Fitoterapia 96 (2014) 65–75

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Advanced glycation inhibition and protection against endothelial dysfunction induced by coumarins and procyanidins from Mammea neurophylla Bach Tai Dang a, Charlotte Gény a, Patricia Blanchard a, Caroline Rouger a, Pierre Tonnerre b, Béatrice Charreau b, Gilbertine Rakolomalala b, Joseph Iharinjaka Randriamboavonjy b, Gervaise Loirand b, Pierre Pacaud b, Marc Litaudon c, Pascal Richomme a, Denis Séraphin a, Séverine Derbré a,⁎ a b c

Université d'Angers, SFR QUASAV, EA 921 SONAS, Angers, France INSERM, U1064, Nantes, France Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles (ICSN), CNRS, Labex LERMIT, Gif sur Yvette Cedex, France

a r t i c l e

i n f o

Article history: Received 12 February 2014 Accepted in revised form 2 April 2014 Available online 13 April 2014 Keywords: 4-(1-Acetoxypropyl)coumarins Advanced glycation end-products (AGEs) Calophyllaceae Endothelial dysfunction Mammea neurophylla 4-Phenylcoumarins

a b s t r a c t Advanced glycation end-products (AGEs) are associated with many pathogenic disorders such as pathogenesis of diabetes or endothelial dysfunction leading to cardiovascular events. Therefore, the identification of new anti-AGE molecules or extracts aims at preventing such pathologies. Many Clusiaceae and Calophyllaceae species are used in traditional medicines to treat arterial hypertension as well as diabetes. Focusing on these plant families, an anti-AGE plant screening allowed us to select Mammea neurophylla for further phytochemical and biological studies. Indeed, both DCM and MeOH stem bark extracts demonstrated in vitro their ability to prevent inflammation in endothelial cells and to reduce vasoconstriction. A bioguided fractionation of these extracts allowed us to point out 4-phenyl- and 4-(1-acetoxypropyl)coumarins and procyanidins as potent inhibitors of AGE formation, potentially preventing endothelial dysfunction. The fractionation steps also led to the isolation of two new compounds, namely neurophyllols A and B from the DCM bark extract together with thirteen known mammea A and E coumarins (mammea A/AA, mammea A/AB, mammea A/BA, mammea A/BB, mammea A/AA cycloD, mammea A/AB cycloD, disparinol B, mammea A/AB cycloE, ochrocarpin A, mammea A/AA cycloF, mammea A/AB cycloF, mammea E/BA, mammea E/BB) as well as δ-tocotrienol, xanthones (1-hydroxy-7-methoxyxanthone, 2-hydroxyxanthone) and triterpenes (friedelin and betulinic acid). During this study, R,S-asperphenamate, previously described from fungal origin was also purified. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The genus Mammea (Calophyllaceae) consists of about 70 species of evergreen trees growing in northern South America and the West Indies (Mammea americana), in India (Mammea longifolia) and Southeast Asia (e.g. Mammea siamensis, Mammea harmandii, Mammea acuminata) as well as in Africa's tropical ⁎ Corresponding author. Tel.: +33 2 41 22 66 69; fax: +33 2 41 48 67 33. E-mail address: [email protected] (S. Derbré).

http://dx.doi.org/10.1016/j.fitote.2014.04.005 0367-326X/© 2014 Elsevier B.V. All rights reserved.

rainforests (Mammea africana) [1–3]. Stem bark of M. africana is used in traditional local medicine in cases of arterial hypertension [4–6] or diabetes [7]. M. siamensis is also described in folk Thai medicine to treat diabetes [8]. High blood pressure and hyperglycemia are closely linked with endothelial dysfunction. In vessels, the endothelium is the innermost layer of cells in direct contact with the blood. This tissue synthesizes and releases substances such as nitric oxide (NO), which regulate the vascular tone. On the one hand, a reduced production of NO by endothelial cells or an increased exposition to reactive

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oxygen species (ROS) may provoke contractions of smooth muscle cells together with an elevation of blood pressure. In particular, superoxide anion radical (O2–.) produced by oxidases degrades NO in peroxynitrite (ONOO−) [9]. On the other hand, hypertension in itself impairs endothelial function [10–12]. As far as diabetes are concerned, chronic hyperglycemia is associated with advanced glycation end-products (AGEs) accumulation in tissues and blood. AGEs are formed during a non-enzymatic reaction involving proteins and sugars called the Maillard or browning reaction [13] and their implication in endothelial dysfunction is now well-known [14,15]. Finally, such an endothelial dysfunction, due to hypertension or hyperglycemia, is implied in atherosclerosis and cardiovascular events [16]. Therefore anti-AGE agents would prevent the endothelial dysfunction and could slow down the cardiovascular alteration then avoiding clinical events [17]. Earlier phytochemical studies on Mammea species have shown that this genus is rich in prenylated 4-phenyl and 4-propylcoumarins, xanthones and benzophenones whereas pentacyclic triterpenes and steroids as well as flavan-3-ols and procyanidins were also reported [18–24]. The bark, leaf and fruit DCM and methanolic extracts from Mammea neurophylla (Schltr.) Kosterm, an endemic neocaledonian shrub, were selected among 37 extracts from 9 plants belonging to Clusiaceae and Calophyllaceae for their anti-AGE activity [25] (Table 1). No phytochemical study of this species was previously reported. Thus, to identify natural products that are able to prevent AGE formation and endothelial dysfunction, biological in vitro studies on these extracts were followed by phytochemical investigations focusing on anti-AGE compounds. 2. Results and discussion 2.1. Anti-AGE potential, anti-inflammatory and vasorelaxing effects of M. neurophylla extracts An anti-AGE activity of substances is often associated with anti-inflammatory properties [25,26] and protective effects towards cardiovascular diseases [27]. The selected extracts were thus tested for their biological effects on endothelial dysfunction which is characterized, at the endothelium level, by pro-inflammatory and prothrombic states and a reduced vasodilatation [28]. The vascular cell adhesion molecule (VCAM-1) is a biomarker of the endothelial dysfunction as it mediates interaction between endothelium and leucocytes. Thus, the anti-inflammatory potential of M. neurophylla extracts Table 1 Anti-AGE activity of M. neurophylla extracts. Organ

Solvent

Bark DCM Bark MeOH Leaf DCM Leaf MeOH Fruit DCM Fruit MeOH a Quercetin Aminoguanidinea a

References.

Anti-AGE activity Vesperlysines-type AGEs IC50 (mg/mL)

Pentosidine-type AGEs IC50 (mg/mL)

0.05 0.2 0.1 0.2 0.15 0.3 0.06 0.3

0.09 0.5 0.1 0.2 0.1 0.2 0.06 0.2

was evaluated by measuring their ability to decrease tumor necrosis factor (TNF)α-induced VCAM-1 expression in endothelial cells (Fig. 1B) [29]. At a concentration of 10 to 100 μg/mL depending on the extract, all of them inhibited the induction of VCAM-1 in response to TNFα with a maximal inhibitory effect close to 90%, which is more important than that obtained with the reference compound pyrrolidine dithiocarbamate (PDTC, 75% at 30 μg/mL). These results were not due to cytotoxic effects since the endothelial cell viability, assessed by 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazodium (MTT) was not really affected by the presence of the same concentration of each extract (Fig. 1A). DCM extracts, in particular leaf and fruit extracts, showed the strongest activity while the bark extract had the lowest impact on endothelial cell viability. Then, the ability of the extracts to produce a concentration-dependent relaxation of rat aorta precontracted with phenylephrine (PhE) was tested in the presence or absence of endothelium. As illustrated in Fig. 1C, DCM and MeOH bark extracts (0.1 to 100 μg/mL) produced a concentration-dependent relaxation. In the case of MeOH bark extract, removal of the endothelium completely abolished the relaxing effect. Effects were less important with leaf and fruit extracts. For that reason, further experiments were undertaken on MeOH bark extract only. In endothelialized rat aortic rings, inhibition of NO synthesis by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), attested by inhibition of carbachol (CCh)-induced relaxation, also totally prevented MeOH bark extract-induced relaxation of PhE contraction (Fig. 1D). In contrast, neither the cyclooxygenase inhibitor indometacin nor the ATP-sensitive potassium channel inhibitor glibenclamide had any effect, suggesting that neither prostacyclin nor hyperpolarizing relaxing factors could be involved in the relaxing effect observed with the MeOH bark extract. These results thus clearly indicated that this extract induced endothelium-dependent vascular relaxation through direct stimulation of NO production. Therefore these data provided evidences for anti-AGE, antiinflammatory and vasorelaxant properties of M. neurophylla extracts. As DCM and MeOH bark extracts of M. neurophylla showed the strongest anti-AGE and vasorelaxant activities, we embarked upon their fractionation, seeking for anti-AGE natural compounds effective on endothelial dysfunction. 2.2. Fractionation of the MeOH bark extract After tannin removal, the bioactive MeOH bark extract was fractionated by centrifugal partition chromatography (CPC) followed, when necessary, by flash chromatographies on silica gel or filtrations on Sephadex LH-20. Those purification steps afforded ten known polyphenols, including two phenolic acids, gallic acid (1) and protocatechuic acid (2), and five flavan-3-ol derivatives, namely epicatechin (5) and procyanidins B2 (3), C1 (4), B5 (6) and A2 (7). The dihydroflavonols neoastilbin (8), astilbin (9) and isoastilbin (10) were also isolated (Chart S1). As illustrated by the HPLC 3D plot chromatogram (Fig. S2A), epicatechin (5) together with procyanidins B2 (3) and C1 (4) appeared as the major products in M. neurophylla MeOH bark extract. The anti-AGE IC50 values [30] obtained for these natural products showed that major products, epicatechin (5) and its dimer and trimer [procyanidins B2 (3) and C1 (4)] were responsible for the anti-AGE activity of the extract (Table 2). The

B.T. Dang et al. / Fitoterapia 96 (2014) 65–75

A

B

140

VCAM-1 expression (%)

Cell viability (%)

120 100 80 60 40 20 0 MeOH

DCM

Bark

DCM

MeOH

60 40 20

DCM

MeOH

DCM

Bark

MeOH

Fruit

100

100

60 40

80 60 40

20

20

0

0

-3

-2

Tension (%)

100

Tension (%)

120

80

-1

80 60 40 20 0

0

log [extract] (mg/ml)

-4 -3 -2 log [extract] (mg/ml)

-1

0

120

100

100

60 40

80 60 40

20

20

0

0 0

-4

-3

-2

-1

Tension (%)

120

100

Tension (%)

120 80

MeOH

Fruit

Leaf 120

-4

DCM

Leaf

120

0

Tension (%)

80

Fruit

Bark

Tension (%)

DCM extracts

MeOH

Leaf

C

MeOH extracts

100

0 DCM

-4 -3 -2 log [extract] (mg/ml)

-1

80 60 40 20 0

0

log [extract] (mg/ml)

D

67

-4 -3 -2 log [extract] (mg/ml)

-1

0

-4

-3

-2

-1

log [extract] (mg/ml)

Relaxation (%)

100 80 60 40 20

***

0 CCh MeOH bark extract L-NAME Indometacin Glibenclamide

+ -

+ + -

*** + -

+ + -

+ + -

+ +

Fig. 1. M. neurophylla extract effects on endothelial cells and on pre-contracted aorta rings. Effect of extracts (0.1 to 100 μg/mL) on endothelial cell viability (A) and TNFα-induced VCAM-1 expression in endothelial cells (B). PDTC (30 μg/mL, 200 μM) and quercetin (30 μg/mL, 100 μM) were used as positive control and reduce by 25 and 70% of VCAM-1 expression respectively. C: Cumulative concentration–response curves for M. neurophylla extracts-induced relaxation of PhE (1 μM)-contracted aorta rings with (black) and without (white) endothelium. D: Relaxing effect of M. neurophylla MeOH bark extract (100 μg/mL) on PhE (1 μM)-contracted pulmonary artery rings in the absence and presence of L-NAME (3 μM), indometacin (10 μM) or glibenclamide (10 μM). Relaxation induced by carbachol (CCh, 10 μM) in the presence and absence of L-NAME was used as control (***P b 0.0001 vs. untreated, n = 3).

ability to prevent AGE formation in general roughly increases with the number of flavan-3-ol units [31,32] but not only as we observed procyanidins B5 (6) N C1 (4) N B2 (3) N epicatechin (5) N procyanidin A2 (7). Indeed, the anti-AGE activity of

such compounds is not strictly correlated with their radical scavenging potential (DPPH) [33]. The ability of polyphenols, especially flavan-3-ols mono- and oligomers to scavenge reactive carbonyl species such as methylglyoxal should also be

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Table 2 Anti-AGE activity of products isolated from M. neurophylla extracts. Product

Gallic acid (1) Protocatechuic acid (2) Procyanidin B2 (3) Procyanidin C1 (4) (−)-Epicatechin (5) Procyanidin B5 (6) Procyanidin A2 (7) Astilbin (9) Mammea A/AA (11) Mammea A/AB cycloE (14) Ochrocarpin A (15) Mammea A/AA cycloF (16) + mammea A/AB cycloF (17) (40/60) Mammea E/BA (18) + mammea E/BB (19) (36/64) Neurophyllol B (20) + neurophyllol A (21) (83/17) 2-Hydroxyxanthone (22) δ-Tocotrienol (23) Mammea A/AA cycloD (24) Mammea A/AB cycloD (25) Mammea A/BA (26) + mammea A/BB (27) (58/42) 1-Hydroxy-7-methoxyxanthone (28) Friedelin (29) Betulinic acid (30) Asperphenamate (31) Quercetina Aminoguanidinea a

Anti-AGE activity Vesperlysines-type AGEs IC50 (mM)

Pentosidine-type AGEs IC50 (mM)

0.1 N3 0.06 0.03 0.08 0.02 0.2 0.5 0.1 0.2 0.1 0.3

0.6 N3 0.2 0.1 0.1 0.06 N3 0.1 0.08 0.1 0.1 0.3

0.3

0.04

0.2

0.08

N3 N3 N3 0.2 0.1

1 N3 0.6 0.1 0.03

N3

N3

N3 N3 Strong interference 0.2 5

N3 N3 N3 0.2 2

References.

taken into account [31,34,35]. As far as dimers were concerned, procyanidin B5 appeared as the most active compound, confirming that carbonyl scavenging dominantly occurs at the C-8 position for flavonoids [36]. The ability of procyanidins to prevent endothelial dysfunction and relax blood vessels through antioxidant mechanisms was already reported [37–41]. However, when compounds 3–5 were tested alone (100 μM, i.e. 58, 87 and 29 μg/mL respectively), neither anti-inflammatory nor vasorelaxant activity could be detected, suggesting a strong synergic effect between mono- and oligomeric flavan-3-ols in M. neurophylla MeOH bark extract. 2.3. Anti-AGE guided fractionation procedure of the DCM bark extract The M. neurophylla DCM bark extract was fractionated by flash chromatography to give 26 fractions (FI to XXVI), which were tested for their anti-AGE properties [30]. Among them (Supplementary data, Fig. S3), only those more active than the natural reference compound quercetin (IC50 ≤ 0.06 mg/mL), were selected for a further bioguided fractionation [30]. Fraction VI was flash chromatographed to give mammea A/AA (11) and mammea A/AB (12). Those major compounds (Fig. S2B) appeared to be responsible for the excellent anti-AGE activity of the crude extract (Table 2). Disparinol B (13), mammea A/AB cycloE (14), ochrocarpin A (15) and a mixture of mammea A/AA

cycloF (16) and mammea A/AB cycloF (17) (40/60) were also isolated. Fraction VIII also induced a strong anti-AGE effect due to mammea E/BA (18) and mammea E/BB (19) isolated as an inseparable mixture (Table 2). Two new compounds 20 and 21 were also isolated as a white, optically active ([α]25 D : − 40°, 0.25, EtOH) inseparable mixture. The HRFABMS exhibited a pseudomolecular [M + H]+ ion at m/z 447.2027 which corresponds to the molecular formula C24H30O8 (calcd for [M + H]+: 447.2013). The UV spectrum (MeOH) (λmax = 222, 295, sh 325 nm) indicated a 4-alkyl-5,7-dihydroxycoumarin exhibiting a 8-acyl substituent [42–44]. The ESI–MS2 in negative mode showed a pseudomolecular [M − H]− ion at m/z = 445 accompanied by a proeminent fragment ion at m/z 385 [M\AcOH\H]−, suggesting an 1-acetoxypropyl group in position 4 (mammea E type coumarin). This was confirmed by the 1H NMR spectrum (Table 3), which exhibited signals at δH = 6.59 (1H, dd, J = 8.0, 2.0 Hz, H-1′), 2.00 (1H, m, H-2′a), 1.67 (1H, m, H-2′b), 1.03 (3H, t, J = 7.5 Hz, H-3′) and 2.17 (3H, s, OCOCH3). The broad doublet at δH = 6.27 (1H, d, J = 0.7 Hz, H-3) showed a long range allylic coupling with H-1′ in the COSY spectrum, indicating the substitution at C-4 by the 1-acetoxypropyl group. The singlets at δH = 10.28 (1H, OH-5) and 14.68 (1H, OH-7) were associated to phenol protons, the latter being hydrogen bonded to a carbonyl group. On the one hand, the presence of a 2-hydroxy-3-methylbut-3-enyl moiety at C-6 was deduced from characteristic signals [45]: two benzylic proton signals at δH = 2.95 (1H, dd, J = 7.5 and 15.0 Hz, H-1″a) and 3.15 (1H, d, J = 15.0 Hz, H-1″b), an oxymethine proton

Table 3 1 H (500 MHz) and 13C (125 MHz) NMR data (δTMS = 0 ppm) for 20 and 21. Neurophyllol B 20

Neurophyllol A 21

Position

δC

δC

2 3 4 4a 5 6 7 8 8a 1′ 2′

159.9 106.2 158.2 101.4 160.3 110.0 166.8 104.0 156.5 74.1 29.0

3′ 1″

10.5 28.6

2″ 3″ 4″

77.8 146.2 110.9

5″ 1‴ 2‴ 3‴ 4‴

18.8 210.8 47.1 16.8 27.4

5‴ 5-OH 7-OH OCOCH3 OCOCH3

11.9

21.3 170.6

δH (mult., J in Hz) 6.28 (d, 0.7)

6.59 1.67 2.00 1.03 2.95 3.15 4.45

(dd, 2.0, 8.0) (m) (m) (t, 7.5) (dd, 7.5, 15.0) (d, 15.0) (d, 7.5)

4.89 (s) 4.98 (s) 1.85 (s) 3.90 (m) 1.25 (d, 6.5) 1.46 (m) 1.89 (m) 0.97 (t, 7.5) 10.28 (s) 14.68 (s) 2.17 (s)

159.9 106.2 158.2 101.4 160.3 110.0 166.7 104.4 156.5 74.1 29.0 10.5 28.6 77.8 146.2 110.9 18.8 206.4 53.7 25.8 22.9 22.9

21.3 170.6

δH (mult., J in Hz) 6.27 (d, 0.7)

6.63 1.67 2.00 1.03 2.95 3.15 4.36

(dd, 1.5, 7.0) (m) (m) (t, 7.5) (dd, 7.5, 15.0) (d, 15.0) (d, 9.0)

4.92 (s) 5.04 (s) 1.87 (s) 3.15 (d, 6.5) 2.26 (m) 1.02 (d, 6.5) 1.02 (d, 6.5) 10.28 (s) 14.68 (s) 2.18 (s)

B.T. Dang et al. / Fitoterapia 96 (2014) 65–75

signal at δH = 4.45 (1H, br d, J = 7.5 Hz, H-2″), two olefinic gem-proton signals at δH = 4.89 and 4.98 (each 1H, s, H-4″), and finally a methyl proton signal at δH = 1.85 (3H, s, H-5″). The presence of this substituent was confirmed by 13C NMR with specific δC at 28.6 (C-1″), 77.8 (C-2″), 146.2 (C-3″), 110.9 (C-4″) and 18.8 (C-5″). On the other hand, the nature of the substituent at C-8 was deduced to be a 1-oxo-2-methylbutyl chain from proton signals at δH = 3.90 (1H, m, H-2‴), 1.25 (3H, d, J = 6.5 Hz, H-3‴), 1.89 (1H, m, H-4‴a), 1.46 (1H, m, H-4‴b) and 0.97 (3H, t, J = 7.5 Hz, H-5‴) and carbon signals at δC = 210.8 (C-1‴), 47.1 (C-2‴), 16.8 (C-3‴), 27.4 (C-4‴) and 11.9 (C-5‴) [44]. The presence and position of these substituents were confirmed by 2D NMR experiments (COSY, HMQC, HMBC) (Fig. 2). This new compound was named neurophyllol B (20), following the nomenclature proposed by Crombie et al. [46]. 1 H and 13C NMR data for 21 appeared as very close to the aforementioned ones (Table 3) since only slight differences could be found concerning the chemical shifts associated with the 8-acyl group. Indeed 21, exhibiting a 3-methylbutyryl group instead of a 1-oxo-2-methylbutyl group at C-8, showed the corresponding proton signals at δH = 3.15 (1H, d, J = 6.5 Hz, H-2‴), 2.26 (1H, m, H-3‴) and 1.02 (6H, d, J = 7.0) and carbon signals at δC = 206.4 (C-1‴), 53.7 (C-2‴), 25.8 (C-3‴), 22.9 (C-4‴ and C-5‴) [44]. The presence and position of this group were firmly confirmed by 2D NMR experiments (COSY, HMQC, HMBC) (Fig. 2). Following Crombie's nomenclature this new compound was named neurophyllol A (21). The absolute configuration at C-2″ in 20 and 21 was determined using the modified Mosher's method [47]. Briefly the MTPA esters were obtained by treating the mixture of 20 and 21 (83/17) with (R)- and (S)-MTPA chloride. Their 1H NMR resonances in pyridine-d5 were then assigned on the basis of an HMQC experiment and by comparison with data obtained in CDCl3. The drift in proton chemical shifts (Δδ = δS − δR)

69

Fig. 3. Difference in the Δδ (δS − δR) values for the (S)- and (R)-MTPA esters of 20 in pyridine-d5.

clearly indicated that 20 and 21 possess the same C-2″ R-configuration. For 20, on the basis of literature data [48], it was also assumed that the configuration at C-2‴ was S. Finally, all mammea E-type coumarins previously isolated had a C-1′ S-configuration [18,48,49]. Thus the absolute configuration of 20 and 21 was expected to be as shown in Fig. 3. Compounds 20 and 21 (83/17) were evaluated for their anti-AGE activity. They inhibited AGE formation in the same range as mammea A/AA (11) (IC50 = 80 μM). Finally, fractions XIII to XV were also implicated in M. neurophylla DCM extract anti-AGE activity. Mammea A/AA cycloF (16) and mammea A/AB cycloF (17) were again isolated from fraction XV and detected as its major compounds. There were also present in large amounts in fractions XIII and XIV, associated with other (anti-AGE) coumarins (data not shown). From fraction XIII, the inactive 2-hydroxyxanthone (22) was also isolated.

Fig. 2. Key COSY (left), HMBC (right) correlations for neurophyllol B (20) and neurophyllol A (21).

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A)

B)

200 140

Cell viability (%)

160 140 120 100 80 60

VCAM-1 expression (%)

180

120 100

0.5 µM 1 µM

80

5 µM 60 40

10 µM 50 µM

40 20 0

20 0

Fig. 4. Mammea A/AA (11) effects on endothelial cells. Effect of extracts (0.5 to 50 μM) on endothelial cell viability (A) and TNFα-induced VCAM-1 expression in endothelial cells (B). PDTC (200 μM) and quercetin (100 μM) were used as positive control and reduce by 25 and 70% of VCAM-1 expression respectively (data not shown).

A systematic phytochemical study was also undertaken on fraction V, less active but representing more than 20% by weight of the extract. Therefore fraction V was fractionated using flash chromatography to give δ-tocotrienol (23) in large amounts, accompanied by 4-phenylcoumarins mammea A/AA cycloD (24), mammea A/AB cycloD (25), mammea A/BA (26), mammea A/BB (27) (58/42) and 1-hydroxy-7methoxyxanthone (28). Finally, inactive triterpenes, friedelin (29) and betulinic acid (30) as well as a derivative of phenylalanine, R, S-asperphenamate (31), directly precipitated respectively from fractions III, IX and XXI. 31 was previously isolated from Penicillium and Aspergillus fungi [50–55] so an endophytic origin is not to be excluded. Mammea coumarins isolated in sufficient amount were individually tested for their anti-AGE potential. It appeared that the more potent compounds (IC50 b 100 μM) always possessed free phenolic hydroxyl groups at C-5 and C-7 (11, 18–21, 26–27) whereas a cyclization decreased the anti-AGE effect (14, 15–17, 24–25) (Table 2 and Chart 1). Concerning the mechanism of action of mammea coumarins, it is known that antioxidants acting by radical scavenging or metal chelation [56–58] and compounds able to trap dicarbonyl species or even break AGEs usually contribute to limit their accumulation [59]. The anti-AGE activity of mammea coumarins may thus be directly linked to their antioxidant potential since noncyclized derivatives were already described as the best antioxidant ones [21]. 2.4. Anti-inflammatory effect on endothelial cells and vasorelaxing potential of mammea A/AA As depicted in Fig. S2B, mammea A/AB (12) and mammea A/AA (11) were the major compounds in the DCM bark extract. 11 exhibiting a strong anti-AGE effect (Table 2) was also evaluated for its anti-inflammatory potential on endothelial cells. At a concentration of 50 μM, 11 inhibited VCAM-1 expression in response to TNFα with a maximal inhibitory

effect ca. 60% (Fig. 4). This apparent anti-inflammatory activity was not associated with a cytotoxic effect. Concerning the role of mammea A/AA in the vasorelaxant activity of M. neurophylla DCM bark extract, it should be noted that a similar effect was already observed for a M. africana extract containing the same coumarin. The role of vascular endothelium was suggested in this previous work [6] and demonstrated in the present study. 3. Conclusion AGEs are implied in endothelial dysfunction associated with pathologies such as high blood pressure and diabetes. Plants belonging to Clusiaceae and Calophyllaceae, in particular from Mammea genus, are used in traditional medicine in cases of arterial hypertension or diabetes. A screening of anti-AGE potential of extracts from these families thus allowed us to select M. neurophylla for further investigations. DCM and MeOH bark extracts, rich in 4-phenyl- and 4-(1-acetoxypropyl)coumarins and procyanidins respectively, demonstrated their ability to prevent inflammation in endothelial cells and vasoconstriction. More particularly, a bioguided fractionation allowed us to point out the interest of mammea coumarins exhibiting free phenol groups as well as procyanidins as inhibitors of AGE formation and their potential to prevent endothelial dysfunction. The anti-AGE-guided fractionation of the DCM bark extract led to the isolation of two new mammea E coumarins, namely neurophyllols A and B (20–21). Thirteen known mammea A and E coumarins were also isolated from DCM bark extract, together with known tocotrienols, xanthones and triterpenes. During this study, R,S-asperphenamate was also purified and suggested to be from an endophytic origin. Finally, bark from this endemic Mammea species from New Caledonia shares a similar composition with that of the Mammea from South and Southeast Asia. Indeed, mammea A and E coumarins, hydroxyxanthones and catechins were also isolated from the bark or twigs of M. acuminata [60], M. harmandii [61], M. longifolia [62] and M. siamensis [63]. However, when mammea A/A coumarins are major compounds

B.T. Dang et al. / Fitoterapia 96 (2014) 65–75

71

Chart 1. Coumarins isolated from M. neurophylla DCM bark extract.

from M. neurophylla bark, M. harmandii twigs rather synthesize mammea A/B coumarins and M. longifolia and M. siamensis bark produce mainly mammea E coumarins. 4. Experimental section 4.1. General experimental procedures Centrifugal partition chromatography (CPC) was performed using a fast centrifugal partition chromatograph FCPC 200 (Kromaton, Angers, France). The total volume of the cell is 275 mL. A valve incorporated in the CPC apparatus allows operation of either descending or ascending mode. The system is equipped with a gradient pump, a UV/vis detector, a rheodyne valve with a 10 mL sample loop and a fraction collector (Kromaton). Flash chromatography purifications were made using an Agilent 971-FP instrument (Agilent Technologies, Massy, France) or a CombiFlash Rf-200 system (Teledyne Isco, Lincoln, USA) containing binary pumps, multi-wavelength

UV detectors and fraction collectors. The samples were adsorbed to silica gel Si60 prior to introduction and a DASi™ sample injection module (Agilent Technologies) was used. For MeOH bark extract, Puriflash PF-30SiHP/4G cartridges (Interchim, Clichy, France) were used and the flow rate was 5 mL/min [64]. For DCM bark extract, various stationary and mobile phases and flow rates were used and are detailed in part 5.4. Purifications were also conducted using open-column chromatography with Sephadex LH-20 (Sigma-Aldrich, St. QuentinFallavier, France). Preparative HPLC were performed onto a PrepStar (Varian S.A., France) assisted by a ProStar/Dynamax software (Varian). HPLC analysis were performed on a Waters 2695 apparatus (Waters, Guyancourt, France) consisting of a pumping system, a vacuum degasser and a DAD detector, assisted by the Empower 2 software (Waters). A 10 μL sample (1 mg/mL) was directly injected onto a Lichrospher 100 RP18 column (150 × 4.6 mm, 5 μm, Merck, Darmstadt, Germany) using an acidic water/MeOH system. For MeOH bark extract, the mobile phase was as follows: water (HCOOH-0.1%)/MeOH

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(95:5 v/v to 40:60 v/v at 30 min, 10:90 v/v at 35 min and 10:90 v/v for 10 min). For DCM bark extract, the mobile phase consisted of a water (HCOOH-0.1%)/MeOH gradient from 20:80 to 0:100 in 25 min, followed by 100% methanol for 10 min. The flow rate was 1 mL/min with UV detection at 235 nm or 280 nm for MeOH bark extract and 290 nm for DCM bark extract. 1H, 13C and 2D NMR spectra were recorded in the appropriate deuterated solvent on a Bruker Avance DRX 500 MHz (Bruker France, Wissembourg, France) or a Jeol GSX 270 MHz (Jeol Europe, Croissy-sur-Seine, France) spectrometer. Mass spectra were recorded on an Esquire 3000 PLUS apparatus (Bruker). 4.2. Plant material M. neurophylla bark, leaves and fruits were collected in November 1998 in the dry forest of “Conservatoire botanique de la forêt de Tiéa”, North Province (New Caledonia). A voucher specimen (LIT-0660) was deposited at the Herbarium of the Botanical and Tropical Ecology, Department of the IRD Center, Noumea (New Caledonia) [25]. 4.3. Extraction Extractions of M. neurophylla bark, leaf and fruit were performed with a Dionex ASE 200 instrument equipped with a solvent controller (Dionex S.A., Voisins Le Bretonneux, France). The system consists of a high pressure pneumatic solvent pump capable of 1500 psi at an elevated flow rate; an extraction solvent pressurized bottle; a carousel for 24 extraction cells of 1–33 mL; a carousel for 40 or 60 mL collection vials; a microprocessor for storing and editing parameters such as temperature, time, and pressure; and infra-red (IR) sensors to detect the fluid arrival into the collection vial and monitor fluid levels during extract collection. In this work, extraction cells of 33 mL were used and filled with 10 g of the vegetable material, reduced in fine powder and mixed with sand. Extractions were performed at 100 °C with DCM and MeOH, at a constant pressure of 100 bars. The static extraction time was 10 min and the purge time was 120 s. Two cycles of operation were applied with a flush of 100%. MeOH extracts (100 mg) were filtered on Upti-Clean polyamide SPE cartridges (Interchim, Montluçon, France). 4.4. Extraction and purification on bark extracts Air-dried and powdered bark (695 g) was extracted with DCM (72 h) and MeOH (72 h) in a Soxhlet apparatus. The DCM and MeOH crude extracts were concentrated under vacuum at 40 °C to yield respectively 29 g and 124 g of dry extracts. 30 g of MeOH extract were partitioned between in EtOAc (3 g) and H2O (27 g). Initial fractionation of the EtOAc extract (1 g × 2) was carried out using centrifugal partition chromatography (CPC) with heptane/EtOAc/MeOH/H2O (1:6:1:6) as a biphasic system [65] in ascending mode to yield 11 fractions (F1 to F11). F2 (113 mg) was subjected to flash chromatography eluted with DCM/EtOAc (100:0 v/v to 70:30 v/v for 30 min and 70:30 to 50:50 v/v for 60 min) to afford 3,4 dihydroxybenzoic acid 2 (22 mg) [66]. F3 (95 mg) was subjected to flash chromatography eluted with DCM/MeOH (96:4 v/v to 90:10 v/v for 70 min) to give 2 (15 mg), (−)-

epicatechin 5 (1 mg) [67,68] and a fraction which gave procyanidin A2 7 (4 mg) [69] and astilbin 9 (10 mg) [70] after filtration on a Sephadex LH-20 column. F4 (167 mg) was subjected to flash chromatography eluted with DCM/MeOH (98:2 v/v to 90:10 v/v for 60 min) to afford 5 (1 mg) and a mixture of neoastilbin 8 and isoastilbin 10 (6 mg) [71]. F5 (154 mg) was subjected to flash chromatography eluted with DCM/MeOH (96:4 v/v to 90:10 v/v for 70 min) to give gallic acid 1 (1 mg) [72], 5 (8 mg) and procyanidin B5 6 (9 mg) [68,73]. DCM bark extract (18 g) was subjected to flash chromatography using a PuriFlash PF-50SiHC/300G cartridge (Interchim) eluted with C6H12/EtOAc (95:5 v/v to 70:30 v/v for 120 min, 50 mL/min) to afford 26 fractions, namely FI to FXXVI in elution order. Fraction V (500 mg × 2) was subjected to reverse flash chromatography (PuriFlash PF-50C18/175G, Interchim) using MeOH/H2O 0.1% HCOOH (80:20 v/v to 90:10 v/v for 110 min, 20 mL/min) to give 1-hydroxy-7-methoxyxanthone (28) (2 mg) [74] a mixture of mammea A/BA (26) and mammea A/BB (27) (51 mg) [75], mammea A/AA cycloD (24) (70 mg), mammea A/AB cycloD (25) (56 mg) [76], and δ-tocotrienol (23) (479 mg) [77]. From fraction VI (7.3 g), a precipitation (1.3 g) occurred in a mixture of cyclohexane/EtOAc. The precipitate (VI-1, 800 mg) was subjected to flash chromatography using a Chromabond® Flash RS80 SiOH cartridge (Macherey-Nagel, GmbH & Co. KG, Germany) eluted with C6H12/EtOAc (100:0 v/v to 96:4 v/v for 190 min, 20 mL/min) to yield mammea A/AA (11) (63 mg) and mammea A/AB (12) (7 mg) [75]. The filtrate (VI-2, 300 mg) was fractionated by flash chromatography using a Chromabond® Flash RS15 SiOH (Macherey-Nagel) eluted with C6H12/EtOAc (95:5 v/v to 90:10 v/v for 20 min and 90:10 v/v to 80:20 v/v for 130 min, 3 mL/min) to obtain 12 fractions, VI-2.1 to VI-2.12. Fraction VI-2.2 was chromatographed over silica gel (DCM–EtOAc, 99:1 v/v) to give disparinol B 13 (4 mg) [44]. The purification of the major compound from fraction VI-2.4 (18 mg) was performed by injecting 5 mL (5 × 1 mL) of a methanolic solution onto an Omnispher C18 (250 × 21.4 mm, 10 μm) column (Varian). Elution was performed using a PrepStar 218 binary pump (Varian) to deliver a constant flow rate of 20 mL/min. The mobile phase consisted of MeOH/water (65:25). Mammea A/AB cycloE (14) (4 mg) [18] was detected at 290 nm using a ProStar 325 UV–vis detector (Varian) and collected with a ProStar collector (Varian). Fraction VI-2.5 (19 mg) contained pure ochrocarpin A (15) [18]. Fraction VI-2.6 (87 mg) contained a mixture of mammea A/AA cycloF (16) and mammea A/AB cycloF (17) [75]. Fraction VIII was subjected to reverse flash chromatography using a PuriFlash PF-50C18HP/35G cartridge (Interchim) eluted with 0.1% HCOOH in water/MeOH (30:70 v/v to 20:80 v/v for 135 min, 20 mL/min) to obtain a mixture of mammea E/BA (18) and mammea E/BB (19) (28 mg) [21]. The residue was purified using a GraceResolv™ silica/24G cartridge (Grace Davison Discovery Sciences, Epernon, France) eluted with C6H12/EtOAc (100:0 v/v to 75:25 v/v for 85 min, 16 mL/min) to afford a mixture of neurophyllol B (20) and neurophyllol A (21). Compounds 29 (55 mg), 30 (20 mg), 22 (10 mg) and 31 (10 mg) precipitated from fraction III (95 mg) in a mixture of DCM/EtOAc and from fractions IX (220 mg), XIII (200 mg) and XXI (200 mg) in a mixture of cyclohexane/EtOAc. They were

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identified as friedelin [78], betulinic acid [79], 2-hydroxyxanthone [80] and R,S-asperphenamate [81] respectively. 4.5. Preparation of (R)-MTPA and (S)-MTPA esters of 20 and 21 A solution of 20 + 21 (83/17) (0.5 mg) in pyridine-d5 (250 μL) was divided into 3 parts. 50 μL were used to register 1 H NMR reference spectrum. To 100 μL, (R)-MTPA chloride (1 μL) or (S)-MTPA chloride was added and mixtures were transferred in two NMR tubes. The reaction was monitored by 1 H NMR (500 MHz) spectroscopy and found to be complete after 4.5 h at 25 °C. 4.6. Anti-AGE assay Inhibition of AGE accumulation was evaluated according to Séro et al. [30]: the 96-well microtiter plate assay was automated on a Freedom Evo® 100 liquid handling workstation (Tecan Lyon, France). The liquid handling (LiHa) arm was equipped with four LiHa standard, fixed, washable tips (Teflon®-coated stainless steel, resistant to DMSO, Tecan). Dispensing steps, i.e. liquid class parameters, were optimized and programmed using the Evoware® software (Tecan). The assay involved incubating bovine serum albumin (BSA, 10 mg/mL) with D-ribose (0.5 M) and the tested compound (3.10−6 to 3.10−3 M) or extract (10−6 to 1 mg/mL) in a phosphate buffer, 50 mM, pH 7.4 (NaN3 0.02%). Solutions (100 μL) were incubated in 96-well microtiter plates at 37 °C for 24 h in a closed system before AGE fluorescence measurement. To avoid quenching phenomena, the fluorescence resulting from the incubation, under the same conditions of BSA (10 mg/mL) and the tested compound (3.10−6 to 3.10−3 M) or extract (10−6 to 1 mg/mL), was subtracted for each measurement. A negative control, i.e. 100% inhibition of AGE formation, consisted of wells with only BSA. A positive control, i.e. no inhibition of AGE formation, consisted of wells with BSA (10 mg/mL) and D-ribose (0.5 M). The final volume assay was 100 μL. Vesperlysines-type and pentosidine-type AGE fluorescence (λexc 370 nm; λem 440 nm and λexc 335 nm; λem 385 nm) was measured using an Infinite M200 (Tecan, Lyon, France) microplate spectrofluorometer and Magellan (Tecan) software. The compound or extract concentration for a 50% inhibition (IC50) was calculated from the data and compared with that of reference compounds quercetin and aminoguanidine. As previously published, this assay can be performed using a single concentration. 4.7. Biological assays 4.7.1. Endothelial cell culture and activation Human primary vascular ECs (HUVECs) were purchased from Lonza (Lonza Verviers SPRL, Verviers, Belgium) and used between passages 2 and 5. ECs were cultured in endothelial cell basal medium (ECBM) supplemented with 10% fetal calf serum (FCS), 0.004 mL/mL ECGS/heparin, 0.1 ng/mL hEGF, 1 ng/mL hbFGF, 1 μg/mL hydrocortisone, 50 μg/mL gentamicin and 50 ng/mL amphotericin B (C-22010, PromoCell, Heidelberg, Germany). For activation, confluent EC monolayers were starved overnight and incubated with recombinant human TNFα (R&D Systems, Lille, France) for the indicated period of time in ECBM supplemented with 2% FCS. When applicable, cells were

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pre-incubated with inhibitors for 1 h before incubation with TNFα (400 U/mL). Inhibitors namely as pyrrolidine dithiocarbamate (PDTC), 4-hydroxy-TEMPO (TEMPOL) and acetovanillone (apocynin), purchased from Sigma-Aldrich (Saint-Louis, MO, USA), were used at 200 μM for PDTC and 2 mM for TEMPOL and 400 μM for apocynin. 4.7.2. Cellular ELISA assays for membrane-bound VCAM-1 For cellular ELISA, ECs were grown to confluence and then treated in triplicates with cytokine (TNFα, 200 U/mL) for 6 h with or without the extracts on 96-well plates (Nunc, Roskilde, Denmark) pre-coated with 1% gelatin. After treatment, cells were fixed in glutaraldehyde. VCAM-1 detection was achieved using anti-VCAM-1 mouse IgG (R&D Systems). Assays were processed using anti-mouse IgG2a-horseradish peroxidase (Cell Sciences™ Inc., Canton, MA, USA) (1:4000) and developed using the ABTS peroxidase substrate reagent (Roche Diagnostics, Indianapolis, IN, USA). Optical density (OD) was measured at 405 nm. Results were expressed as a percentage of inhibition of VCAM-1 expression relative to the positive control with TNFα alone. 4.7.3. Cellular viability assay Cell toxicity was quantified by MTT colorimetric assays. ECs were plated onto 96-well plates (Nunc) pre-coated with 1% gelatin at 1 × 104 cells/well. After treatment, cell viability was assessed by incubation with 1 mg/mL MTT (3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazodium) for 4 h at 37 °C and recording the OD at 570 nm. Experiments were performed in triplicates, and results are expressed as a percentage ± SEM values. The relationship between OD and cell number was determined to be linear by the regression curve and the equation of the curve allowed us to determine the cell number for each treatment. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. 4.7.4. Contraction experiments Male Wistar rats (250 g) were used. Rat aortas and pulmonary arteries were collected in physiological saline solution (in mM: 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, 10 Tris, pH 7.4 with HCl) and cut in rings. Arterial rings were then suspended under isometric conditions, connected to a force transducer (Pioden Controls Ltd., Canterbury, UK) in a Krebs– Henseleit solution at 37 °C bubbled with 95% O2–5% CO2. After equilibration, the response to KCl 60 mM was determined. The presence and the functionality of the endothelium were checked by adding carbachol (CCh, 10 μM) to rings pre-contracted by phenylephrine (PhE, 1 μM). Similarly, the relaxing effect of extracts was tested by adding increasing concentrations of M. neurophylla extracts to rings precontracted by PhE (1 μM). In some experiments performed in de-endothelialized, the endothelium was removed by a gentle mechanical rubbing of the intimal surface. All experiments were conducted in accordance with international guidelines for the care and use of laboratory animals. 4.7.5. Statistical analysis Values are expressed as mean ± SEM of n independent experiments or samples. Statistical analysis was performed

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by two tailed, unpaired Student's t test. A P value of P b 0.05 was considered significant. Acknowledgment This work was supported by a grant from the Vietnamese Government (BTD). PT was supported by “PROVASC”, a grant from la “Région Pays de la Loire”. We thank B. Siegler and Dr. I. Freuze for their assistance in NMR spectroscopy and EIMS respectively. We are very grateful to the North and South Provinces of New Caledonia who facilitated our field investigation. We express our thanks to Dr. J.-M. Veillon of the Botany and Plant Ecology Department, Institut de Recherche pour le Développement (IRD), Nouméa, for his assistance in the botanical determination and B. Fogliani for the plant collection. Appendix A. Supplementary data Figures S1 to S3, Table S1, and Charts S1 and S2 as well as NMR spectra of compounds 20 and 21 can be found, in the online version at http://dx.doi.org/10.1016/j.fitote.2014.04.005. References [1] IPNI. The International Plant Names Index. 06-02-2013. Online Source, Available at: http://www.ipni.org/; 2012. [2] Stevens PF. Angiosperm Phylogeny Website, Version 12. 06-02-2013. Online Source, Available at: http://www.mobot.org/MOBOT/Research/ APweb/; 2012. [3] Ruhfel BR, Bittrich V, Bove CP, Gustafsson MH, Philbrick CT, Rutishauser R, et al. Phylogeny of the clusioid clade (Malpighiales): evidence from the plastid and mitochondrial genomes. Am J Bot 2011;98:306–25. [4] Nguelefack-Mbuyo EP, Dongmo AB, Nguelefack TB, Kamanyi A, Kamtchouing P, Dimo T. Endothelium/nitric oxide mediates the vasorelaxant and antihypertensive effects of the aqueous extract from the stem bark of Mammea africana Sabine (Guttiferae). J Evid Based Complement Altern Med 2012;2012:1–8 (Article 961741). [5] Nguelefack-Mbuyo PE, Nguelefack TB, Dongmo AB, Afkir S, Azebaze AGB, Dimo T, et al. Anti-hypertensive effects of the methanol/methylene chloride stem bark extract of Mammea africana in L-NAME-induced hypertensive rats. J Ethnopharmacol 2008;117:446–50. [6] Dongmo AB, Azebaze AGB, Nguelefack TB, Ouahouo BM, Sontia B, Meyer M, et al. Vasodilator effect of the extracts and some coumarins from the stem bark of Mammea africana (Guttiferae). J Ethnopharmacol 2007;111:329–34. [7] Tchamadeu MC, Dzeufiet PDD, Nouga CCK, Azebaze AGB, Allard J, Girolami JP, et al. Hypoglycaemic effects of Mammea africana (Guttiferae) in diabetic rats. J Ethnopharmacol 2010;127:368–72. [8] Steinrut L, Itharat A, Ruangnoo S. Free radical scavenging and lipid peroxidation of Thai medicinal plants used for diabetic treatment. J Med Assoc Thai 2011;94:S178–82. [9] Paravicini TM, Touyz RM. Redox signaling in hypertension. Cardiovasc Res 2006;71:247–58. [10] Higashi Y, Kihara Y, Noma K. Endothelial dysfunction and hypertension in aging. Hypertens Res 2012;35:1039–47. [11] Tang EH, Vanhoutte PM. Endothelial dysfunction: a strategic target in the treatment of hypertension? Pflugers Arch 2010;459:995–1004. [12] Paniagua OA, Bryant MB, Panza JA. Transient hypertension directly impairs endothelium-dependent vasodilation of the human microvasculature. Hypertension 2000;36:941–4. [13] Maillard LC. Action des acides aminés sur les sucres; formation des mélanoïdes par voie méthodique. C R Acad Sci 1912;154:66–7. [14] Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813–20. [15] Versari D, Daghini E, Virdis A, Ghiadoni L, Taddei S. Endothelial dysfunction as a target for prevention of cardiovascular disease. Diabetes Care 2009;32: S314–21. [16] Widlansky ME, Gokce N, Keaney Jr JF, Vita JA. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 2003;42:1149–60.

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