Profile and quantification of glucosinolates in Pentadiplandra brazzeana Baillon

Phytochemistry 73 (2012) 51–56

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Profile and quantification of glucosinolates in Pentadiplandra brazzeana Baillon Gina R. De Nicola a, Maximilienne Nyegue b, Sabine Montaut c,⇑, Renato Iori a, Chantal Menut d, Arnaud Tatibouët e, Patrick Rollin e, Chantal Ndoyé b, Paul-Henri Amvam Zollo b a

Agricultural Research Council – Industrial Crop Research Centre (CRA-CIN), Via di Corticella 133, 40128 Bologna, Italy Department of Biochemistry, University of Yaoundé I, B.P. 812, Yaoundé, Cameroon c Department of Chemistry & Biochemistry, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, Canada P3E 2C6 d IBMM, UMR 5247 UM2-UM1, 15 avenue Charles Flahault, B.P. 14491, 34093 Montepellier, France e Institut de Chimie Organique et Analytique, UMR-CNRS 6005, Université d’Orléans, B.P. 6759, F-45067 Orléans Cedex 2, France b

a r t i c l e

i n f o

Article history: Received 11 January 2011 Received in revised form 1 August 2011 Available online 10 October 2011 Keywords: Pentadiplandra brazzeana Pentadiplandraceae Isolation Desulfoglucosinolates Desulfoglucotropaeolin Desulfoglucolimnanthin Desulfoglucoaubrietin Isothiocyanates

a b s t r a c t Glucosinolates (GLs) present in root, seed, and leaf extracts of Pentadiplandra brazzeana Baillon were characterized and quantified according to the ISO 9167-1 method based on the HPLC analysis of desulfo-GLs. The analyses were complemented by GC–MS analyses of the isothiocyanates (ITCs) generated from GL degradation by myrosinase. Glucotropaeolin (1a), glucolimnanthin (2a), and glucoaubrietin (3a) were shown to be present in the root extract, whereas the seed mainly contained 3a. 3,4-Dimethoxybenzyl GL (4a), glucobrassicin (5a) and traces of 1a were detected in the leaf extract. The products were fully characterized as their desulfo-counterparts by spectroscopic techniques. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The plant family Pentadiplandraceae possesses only one genus, Pentadiplandra, which contains a single species, Pentadiplandra brazzeana Baillon (Baillon, 1886; Hutchinson and Daziel, 1958; Villiers, 1973). According to Mabberley, P. brazzeana belongs to the plant family Capparaceae (Mabberley, 1990). A study based on the morphology has linked Pentadiplandra to the Caricaceae (Rodman, 1991). Furthermore, a macromolecular study of the rbcL chloroplast gene and 18S ribosomal RNA gene has shown a phylogenetic similarity of Pentadiplandra with Capparales (Rodman et al., 1996, 1998). The genus was then placed either in a clade with Tovariaceae or as an unresolved clade with Capparaceae and Tovariaceae (Rodman et al., 1996, 1998). Recent botanical studies on the development and anatomy of Pentadiplandra indicated that this genus has a strong phylogenetic similarity with the glucosinolate-containing Tovaria pendula Ruiz and Pavón (Tovariaceae) and that it represents a relict genus in a separate family at the evolutionary base of the Brassicales (Ronse De Craene, 2002). However, analyses using rbcL, ndhF, and matK sequence showed a weak resolution of the relationship of Pentadiplandraceae and Tovariaceae (Hall et al., 2004). These past investigations show that P. brazzeana ⇑ Corresponding author. Tel.: +1 705 6751151x2185; fax: +1 705 6754844. E-mail address: [email protected] (S. Montaut). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.09.006

is a distinct and unique plant, difficult to place with certainty within the Brassicales. Therefore, it is of great interest to investigate in detail the metabolites of P. brazzeana and their biological activities. Commonly called ‘‘oubli’’ (Dounias, 2009) or ‘‘liane blanche’’ (Titanji et al., 2008) in French, ‘‘ndifeu’’ in the Bangangte region in Cameroon (Jiofack et al., 2009), and ‘‘kenge kiese’’ by the Kikongo tribe in the Democratic Republic of Congo (Makumbelo et al., 2008), P. brazzeana is a plant found in Western and Central Africa (Titanji et al., 2008; Jiofack et al., 2009; Makumbelo et al., 2008). The root of this climber is used for example as a condiment by populations of Central Africa (Tsopmo et al., 1999), as an aphrodisiac (Noumi et al., 1998), for toothache (Betti, 2004), and for peptic ulcer in Cameroon (Noumi and Dibakto, 2000). In the Democratic Republic of Congo, the root is used against stomach pain and haemorrhoids (Makumbelo et al., 2008). Additionally, the leaves are employed against diarrhoea in the traditional pharmacopoeia in Cameroon (Jiofack et al., 2009). In other respects, P. brazzeana berries are used as sweeteners (Tsopmo et al., 1999). In previous studies, the aqueous extract of P. brazzeana root was shown to possess androgenic activity (Kamtchouing et al., 2002). Additionally, the crude extract of P. brazzeana root as well as the compounds isolated from the extract (three thioureas and three ureas) have exhibited a moderately strong antiplasmodial activity against two Plasmodium falciparum strains (chloroquine-resistant Indochina W-2 and chloroquine-sensitive Sierra Leone D-6)

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(Ngamba, 2004). Compared with the extract, the compounds showed a better activity, even though they are less potent than chloroquine. They were also as effective against the chloroquineresistant as the chloroquine-sensitive strain of the parasite. In addition, the essential oil obtained from the root was shown to possess free-radical scavenging, anti-inflammatory, antibacterial and antifungal activities (Nyegue et al., 2009). Previous phytochemical investigations have focused on brazzein and pentadin – sweet-tasting proteins isolated from the fruits of P. brazzeana (Ming and Hellekant, 1994; van der Wel et al., 1989). In other respects, some arylalkyl isothiocyanates (ITCs) and related thiocarbamates and thioureas were isolated from root extracts (El Migirab et al., 1977; Tsopmo et al., 1999). The isolated arylalkyl ITCs were benzyl ITC, 4-methoxybenzyl ITC, and 2-(4methoxyphenyl)-2,2-dimethylethyl ITC, which would indicate the presence of benzyl-, 4-methoxybenzyl-, and 2-(4-methoxyphenyl)-2,2-dimethylethyl GL, respectively, in the root of P. brazzeana. Choline was also found in P. brazzeana (McLean et al., 1996). More recently, hydrodistillation of the root produced an essential oil, the major constituents of which were benzyl ITC and benzyl cyanide (78% and 17%, respectively) (Nyegue et al., 2009; Koudou et al., 2001) indicating the presence of glucotropaeolin (1a) in the plant. Finally, Mithen et al. detected, by LC–MS, low levels of indole GLs (numbers and names not mentioned) in herbarium tissue of P. brazzeana, low levels of 1a in fresh air-dried young leaf, and traces of 1a and higher concentrations (exact quantification not given) of two methoxybenzyl GLs in the root (Mithen et al., 2010). The presence of ITCs in processed parts of the plant led us to investigate the GL profile in P. brazzeana root, seed, and leaf. In the scope of our continued interest in the chemistry of Brassicales (Montaut et al., 2009, 2010) the GLs present in the root, seed, and leaf of P. brazzeana were determined herein. A systematic investigation using a standard HPLC-UV spectrophotometry protocol for desulfo-GLs (DS-GLs) was performed. In order to complete this study, the ITCs generated by myrosinase-catalyzed degradation of GLs were analyzed by GC–MS. The results obtained from this phytochemical study – which is not fully consistent with previous findings - are presented and discussed.

2. Results and discussion First, the GL content in root, seed, and leaf of P. brazzeana was determined, according to the EU official method ISO 9167-1, based on the HPLC analysis of DS-GLs (EEC, 1990). The HPLC chromatogram showed the presence of one major peak at tR = 15.4 min and two minor peaks at tR = 13.7 and 15.9 min in the extract of P. brazzeana root. The peak at tR = 13.7 min was assigned to DSbenzyl GL (1b), on the basis of the retention time and the UV spectrum of a spiked standard which indicated that P. brazzeana root originally contained glucotropaeolin (GTL, 1a) (Fig. 1). The compound at tR = 15.9 min displayed spectroscopic data identical to those of a standard sample of DS-m-methoxybenzyl GL (2b) obtained from Limmanthes alba Hartweg ex. Benth. Our spectroscopic data were also compared with those reported in the literature for m-methoxybenzyl GL (Stevens et al., 2009). The UV spectrum was characteristic of an aromatic compound with a kmax at 273 nm. From the mass spectrum (MS), the mass of the compound was established to m/z 359. The 1H NMR spectroscopic data indicated the presence of a meta-disubstituted aromatic ring bearing one methoxy group (dH 3.87 ppm) and one benzylic methylene group (dH 4.05 ppm) (Table 1). Signals attributable to a b-linked glucopyranosyl moiety were observed (Table 1), including the doublet of the anomeric proton (dH-10 4.74 ppm, Jvic = 9.5 Hz) (Montaut et al., 2009). The assignment of carbons and protons was achieved by analysis of HMBC and HMQC 2D shift correlations.

This is the first time that 2b has been obtained from P. brazzeana root and spectroscopically characterized. This result is indicative of the presence of m-methoxybenzyl GL (2a), also known as glucolimnanthin (GLI), in the plant (Fig. 1). The compound at tR = 15.4 min 3b was obtained from a seed extract of P. brazzeana, via desulfation using Helix pomatia sulfatase EC. 3.1.6.1 and DEAE Sephadex A-25 anion exchange resin. The UV spectrum of 3b exhibited a maximum absorption at 226 nm and a second one with a lower intensity at 275 nm, which is similar to the reported data for p-methoxybenzyl isothiocyanate (Kjær et al., 1956). The 1H NMR spectroscopic data indicated the presence of a para-disubstituted aromatic ring displaying two broad doublets (dH 7.31 and 7.04 ppm), one methoxy group (dH 3.85 ppm) and one benzylic methylene group (dH 3.99 ppm) (Table 1). Similarly to 2b, signals attributable to a b-linked glucopyranosyl moiety (dH-10 4.74 ppm, Jvic = 9.4 Hz) were observed (Table 1) (Montaut et al., 2009). The assignment of carbons and protons, ascertained by HMBC and HMQC 2D experiments, led to the structure of DS-p-methoxybenzyl GL for compound 3b (Fig. 1). This is the first time that 3b has been obtained from P. brazzeana seed and spectroscopically characterized. This result is indicative of the presence of p-methoxybenzyl GL (3a), also known as glucoaubrietin (GAU), in the plant. Additionally, a GC–MS analysis of the ITCs, obtained from the myrosinase (thioglucoside glucohydrolase, EC. 3.2.1.147)-catalyzed hydrolysis of the GL-containing extract of the root and the one of the seed, in phosphate buffer, confirmed the profiles observed for DS-GLs. The DS-GLs HPLC chromatogram of the leaf extract showed one major peak at tR = 14.2 min and other minor peaks eluting at tR = 16.1, 22.7, 23.5 and 23.8 min. The UV spectrum of the first peak was similar to the one of a para-substituted arylalkyl GL (Wathelet et al., 2004) with a kmax at 228 nm and another one at 280 nm. Retention time and UV spectrum did not correspond to those of the DS-GL standards available in our laboratory. The identification was therefore based on the GC–MS analysis of the ITCs obtained after treatment by myrosinase. 3,4-Dimethoxybenzyl ITC and traces of benzyl ITC were detected, indicating that 3,4-dimethoxybenzyl GL (4a) and traces of GTL (1a) exist in the leaf of P. brazzeana. In addition, the GC–MS analysis showed a large quantity of 3,4-dimethoxybenzaldehyde and traces of 3,4-dimethoxybenzyl cyanide and 3,4-dimethoxybenzylalcohol. However, the latter three compounds were also detected by GC–MS in the leaf extract before enzyme treatment. 3,4-Dimethoxybenzyl cyanide may originate from the hydrolysis of 3,4-dimethoxybenzyl GL, whereas 3,4-dimethoxybenzyl alcohol may result from the degradation of 3,4-dimethoxybenzyl ITC. This could also mean that 3,4-dimethoxybenzyl GL has suffered hydrolysis during harvest and/or storage of the leaf sample. The second peak present in the DS-GL HPLC chromatogram of the leaf extract was assigned to desulfoglucobrassicin (DS-GBS) (5b) by comparison of the retention time and the UV spectrum with those of a purified standard, indicating that P. brazzeana leaf originally contained GBS (5a). The three other minor peaks with retention times typical of those of desulfo-indole GLs remain unidentified. The contents of individual and total GLs in the root, seed and leaf were evaluated by using a defined external calibration curve of a DS-sinigrin (DS-SIN) standard, considering the following relative proportionality factor (RPF) values: 0.95 for DS-GTL (1b) and 0.29 for DS-GBS (5b) as reported in the literature (Wathelet et al., 2004), 0.46, and 0.55 for DS-GAU (3b) and DS-GLI (2b), respectively, as evaluated in this study for the first time. The desulfo-3,4-dimethoxybenzyl GL (4b) present in the leaf was quantified using an arbitrary RPF value equal to 1 because its isolation and purification was not achieved, given the small amount found in the leaf. The results of the quantification are reported in Table 2. The root of P. brazzeana contained 81–82% of GAU, 15–16% of

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Fig. 1. GLs and corresponding DS-GLs from P. brazzeana.

Table 1 NMR Spectroscopic Data (D2O) for DS-glucolimnanthin (2b) and DS-glucoaubrietin (3b). Position

2b

3b dH (J in Hz)

dC Glucose moiety 10 20 30 40 50 60

80.8 71.6 76.7 68.5 79.4 60.0

Arylaliphatic moiety 1 2 3 4 5 6 7 8 OCH3

dC

4.74, d (9.5) 3.36, m 3.36, m 3.44, m 3.25, ddd (1.9, 4.8, 6.2) H-60 a 3.70, dd, (2.0, 12.5) H-60 b 3.67, dd, (3.5, 12.5)

158.9 37.6 137.4 130.0 154.0 120.5 113.3 112.4 55.0

– 4.05, – 7.39, – 7.00, 7.00, 7.00, 3.87,

81.3 72.1 77.2 68.9 79.9 60.2

158.2 37.5 128.0 129.4 114.6 154.9 114.6 129.4 55.5

s t (7.9) m m m s

dH (J in Hz) 4.74, d (9.4) 3.34, m 3.34, m 3.42, m 3.25, ddd (2.5, 4.8, 7.6) H-60 a 3.70, dd (2.5, 12.5) H-60 b 3.65, dd, (4.8, 12.5) – 3.99, – 7.31, 7.04, – 7.04, 7.31, 3.85,

s d (8.6) d (8.6) d (8.6) d (8.6) s

Table 2 GL content of P. brazzeana root, seed, and leaf. Glucosinolates (lmol/g dry weight)

Root Seed Leaf

GTL (1a)

GLI (2a)

GAU (3a)

3,4-Dimethoxybenzyl GL (4a)

GBS (5a)

Total GLs

8.1 ± 0.9 – –

1.6 ± 0.2 – –

38.3 ± 2.0 109.1 ± 5.0 –

– – 2.1 ± 0.1

– – 0.1 ± 0.0

48.0 ± 3.1 109.1 ± 5.0 2.2 ± 0.1

GTL, and 3–4% of GLI. Quite interestingly, the seed extract of P. brazzeana was shown, by HPLC measurements, to contain exclusively GAU in a considerable amount (5.2% w/w based on the whole non-defatted seed). The above results apparently lacked consistency with data previously obtained from hydrodistillation experiments (Nyegue et al., 2009). Therefore, this discrepancy led us to repeat the hydrodistillation process on a portion of the sample of P. brazzeana root used for the quantification of GLs. The essential oil obtained (yield 0.07%) was shown by GC-FID and GC–MS analyses to contain 57% of benzyl ITC, 10% of benzyl cyanide and only 5% of p-methoxybenzyl ITC. This disconcerting selective recovery of ITCs could be interpreted through a marked difference in volatility between the benzyl and the p-methoxybenzyl species. Additionally, a possible partial hydrolytic degradation of p-methoxybenzyl ITC during the process can be hypothesized. However, it has to be noted that to date no conclusive literature

data can support our assumption regarding the stability of p-methoxybenzyl ITC in aqueous media. It is also worth mentioning that the presence of 2-(4-methoxyphenyl)-2,2-dimethylethyl GL anticipated from the isolation of the corresponding ITC in the root of P. brazzeana (El Migirab et al., 1977) was not confirmed in our analysis of the DS-GLs. T. pendula and P. brazzeana do not show very similar GL profiles. In T. pendula, isopropyl GL, benzyl GL, glucobrassicin (5a), 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, and N-acetylglucobrassicin were identified, whereas in our study, no alkyl GL could be detected (Mithen et al., 2010; Schraudolf and Bäuerle, 1986; Christensen et al., 1982; Schraudolf, 1965). Only traces of 5a were detected in the leaf of P. brazzeana. Therefore, our results do not support the claim, based on botanical observations, of strong phylogenetic similarity between T. pendula and P. brazzeana.

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Plants from the Caricaceae family contain only benzyl GL, while Capparaceae species contain several aliphatic, sulfur-containing, and indole GLs. Arylaliphatic glucosinalbin (4-hydroxybenzyl GL) and glucotropaeolin have also been identified in Capparaceae (Fahey et al., 2001). Thus P. brazzeana does not seem to have such a close phylogenetic link with the Caricaceae and the Capparaceae. A closer relationship would appear between P. brazzeana and Tropaeolum sp., in which benzyl- and 4-methoxybenzyl GLs predominate (Fahey et al., 2001). Our results confirm the singularity of P. brazzeana and support its classification in the separate Pentadiplandraceae family. 3. Conclusions In summary, for the first time, the GL composition in the root of P. brazzeana is described. In addition, the GL content in the seed was investigated and thus showed that this plant organ contains exclusively GAU (3a). Furthermore, the spectroscopic data of DS-GAU (3b) and DS-GLI (2b) are reported, as well as establishing the presence of 3,4-dimethoxybenzyl GL (4a), GBS (5a) and GTL (1a) for the first time in the leaf. The total GL content was also determined and each GL was quantified in the different plant organs of P. brazzeana. In the literature, p-methoxybenzyl ITC was reported to possess antibacterial activity against sexually transmitted diseases Neisseria gonorrhoeae, Haemophilus ducreyi, Candida albicans and the antibiotic sensitive and resistant organisms (Swart et al., 2002). Now that a good source of 3a has been identified, further bio-activity investigations could be envisaged with 3a, as the precursor of the above ITC. 4. Experimental 4.1. General experimental procedures Deuterium oxide (D2O) was purchased from Carlo Erba ReactifsSDS (Val de Reuil, France). HPLC analyses of DS-GLs were performed on an Agilent model 1100 equipped with a diode array detector. Optical rotations [a]D were measured at 25 °C with a Perkin–Elmer 141 polarimeter. The specific rotation is expressed in units of 101° cm2 g1 and concentration (c) in g/100 mL. UV spectra were determined on a computerized Varian Cary 300 Bio UV/Visible spectrophotometer equipped with a dual cell peltier accessory. NMR spectra were recorded at 400 MHz (1H) and 100 MHz (13C) on a Bruker Avance 2 spectrometer, d values being referenced to D2O at 4.80 ppm. Mass spectra were recorded on a Perkin-Elmer SCIEX API-300 spectrometer (electrospray, positive mode). HRMS spectra were recorded on a Bruker Maxis spectrometer (electrospray, positive mode). 4.2. Plant material P. brazzeana root, leaf and seed were harvested on February 5th 2010 at Elounden (Yaoundé, Cameroon) by Mrs. Ada, botanist at the National Herbarium of Cameroun in Yaoundé. A voucher specimen is kept at the National Herbarium of Cameroon in Yaoundé (Identification No. 6538NM/01). 4.3. HPLC analysis and quantification of desulfoglucosinolates GLs were extracted as previously reported with some modifications (Barillari et al., 2005). Leaf, seed and root were reduced to a fine powder. Samples of ca. 500 mg were extracted for 5 min at 80 °C twice with EtOH–H2O (5 mL, 70:30 v/v), using a U-Turrax (IKA T25) homogenizer and then centrifuged. Supernatants were combined to reach a final volume of 10 mL. Each extract (1 mL) was loaded onto a mini-column filled with DEAE-Sephadex A-25 anion-exchange resin (0.6 mL, GE Healthcare) conditioned with 25 mM sodium acetate buffer (pH 5.6). After washing with buffer (3 mL), puri-

fied sulfatase (200 lL, 5.83 nkat/mL) (Leoni et al., 1998) was loaded onto the mini-column which was left overnight at 30 °C. The DS-GLs were then eluted with ultra pure H2O (3 mL) and finally injected into an HPLC. The DS-GLs were analyzed on an Agilent 1100 HPLC system equipped with an Inertsil ODS-3 column (250  3.0 mm, 5 lm particle size), thermostated at 30 °C, and having a diode array as detector. The chromatography was performed at a flow rate of 1 mL min1 eluting with a gradient of H2O (A) and CH3CN (B) following the program: 1 min 1% B; 22 min linear gradient up to 22% B; 3 min linear gradient down to 1% B. Desulfo-GLs were detected by absorbance monitoring at 229 nm. The amount of GL was quantified by using a calibration curve of pure DS-SIN solution (range from 0.14 to 1.4 mM) and the RPFs of each individual DS-GLs (Wathelet et al., 2004). The RPFs for DS-GAU (3b) and DS-GLI (2b), unreported in the literature, were thus determined experimentally. Serial water dilutions of pure 3b (range from 0.06 to 1.42 mM) and 2b (range from 0.05 to 2.48 mM) were used to construct a reference HPLC calibration curve for each compound. The RPFs were then calculated relatively to DS-SIN as the ratio between the slope values of the calibration curve obtained for pure DS-SIN and the one for the tested compound. Identification of the peaks was performed on the basis of retention times and UV spectra of spiked DS-GTL (1b) and DS-GBS (5b) pure standards available in our laboratory (Leoni et al., 1998) and DS-GLI (2b), purified in the present study (see Subsection 4.4). 4.4. Preparation of desulfoglucolimnanthin (2b) A sample of standard 2b was prepared according to Leoni et al. (1998) from intact GLI (2a), previously purified from Limnanthes alba, by a one-step anion exchange chromatography (Visentin et al., 1992; Barillari et al., 2001). 4.4.1. Desulfoglucolimnanthin (2b) White amorphous powder; HPLC, tR = 15.9 min; ½a25 D 37 (c 1.0, H2O); UV (H2O) kmax 273 nm; for 1H NMR (D2O) and 13C NMR (D2O) spectroscopic data, see Table 1; ESI+-MS m/z 720 [2M+H]+ (4), 398 [M+K]+ (12), 382 [M+Na]+ (18), 360 [M+H]+ (25), 198 [M+HC6H11O5]+ (100), 121 [M-C7H12NO6S]+ (59); HRESIMS m/z 382.0903 [M+Na]+ (calcd. for C15H21NO7SNa, 382.0931). 4.5. Extraction of glucoaubrietin (3a) and preparation of desulfoglucoaubrietin (3b) Ripe seeds (4 g) of P. brazzeana were extracted twice with boiling EtOH–H2O (170 mL, 70:30 v/v). After centrifugation, the solvent was eliminated with a rotary evaporator to afford crude extract (1 g). This was dissolved in H2O (5 mL) and loaded onto five mini-columns filled with DEAE-Sephadex A-25 anion-exchange resin (1.8 mL) conditioned with H2O. After washing with H2O, purified sulfatase (500 lL, 12.2 nkat/mL) was loaded onto the columns and left overnight at 30 °C. Elution ultra pure H2O (4 mL) followed by freeze-drying yielded pure DS-GAU (80 mg) (3b). 4.5.1. Desulfoglucoaubrietin (3b) White amorphous powder; HPLC, tR = 15.4 min; ½a25 D 36 (c 0.3, MeOH) (Cassel et al., 1998); UV(H2O) kmax 226 and 275 nm; for 1H NMR (D2O) and 13C NMR (D2O) spectroscopic data, see Table 1; ESI+-MS m/z 757 [2M+K]+ (5), 741 [2M+Na]+ (13), 719 [2M+H]+ (4), 398 [M+K]+ (25), 382 [M+Na]+ (100), 360 [M+H]+ (32), 198 [M+H-C6H11O5]+ (66), 121 [M-C7H12NO6S]+ (87); HRESIMS m/z 382.0903 [M+Na]+ (calcd. for C15H21NO7SNa, 382.0931). 4.6. Hydrodistillation and chemical analysis The essential oil was extracted and analyzed according to the previously published procedure (Nyegue et al., 2009). Fresh roots

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(5 g) were submitted to hydrodistillation for 5 h using a Clevengertype apparatus. The resulting volatile extract was recovered in a 0.08% solution of tridecane in hexane (5 mL) previously introduced in the decantation part of the Clevenger apparatus. The organic phase was collected, dried over anhydrous Na2SO4 and immediately analyzed by GC-FID and GC–MS. The identification of the constituents was based on the comparison of their relative retention times and mass spectra with those obtained from authentic samples and/or spectra of the NBS 75K and Wiley 7th NIST 98 EPA/ NIH libraries and literature data (Adams, 2007). The percentage composition of the oil constituents was obtained from electronic integration measurements of GC-FID peak areas without use of response factors correction. The essential oil yield was calculated relatively to the tridecane peak area given by GC-FID analysis.

4.7. Conversion of glucosinolates into isothiocyanates and chemical analysis GLs from freeze-dried samples of P. brazzeana root, seed or leaf were extracted twice as described in Section 4.3. The hydroalcoholic extract was evaporated to dryness with a rotary evaporator and the residue dissolved in potassium phosphate buffer (2 mL) 50 mM pH 7. ITCs were produced via enzymatic hydrolysis of GLs from the crude extract by the addition of myrosinase (200 lL, 0.2 lkat/mL) to the buffered solution. After 30 min at 37 °C, the resulting ITCs were extracted with CH2Cl2 (5 mL) and analyzed by GC–MS. The myrosinase (b-thioglucoside glucohydrolase, EC 3.2.1.147) we used was isolated from seeds of Sinapis alba L. as described by Pessina et al. (1990) with some modifications. The enzyme solution was stored at 4 °C in sterile distilled H2O until use. One myrosinase katal was defined as the amount of enzyme able to hydrolyze sinigrin (1 mol) per second at pH 6.5 and 37 °C (Palmieri et al., 1987). GC–MS analyses of ITCs were carried out using a Hewlett–Packard GCD System model G1800A equipped with a 30 m  0.25 mm capillary column HP-5 ms. The flow rate of the carrier gas (He) was 1 mL min1. The column temperature was 40 °C at the beginning and 230 °C at the end, with a rate of 10 °C min1. The temperatures of the injector and of the detector were 180 and 280 °C, respectively. The identification of mmethoxybenzyl- and p-methoxybenzyl ITCs was assigned on the basis of retention times and mass spectra of ITCs obtained by myrosinase-catalyzed degradation of pure 2a available in our laboratory and 3a extracted in this study, respectively. 3,4-Dimethoxybenzyl ITC was identified using a standard synthesized in the laboratory, as previously reported (Goodyer et al., 2003) whereas benzyl ITC was assigned using a standard purchased from Fluka. 3,4-Dimethoxybenzaldehyde, 3,4-dimethoxybenzyl cyanide and 3,4-dimethoxybenzyl alcohol detected in the leaf extract were identified by matching the recorded mass spectra with the NBS75K.L (NIST, 1992 version) mass spectrum library of the GC– MS data system.

4.7.1. Benzyl isothiocyanate EIMS 70 eV m/z (rel. int.): 149 [M]+ (3), 91 [M-NCS]+ (100), 77 [M-CH2NCS]+ (1), 65 (13), 51 (4). The observed data are in agreement with the ones described by El Migirab et al. (1977).

4.7.2. m-Methoxybenzyl isothiocyanate EIMS 70 eV m/z (rel. int.): 179 [M]+ (33), 121 [M-NCS]+ (100), 91 (26), 77 [M-CH2NCS-OCH2]+ (12), 65 (6), 51 (6). The observed data are in agreement with the ones described by Vaughn and Berhow (2005).

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4.7.3. p-Methoxybenzyl isothiocyanate EIMS 70 eV m/z (rel. int.): 179 [M]+ (7), 121 [M-NCS]+ (100), 91 (5), 77 [M-CH2NCS-OCH2]+ (10), 65 (2), 51 (4). The observed data are in agreement with the ones described by El Migirab et al. (1977). 4.7.4. 3,4-Dimethoxybenzyl isothiocyanate EIMS 70 eV m/z (rel. int.): 209 [M]+ (17), 151 [M-NCS]+ (100), 135 (6), 107 [C7H7O]+ (16), 91 (5), 77 (5), 65 (7), 51(9). The observed data in agreement with the ones described by Radulovic´ et al. (2008). Acknowledgements Financial supports from the Natural Sciences and Engineering Research Council of Canada (Discovery grant, S.M.) and l’Agence Universitaire de la Francophonie (M.N.) are gratefully acknowledged. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, fourth ed. Allured Publishing Corporation, Carol Stream, Il 60188-2787, SA. Baillon, H., 1886. Quelques nouveaux types de la flore du Congo. Bull. Mens. Soc. Linn. Paris 1, 609–612. Barillari, J., Gueyrard, D., Rollin, P., Iori, R., 2001. Barbarea verna as a source of 2-phenylethyl glucosinolate, precursor of cancer chemopreventive phenylethyl isothiocyanate. Fitoterapia 72, 760–764. Barillari, J., Iori, R., Rollin, P., Hennion, F., 2005. Glucosinolates in the subantarctic crucifer Kerguelen cabbage (Pringlea antiscorbutica). J. Nat. Prod. 68, 234–236. Betti, J.L., 2004. An ethnobotanical study of medicinal plants among the Baka pygmies in the Dja biosphere reserve, Cameroon. Afr. Study Monogr. 25, 1–27. Cassel, S., Casenave, B., Déléris, G., Latxague, L., Rollin, P., 1998. Exploring an alternative approach to the synthesis of arylalkyl and indolylmethyl glucosinolates. Tetrahedron 54, 8515–8524. Christensen, B.W., Kjær, A., Øgaard Madsen, J., Olsen, C.E., Olsen, O., Sørensen, H., 1982. Mass-spectrometric characteristics of some pertrimethylsilylated desulfoglucosinolates. Tetrahedron 38, 353–357. Dounias, E. Pentadiplandra brazzeana Baill. Record from Protobase, In: Schemelzer, G.H., Gurib Fakim, A. (Eds.), PROTA (Plant Resources of Tropical Africa/ Ressources végétales de l’Afrique tropicale), Wageningen, Netherlands. Available from: . (accessed 14.04.09). EEC Regulation No. 1864/90, Enclosure VIII. 1990. Oilseeds – determination of glucosinolates – high performance liquid chromatography. Off. J. Eur. Communities L170, 27-34. El Migirab, S., Berger, Y., Jadot, J., 1977. Isothiocyanates, thiourées et thiocarbamates isolés de Pentadiplandra brazzeana. Phytochemistry 16, 1719–1721. Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5–51. Goodyer, C.L.M., Chinje, E.C., Jaffar, M., Stratford, I.J., Threadgill, M.D., 2003. Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase. Bioorg. Med. Chem. 11, 4189–4206. Hall, J.C., Iltis, H.H., Sytsma, K.J., 2004. Molecular phylogenetics of core Brassicales, placement of orphan genera Emblingia, Forchhammeria, Tirania, and character evolution. Syst. Bot. 29, 654–669. Hutchinson, J., Daziel, J.M., 1958. Pentadiplandraceae, in: Flora of West Tropical Africa 1. The Crown Agents for the Colonies, London, U.K., pp. 649–651. Jiofack, T., Ayissi, I., Fokunang, C., Guedje, N., Kemeuze, V., 2009. Ethnobotany and phytomedicine of the upper Nyong valley forest in Cameroon. Afr. J. Pharm. Pharmacol. 3, 144–150. Kamtchouing, P., Mbongue Fandio, G.Y., Dimo, T., Jantsa, H.B., 2002. Evaluation of androgenic activity of Zingiber officinale and Pentadiplandra brazzeana in male rats. Asian J. Androl. 299–301. Kjær, A., Gmelin, R., Jensen, R.B., 1956. Isothiocyanates XV. P-Methoxybenzyl isothiocyanate, a new natural mustard oil occurring as glucoside (glucoaubrietin) in Aubrieta species. Acta Chem. Scand. 10, 26–31. Koudou, J., Sakanga, O., Menut, C., Bessière, J.M., 2001. Constituants volatils de l’huile essentielle de Pentadiplandra brazzeana Baillon de Centrafrique. Pharm. Med. Trad. Afr. 11, 31–35. Leoni, O., Iori, R., Haddoum, T., Marlier, M., Wathelet, J.-P., Rollin, P., Palmieri, S., 1998. Approach to the use of immobilized sulphatase for analytical purposes and for the production of desulfo-glucosinolates. Ind. Crops Prod. 7, 335–343. Mabberley, D.J., 1990. The plant book. A portable dictionary of higher plant. Cambridge University Press, Cambridge, pp. 130–131. Makumbelo, E., Lukoki, L., Paulus, J.J.S.J., Luyindula, N., 2008. Stratégie de valorisation des espèces ressources des produits non ligneux de la savane des environs de Kinshasa:II. Enquête ethnobotanique (aspects médicinaux). Tropicultura 26, 129–134.

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