a published method.⁴⁹ Samples were prepared at concentrations of 572, 286, 143, 71.5, 35.75, 17.87, and 8.94 µg/mL and dissolved in H₂O/DMSO (9/1, v/v). A volume of 45 µL of each diluted sample was added to a premixed reaction mixture containing cupric chloride (CuCl₂; 90 µL, 10 mM), freshly prepared neocuproine (90 µL, 7.5 mM) dissolved in distilled H₂O and ethanol in proportion 8/2 (v/v), and NH₄Ac buffer (90 µL, 1M, pH 7.0). Similarly, a blank test mixture was prepared by adding a sample solution (45 µL) to the premixed reaction mixture (270 µL) without CuCl₂. The reaction proceeded for 30 minutes at room temperature on a 96-well microplate and the absorbance was then read at 450 nm. Ascorbic acid, quercetin, and trolox were used as positive controls. The power cupric ion reducing was calculated as follows: [1 – A ₀/(A ₁ – A ₂)] × 100, where A ₀ is the absorbance of the control (without sample), A ₁ is the absorbance in the presence of the sample, and A ₂ is the absorbance of the blank. All the tests were conducted in triplicate and IC₅₀ were determined by interpolation of concentration versus inhibition curves obtained by an MSExcel calculation sheet.

Elastase Enzyme Assay

Extracts and fractions were evaluated for their ability to inhibit human leukocyte elastase (HLE) (Merck Biosciences). Tests were performed in 96-well microplates precoated with 1% bovine serum albumin. HLE (0.8 µM) was incubated for 1 hour at 27°C in Tris buffer (50 mM Tris–hydrochloric acid pH 7.5 containing 500 mM sodium chloride) containing 0.1–1000 µg/mL of the tested sample. The pure solvent was used as a control sample. The assay was initiated by adding HLE fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-AMC (λ _exc = 380 nm/λ _em = 460 nm) at a final concentration of 80 µM. The rate of substrate cleavage was measured in triplicate for each sample concentration with 1 measurement per minute for 60 minutes. HLE activity was calculated according to: % HLE activity = (Slope_sample × 100)/slope_control, where slope_sample and slope_control are the slope of the curve in the graph of fluorescence intensity versus time. The IC₅₀ values were calculated by non-linear regression analysis with the Graphpad software (La Jolla, USA).

Supplemental Material

Supplementary material - Supplemental material for Investigation of Antioxidant and Elastase Inhibitory Activities of Geum urbanum Aerial Parts, Chemical Characterization of Extracts Guided by Chemical and Biological Assays

Supplemental material, Supplementary material, for Investigation of Antioxidant and Elastase Inhibitory Activities of Geum urbanum Aerial Parts, Chemical Characterization of Extracts Guided by Chemical and Biological Assays by Marie Schmitt, Abdulmagid Alabdul Magid, Jean-Marc Nuzillard, Jane Hubert, Nicolas Etique, Laurent Duca and Laurence Voutquenne-Nazabadioko in Natural Product Communications

Footnotes

Acknowledgments

Dr Marie Schmitt gratefully acknowledges ICMR and MEDyC laboratories as well as URCA PlAneT platform (University Reims Champagne-Ardenne) for giving the possibility to perform all the necessary manipulations for the realization of this publication.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors are grateful to Grand Est region in France and EU-program FEDER for their financial support of this thesis CHAVIC project.

ORCID iD

Laurence Voutquenne-Nazabadioko

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