A tocotrienol series with an oxidative terminal prenyl unit from Garcinia amplexicaulis

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A tocotrienol series with an oxidative terminal prenyl unit from Garcinia amplexicaulis

Alexis Lavaud

^a,b

, Pascal Richomme

^a

, Julia Gatto

^a

, Marie-Christine Aumond

^a

, Cyril Poullain

^c

, Marc Litaudon

^c

, Ramaroson Andriantsitohaina

^b

, David Guilet

^a,^⇑

aUniversité d’Angers, Laboratoire SONAS, IFR Quasav, 49100 Angers, France

bINSERM UMR U1063, IBS-IRIS, Université d’Angers, 49100 Angers, France

cCentre de Recherche de Gif, Institut de Chimie des Substances Naturelles (ICSN), CNRS, Labex LERMIT, 91198 Gif sur Yvette Cedex, France

a r t i c l e i n f o

Article history:

Received 21 July 2014

Received in revised form 17 October 2014 Available online 13 November 2014

Keywords:

Garcinia amplexicaulis Clusiaceae

Tocotrienols Chromanols

Inhibition of lipid peroxidation

a b s t r a c t

Ten tocotrienol derivatives, i.e., amplexichromanols (1–10), were isolated from stem bark ofGarcinia amplexicaulis Vieill. ex Pierre collected in Caledonia. The structures of the compounds 1–5 were determined to be chromanol derivatives substituted by a polyprenyl chain oxidized in terminal position.

The remaining compounds6–10are the corresponding dimeric derivatives. Eleven known compounds, including xanthones, tocotrienol derivatives, triterpenes and phenolic compounds, were also isolated.

Their structures were mainly determined using one and two-dimensional NMR and mass spectroscopy analysis. The compounds and some amplexichromanol molecules formerly isolated fromG. amplexicaulis exhibited signiﬁcant antioxidant activity against lipid peroxidation and in the ORAC assay.

1. Introduction

Tocotrienols and their derivatives, consisting of a polyprenyl chain attached to a chromanol moiety, are widely distributed among plant species and marine organisms. Palm oil, rice bran and annatto seeds are industrially processed to obtain supplies of natural tocotrienols to be used as food additives and in cosmeceu- ticals (Frega et al., 1998; Harinantenaina, 2008). Tocotrienols and tocopherols have long been only associated with vitamin E, i.e., the main lipid-soluble antioxidant in tissues. However, in the last 10 years, only tocotrienols have been reported to possess a pleio- tropic range of biological activities, including inhibition of cholesterol biosynthesis (Pearce et al., 1994), antiangiogenic (Miyazawa et al., 2004) and proapoptotic effects (Agarwal et al., 2004).

In a previous investigation onGarciniaplants,d-amplexichromanol and

c

-amplexichromanol, the two major tocotrienol derivatives from lipophilic extracts ofGarcinia amplexicaulis, were found to be antiangiogenic agents in the low nanomolar range (Lavaud et al., 2013). Intensive phytochemical investigation ofG.

amplexicauliswas undertaken as a follow-up to these studies and in the light of the signiﬁcant antioxidant activity against lipid peroxidation measured for its dichloromethane extract. Here we describe the structural elucidation and bioactivity of ten new

tocotrienol-like compounds, i.e., amplexichromanols (1–10), while eleven known compounds (11–21) were also isolated.

Amplexichromanols (1–5) are chromanols with an oxidative terminal prenyl unit (carboxylic acid, aldehyde or alcohol), while others (6–10) are dimeric structures. The modes of connection between tocotrienol units of bi-amplexichromanols (9–10) are unprecedented in such lipid metabolites.

2. Results and discussion 2.1. Structural elucidation

Dried stem bark ofG. amplexicaulisVieill. ex Pierre, a Clusiaceae species were extracted with dichloromethane (DCM) and then methanol. The DCM extract, which showed signiﬁcant inhibitory activity against lipid peroxidation (Table 5), was fractionated using normal- and reverse-phase ﬂash chromatography followed by preparative HPLC. Through the phytochemical investigation, ten novel tocotrienol-like compounds (1–10) were isolated, elucidated through NMR and mass spectrometry analysis and then partially assessed for their antioxidant activityFig. 1.

c

-(E)-deoxy-amplexichromanal (1) was isolated as a pale yellow oil and its molecular formula was established as C28H40O3

based on the [M+Na]⁺ quasimolecular ion peak observed in the HR-ESIMS spectrum. The spectral feature of1appeared to be very

⇑ Corresponding author. Tel.: +33 241 226 676; fax: 33 241 226 634.

E-mail address:david.guilet@univ-angers.fr(D. Guilet).

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Phytochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h y t o c h e m

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similar to that of

c

-amplexichromanol (Lavaud et al., 2013), which was isolated from the same plant extract. Indeed, typical signals of the farnesyl side chain were noted in the ¹H NMR spectrum (Table 1), with three oleﬁnic protons at d_H 5.12 (1H, t, J= 6.8), 5.14 (1H, t,J= 6.8), and 6.46 (1H, t,J= 7.4), while the chromanol ring was characterized with signals for one aromatic proton atdH

6.37 (1H, s, H-5) and a benzylic methylene at d_H 2.68 (2H, t, J= 6.2, H-4). However,¹H NMR and HMQC spectra revealed that 1 differed from

c

-amplexichromanol by the additional presence of a methyl group atd_H1.73 (3H, s, H-22) and an aldehyde atd_H 9.37 (1H, s, H-21) anddC195.4 (CH), instead of the two oxymethylene groups arounddH4.10–4.30 (2H, s, H-21/H-22) at the terminal isoprene unit. This structural hypothesis was conﬁrmed by the HMBC experiments showing long range correlations between H- 21/C-19, C-20, C-22 and H-22/C-19, C-20, C-21. The Egeometry was assigned on the basis of the NOESY cross-peaks H-21/H-19.

The C16H20O3 molecular formula of compound 2 was determined by HR-ESIMS. The¹H and¹³C NMR data of2showed signal features of the d-chromanol moiety: two aromatic protons atdH

6.40 and 6.50, two oxygenated aromatic carbons atd_C145.3 and 148.2 and two methyl groups atdC24.5 and 16.0 ppm (Table 1).

The peak corresponding to a carbonyl carbon atdC198.4 coupled with UV maximum at 219 nm was indicative of an

a

,b-unsaturated ketone in the prenyl chain. The short prenyl chain of this compound was conﬁrmed by the presence of only two oleﬁnic carbons atd_C143.5 and 134.1 conjugated to the ketone. HMBC long-range correlations of the carbonyl carbon at C-12 with H-10, H-11, H-13 and also the vinyl methylene protons H-9 with C-2, C-3, C-10, C-11 and C-14 correlations allowed the structural elucidation. On the basis of above evidences, 2 was therefore proposed as (2R)-2,8- dimethyl-2-[(2E)-4-oxo-2-penten-1-yl]-chroman-6-ol.

The molecular formula ofd-(E)-deoxy-amplexichromanol (3), i.e., C₂₇H₄₀O₃, was determined by HR-ESIMS and¹³C NMR spectrometry (Table 1). The¹³C NMR data for this compound was very similar to that of d-amplexichromanol, previously isolated from the same plant (Lavaud et al., 2013). Indeed, the main difference concerns the replacement of an oxymethylene with a methyl carbon C-22 atdC13.7 ppm (dH1.66). TheEgeometry of the last double bond was assigned on the basis of NOESY cross-peaks H-21/H-19.

The molecular formula ofd-dihydroxy-amplexichromanol (4) was deduced as C27H42O6by HR-ESIMS associated with¹³C NMR analyses (Table 1). Compound 3 and d-amplexichromanol had eight degrees of unsaturation for the three double bonds and the chromanol ring. In this case, the¹³C NMR data, combined with the fact that there were only seven degrees of unsaturation inher- ent to the molecular formula, showed that compound4lost one of these double bonds. Indeed, this hypothesis was conﬁrmed by the appearance of carbon signals atd_C75.3 and 78.0, and the presence of ten oleﬁnic carbons arounddC110–150 ppm. Interpretation of a proton spin system containing the oxymethine proton H-15 atdH

3.23 (t,J= 10.6), facilitated by long-range correlations between this methine (dC 78.0) and a neighboring methyl group (dH 1.06, dC

21.8), located a hydroxyl substituent at C-15 of the farnesyl chain.

Another nearby hydroxyl substituent was also present at the quaternary carbon C-16 (dC75.4) and conﬁrmed by the HMBC correlations H-15/C-16, H-23/C-16 and H-23/C-17. On the basis of COSY cross-peaks H-14/H-15, the dihydroxylation of the second double bond of the farnesyl chain at C-15 was established. HMBC long-range correlations of the oleﬁnic proton H-11 (dH5.17) with C-24, C-13, C-10, and C-9 and also between the methyl protons H-25 (d_H 1.21) and the same alkyl carbon C-9 corroborated the substitution of the chromanol ring by the prenyl moiety sensu stricto.

d-(E)-Amplexichromanal (5) was isolated as a yellow oil, which was analyzed for C27H38O4by combined HRESIMS and¹³C NMR

spectrometry. The spectral features of5were very similar to that of d-amplexichromanol. Indeed, typical resonances of the chromanol ring and the farnesyl chain were noted in the¹H NMR spectrum (Table 1). However, the¹³C NMR data showed that one primary alcohol function disappeared to the beneﬁt of an aldehyde function (dC 195.9). On the basis of long-range correlations, the carbonyl carbon C-21 was assigned at the terminal isoprene unit.

The geometryEwas determined via combined HMBC correlations (H-19 with C-18, C-20, C-21, C-22) and NOESY cross-peaks (H-21/

H-19, and H-22/H-18).

The molecular formulas ofd,d-bi-O-amplexichromanol (6) and d,

c

-bi-O-amplexichromanol (7), C54H78O8 and C55H80O8, respectively, were determined by HREIMS and¹³C NMR spectrometry.

On the basis of 2D NMR analyses (Table 2), these two dimeric compounds were found to be very similar, with the same modified farnesyl side chain for its monomeric units composed of primary alcohol functions (dC= 60.0 and 67.6) at each terminal isoprene unit. Indeed, the dimeric structure was also confirmed in the¹H NMR spectrum by a comparison of the integration between aromatic protons (e.g., for compound6,d_H= 6.35, 6.53 and 6.70, each signal integrated for one proton) and oxymethylene protons (e.g., for compound6,dH= 4.19 and 4.28, each signal integrated for four protons). The modified farnesyl side chains of compounds6and7 were similar, which suggested that each amplexichromanol unit was connected by the chromanol ring. Indeed, a similar compound, i.e.,

c

,d-bi-O-amplexichromanol, was previously isolated from the same plant extract (Lavaud et al., 2013) and presented the same chemical shifts for the aromatic carbons. Compounds6and7were thus oxidative dimers of d-amplexichromanol and

c

-amplexichromanol. It should be noted that at the difference of their analogous

c

,d-bi-O-amplexichromanol, spatial relationships between the two monomeric sub-units were not observed in NOESY spectra of6 and 7. The aromatic region of the¹H NMR spectrum of 6 showed a singlet (dH 6.70) due to an isolated proton H-7, while two doublets (dH 6.35 and 6.53, both J= 2.6) were due to two meta-related protons, i.e., H-5⁰and H-7⁰ (Table 2). The two units were thus linked through an aromatic carbon at C-5 (dC= 136.9) of one monomer and the oxygen of the hydroxyl at C-6⁰ (dC= 149.6) of the other monomer. Bothd-amplexichromanol units were connected to form compound6. Regarding compound7, the aromatic region of the¹H NMR spectrum still showed a singlet (dH

6.71) due to the isolated proton H-7, but only one singlet (dH6.06) due to the aromatic proton H-5⁰. Compound7differed from com- pound6, with an aromatic proton replaced at C-7⁰ by a methyl group (dC= 12.0, dH= 2.31). Indeed, these spectral features were conﬁrmed by long-range correlations H-27⁰ with C-6⁰/C-7⁰/C-8⁰ and H-5⁰with C-4⁰/C-6⁰/C-8a⁰.

An analogous metabolite, i.e.,d,

c

-biamplexichromanol (8), was analyzed for C₅₅H₈₀O₈by HRESIMS and¹³C NMR spectrometry. The NMR data obtained for this compound were reminiscent of those from compound7(Table 2). Indeed, the¹H and¹³C NMR data of the farnesyl side chain with oxymethylene at the terminal isoprene unit were similar. However, the¹H NMR spectrum revealed only one aromatic proton (dH 6.71) due to an isolated proton H-7. As compared with the¹H NMR and¹³C NMR signals of7and8, the aromatic carbons atdC 137.4 and 109.5 respectively for the C-5 and C-5⁰of7were replaced bydC116.4 and 122.2 in8. On the basis of HMBC experiments, the chromanol assignments were unambig- uously assigned by long-range correlations between H-4/H-4⁰and the quaternary carbons C-5/C-5⁰, and between H-26/H-26⁰ and C-4a/C-4a⁰, and C-7/C-7⁰. These spectral data were quite similar to those of

c

-tocopherol biphenyl dimer (Goh et al., 1990) suggesting that 8 possessed the same carbon–carbon linkage (C-5–C-5⁰) between the monomer units. Thus, compound 8 was determined to be a biphenyl dimer of d-amplexichromanol and

c

-amplexichromanol.

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The molecular formula ofd,d-biamplexichromanoate A (9) was deduced as C₅₄H₇₈O₇by HREIMS analyses and¹³C NMR spectrometry. On the basis of the NMR data and integration of protons in the

1H NMR spectrum, compound 9 appeared as dimeric structures with both tocotrienol units (Table 3). Chemical shifts in the chromanol rings were very similar between the two monomeric units, which probably implicated a linkage by the terminal isoprene units. Indeed, two oxymethylenes (dH 4.08 and 4.70,d_C65.8 and 60.0 ppm) and a carbonyl signal (dC177.4) used as connection were found to be present on the basis of the ¹³C NMR data and

long-range correlations. Then the position of the ester bridge between both units was conﬁrmed by combined HMBC correlations between the methine proton H-20⁰/C-22⁰, methyl protons H-21⁰/C-22⁰ and oxymethylene protons H-21/C-22⁰. Spectral and stereochemical assignments were then achieved through HMBC and NOESY experiments with for examples long-range correlations H-22/C-22⁰ and NOESY cross-peaks H-21/H-19 observations. No other natural tocotrienol derivatives with such an ester bridge are known. All dimers of tocotrienols were only connected by the chromanol ring. In this case, the monomer bearing the carbonyl O

HO

R2

R3

CH3

R₁

2 4a

27 25

12 16

22 21 26

8a 9

24 23

3 5 4

R

1

R

2

R

3

δ -amplexichromanol -H -CH

2

OH -CH

2

OH γ -amplexichromanol -CH

3

-CH

2

OH -CH

2

OH γ -(Z)-deoxy-amplexichromanol -CH

3

-CH

3

-CH

2

OH

1 -CH

3

-CHO -CH

3

3 -H -CH

2

OH -CH

3

5 -H -CHO -CH

2

OH

O HO

O CH₃

2 4a

14

12 15

8a 9

7 5 13

O HO

CH2OH CH2OH CH3

2 4a

25

12 16

22 21 26

8a 9

24 23

OHOH

2 4

O HO

CH₂OH CH₂OH

O O

CH₂OH CH₂OH R

24 23 22

2 4a 7

25

12 16 21

26

8a 9

2' 4a'

27'

25'

12' 16'

22'

21' 26'

8a' 9'

24' 23'

19

19'

O

HO

CH2OH CH₂OH

O HO

CH₂OH CH2OH

24 23 22

2 4a 7

25

12 16 21

26

8a 9

2' 4a'

27' 25'

12' 16'

22'

21' 26'

8a' 9'

24' 23'

19

19'

6 R = -H 8

7 R = -CH

3

22

24 23

2 4a

25

12 16 21

26

8a 9

2' 4a'

25'

12' 16'

22' 21' 26'

8a' 9'

24' 23'

19

19' O

HO

CH2OH H₂C

O

HO O O

O

HO

O HO

O O O O

22

24 23

2 4a

25

12 16 21

26

8a 9

2' 4a'

25'

12' 16'

22' 21' 26'

8a' 9'

24' 23'

19

19'

9 10

Fig. 1.Structures of compounds1–10.

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might have been previously metabolized via oxidative degradation of the prenyl side chain of ad-tocotrienol. This mechanism involves cytochrome P-450-catalyzedx-hydroxylation and oxidation of the

terminal isoprene unit to form a carboxy-tocotrienol derivative (Freiser and Jiang, 2009; Sontag and Parker, 2002). Indeed, this enzyme, which is present in plant species, could act on tocotrienols Table 1

1H (500 MHz) and¹³C (125 MHz) NMR data of compounds1–5(in CDCl3except for4in methanol-d4,din ppm,Jin Hz in parenthesis).

No 1 2 3 4 5

dC dH dC dH dC dH dC dH dC dH

2 75.1 75.0 75.3 76.2 75.2

3 31.3 1.68–1.76, m 31.4 1.79, m 31.4 1.75–1.81, m 32.8 1.71, m 31.4 1.76, m

4 22.1 2.68, t (6.2) 22.3 2.72, m 22.5 2.69, t (6.7) 23.5 2.64, t (6.8) 22.4 2.68, td (6.8 & 2.0)

4a 118.2 120.9 121.2 122.3 121.2

5 112.1 6.37, s 112.6 6.40, d (2.8) 112.6 6.38, d (2.8) 113.6 6.28, d (2.4) 112.5 6.37, d (2.8)

6 146.2 148.2 147.8 150.4 147.8

7 121.6 115.9 6.50, d (2.8) 115.6 6.47, d (2.8) 116.6 6.36, d (2.4) 115.6 6.47, d (2.8)

8 125.7 127.4 127.3 127.8 127.3

8a 145.6 145.3 145.9 146.4 145.8

9 39.7 1.54–1.66, m 42.7 2.45, dd (14.0 & 7.5) 39.3 1.52–1.65, m 40.5 1.50–1.60, m 39.2 1.52–1.63, m 2.55, dd (14.0 & 8.0)

10 22.2 2.12, m 143.5 6.88, ddd (16.0, 8.0 & 7.5) 22.2 2.11, m 23.3 2.10, m 22.1 2.11, m

11 124.4 5.12, t (6.8) 134.1 6.10, d (16.0) 124.4 5.12, t (7.0) 125.8 5.17, t (7.4) 124.5 5.11, t (7.0)

12 134.8 198.4 135.0 136.2 134.7

13 39.5 1.97, m 26.9 2.26, s 39.5 1.98, m 38.0 2.20, m 39.4 1.96, m

14 26.4 2.08, m 24.5 1.28, s 26.4 1.96–2.05, m 30.4 1.30 & 1.73, m 26.4 2.07, m

15 125.5 5.14, t (6.8) 16.0 2.13, s 124.4 5.09, t (7.0) 78.0 3.23, t (10.6) 125.9 5.12, t (7.0)

16 133.3 134.6 75.4 132.9

17 37.9 2.15, m 39.4 1.98–2.07, m 39.4 1.56 & 1.93, m 38.1 2.15, m

18 27.4 2.44, q (7.4) 26.2 2.05–2.10, m 22.3 1.20–1.25, m 27.2 2.48, m

19 154.6 6.46, t (7.4) 126.3 5.38, t (7.0) 131.1 5.55, t (7.4) 156.7 6.58, t (7.5)

20 139.2 134.5 139.0 141.2

21 195.4 9.37, s 69.1 4.00, s 65.6 4.05, s 195.9 9.40, s

22 9.2 1.73, s 13.7 1.66, s 58.2 4.14, s 55.9 4.35, s

23 15.9 1.61, s 16.0 1.59, s 21.8 1.06, d (1.2) 15.8 1.59, s

24 15.8 1.59, s 15.9 1.59, s 16.0 1.57, s 15.8 1.58, s

25 24.0 1.26, s 24.2 1.26, s 24.5 1.21, s 24.2 1.26, s

26 11.9 2.11, s 16.1 2.12, s 16.4 2.03, s 16.1 2.12, s

27 11.9 2.13, s

Table 2

1H NMR data (500 MHz) of compounds6–8(CDCl3,din ppm,Jin Hz in parenthesis).

No 6 7 8

n n⁰ n n⁰ n n⁰

dC dH dC dH dC dH dC dH dC dH dC dH

2 75.0 75.5 75.0 75.4 75.0 75.0

3 30.5 1.64–1.68, m 31.2 1.72–1.78, m 30.5 1.76, m 31.2 1.67, m 31.2 1.61, m 31.3 1.69–1.76, m

4 17.6 2.48, m 22.5 2.69, m 22.3 2.53, m 22.3 2.53, m 20.5 2.12/2.31, m 20.6 2.12/2.31, m

4a 115.2 121.3 123.5 118.1 117.4 120.5

5 136.9 111.8 6.35, d (2.6) 137.4 109.5 6.06, s 116.4 122.2

6 141.2 149.6 141.2 148.0 146.2 146.2

7 115.4 6.70, s 115.0 6.53, d (2.6) 115.2 6.71, s 123.5 115.4 6.71, s 115.7

8 123.4 127.6 114.9 126.2 128.3 126.7

8a 145.5 147.0 145.6 145.6 145.7 144.6

9 39.5 1.52–1.60, m 39.5 1.52–1.60, m 39.5 1.52–1.60, m 39.5 1.52–1.60, m 39.5 1.52–1.64, m 39.5 1.52–1.64, m

10 22.1 2.10, m 22.1 2.10, m 22.0 2.09, m 22.1 2.09, m 22.2 2.06–2.12, m 22.2 2.06–2.12, m

11 124.3 5.11, m 124.3 5.11, m 124.3 5.10, m 124.3 5.10, m 124.4 5.10, m 124.4 5.10, m

12 134.9 135.0 134.9 135.0 134.8 134.8

13 39.3 2.03, m 39.3 2.03, m 39.2 2.01–2.04, m 39.2 2.01–2.04, m 39.0 2.03, m 39.0 2.03, m

14 26.4 2.05, m 26.4 2.05, m 26.4 2.05, m 26.4 2.05, m 26.2 2.06, m 26.2 2.06, m

15 124.9 5.08, m 124.9 5.08, m 124.9 5.09, m 124.9 5.10, m 124.9 5.08, m 78.0 5.08, m

16 134.1 134.1 134.0 134.1 134.0 134.0

17 39.0 1.95, m 39.0 1.95, m 39.1 1.95, m 39.1 1.95, m 39.2 1.99, m 39.2 1.99, m

18 25.9 2.15, m 25.9 2.15, m 25.9 2.15, m 25.9 2.15, m 25.9 2.14, m 25.9 2.14, m

19 130.9 5.52, m 130.9 5.52, m 130.8 5.52, t (7.4) 130.8 5.52, t (7.4) 130.8 5.50, t (7.1) 130.8 5.50, t (7.1)

20 136.9 136.9 137.0 137.0 136.9 136.9

21 67.6 4.19, s 67.6 4.19, s 67.6 4.17, s 67.6 4.20, s 67.5 4.15, s 67.5 4.14, s

22 60.0 4.28, s 60.0 4.28, s 60.0 4.27, s 60.0 4.29, s 59.9 4.25, s 59.9 4.24, s

23 15.9 1.56, s 15.9 1.58, s 15.9 1.58, s 15.9 1.58, s 16.0 1.58, s 16.0 1.58, s

24 15.8 1.58, s 15.8 1.58, s 15.8 1.57, s 15.8 1.57, s 15.8 1.59, s 15.8 1.59, s

25 24.2 1.26, s 24.1 1.24, s 23.8 1.22, s 23.8 1.25, s 24.1 1.26, s 24.1 1.26, s

26 15.8 2.16, s 16.2 2.11, s 15.8 2.17, s 11.9 2.16, s 16.3 2.20, s 12.0 2.17, s

27 12.0 2.31, s 12.3 2.19, s

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to metabolize carboxychromanol derivatives. Then this carboxychromanol might react with an amplexichromanol to form com- pound9.

d,d-Amplexichromanol peroxide (10) was analyzed for C54H76O8

by combined HREIMS and ¹³C NMR spectrometry. The spectral feature of10appeared to be very similar to that of compound9.

Indeed, the NMR data regarding the chromanol ring and the prenyl side chain of each monomeric unit were very similar (Table 3). The differences concerned the terminal isoprene unit and especially the connection between both monomeric units. In comparison to com- pound9, the NMR data revealed the same carboxychromanol unit.

However, the carbonyl signal C-22⁰(dC182.0) was shifted, indicat- ing a different linkage than for10. No oxymethylene carbon around dC60 ppm was present in the¹³C NMR spectrum. However, on the basis of 2D NMR analyses, the oxymethylene carbons of10were replaced by a methyl group (dH1.90,d_C20.5) and a carbonyl signal atdC172.5 ppm. The difference in chemical shifts between the two carbonyl signals (C-22 and C-22⁰) was due to the presence of the double bond at C-19. Indeed, the carbonyl at C-22 was an

a

,b- unsaturated form while the carbonyl at C-22⁰was not conjugated.

The two monomeric units were thus connected by a peroxide bridge, which is unprecedented in natural products. The structural elucidation was conﬁrmed by high-resolution mass spectrometry analysis.

Eleven known compounds were also isolated from the DCM extract ofG. amplexicaulisand identiﬁed as the xanthones, cudrax- anthone G11(Ito et al., 1996), 1,3,5-trihydroxy-4-prenylxanthone 14 (Helesbeux et al., 2004), nigrolineaxanthone F 16 (Rukachaisirikul et al., 2003), and 1,3,7-trihydroxy-2-prenylxanthone 17 (Garcia Cortez et al., 1998), the chromene compounds, garcinal 12 (Terashima et al., 1997), and sargachromanol A 15 (Jang et al., 2005), the phenolic compounds, syringaldehyde 13 (Jalali-Heravi et al., 2004), and naringenin21, and the three known triterpenoids, cabraleadiol18(Nakamura et al., 1997), (205,23E)- eupha-8,23-diene-3b,25-diol19 (Leong and Harrison, 1999) and (3b,11b)-3,11-dihydroxylanosta-8,24-dien-7-one 20 (Lu et al.,

2007). The structures of these compounds were determined on the basis of an analysis of their 1D, 2D NMR and mass spectrometry data as well as on comparisons with literature data.

2.2. Oxidation and lipid peroxidation

Tocotrienol derivatives have been reported to exhibit moderate to signiﬁcant antioxidant activities (Jang et al., 2005; Merza et al., 2004) depending on the free radical scavenger assays used. The antioxidant activity of the isolated compounds (in sufﬁcient quan- tity) was thus assessed by two different methods, i.e. the ORAC assay and TBARS assay against lipid peroxidation (Tables 4 and 5, respectively). In the ORAC assay, tocotrienol isoforms (

a

-,b-,

c

-) were also evaluated for their antioxidant activity to compare the Table 3

1H NMR data (500 MHz) of compounds9–10(CDCl3,din ppm,Jin Hz in parenthesis).

No 9 10

n n⁰ n n⁰

dC dH dC dH dC dH dC dH

2 75.3 75.3 75.3 75.3

3 31.3 1.74–1.84, m 31.3 1.74–1.84, m 31.3 1.74–1.84, m 31.3 1.74–1.84, m

4 22.4 2.68, m 22.4 2.68, m 22.4 2.69, m 22.4 2.69, m

4a 121.2 121.2 121.2 121.2

5 112.5 6.37, br s 112.5 6.37, br s 112.5 6.38, d (2.8) 112.5 6.38, d (2.8)

6 147.8 147.8 147.7 147.7

7 115.6 6.47, br s 115.6 6.47, br s 115.6 6.48, d (2.8) 115.6 6.48, d (2.8)

8 127.3 127.3 127.3 127.3

8a 145.8 145.8 145.9 145.9

9 39.2 1.52–1.64, m 39.2 1.52–1.64, m 39.4 1.54–1.66, m 39.4 1.54–1.66, m

10 22.1 2.10, m 22.1 2.10, m 22.1 2.10, m 22.1 2.10, m

11 124.4 5.11, m 124.4 5.11, m 124.5 5.11, m 124.5 5.11, m

12 134.9 134.9 135.0 135.0

13 39.4 2.01–2.04, m 39.4 2.01–2.04, m 39.5 1.97, m 39.5 1.97, m

14 26.4 2.05, m 26.4 2.05, m 26.5 2.06, m 26.5 2.06, m

15 124.9 5.06, m 124.4 5.11, m 125.0 5.08, m 124.3 5.11, m

16 134.5 134.8 134.0 134.5

17 39.5 1.95, m 26.4 2.05, m 39.0 1.97, m 26.4 2.06, m

18 26.1 2.18, m 25.4 1.92, m 28.1 2.60, q (7.0) 25.3 1.41, m

19 133.4 5.66, t (7.2) 33.3 1.35, m 146.4 6.06, td (7.0 and 1.3) 33.0 1.39, m

20 134.0 39.4 2.44, m 125.9 39.1 2.46, m

21 65.8 4.08, s 17.1 1.13, d (7.0) 20.5 1.90, s 16.9 1.17, d (7.0)

22 60.0 4.70, s 177.4 172.5 182.0

23 15.8 1.58, s 15.9 1.56, s 15.8 1.57, s 15.8 1.57, s

24 15.9 1.58, s 15.9 1.58, s 15.8 1.58, s 15.8 1.58, s

25 24.2 1.26, s 24.2 1.26, s 24.0 1.26, s 24.0 1.26, s

26 16.1 2.12, s 16.1 2.12, s 16.1 2.12, s 16.1 2.12, s

Table 4

ORAC assay results of tocotrienol derivatives.

IC50(lmol TE/lmol) S.D.^c(%) DCM extract G. amplexicaulis 1635^a

a-Tocotrienol n.a.^b –

d-Tocotrienol n.a. –

c-Tocotrienol n.a. –

3 0.74 3.7

4 1.76 1.5

5 0.50 8.1

6 n.a. –

8 n.a. –

9 n.a. 8.7

10 1.80 –

d-Amplexichromanol 1.45 2.8

c-Amplexichromanol 0.92 1.8

d-(Z)-Deoxy-amplexichromanol 1.61 6.1

c-(Z)-Deoxy-amplexichromanol 0.80 8.5

Garcinoic acid 0.64 1.4

aExpressed inlmol TE (Trolox Equivalent)/g extract.

bNo activity recorded.

c Standard deviation.

(6)

result to monomers of tocotrienol derivatives. No free radical scavenger capacity was detected for

a

-,b-,

c

-tocotrienol, while the new compounds3–5,d-amplexichromanol,

c

-amplexichromanol,d-(Z)- deoxy-amplexichromanol,

c

-(Z)-deoxy-amplexichromanol and garcinoic acid displayed significant antioxidant activity in comparison to the Trolox^Òreference, which is an hydrosoluble form of vitamin E. In this assay, the oxidative function at the terminal isoprene unit of the side chain was essential to exhibit antioxidant activity. However, the different oxidative states between compounds had little effect on their antioxidant activity. These results were in compliance with previous results which suggested that only the length of the polyprenyl side chain influenced the potency (Terashima et al., 2002). More interestingly, all amplexichromanol dimers, except compound10, lost their capacity to scavenge oxygen free radicals. Indeed, d,d-amplexichromanol peroxide (10) showed significant antioxidant activity, which was probably due to their poor stability in solution. Its antioxidant activity might result from cleavage of the peroxide bridge and the formation of two monomers. In line with the antioxidant effects of tocotrienols, tocotrienol supplementation reduces blood levels of lipid perox- ides while enhancing blood flow in patients with carotid athero- sclerosis (Tomeo et al., 1995). Indeed, peroxidation of polyunsaturated fatty acids can result in deterioration of biological membranes and production of secondary products, namely reactive carbonyl compounds (RCCs), which are precursors of advanced lipid peroxidation end-products (ALEs) (Negre-Salvayre et al., 2008). ALEs accumulated during ageing, neurodegenerative and oxidative stress related diseases form crosslinks on tissular proteins (carbonyl stress), thus progressively inducing dysfunction and damage in all tissues. Reducing the accumulation of lipid peroxidation products like RCCs may therefore prevent the patholog- ical consequences. Among RCCs, malondialdehyde (MDA) is one of the most abundant aldehydes resulting from the oxidation of polyunsaturated fatty acids. In the assay used to assess the potency of new tocotrienols against lipid peroxidation, MDA reacted with two molecules of thiobarbituric acid (TBA) yielding a pinkish red chro- mogen (thiobarbituric acid reactive substance, TBARS) with an absorbance peak at 532 nm. The DCM extract ofG. amplexicaulis showed 6-fold greater protection against lipid peroxidation than rosmarinic extract, which is used industrially to protect food against oxidative damage. This result justified assessment of the potency of compounds isolated from this extract against lipid peroxidation. The antioxidant activity against lipid peroxidation clearly resulted from the tocotrienol derivatives present in the

extract (Table 5). Indeed, all tested compounds showed an equivalent or 2-fold greater antioxidant activity than

a

-tocopherol, the main vitamin E form used to protect food and cosmetic prepara- tions. Compared to the ORAC assay, dimers6–10did not lose their potency and compound10still had high antioxidant activity. Only one compound showed poor activity against lipid peroxidation.

The 10-fold decrease in radical-scavenging activity of compound 4relative to the other tested tocotrienols highlighted the role of the double bond in this activity. It was previously suggested that the mechanism of the antioxidant activity of tocotrienols involves a phenoxyl radical, which is formed via the phenolic hydroxyl group at the 6-position, and inhibits lipid autoxidation (Kamal- Eldin and Appelqvist, 1996). The loss of activity of compound 4 underscores the role of the double bond in the antioxidant activity of tocotrienols against lipid peroxidation.

3. Concluding remarks

So far more than 40 tocotrienol-like compounds, associating the chroman-6-ol skeleton and at least two prenyl units, have been isolated from plant species, marine organisms and animal tissues (Dunlap et al., 2002; Iwashima et al., 2008; Jang et al., 2005;

Merza et al., 2004; Silva et al., 2001; Terashima et al., 1997). Most of the structural diversity originates from brown seaweed,Sargas- sumsp., with around 20 analogues described among plant species, while tocotrienols and derivatives have been mainly described in Pinaceous, Canellaceous, Poaceous and Clusiaceous species (Harinantenaina, 2008). The phytochemical investigation of G.

amplexicaulis, a Clusiaceae species, revealed the high tocotrienol derivative content in this endemic New Caledonian shrub. From a chemical standpoint, natural tocotrienols with two primary alcohol functions located at the terminal part of the farnesyl chain are unique to this day. From a biochemical standpoint, with 15 different bio-synthesized tocotrienols,G. amplexicaulisexhibits one of the highest degrees of structural diversity in this class of compounds in angiosperms.

4. Experimental 4.1. General

Optical rotations were recorded on a Schmidt–Haensch polar- tronic-D polarimeter, UV spectra on a Varian spectrophotometer and IR spectra on a Bruker FT IR Vector 22 using liquid ﬁlms.¹H and¹³C NMR were obtained on a Bruker Avance DRX 500 MHz (500 and 125 MHz, respectively) spectrometer in CDCl3or methanol-d4with TMS as internal standard. Mass spectrometry analyses were performed on a JMS-700 (JEOL LTD, Akishima, Japan) double- focusing mass spectrometer with reversed geometry, equipped with a pneumatically assisted electrospray ionization (ESI) source.

Chromatographic separations such as ﬂash chromatography Intel- liFlash 310 (Analogix, Burlington, USA) using pre-packed C18(Inter- chim, Montluçon, France) or a silica gel column Chromabond^Òﬂash RS column (Macherey–Nagel, Düren, Germany), along with a preparative chromatography Varian ProStar 210 and PrepStar 218 sol- vent delivery module (Agilent, Santa Clara, USA) with a C18Varian column (5

l

m; 25021.4 mm), were used to purify compounds.

HPLC-UV analyses were performed using a Waters Alliance system (Milford, USA) equipped with a quaternary HPLC pump, degasser, autosampler and PDA diode array detector (Milford, USA).

4.2. Plant material

Stem bark ofG. amplexicaulisVieill. ex Pierre was collected in July 1998, in the Forêt Cachée region in southern New Caledonia Table 5

Inhibitory activity of lipid peroxidation of tocotrienol derivatives.

IC50(lM) S.D.^b(%)

Rosmarinic extract 30.3^a 20.4

DCM extractG. amplexicaulis 4.6^a 21.6

a-Tocopherol 8.8 11.5

b-Tocotrienol 4.2 11.9

d-Tocotrienol 4.7 3.3

c-Tocotrienol 3.0 6.9

3 3.8 14.7

4 38.9 0.5

5 8.9 10.1

6 9.9 14.2

8 8.7 11.0

9 5.1 23.7

10 1.7 9.7

d-Amplexichromanol 4.7 8.0

c-Amplexichromanol 3.2 6.4

d-(Z)-Deoxy-amplexichromanol 4.5 3.1

c-(Z)-Deoxy-amplexichromanol 2.2 4.5

Garcinoic acid 5.1 4.9

aExpressed inlg/mL.

b Standard deviation.

(7)

and identiﬁed by Marc Litaudon. A specimen (LIT-0554) was deposited at the Laboratoire des Plantes Médicinales (CNRS), Nou- mea, New Caledonia.

4.3. Extraction and isolation

Dried stem bark (270 g) ofG. amplexicauliswas extracted with 3 L of dichloromethane (DCM) using a Soxhlet apparatus for 24 h, with further extraction using another Soxhlet apparatus for 24 h with 3 L of methanol (MeOH). 30 g of DCM and MeOH extract was obtained after each extraction. DCM extract (20 g) was separated by silica gel normal-phase vacuum flash column chromatography (m= 400 g silica gel) using a DCM/acetone mixture to yield 36 fractions (F1–36) on the basis of the TLC analysis results. Frac- tion F11 (300 mg) was purified by RP-18 vacuum flash chromatography using an MeOH/H2O mixture (1:1 to 1:0) as mobile phase to yield compound1(3.8 mg). The combined purification of fractions F18 and F19 (500 mg) generated compounds 2(2.6 mg) and 17 (19.2 mg) in two steps using silica gel normal-phase flash chromatography (cyclohexane/EtOAc mixture) followed by RP-18 (4 g column) vacuum flash chromatography (MeOH/H2O mixture).

Fraction FC21 (290 mg) was purified by normal-phase vacuum flash chromatography using a cyclohexane/EtOAc mixture (95:5 to 1:1) to yield compound 3 (14.0 mg). Fraction F23 (300 mg) was separated by normal-phase flash chromatography with a cyclohexane/EtOAc mixture (9:1 to 1:1). Compound18 (6.2 mg) was isolated after this step. Compounds 19 (27.6 mg) and 10 (6.0 mg) were obtained from further purification by RP-18 (4 g column) flash chromatography (MeOH/H2O mixture) of fraction F23.

Fraction F5 (80 mg) was separated by normal-phase (15 g silica gel column) vacuum ﬂash chromatography with a cyclohexane/

ethyl acetate (EtOAc) mixture (95:5 to 10:90) to obtain compound 11(5.6 mg). Fractions F14 (300 mg) and F25 (200 mg) were respectively separated using normal-phase vacuum flash chromatography with a cyclohexane/EtOAc mixture (1:0 to 1:1) to afford compound 12 (4.2 mg) and21 (10.0 mg). Fraction F15 (300 mg) was separated by RP-18 (4 g column) flash chromatography (MeOH/H2O mixture) to isolate compounds 13 (1.9 mg), 14 (5.9 mg),15(2.4 mg) and16(4.2 mg). Fraction F27 (550 mg) was fractionated by normal-phase flash chromatography with a cyclohexane/EtOAc mixture (1:0 to 1:1) and was further subjected to RP-18 flash chromatography (MeOH/H2O mixture) to afford com- pounds5(8.0 mg),20(6.3 mg), and9(9.2 mg). Fraction F35 (2 g) was fractionated by RP-18 (150 g column) flash chromatography (MeOH/H2O mixture) and was further subjected to preparative HPLC using an isocratic mixture of MeOH and H2O (65%) to yield compounds4(16.9 mg) and6(17.2 mg), and using isocratic 92%

aqueous MeOH to afford7(6.2 mg) and8(6.8 mg).

4.3.1.

c

-(E)-Deoxy-amplexichromanal (1) Pale yellow oil; [

a

]D22

16.7°(c0.03, MeOH); UV (MeOH)kmax

(log

e

) 296.9 (3.41), 272.0 (3.24), 220.0 (4.11), 202.1 (4.42) nm;

1H and¹³C NMR, seeTable 1; HRESIMS:m/z 447.2874 [M+Na]⁺ (calcd for C28H40O3Na, 447.2870).

4.3.2. (2R)-2,8-Dimethyl-2-[(2E)-4-oxo-2-penten-1-yl]-chroman-6-ol (2)

Pale yellow oil; [

a

]_D²²12.0°(c0.025, MeOH); UV (MeOH)k_max (log

e

) 296.9 (3.27), 263.0 (2.67), 219.0 (4.05), 202.1 (4.30) nm;¹H and¹³C NMR, seeTable 1; HRESIMS:m/z283.1306 [M+Na]⁺(calcd for C₁₆H₂₀O₃Na, 283.1305).

4.3.3.d-(E)-Deoxy-amplexichromanol (3) Pale yellow oil; [

a

]D23

16.4°(c0.07, MeOH); UV (MeOH)k_max (log

e

) 296.0 (3.42), 260.0 (3.17), 203.0 (4.44), 202.1 (4.43) nm;¹H

and¹³C NMR, seeTable 1; HRESIMS:m/z411.2895 [MH](calcd for C27H39O3, 411.2905).

4.3.4.d-Dihydroxy-amplexichromanol (4)

Yellow oil; [

a

]D23+1.5°(c0.15, MeOH); UV (MeOH)kmax(log

e

) 296.9 (3.37), 269.0 (3.13), 201.0 (4.49) nm;¹H and¹³C NMR, see Table 1; HRESIMS:m/z485.2870 [M+Na]⁺(calcd for C₂₇H₄₂O₆Na, 485.2874).

4.3.5.d-(E)-Amplexichromanal (5)

Pale yellow oil; [

a

]D23 4.0°(c 0.07, MeOH); UV (MeOH)kmax

(log

e

) 296.9 (3.44), 271.0 (3.06), 219.0 (4.14), 206.0 (4.64) nm;

1H and ¹³C NMR, seeTable 1; HRESIMS:m/z 449.2660 [M+Na]⁺ (calcd for C27H38O4Na, 449.2657).

4.3.6.d,d-Bi-O-amplexichromanol (6)

Pale yellow oil; [

a

]D23+18.7°(c0.15, MeOH); UV (MeOH)kmax

(log

e

) 295.0 (3.73), 261.0 (3.16), 206.0 (4.87), 205.1 (4.79) nm;

1H and ¹³C NMR, seeTable 2; HRESIMS: m/z 877.5572 [M+Na]⁺ (calcd for C₅₄H₇₈O₈Na, 877.5556).

4.3.7.d,

c

-Bi-O-amplexichromanol (7)

Pale yellow oil; [

a

]_D²²+23.7°(c0.03, MeOH); UV (MeOH)k_max (log

e

) 293.0 (3.68), 203.0 (4.73) nm;¹H and¹³C NMR, seeTable 2;

HRESIMS:m/z891.5725 [M+Na]⁺(calcd for C55H80O8Na, 891.5745).

4.3.8.d,

c

-Biamplexichromanol (8)

Pale yellow oil; [

a

]D22+22.5°(c0.06, MeOH); UV (MeOH)kmax

(log

e

) 301.0 (3.80), 264.0 (3.40), 206.0 (4.85), 205.1 (4.82) nm;

1H and ¹³C NMR, seeTable 2; HRESIMS: m/z 891.5719 [M+Na]⁺ (calcd for C55H80O8Na, 891.5745).

4.3.9.d,d-Biamplexichromanoate A (9)

Pale yellow oil; [

a

]D22 1.0°(c 0.06, MeOH); UV (MeOH)kmax

(log

e

) 296.9 (3.73), 261.0 (3.12), 206.0 (4.80), 203.0 (4.82) nm;

4.3.10.d,d-Amplexichromanol peroxide (10)

Pale yellow oil; [

a

]D2213.2°(c0.06, MeOH); UV (MeOH)kmax

(log

e

) 296.9 (3.40), 263.0 (2.80), 206.0 (4.51), 203.0 (4.53) nm;

4.4. Oxygen radical absorbance capacity (ORAC) assay

ORAC assays were carried out according to the method of (Huang et al., 2002) with some modifications. This assay measures the ability of antioxidant compounds to inhibit the decline in fluorescein (FL) fluorescence induced by a peroxyl radical genera- tor, namely 2,2⁰-azobis(2-methylpropionamidine)dihydrochloride (AAPH). The assay was performed in a 96-well plate. The reaction mixture contained 100

l

L of 75 mM phosphate buffer (pH 7.4), 100

l

L of freshly prepared FL solution (0.1

l

m in phosphate buffer), 50

l

L of freshly prepared AAPH solution (51.6 mg/mL in phosphate buffer), and 20

l

L of sample per well. Samples were analysed in triplicate and diluted at different concentrations (25

l

g/mL, 12.5

l

g/mL, 6.25

l

g/mL and 3.12

l

g/mL) from stock solutions at 1 mg/mL in DMSO. FL, phosphate buffer, and samples were prein- cubated at 37°C for 10 min. The reaction was started by the addi- tion of AAPH using the microplate reader’s injector (Inﬁnite^Ò200, Tecan, France). Fluorescence was then measured and recorded for 40 min at excitation and emission wavelengths of 485 and 520 nm, respectively. The 75 mM phosphate buffer was used as blank, and 12.5, 25, 50, and 75

l

M of Trolox (hydrophilic

a

-tocopherol analog) were used as calibration solutions. A sample of 8.8

l

M

(8)

chlorogenic acid was used as quality control. The ﬁnal ORAC values were calculated by using a regression equation between the Trolox concentration and the net area under the FL decay curve and expressed as micromoles of Trolox equivalents per micromole of tested compounds. The area under the curve was calculated using Magellan™ data analysis software (Tecan, France). d-amplexichromanol,

c

-amplexichromanol, d-(Z)-deoxy-amplexichromanol,

c

-(Z)-deoxy-amplexichromanol and garcinoic acid were previously isolated fromG. amplexicaulis(Lavaud et al., 2013). Reference products (

a

,b,

c

)-tocotrienol were purchased from Sigma–Aldrich.

4.5. Inhibitory activity of lipid peroxidation (TBARS assay)

As a lipid source, pasteurized dried egg yolk powder (mainly containing phospholipids, triacylglycerols and proteins) was used and peroxidation was induced by iron through the indirect formation of an HO-type oxidant (North et al., 1992). The thiobarbituric acid reactive species (TBARS) assay was carried out according to the method of (Viuda-Martos et al., 2011) with some modiﬁca- tions. The reaction mixture for inducing lipid peroxidation contained 300

l

L fowl egg yolk emulsiﬁed with 0.1 M phosphate buffer, pH 7.4 (25 g/L), and 30

l

L of Fe²⁺(1 mM). 30

l

L of sample at different concentrations (range 1–50

l

m, ﬁnal concentration) was added to the above mixture and incubated at 37°C for 1 h, after which it was treated with 150

l

L of 15% TCA and 300

l

L of 1% TBA. The reaction tubes were kept in a boiling water bath for 20 min. Upon cooling, the tubes were centrifuged at 12,000g for 10 min to remove precipitated protein. TBARS formation was measured at 532 nm absorbance. The control ‘‘TBARS max’’ was buf- fered egg with Fe²⁺ alone. The percentage inhibition ratio was calculated by the following equation:

%inhibition¼ ½ðA\Tbarsmax"AsampleÞ=A\Tbarsmax" 100

To determine the concentration needed to achieve 50% inhibition of phospholipid oxidation in egg yolk (IC50), the percentage of lipid peroxidation inhibition was plotted against the sample concentration. Reference rosemary extract (E392) was prepared according to directive 2010/67/EU.

Acknowledgments

We thank Angers Loire Métropole for granting a Ph.D. scholar- ship to A.L. We thank Dr. I. Freuze and B. Siegler from Plateforme d’Imagerie et d’Analyses Moléculaires (PIAM), Université d’Angers, for their assistance in HREIMS and NMR analysis. The authors are grateful to South Province of New Caledonia which facilitated our ﬁeld investigation.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.phytochem.2014.

10.024.

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