Cyperaceae Species Are Potential Sources of ... - ACS Publications

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Cyperaceae Species Are Potential Sources of Natural Mammalian Arginase Inhibitors with Positive Effects on Vascular Function Kamel Arraki,† Perle Totoson,† Alain Decendit,‡ Alain Badoc,‡ Andy Zedet,† Julia Jolibois,† Marc Pudlo,† Céline Demougeot,† and Corine Girard-Thernier*,† †

PEPITE EA4267, University of Bourgogne Franche-Comté, 25000 Besançon, France MIB−UR Œnologie, EA 4577, USC 1366 INRA, University of Bordeaux, ISVV, 33882 Villenave-d’Ornon, France

‡

ABSTRACT: The inhibition of arginase is of substantial interest for the treatment of various diseases of public health interest including cardiovascular diseases. Using an ex vivo experiment on rat aortic rings and an in vitro assay with liver bovine purified arginase, it was demonstrated that several polyphenolic extracts from Cyperus and Carex species possess vasorelaxant properties and mammalian arginase inhibitory capacities. Phytochemical studies performed on these species led to the identification of eight compounds, including monomers, dimers, trimers, and tetramers of resveratrol. The potential of these stilbenes as inhibitors of mammalian arginase was assessed. Five compounds, scirpusin B (5), εviniferin (4), cyperusphenol B (6), carexinol A (7), and the new compound virgatanol (1), showed significant inhibition of arginase, with percentage inhibition ranging from 70% to 95% at 100 μg/mL and IC50 values between 12.2 and 182.1 μM, confirming that these stilbenes may be useful for the development of new pharmaceutical products.

A

inhibitors.8 Among naturally occurring compounds, stilbenoids form a group of phenolic compounds, whose distribution within the plant kingdom is limited to species that have acquired the ability to synthesize this kind of molecule during their evolution.9 Stilbenes are polyphenols containing resveratrol as a basic subunit. These compounds occur in numerous plants belonging to different botanical families.9 These compounds have received much attention because of their cardioprotective effects,10 but they also display anti-inflammatory, antioxidative,11 and antimicrobial activities.12 They are also known as anticancer and cancer-chemopreventive agents.13−15 Recently, the stilbene piceatannol-3′-O-β-D-glucopyranoside was reported as an active constituent of the Asian medicinal plant Rheum undulatum L., possessing potent rat arginase inhibitory activity.16 Consistent with the efficacy of stilbenes as arginase inhibitors, we recently demonstrated that resveratrol and piceatannol possessed in vitro inhibitory activity against liver bovine arginase.17 Cyperus and Carex genera, from the Cyperaceae family, are renowned sources of biologically active oligostilbenoids including piceatannol and resveratrol oligomers.18 Herein the vasorelaxant and mammalian arginase inhibitory effects of extracts from Cyperus (Cy.) eragrostis and two Carex (C. appressa var. virgata and C. cuprina) are discussed.

rginases (amidinohydrolase, EC 3.5.3.1) are trimeric metalloenzymes, characterized by an unusual binuclear active site, containing two manganese ions bridged by a hydroxy group. These enzymes catalyze the hydrolysis of Larginine to urea and L-ornithine.1,2 In mammals, two isoforms, type I arginase (Arg I) and type II arginase (Arg II), are known. Arg I plays a crucial role in the urea cycle in the liver, while Arg II is preferentially involved in L-arginine homeostasis in nonhepatic tissues. This enzyme plays a crucial role because the produced ornithine is the precursor of polyamines involved in cell proliferation and proline involved in collagen production.3,1 Moreover, by substrate competition via the consumption of L-arginine, Arg II could decrease L-arginine available to nitric oxide (NO) synthase, which breaks down Larginine into L-citrulline and NO, a key mediator of endothelial function.4 Consequently, increased vascular arginase activity is involved in endothelial dysfunction associated with several pathologies. The mechanism involves two pathways: the decrease in the amount of L-arginine necessary to produce NO via NOS and the increase in L-ornithine production, leading to vascular dysfunction.5 Previous studies conducted in animal models or in humans showed that inhibition of arginase enhanced NO bioavailability, thereby restoring normal vascular function.6 In spite of the availability of some synthesized arginase inhibitors, no drug has yet been developed, in particular because of problematic pharmacokinetic parameters.7 Finding new arginase inhibitors suitable for the treatment of cardiovascular diseases in humans still poses challenges. Natural products constitute a significant source of promising arginase © 2017 American Chemical Society and American Society of Pharmacognosy

Received: March 7, 2017 Published: August 24, 2017 2432

DOI: 10.1021/acs.jnatprod.7b00197 J. Nat. Prod. 2017, 80, 2432−2438

Journal of Natural Products

Article

Figure 1. (A) Concentration−response curves of extracts from Cyperus eragrostis, Carex apressa var. virgata, and Carex cuprina on intact and endothelium-denuded aortic rings preconstricted with 10−6 M phenylephrine (PE). (B) Vasorelaxant effect of each extract in the presence or not of the NOS inhibitor L-NAME (10−4 M). Data are expressed as means ± SEM, n = 6−10 rings from 10 rats, ***p < 0.001.

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RESULTS AND DISCUSSION The underground parts of three Cyperaceae species, Cyperus eragrostis (seeds), Carex appressa var. virgata (seeds), and Carex cuprina (roots), were extracted with MeOH, and the extracts dried and subsequently subjected to solid phase extraction (SPE) in order to recover polyphenolic compounds. These polyphenolic-enriched extracts were first evaluated for their vasorelaxant activity on rat aortic ring. Consistent with a positive effect of the extracts on endothelial function, the polyphenolic fractions from Carex and Cyperus species induced significant vasorelaxant effects that were suppressed when endothelium was removed (Figure 1). EC50 values of the phenolic fractions of Carex cuprina (C. cuprina), Carex appressa var. virgata (C. appressa var.

virgata), and Cyperus eragrostis (Cy. eragrostris) were 4.3, 3.6, and 3.9 μg/mL, respectively, with Emax reaching more than 90% (Figure 1). The endothelium-dependent vasorelaxant effects of the extracts were the consequence of increased nitric oxide synthase (NOS) activity, as attested by the dramatic reduction of relaxation induced by the NOS inhibitor L-NAME. Given that NOS and arginase compete for their common substrate, these results show evidence of an inhibitory effect of the extracts on vascular arginase. In agreement with this hypothesis, the biological evaluation of the polyphenolic fractions of Cy. eragrostis, C. cuprina, and C. appressa var. virgata on an enzymatic in vitro assay for arginase activity17 revealed that these extracts were able to inhibit arginase (more than 50% inhibition at 100 μg/mL) (Figure 2). 2433



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Table 2. 1H (Acetone-d6, 400 MHz) and 13C (Methanol-d4, 100 MHz) NMR Spectroscopic Data for Compound 1 position 1a 2a, 6a 3a, 5a 4a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b, 6b 3b, 5b 4b 7b 8b 9b 10b 11b 12b 13b 14b

Figure 2. Arginase inhibition (at 10 and 100 μg/mL) of the methanolic extracts of Carex appressa var. virgata, Carex cuprina, and Cyperus eragrostis. Results are expressed as means ± SD obtained from three distinct experiments performed in duplicate.

δH (m, J in Hz)

δC, type 130.6, 131.5, 117.1, 159.8, 89.9, 50.8, 142.1, 118.1, 156.4, 101.9, 158.1, 107.1, 134.5, 130.6, 116.2, 156.4, 42.5, 49.5, 139.7, 120.5, 157.8, 96.2, 157.4, 112.7,

7.18 (2 H, d, J = 8.8) 6.83 (2 H, d, J = 8.8) 5.79 (1 H, d, J = 12.0) 4.27 (1 H, d, J = 12.0)

6.59 (1 H, br s) 6.34 (1 H, d, J = 1.6) 6.95 (2 H, d, J = 8.0) 6.60 (2 H, d, J = 8.0) 5.85 (1 H, br s) 3.97 (1 H, br s)

C CH CH C CH CH C C C CH C CH C CH CH C CH CH C C C CH C CH

In order to isolate active compounds from the extracts, reverse-phase preparative liquid chromatography (PLC) has been used and afforded eight stilbenes, including a new resveratrol dimer (1) and seven known resveratrol monomers and oligomers (2−8). This is the first study of the seeds of C. appressa var. virgata and Cy. eragrostis. The isolated compounds were identified through ESIMS analysis (Table 1) and by comparison of published spectroscopic data.

a

Table 1. Identification of Stilbenoids Using Retention Times and ESIMS Data

Table 3. Major HMBC Correlations for Compound 1

compound

tR (min)

Carex appressa var. virgata 1 23.0 2 10.2 3 18.5 4 25.9 Cyperus eragrostis 5 21.1 6 32.3 Carex cuprina 7 22.9 8 24.5

[M + H]

+

compound 1 resveratrol diglucoside piceatannol ε-viniferin

487 729

scirpusin B cyperusphenol B

941 925

carexinol A kobophenol A

5.21 (1 H, br d, J = 2.0)

The coupling constants (J) are given in parentheses and reported in Hz; chemical shifts (δ) are given in ppm.

identification

471 553 245 455

5.77 (1 H, br d, J = 2.0)

The new compound 1 was isolated as a pale brown powder. Its molecular formula was established as C28H22O7 by means of ESIMS (m/z 471.14321 [M + H]+, 470.13592 [M]). All 1H and 13C NMR assignments for 1 were performed by using 2D NMR spectroscopic data (HSQC, HMBC, COSY, NOESY). The 1H and 13C NMR data (Tables 2 and 3) are in line with a dimer of resveratrol. These data showed the presence of two 4hydroxyphenyl groups (A1 and B1) and two tetrasubstituted benzene units (A2 and B2). The 1H NMR spectrum of compound 1 is consistent with the presence of (i) two groups of meta-coupled aromatic protons belonging to rings A2 [δ 6.34 (1H, d, J = 1.6 Hz, H-14a), δ 6.59 (1H, br s, H-12a)] and B2 [δ 5.21 (1H, br d, J = 2.0 Hz, H-14b), δ 5.77 (1H, br d, J = 2.0 Hz, H-12b)] and (ii) two groups of ortho-coupled aromatic protons belonging to hydroxyphenyl moieties [A1: δ 7.18 (2H, d, J = 8.8 Hz, H-2a and 6a), δ 6.83 (2H, d, J = 8.8 Hz, H-3a and 5a), and B1: δ 6.95 (2H, d, J = 8.0 Hz, H-2b and 6b), δ 6.60 (2H, d, J = 8.0 Hz, H-3b and 5b)]. The NMR data also exhibited the

position

HMBC

position

HMBC

1a 2a, 6a 3a, 5a 4a 7a 8a 9a 10a 11a 12a 13a 14a

2(6)a, 7a 3(5)a, 7a, 8a 2(6)a 3(5)a, 2(6)a 2(6)a, 8a 7a, 14a 7a, 8a, 14a, 7b 8a, 12a, 14a, 7b, 8b 12a, 7b 14a 12a, 14a 8a, 12a

1b 2b, 6b 3b, 5b 4b 7b 8b 9b 10b 11b 12b 13b 14b

3(5)b, 7b 3(5)b, 7b 2(6)b 3(5)b, 2(6)b 2(6)b, 8b 7b, 14b 7b, 8b, 14b, 8a 8b, 14b, 12b, 7a, 8a 7a, 12b 14b 12b, 14b 12b, 8b

signals of two pairs of aliphatic methine protons coupled as shown in Table 2: H-7a (δ 5.79, d, J = 12.0 H2) and H-8a (δ 4.27, d, J = 12.0 H2); H-7b (δ 5.85, br s) and H-8b (δ 3.97, br s). NOESY data facilitated the verification of the orientations of the methine protons at C-8a, C-7b, and C-8b on the cycloheptane ring. The absence of an NOE between H-8a and H-7b revealed that H-8a and H-7b were on opposite sides, whereas the NOE observed between H-8b and H-7a and H-8a revealed that H-8b and H-7a/H-8a were cofacial. Moreover, these data confirmed that H-7b and H-8b were trans-oriented, whereas H-7a and H8a were cis-oriented (Figure 3). Since H-7a, H-8a, H-7b, and H8b of 1 were cis- and trans-oriented based on the NOESY data, the orientations of H-7a, H-8a, H-7b, and H-8b were β, β, α, and β. Other significant NOEs in support of these observations were between H-8a β/H-2b(6b), H-8b β/H-7a β, and H-8b β/ 2434



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Figure 3. Structure (a) and NOESY (b) correlations for compound 1.

Table 4. Chemical Shifts of Aliphatic Proton Pairs of H-7a/H-8a and H-7b/H-8b for Compound 1, Acuminatol,22 (+)-Ampelopsin,19 (−)-Hemsleyanol A,21 and (+)-Balanocarpol20 δH (m, J in Hz) compound virgatanol (1)a acuminatola (+)-ampelopsin Ab (−)-hemseyanol Aa (+)-balanocarpola a

7a 5.79 5.67 5.77 5.75 5.69

(d, (d, (d, (d, (d,

8a 12.0) 11.7) 11.7) 9.8) 9.3)

4.27 4.19 4.17 5.41 5.16

(d, 12.0) (d, 11.7) (br d, 11.7) (d, 9.8) (br d, 9.3)

7b 5.85 5.46 5.45 5.07 4.90

(br s) (br s) (d, 5.0) (d, 5.9) (br s)

8b 3.97 5.09 5.42 4.76 5.40

(br (br (br (br (br

s) s) s) d) s)

Measured in acetone-d6 (400 MHz). bmeasured in acetone-d6 (500 MHz).

Figure 4. Structures of compounds 1−8.

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Figure 5. Arginase inhibition (at 10 and 100 μM) of the compounds 1−8 and nor-NOHA (control). Results are expressed as means ± SEM obtained from three distinct experiments, performed in duplicate.

and antibacterial activities,26 as well as neuroprotective effects.27 Piceatannol is now well known for its pharmacological effects on the cardiovascular system.28 Piceatannol was recently described as a mammalian arginase inhibitor and is used as a natural reference inhibitor.17 Compounds 1−8 were screened for their arginase inhibitory activity at two concentrations (10 and 100 μM) using purified bovine liver arginase17 (Figure 5). The results showed that, except for kobophenol A and resveratrol diglucoside, the isolated stilbenes were able to inhibit arginase in the range 80% to 90% at 100 μM. Their IC50 values are collected in Table 5.

H-14b. In the HMBC spectrum (Table 3, Figure 3), significant correlations were observed between C-2a(6a)/H-7a, C-8a/H7a, C-9a/H-7a, C-9a/H-8a, and C-10b/H-8a, indicating that the H-7aβ and H-8aβ benzylic methine protons could be assigned to the protons of the dihydrofuran ring. There were significant correlations observed between C-9b/ H-7b, C-1b/H-7b, C-11a/H-7b, C-10a/H-7b, and C-8b/H-7b. These correlations proved that there is a pair of methine protons attached to C-7b (H-7bα) and C-8b (H-8bβ). The relative configuration of compound 1 was confirmed as shown in Figure 3. It should be noted that four stereoisomers of compound 1 were previously isolated and identified. (+)-Ampelopsin A was isolated from Ampelopsis brevipedunculata var. hancei (Vitaceae family),19 (+)-balanacarpol from Hopea parvifolia (Dipterocarpaceae family),20 (−)-hemsleyanol A from Shorea hemsleyana,21 and acuminatol from S. accuminata (Dipterocarpaceae family).22 Despite the fact that these four stereoisomers have the same 2D skeleton in common, the chemical shifts of H-7a, H-8a, H-7b, and H-8b were quite different (Table 4). It has been suggested that these different values are mainly due to the A1 and B1 rings, considering their energy-optimized conformations. NOE correlations (Figure 3) between H-8a/H-2b(6b), H8b/H-2b(6b), and H-8a/H-7a in compound 1 suggested that H-7a, H-8a, H-8b, and ring B1 were cofacial. Taking into account NMR and MS data, it was concluded that compound 1 was a new resveratrol dimer and assigned the trivial name virgatanol. The specific rotation of 1 was [α]D = −369 (c 0.04, MeOH). Seven known compounds (2−8) were characterized as (E)resveratrol 3,5-O-β-diglucoside (2), (E)-piceatannol (3), εviniferin (4), (E)-scirpusin B (5), cyperusphenol B (6), carexinol A (7), and kobophenol A (8) (Figure 4). Table 1 shows the chromatographic data set and the m/z values of the isolated stilbenes. NMR and ESIMS data were used to identify compounds 2 to 8 as known stilbenes from Carex and Cyperus genera. This is the first study of the seeds of C. appressa var. virgata and Cy. eragrostis. Piceatannol (3), cyperusphenol B (6), and scirpusin B (5) were previously isolated from Cyperus rotundus.23 Kobophenol A (8) was isolated from Carex kobomugi, which also contains ε-viniferin (4),24 and from Carex folliculata.25 These constitute interesting compounds in view of their potential biological properties. Biological activities of kobophenol A are various: antioxidant

Table 5. Arginase Inhibitory Activity of Compounds 1−8a compound nor-NOHA 1 2 3 4 5 6 7 8

arginase inhibition, IC50 (μM) 1.7 182.1 n.d.c 12.6 27.8 22.6 12.2 25.3 n.d.

± 0.2 ± 19.4b ± ± ± ± ±

0.6 2.3b 3.0b 0.9 2.3b

Values are means ± SEM and were obtained from three distinct experiments performed in triplicate. bp < 0.05, significantly different from piceatannol (natural reference inhibitor, compound 3). cn.d.: not determined. a

The synthesized compound Nω-hydroxy-nor-L-arginine (norNOHA), a well-known reference inhibitor, was used as a positive control. Even though all the evaluated compounds remained less active than nor-NOHA, it is noteworthy that cyperusphenol B is as active as the natural reference inhibitor piceatannol, one of the most active natural compounds on mammalian arginase.8,17 In summary, three species of Cyperaceae, Carex cuprina, C. appressa (var. virgata), and Cyperus eragrostis, were used in these biological and phytochemical studies. Eight stilbenes were isolated, among which compound 1 is a new structure and epimeric with (+)-ampelopsin A, (+)-balanacarpol, (−)-hemsleyanol A, and acuminatol. It was named virgatanol and was isolated from the seeds of C. appressa var. virgata. The phenolic extracts exhibited endothelium-dependent vasorelaxant properties that are congruent with the arginase inhibitory effect of the 2436



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(20 mg, tR 25.9 min), 5 (17 mg, tR 21.1 min), 6 (7 mg, tR 32.3 min), 7 (25 mg, tR 22.9 min), and 8 (15 mg, tR 24.5 min). Compound purity was controlled by analytical HPLC. Compound 2 [(E)-resveratrol 3,5-O-β-diglucoside] was isolated from C. appressa var. virgata. Its molecular formula was established as C26H33O13 by means of ESIMS (m/z 453.2 [M + H]+). 1H and 13C NMR data were consistent with reported data.18 Compound 3 (piceatannol) was isolated from C. appressa var. virgata. Its molecular formula was established as C14H12O4 by means of ESIMS (m/z 245.1 [M + H]+). 1H and 13C NMR data were consistent with reported data.28 Compound 4 (ε-viniferin) was isolated from C. appressa var. virgata. Its molecular formula was established as C28H22O6 by means of ESIMS (m/z 455.1 [M + H]+). 1H and 13C NMR data were consistent with reported data.29 Compound 5 (scirpusin B) was isolated from Cy. eragrostis. Its molecular formula was established as C28H22O8 by means of ESIMS (m/z 487.1 [M + H]+). 1H and 13C NMR data were consistent with reported data.30 Compound 6 (cyperusphenol B) was isolated from Cy. eragrostis. Its molecular formula was established as C42H32O12 by means of ESIMS (m/z 729.1 [M + H]+). 1H and 13C NMR data were consistent with reported data.23 Compound 7 (carexinol A) was isolated from C. cuprina. Its molecular formula was established as C56H45O14 by means of ESIMS (m/z 941.3 [M + H]+). 1H and 13C NMR data were consistent with reported data.31 Compound 8 (kobophenol A) was isolated from C. cuprina. Its molecular formula was established as C56H45O13 by means of ESIMS (m/z 925.3 [M + H]+). 1H and 13C NMR data were consistent with reported data.32 Vascular Reactivity Studies. Experiments were conducted on 10 male Sprague−Dawley rats (7−8 weeks old), purchased from Janvier (Le Genest Saint Isle, France). The experimental procedures were approved by the local ethics committee for animal experimentation No. 2015/001-CD/5PR of Franche-Comté University (Besançon, France) and complied with the “Animal Research: Reporting in Vivo Experiments” ARRIVE guidelines. After anesthesia with sodium pentobarbital (60 mg/kg, intraperitoneally), the thoracic aorta was excised, cleaned of connective tissue, and cut into rings of approximately 2 mm in length. The rings were suspended in Krebs solution (M: NaCl 118, KCl 4.65, CaCl2 2.5, KH2PO4 1.18, NaHCO3 24.9, MgSO4 1.18, glucose 12, pH 7.4), maintained at 37 °C, and continuously aerated with 95% O2 and 5% CO2, for isometric tension recording in organ chambers, as previously described.33 After a 90 min equilibration period under a resting tension of 2 g, the presence of functional endothelium was verified by the ability of the endotheliumdependent agonist acetylcholine (Ach, 10−6 M) to induce more than 80% relaxation in rings preconstricted with phenylephrine (PE, 10−6 M). In some rings, endothelium was mechanically removed and confirmed by the absence of relaxation to acetylcholine. Water solubility of each extract at 10 g/L (the highest required concentration) was checked before the study. Intact and endothelium-denuded rings were preconstricted with PE (10−6 M) and relaxed with cumulative concentrations of extracts from Cy. eragrostis, C. appressa var. virgata, and C. cuprina dissolved in distilled H2O (10−5 to 10−2 g/L). In order to determine the contribution of NOS to the extract-induced vasorelaxation, rings were preincubated for 30 min with the NOS inhibitor NΩ-nitro-L-arginine methyl ester (L-NAME, 10−4 M). The extract-induced relaxation was expressed as the percent decrease of the preconstriction to PE. Enzyme Inhibition Assay. Urea amount produced by the hydrolysis of L-arginine by arginase [purified liver bovine arginase (b-ARGI)] can be detected using a color reactant (α-isonitrosopropiophenone) followed by a colorimetric assay,17 as described below. In each well of a 96-well microplate the following solutions were added in this order: (1) buffer containing Tris-HCl (50 mM, pH 7.5) and 0.1% of bovine serum albumin (TBSA buffer) (10 μL), with or without (control) arginase (0.025 U/μL), (2) Tris-HCl solution (50 mM, pH 7.5) containing 10 mM MnCl2 as a cofactor (30 μL), a solution

isolated compounds. Cyperusphenol B inhibited arginase with an IC50 close to that of piceatannol. Collectively, these results extend the natural product pipeline of new arginase inhibitors for treating vascular diseases.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotation was recorded on an Anton Paar MC300 polarimeter in MeOH. Identification and structural elucidation of the purified compounds were carried on a mass spectrometer (high-resolution electrospray ionization mass spectra, or HRESIMS) and an NMR spectrometer. HRESIMS data were acquired on an SCA Illkirch QToF instrument. 1 H NMR at 400 MHz and 13C NMR data at 100 MHz were acquired using a Bruker AC300 spectrometer (Bruker BioSpin). All the compounds were dissolved in methanol-d4 or acetone-d6 for 1D NMR and 2D NMR measurements (including COSY, HSQC, NOESY, and HMBC). Chemical shifts (δ) were reported in parts per million (ppm) relative to the residual solvent signals. Coupling constants (J) were reported in Hz. Data were presented as follows: chemical shift (δ, ppm), multiplicity (s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet), coupling constant (J, Hz), integration. All reagents were from Sigma-Aldrich (Saint-Quentin Fallavier, France). They were used without further purification, except for purified liver bovine arginase 1, which was purchased from MP Biomedicals (one unit (1U) of bovine arginase corresponds to the amount of enzyme able to convert 1 μmol of L-arginine to urea and Lornithine per minute at pH 9.5 and 37 °C). MeOH, MeCN, and DMSO were obtained from Carlo Erba Reagents (Val de Reuil, France) and VWR Chemicals (Fontenay sous Bois, France). Deuterated solvents and trifluoroacetic acid (TFA) were purchased from Eurisotop and Fisher Scientific, respectively. Water was purified (resistivity >18 mΩ/cm) using an ELGA water purification system (ELGA LabWater). Plant Material. Cyperus eragrostis Lam. (IPEN code FR-0-TAL20070521W) seeds, Carex appressa R.Br. var. virgata (Sol. ex Boott) Kük. (XX-0-TAL-20110326W) seeds, and Carex cuprina (Sándor ex Heuff.) Nendtv. ex A.Kern. (FR-0-TAL-20090304W) whole plant underground parts were collected in the Botanical Garden of Talence (France) between 2012 and 2015. Each plant was authenticated by one of the authors (A.B.). The samples were well-dried and kept away from moisture. Extraction and Isolation. All parts of Carex cuprina and seeds of Carex appressa var. virgata and Cyperus eragrostis were ground into a powder. A sample of each (12 g) was macerated in 100% MeOH (about 150 mL) for 24 h with stirring at 4 °C. The extraction procedure was repeated twice. The methanolic solutions were recovered by filtration, pooled, and concentrated under reduced pressure to obtain dry extracts. These extracts were dissolved in 30% MeOH (1 g of extract in 600 μL of MeOH and 1.4 mL of H2O) using vortexing and sonicating. Each extract was prepurified by using an SPE mini-column Strata C18-E (55 μm, 70 Å). Each sample (2 mL) was loaded onto the C18 mini-column, washed with 4 mL of H2O, and eluted with 90% MeOH. The recovered solution contained polyphenols (flavonoids and stilbenes). Each extract was evaporated to dryness with the same vacuum evaporator. Before HPLC analyses, the dried extract was redissolved in 50% MeOH HPLC grade by vortexing and sonicating before filtration through an Acrodisc (25 mm syringe filters) 0.2 μm nylon membrane (HPLC certified). The purification was done by PLC. Polyphenolic extracts were separated on a Gilson PLC 2020 Kinetex EVO reversed-phase C18 column (250 × 21.2 mm, 5 μm). The solvent system used was ultrapure H2O acidified with 0.1% TFA (solvent A) and MeCN acidified with 0.1% TFA (solvent B). The elution program at 20 mL/ min was 10% B (0−5 min), 10−60% B (5−35 min), and 60% B (35− 40 min) followed by a 5 min wash with 100% B and a 5 min reequilibration step. The injections were 500 μL. The chromatograms were registered at 286 and 306 nm. PLC yielded compounds 1 (29 mg, tR 23.0 min), 2 (5.5 mg, tR 10.2 min), 3 (16 mg, tR 18.5 min), 4 2437



Article

containing an inhibitor or its solvent (as a control) (10 μL), (4) a solution of L-arginine (pH 9.7, 0.05 M) (20 μL). The microplate was incubated for 60 min in a 37 °C water bath after covering with a plastic sealing film. Addition of 120 μL of H2SO4/H3PO4/H2O (1:3:7) quenched the reaction. The microplate was left on ice for 5 min. Thereafter, 10 μL of α-isonitrosopropiophenone (5% in absolute EtOH) was added, and the microplate was heated in an oven at 100 °C for 45 min after covering with an aluminum sealing film. The colored product being photosensitive, the microplate was kept in the dark until reading. After 5 min of centrifugation and cooling for another 10 min, the microplate was shaken for 2 min and the absorbance was read at 550 nm and 25 °C using a spectrophotometer (Synergy HT BioTeck). The level of arginase activity was expressed as relative to the “100% arginase activity”. The experiment was repeated three times with each microplate under similar experimental conditions (e.g., various inhibitor concentrations. Determination Percentages of Arginase Inhibition and IC50 Values. For each compound a stock solution (70 mM) was prepared in DMSO and stored at −26 °C. These stock solutions were extemporaneously successively diluted in ultrapure H2O to afford the following concentrations: 7000, 2100, 700, 210, 70, 21, 7, 2.1, and 0.7 μM, corresponding to final concentrations in the wells of 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 μM, respectively. For a first screening, compounds were tested at final concentrations of 10 and 100 μM. Each solution was incubated with arginase for 1 h, as described above. The percentage of arginase inhibition was calculated by conversion of the resulting absorbance [relative to the absorbance of controls with only solvent (“100% arginase activity”)] and plotted on a semilogarithmic scale. The IC50 values were estimated by nonlinear sigmoidal curve-fitting by using Prism (GraphPad Software, version 5.0.3). Data and Statistical Analysis. Values were presented as means ± SEM. Data were analyzed with Prism (GraphPad Software, version 5.0.3). Comparison between two values was assessed by unpaired Student’s t test or Mann−Whitney U test when data were not normally distributed. Concentration−response curves were compared by two-way analysis of variance (ANOVA) for repeated measures. A p < 0.05 was considered significant.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +33 3 81 66 55 59. Fax: +33 3 81 66 56 92. E-mail: [email protected]. ORCID

Corine Girard-Thernier: 0000-0002-5116-1395 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Botanical Gardens of Caen and Nantes for the supply of Carex seeds to the Botanical Garden of Talence, and M. Coster for English language revision.

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