Phytochemistry 109 (2015) 103–110

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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

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,⇑ a b c

Université d’Angers, Laboratoire SONAS, IFR Quasav, 49100 Angers, France INSERM UMR U1063, IBS-IRIS, Université d’Angers, 49100 Angers, France Centre 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 of Garcinia 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 compounds 6–10 are 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 from G. amplexicaulis exhibited significant antioxidant activity against lipid peroxidation and in the ORAC assay. Ó 2014 Elsevier Ltd. All rights reserved.

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 cosmeceuticals (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 pleiotropic 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 on Garcinia plants, d-amplexichromanol and c-amplexichromanol, the two major tocotrienol derivatives from lipophilic extracts of Garcinia amplexicaulis, were found to be antiangiogenic agents in the low nanomolar range (Lavaud et al., 2013). Intensive phytochemical investigation of G. amplexicaulis was 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 ⇑ Corresponding author. Tel.: +33 241 226 676; fax: 33 241 226 634. E-mail address: [email protected] (D. Guilet). http://dx.doi.org/10.1016/j.phytochem.2014.10.024 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

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 of G. amplexicaulis Vieill. 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 activity Fig. 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 of 1 appeared to be very

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A. Lavaud et al. / Phytochemistry 109 (2015) 103–110

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 dH 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 at dH 6.37 (1H, s, H-5) and a benzylic methylene at dH 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 at dH 1.73 (3H, s, H-22) and an aldehyde at dH 9.37 (1H, s, H-21) and dC 195.4 (CH), instead of the two oxymethylene groups around dH 4.10–4.30 (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 H21/C-19, C-20, C-22 and H-22/C-19, C-20, C-21. The E geometry 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. The 1H and 13C NMR data of 2 showed signal features of the d-chromanol moiety: two aromatic protons at dH 6.40 and 6.50, two oxygenated aromatic carbons at dC 145.3 and 148.2 and two methyl groups at dC 24.5 and 16.0 ppm (Table 1). The peak corresponding to a carbonyl carbon at dC 198.4 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 at dC 143.5 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)-2,8dimethyl-2-[(2E)-4-oxo-2-penten-1-yl]-chroman-6-ol. The molecular formula of d-(E)-deoxy-amplexichromanol (3), i.e., C27H40O3, was determined by HR-ESIMS and 13C NMR spectrometry (Table 1). The 13C 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 at dC 13.7 ppm (dH 1.66). The E geometry of the last double bond was assigned on the basis of NOESY cross-peaks H-21/H-19. The molecular formula of d-dihydroxy-amplexichromanol (4) was deduced as C27H42O6 by HR-ESIMS associated with 13C 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, the 13C NMR data, combined with the fact that there were only seven degrees of unsaturation inherent to the molecular formula, showed that compound 4 lost one of these double bonds. Indeed, this hypothesis was confirmed by the appearance of carbon signals at dC 75.3 and 78.0, and the presence of ten olefinic carbons around dC 110–150 ppm. Interpretation of a proton spin system containing the oxymethine proton H-15 at dH 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 (dC 75.4) 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 (dH 5.17) with C-24, C-13, C-10, and C-9 and also between the methyl protons H-25 (dH 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 C27H38O4 by combined HRESIMS and 13C NMR

spectrometry. The spectral features of 5 were very similar to that of d-amplexichromanol. Indeed, typical resonances of the chromanol ring and the farnesyl chain were noted in the 1H NMR spectrum (Table 1). However, the 13C 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 geometry E was 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 of d,d-bi-O-amplexichromanol (6) and d,c-bi-O-amplexichromanol (7), C54H78O8 and C55H80O8, respectively, were determined by HREIMS and 13C 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 the 1H NMR spectrum by a comparison of the integration between aromatic protons (e.g., for compound 6, dH = 6.35, 6.53 and 6.70, each signal integrated for one proton) and oxymethylene protons (e.g., for compound 6, dH = 4.19 and 4.28, each signal integrated for four protons). The modified farnesyl side chains of compounds 6 and 7 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. Compounds 6 and 7 were 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 of 6 and 7. The aromatic region of the 1H 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-50 and 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. Both d-amplexichromanol units were connected to form compound 6. Regarding compound 7, the aromatic region of the 1H NMR spectrum still showed a singlet (dH 6.71) due to the isolated proton H-7, but only one singlet (dH 6.06) due to the aromatic proton H-50 . Compound 7 differed from compound 6, 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-50 with C-40 /C-60 /C-8a0 . An analogous metabolite, i.e., d,c-biamplexichromanol (8), was analyzed for C55H80O8 by HRESIMS and 13C NMR spectrometry. The NMR data obtained for this compound were reminiscent of those from compound 7 (Table 2). Indeed, the 1H and 13C NMR data of the farnesyl side chain with oxymethylene at the terminal isoprene unit were similar. However, the 1H NMR spectrum revealed only one aromatic proton (dH 6.71) due to an isolated proton H-7. As compared with the 1H NMR and 13C NMR signals of 7 and 8, the aromatic carbons at dC 137.4 and 109.5 respectively for the C-5 and C-50 of 7 were replaced by dC 116.4 and 122.2 in 8. On the basis of HMBC experiments, the chromanol assignments were unambiguously assigned by long-range correlations between H-4/H-40 and 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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A. Lavaud et al. / Phytochemistry 109 (2015) 103–110

26

27

CH3

R1

25

8a

21

9

O

12

16

24

23

R2

2

HO

5

3

4a

4

R2 -CH2OH -CH2OH -CH3 -CHO -CH2OH -CHO

R1 -H -CH3 -CH3 -CH3 -H -H

δ-amplexichromanol γ-amplexichromanol γ-(Z)-deoxy-amplexichromanol 1 3 5 15

CH3 8a

7

9

12

CH3

O

O

8a

5

13

4a

HO

OH OH

25

2

HO

9 2

24

4a

7

9

O

12

4a

24

O

CH2OH 22

24'

CH2OH

R

O

8a'

16'

12'

9'

26'

26

9

12

16

4a

24

23

4a'

24'

23'

8a' 26'

O

24

23

4a'

24'

23'

12'

16'

8a' O

27'

19

22

CH2OH

22'

CH2OH

9'

CH2OH

19'

21'

25

21

CH2OH

8a

9

O

22

O

O

19'

12

16

19

24

23

O

24'

23'

O

2

H2C

HO

4a

HO

4a' 2'

16'

CH2OH

2' 25'

9' 12'

21

19

8

22' 2'

4a

26

25

O

16

R = -H R = -CH3

2

HO HO

21'

12

26'

6 7

8a

CH2OH

19'

25'

9

O 2

HO HO

22'

23'

2' 27'

22

25 8a

7

CH2OH

23

4a'

26

21

19

16

2

HO

CH2OH

23

4

25 8a

21

CH2OH

16

12

2 26

R3 -CH2OH -CH2OH -CH2OH -CH3 -CH3 -CH2OH

26

14

O

R3

22

8a'

21'

25'

O

26'

9

21 22

O O

22' 9' 12'

16'

19'

21'

25'

10 Fig. 1. Structures of compounds 1–10.

The molecular formula of d,d-biamplexichromanoate A (9) was deduced as C54H78O7 by HREIMS analyses and 13C NMR spectrometry. On the basis of the NMR data and integration of protons in the 1 H 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, dC 65.8 and 60.0 ppm) and a carbonyl signal (dC 177.4) 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

106

A. Lavaud et al. / Phytochemistry 109 (2015) 103–110

Table 1 H (500 MHz) and

1

No

13

C (125 MHz) NMR data of compounds 1–5 (in CDCl3 except for 4 in methanol-d4, d in ppm, J in Hz in parenthesis).

1 dC

2 3 4 4a 5 6 7 8 8a 9

75.1 31.3 22.1 118.2 112.1 146.2 121.6 125.7 145.6 39.7

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

22.2 124.4 134.8 39.5 26.4 125.5 133.3 37.9 27.4 154.6 139.2 195.4 9.2 15.9 15.8 24.0 11.9 11.9

2 dH

3

dC

dH

75.0 31.4 22.3 120.9 112.6 148.2 115.9 127.4 145.3 42.7

1.68–1.76, m 2.68, t (6.2) 6.37, s

1.54–1.66, m 2.12, m 5.12, t (6.8)

75.3 31.4 22.5 121.2 112.6 147.8 115.6 127.3 145.9 39.3

1.79, m 2.72, m 6.40, d (2.8) 6.50, d (2.8)

2.45, 2.55, 6.88, 6.10,

143.5 134.1 198.4 26.9 24.5 16.0

1.97, m 2.08, m 5.14, t (6.8)

dC

dd (14.0 & 7.5) dd (14.0 & 8.0) ddd (16.0, 8.0 & 7.5) d (16.0)

22.2 124.4 135.0 39.5 26.4 124.4 134.6 39.4 26.2 126.3 134.5 69.1 13.7 16.0 15.9 24.2 16.1

2.26, s 1.28, s 2.13, s

2.15, m 2.44, q (7.4) 6.46, t (7.4) 9.37, 1.73, 1.61, 1.59, 1.26, 2.11, 2.13,

s s s s s s s

4 dH

dC 76.2 32.8 23.5 122.3 113.6 150.4 116.6 127.8 146.4 40.5

1.75–1.81, m 2.69, t (6.7) 6.38, d (2.8) 6.47, d (2.8)

1.52–1.65, m 2.11, m 5.12, t (7.0)

23.3 125.8 136.2 38.0 30.4 78.0 75.4 39.4 22.3 131.1 139.0 65.6 58.2 21.8 16.0 24.5 16.4

1.98, m 1.96–2.05, m 5.09, t (7.0) 1.98–2.07, m 2.05–2.10, m 5.38, t (7.0) 4.00, 1.66, 1.59, 1.59, 1.26, 2.12,

s s s s s s

5 dH

dC

1.71, m 2.64, t (6.8) 6.28, d (2.4) 6.36, d (2.4)

1.50–1.60, m 2.10, m 5.17, t (7.4) 2.20, m 1.30 & 1.73, m 3.23, t (10.6) 1.56 & 1.93, m 1.20–1.25, m 5.55, t (7.4) 4.05, 4.14, 1.06, 1.57, 1.21, 2.03,

dH

75.2 31.4 22.4 121.2 112.5 147.8 115.6 127.3 145.8 39.2

s s d (1.2) s s s

1.76, m 2.68, td (6.8 & 2.0) 6.37, d (2.8) 6.47, d (2.8)

1.52–1.63, m

22.1 124.5 134.7 39.4 26.4 125.9 132.9 38.1 27.2 156.7 141.2 195.9 55.9 15.8 15.8 24.2 16.1

2.11, m 5.11, t (7.0) 1.96, m 2.07, m 5.12, t (7.0) 2.15, m 2.48, m 6.58, t (7.5) 9.40, 4.35, 1.59, 1.58, 1.26, 2.12,

s s s s s s

Table 2 H NMR data (500 MHz) of compounds 6–8 (CDCl3, d in ppm, J in Hz in parenthesis).

1

No

6

7 n0

n dC 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

75.0 30.5 17.6 115.2 136.9 141.2 115.4 123.4 145.5 39.5 22.1 124.3 134.9 39.3 26.4 124.9 134.1 39.0 25.9 130.9 136.9 67.6 60.0 15.9 15.8 24.2 15.8

dH

dC

1.64–1.68, m 2.48, m

6.70, s

1.52–1.60, m 2.10, m 5.11, m 2.03, m 2.05, m 5.08, m 1.95, m 2.15, m 5.52, m 4.19, 4.28, 1.56, 1.58, 1.26, 2.16,

s s s s s s

75.5 31.2 22.5 121.3 111.8 149.6 115.0 127.6 147.0 39.5 22.1 124.3 135.0 39.3 26.4 124.9 134.1 39.0 25.9 130.9 136.9 67.6 60.0 15.9 15.8 24.1 16.2

8 n0

n dH

dC

1.72–1.78, m 2.69, m 6.35, d (2.6) 6.53, d (2.6)

1.52–1.60, m 2.10, m 5.11, m 2.03, m 2.05, m 5.08, m 1.95, m 2.15, m 5.52, m 4.19, 4.28, 1.58, 1.58, 1.24, 2.11,

s s s s s s

75.0 30.5 22.3 123.5 137.4 141.2 115.2 114.9 145.6 39.5 22.0 124.3 134.9 39.2 26.4 124.9 134.0 39.1 25.9 130.8 137.0 67.6 60.0 15.9 15.8 23.8 15.8

dH

dC

1.76, m 2.53, m

6.71, s

1.52–1.60, m 2.09, m 5.10, m 2.01–2.04, m 2.05, m 5.09, m 1.95, m 2.15, m 5.52, t (7.4) 4.17, 4.27, 1.58, 1.57, 1.22, 2.17,

s s s s s s

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

75.4 31.2 22.3 118.1 109.5 148.0 123.5 126.2 145.6 39.5 22.1 124.3 135.0 39.2 26.4 124.9 134.1 39.1 25.9 130.8 137.0 67.6 60.0 15.9 15.8 23.8 11.9 12.0

n0

n dH

dC

1.67, m 2.53, m 6.06, s

1.52–1.60, m 2.09, m 5.10, m 2.01–2.04, m 2.05, m 5.10, m 1.95, m 2.15, m 5.52, t (7.4) 4.20, 4.29, 1.58, 1.57, 1.25, 2.16, 2.31,

s s s s s s s

75.0 31.2 20.5 117.4 116.4 146.2 115.4 128.3 145.7 39.5 22.2 124.4 134.8 39.0 26.2 124.9 134.0 39.2 25.9 130.8 136.9 67.5 59.9 16.0 15.8 24.1 16.3

dH

dC

1.61, m 2.12/2.31, m

6.71, s

1.52–1.64, m 2.06–2.12, m 5.10, m 2.03, m 2.06, m 5.08, m 1.99, m 2.14, m 5.50, t (7.1) 4.15, 4.25, 1.58, 1.59, 1.26, 2.20,

s s s s s s

75.0 31.3 20.6 120.5 122.2 146.2 115.7 126.7 144.6 39.5 22.2 124.4 134.8 39.0 26.2 78.0 134.0 39.2 25.9 130.8 136.9 67.5 59.9 16.0 15.8 24.1 12.0 12.3

dH 1.69–1.76, m 2.12/2.31, m

1.52–1.64, m 2.06–2.12, m 5.10, m 2.03, m 2.06, m 5.08, m 1.99, m 2.14, m 5.50, t (7.1) 4.14, 4.24, 1.58, 1.59, 1.26, 2.17, 2.19,

s s s s s s s

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

107

A. Lavaud et al. / Phytochemistry 109 (2015) 103–110 Table 3 H NMR data (500 MHz) of compounds 9–10 (CDCl3, d in ppm, J in Hz in parenthesis).

1

No

9

10 0

n dC 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

75.3 31.3 22.4 121.2 112.5 147.8 115.6 127.3 145.8 39.2 22.1 124.4 134.9 39.4 26.4 124.9 134.5 39.5 26.1 133.4 134.0 65.8 60.0 15.8 15.9 24.2 16.1

n dH

dC

1.74–1.84, m 2.68, m 6.37, br s 6.47, br s

1.52–1.64, m 2.10, m 5.11, m 2.01–2.04, m 2.05, m 5.06, m 1.95, m 2.18, m 5.66, t (7.2) 4.08, 4.70, 1.58, 1.58, 1.26, 2.12,

s s s s s s

75.3 31.3 22.4 121.2 112.5 147.8 115.6 127.3 145.8 39.2 22.1 124.4 134.9 39.4 26.4 124.4 134.8 26.4 25.4 33.3 39.4 17.1 177.4 15.9 15.9 24.2 16.1

n0

n dH

dC 75.3 31.3 22.4 121.2 112.5 147.7 115.6 127.3 145.9 39.4 22.1 124.5 135.0 39.5 26.5 125.0 134.0 39.0 28.1 146.4 125.9 20.5 172.5 15.8 15.8 24.0 16.1

1.74–1.84, m 2.68, m 6.37, br s 6.47, br s

1.52–1.64, m 2.10, m 5.11, m 2.01–2.04, m 2.05, m 5.11, m 2.05, 1.92, 1.35, 2.44, 1.13,

m m m m d (7.0)

1.56, 1.58, 1.26, 2.12,

s s s s

to metabolize carboxychromanol derivatives. Then this carboxychromanol might react with an amplexichromanol to form compound 9. d,d-Amplexichromanol peroxide (10) was analyzed for C54H76O8 by combined HREIMS and 13C NMR spectrometry. The spectral feature of 10 appeared to be very similar to that of compound 9. 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 compound 9, the NMR data revealed the same carboxychromanol unit. However, the carbonyl signal C-220 (dC 182.0) was shifted, indicating a different linkage than for 10. No oxymethylene carbon around dC 60 ppm was present in the 13C NMR spectrum. However, on the basis of 2D NMR analyses, the oxymethylene carbons of 10 were replaced by a methyl group (dH 1.90, dC 20.5) and a carbonyl signal at dC 172.5 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,bunsaturated form while the carbonyl at C-220 was 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 of G. amplexicaulis and identified as the xanthones, cudraxanthone G 11 (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 naringenin 21, and the three known triterpenoids, cabraleadiol 18 (Nakamura et al., 1997), (205,23E)eupha-8,23-diene-3b,25-diol 19 (Leong and Harrison, 1999) and (3b,11b)-3,11-dihydroxylanosta-8,24-dien-7-one 20 (Lu et al.,

dH

dC 75.3 31.3 22.4 121.2 112.5 147.7 115.6 127.3 145.9 39.4 22.1 124.5 135.0 39.5 26.5 124.3 134.5 26.4 25.3 33.0 39.1 16.9 182.0 15.8 15.8 24.0 16.1

1.74–1.84, m 2.69, m 6.38, d (2.8) 6.48, d (2.8)

1.54–1.66, m 2.10, m 5.11, m 1.97, m 2.06, m 5.08, m 1.97, m 2.60, q (7.0) 6.06, td (7.0 and 1.3) 1.90, s 1.57, 1.58, 1.26, 2.12,

s s s s

dH 1.74–1.84, m 2.69, m 6.38, d (2.8) 6.48, d (2.8)

1.54–1.66, m 2.10, m 5.11, m 1.97, m 2.06, m 5.11, m 2.06, 1.41, 1.39, 2.46, 1.17,

m m m m d (7.0)

1.57, 1.58, 1.26, 2.12,

s s s s

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 quantity) 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 4 ORAC assay results of tocotrienol derivatives.

DCM extract G. amplexicaulis

a-Tocotrienol d-Tocotrienol c-Tocotrienol 3 4 5 6 8 9 10 d-Amplexichromanol c-Amplexichromanol d-(Z)-Deoxy-amplexichromanol c-(Z)-Deoxy-amplexichromanol Garcinoic acid a b c

IC50 (lmol TE/lmol)

S.D.c (%)

1635a n.a.b n.a. n.a. 0.74 1.76 0.50 n.a. n.a. n.a. 1.80 1.45 0.92 1.61 0.80 0.64

– – – 3.7 1.5 8.1 – – 8.7 – 2.8 1.8 6.1 8.5 1.4

Expressed in lmol TE (Trolox Equivalent)/g extract. No activity recorded. Standard deviation.

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Table 5 Inhibitory activity of lipid peroxidation of tocotrienol derivatives. IC50 (lM) Rosmarinic extract DCM extract G. amplexicaulis a-Tocopherol b-Tocotrienol d-Tocotrienol c-Tocotrienol 3 4 5 6 8 9 10 d-Amplexichromanol c-Amplexichromanol d-(Z)-Deoxy-amplexichromanol c-(Z)-Deoxy-amplexichromanol Garcinoic acid a b

a

30.3 4.6a 8.8 4.2 4.7 3.0 3.8 38.9 8.9 9.9 8.7 5.1 1.7 4.7 3.2 4.5 2.2 5.1

S.D.b (%) 20.4 21.6 11.5 11.9 3.3 6.9 14.7 0.5 10.1 14.2 11.0 23.7 9.7 8.0 6.4 3.1 4.5 4.9

Expressed in lg/mL. Standard deviation.

result to monomers of tocotrienol derivatives. No free radical scavenger capacity was detected for a-, b-, c-tocotrienol, while the new compounds 3–5, d-amplexichromanol, c-amplexichromanol, d-(Z)deoxy-amplexichromanol, c-(Z)-deoxy-amplexichromanol and garcinoic acid displayed significant antioxidant activity in comparison to the TroloxÒ reference, 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 compound 10, 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 peroxides while enhancing blood flow in patients with carotid atherosclerosis (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 pathological 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 chromogen (thiobarbituric acid reactive substance, TBARS) with an absorbance peak at 532 nm. The DCM extract of G. 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 preparations. Compared to the ORAC assay, dimers 6–10 did not lose their potency and compound 10 still had high antioxidant activity. Only one compound showed poor activity against lipid peroxidation. The 10-fold decrease in radical-scavenging activity of compound 4 relative 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 (KamalEldin 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, Sargassum sp., 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. amplexicaulis exhibits 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 polartronic-D polarimeter, UV spectra on a Varian spectrophotometer and IR spectra on a Bruker FT IR Vector 22 using liquid films. 1H and 13C NMR were obtained on a Bruker Avance DRX 500 MHz (500 and 125 MHz, respectively) spectrometer in CDCl3 or methanol-d4 with TMS as internal standard. Mass spectrometry analyses were performed on a JMS-700 (JEOL LTD, Akishima, Japan) doublefocusing mass spectrometer with reversed geometry, equipped with a pneumatically assisted electrospray ionization (ESI) source. Chromatographic separations such as flash chromatography IntelliFlash 310 (Analogix, Burlington, USA) using pre-packed C18 (Interchim, Montluçon, France) or a silica gel column ChromabondÒflash RS column (Macherey–Nagel, Düren, Germany), along with a preparative chromatography Varian ProStar 210 and PrepStar 218 solvent delivery module (Agilent, Santa Clara, USA) with a C18 Varian column (5 lm; 250  21.4 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 of G. amplexicaulis Vieill. ex Pierre was collected in July 1998, in the Forêt Cachée region in southern New Caledonia

A. Lavaud et al. / Phytochemistry 109 (2015) 103–110

and identified by Marc Litaudon. A specimen (LIT-0554) was deposited at the Laboratoire des Plantes Médicinales (CNRS), Noumea, New Caledonia. 4.3. Extraction and isolation Dried stem bark (270 g) of G. amplexicaulis was 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. Fraction 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 compound 1 (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). Compound 18 (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) and 21 (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) and 16 (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 compounds 5 (8.0 mg), 20 (6.3 mg), and 9 (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 compounds 4 (16.9 mg) and 6 (17.2 mg), and using isocratic 92% aqueous MeOH to afford 7 (6.2 mg) and 8 (6.8 mg). 4.3.1. c-(E)-Deoxy-amplexichromanal (1) Pale yellow oil; [a]22 D 16.7° (c 0.03, MeOH); UV (MeOH) kmax (log e) 296.9 (3.41), 272.0 (3.24), 220.0 (4.11), 202.1 (4.42) nm; 1 H and 13C NMR, see Table 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]22 D 12.0° (c 0.025, MeOH); UV (MeOH) kmax (log e) 296.9 (3.27), 263.0 (2.67), 219.0 (4.05), 202.1 (4.30) nm; 1H and 13C NMR, see Table 1; HRESIMS: m/z 283.1306 [M+Na]+ (calcd for C16H20O3Na, 283.1305). 4.3.3. d-(E)-Deoxy-amplexichromanol (3) Pale yellow oil; [a]D23 16.4° (c 0.07, MeOH); UV (MeOH) kmax (log e) 296.0 (3.42), 260.0 (3.17), 203.0 (4.44), 202.1 (4.43) nm; 1H

109

and 13C NMR, see Table 1; HRESIMS: m/z 411.2895 [MH] (calcd for C27H39O3, 411.2905). 4.3.4. d-Dihydroxy-amplexichromanol (4) Yellow oil; [a]D23 +1.5° (c 0.15, MeOH); UV (MeOH) kmax (log e) 296.9 (3.37), 269.0 (3.13), 201.0 (4.49) nm; 1H and 13C NMR, see Table 1; HRESIMS: m/z 485.2870 [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; 1 H and 13C NMR, see Table 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° (c 0.15, MeOH); UV (MeOH) kmax (log e) 295.0 (3.73), 261.0 (3.16), 206.0 (4.87), 205.1 (4.79) nm; 1 H and 13C NMR, see Table 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° (c 0.03, MeOH); UV (MeOH) kmax (log e) 293.0 (3.68), 203.0 (4.73) nm; 1H and 13C NMR, see Table 2; HRESIMS: m/z 891.5725 [M+Na]+ (calcd for C55H80O8Na, 891.5745). 4.3.8. d,c-Biamplexichromanol (8) Pale yellow oil; [a]D22 +22.5° (c 0.06, MeOH); UV (MeOH) kmax (log e) 301.0 (3.80), 264.0 (3.40), 206.0 (4.85), 205.1 (4.82) nm; 1 H and 13C NMR, see Table 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; 1 H and 13C NMR, see Table 3; HRESIMS: m/z 861.5639 [M+Na]+ (calcd for C54H78O7Na, 861.5640). 4.3.10. d,d-Amplexichromanol peroxide (10) Pale yellow oil; [a]22 D 13.2° (c 0.06, MeOH); UV (MeOH) kmax (log e) 296.9 (3.40), 263.0 (2.80), 206.0 (4.51), 203.0 (4.53) nm; 1 H and 13C NMR, see Table 3; HRESIMS: m/z 875.5433 [M+Na]+ (calcd for C54H76O8Na, 875.5432). 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 generator, namely 2,20 -azobis(2-methylpropionamidine)dihydrochloride (AAPH). The assay was performed in a 96-well plate. The reaction mixture contained 100 lL of 75 mM phosphate buffer (pH 7.4), 100 lL of freshly prepared FL solution (0.1 lm in phosphate buffer), 50 lL of freshly prepared AAPH solution (51.6 mg/mL in phosphate buffer), and 20 lL of sample per well. Samples were analysed in triplicate and diluted at different concentrations (25 lg/mL, 12.5 lg/mL, 6.25 lg/mL and 3.12 lg/mL) from stock solutions at 1 mg/mL in DMSO. FL, phosphate buffer, and samples were preincubated at 37 °C for 10 min. The reaction was started by the addition of AAPH using the microplate reader’s injector (InfiniteÒ 200, 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 lM of Trolox (hydrophilic a-tocopherol analog) were used as calibration solutions. A sample of 8.8 lM

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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 from G. 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 modifications. The reaction mixture for inducing lipid peroxidation contained 300 lL fowl egg yolk emulsified with 0.1 M phosphate buffer, pH 7.4 (25 g/L), and 30 lL of Fe2+ (1 mM). 30 lL of sample at different concentrations (range 1–50 lm, final concentration) was added to the above mixture and incubated at 37 °C for 1 h, after which it was treated with 150 lL of 15% TCA and 300 lL 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 buffered egg with Fe2+ alone. The percentage inhibition ratio was calculated by the following equation:

% inhibition ¼ ½ðA\Tbarsmax"  Asample Þ=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. scholarship 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, at http://dx.doi.org/10.1016/j.phytochem.2014. 10.024. References Agarwal, M.K., Agarwal, M.L., Athar, M., Gupta, S., 2004. Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association. Cell Cycle 3, 205–211. Dunlap, W.C., Fujisawa, A., Yamamoto, Y., Moylan, T.J., Sidell, B.D., 2002. Notothenioid fish, krill and phytoplankton from Antarctica contain a vitamin E constituent (alpha-tocomonoenol) functionally associated with cold-water adaptation. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 133, 299–305. Frega, N., Mozzon, M., Bocci, F., 1998. Identification and estimation of tocotrienols in the annatto lipid fraction by gas chromatography-mass spectrometry. J. Am. Oil Chem. Soc. 75, 1723–1727.

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