The key role of 13 C NMR analysis in the identification of individual...

Special Issue: Research Article

Received: 19 May 2014, Revised: 16 July 2014, Accepted: 17 August 2014 Published online in Wiley Online Library: 2 October 2014

(wileyonlinelibrary.com) DOI 10.1002/ffj.3215

The key role of 13C NMR analysis in theidentification of individual components ofPolyalthia longifolia leaf oil†

Zana A. Ouattara,a,b Jean Brice Boti,a Antoine Coffy Ahibo,b

Sylvain Sutour,a Joseph Casanova,a Félix Tomia and Ange Bighellia*

ABSTRACT: Polyalthia longifolia produces sesquiterpene-rich essential oils (EOs) whose compositions varied substantiallyfrom sample to sample depending on the origin of the plant (Nigeria and Vietnam). Nothing is known about the phytochem-istry of Ivoirian P. longifolia. The aim of the present study was to characterize Ivoirian P. longifolia through the chemical com-position of the leaf oil and to develop a strategy that allows the identification of minor oxygenated sesquiterpenes whose MSdata are not compiled in commercial or laboratory-constructed MS libraries. The EO was submitted to gas chromatography(GC) retention index (RI), GC-mass spectrometry (MS) and 13C nuclear magnetic resonance (NMR) analysis. Then hydrocarbonsand oxygenated components were separated and the oxygenated fraction was chromatographed on silica gel. The fractionswere analysed by GC(RI) and 13C NMR. Seventy compounds accounting for 91.8% of the EO were identified. Sesquiterpenehydrocarbons, (E)-β-caryophyllene (27.8%), α-zingiberene (20.0%) and allo-aromadendrene (15.0%), were the major compo-nents. Various oxygenated sesquiterpenes whose MS data were not compiled in commercial and laboratory-made MS librar-ies were identified by comparison of their chemical shift values in the spectrum of the fraction of CC with those reported inthe literature and compiled in a laboratory-constructed 13C NMR data library. The composition of the investigated Ivoirian P.longifolia oil sample presented similarities and differences with Nigerian and Vietnamese oils. Combined analysis of IvoirianP. longifolia EO by chromatographic and spectroscopic techniques including 13C NMR without isolation of the components,appeared particularly efficient to identify minor components of EOs, whose MS spectra are insufficiently differentiated orMS data are not compiled in commercial and lab-constructed MS libraries. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: Polyalthia longifolia; Annonaceae; 13C NMR analysis; essential oil composition; Ivory Coast

* Correspondence to: Ange Bighelli, Université de Corse-CNRS, UMR 6134SPE, Equipe Chimie et Biomasse, Route des Sanguinaires, 20000 Ajaccio,France. E-mail: [email protected]

† This article is part of the virtual special issue of the Flavour and FragranceJournal entitled “Essential oils: chemical analysis and biological properties”edited by Patrizia Rubiolo and Paula Dugo.

a Université de Corse-CNRS, UMR 6134 SPE, Equipe Chimie et Biomasse,Route des Sanguinaires, 20000 Ajaccio, France

b Laboratoire de Chimie Organique Biologique, UFR-SSMT, Université FélixHouphouët-Boigny, BPV 34 Abidjan, Ivory Coast

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IntroductionThe genus Polyalthia belongs to the Annonaceae family, withmorethan 70 species, this is one of the few genera of Annonaceae com-mon to tropical parts of Africa, Madagascar, Oceania and especiallySouth-East Asia. Polyalthia longifolia, native to India and Sri Lanka,has been introduced in many countries in tropical climates, partic-ularly in the Ivory Coast. It is a tree with a straight bole, small-to-medium sized and it reaches 25m in height. The leaves areovate-oblong to narrowly lanceolate. They measure 11 to 22 cmlong and 2 to 6 cm wide. The flowers are in the axils of leaves orfallen leaves. Polyalthia longifolia is planted as an ornamentalshade tree and along the roadside.[1] The bark is used against skindiseases, fever, diabetes and hypertension. The antimicrobialactivity of leaves has been demonstrated.[2]

Various compounds were isolated from extracts ofP. longifolia, mainly clerodane-type diterpenes[3–5] and alka-loids.[6–8] They have recently been reviewed.[9] In contrast, onlya few studies addressed the volatile compounds of P. longifolia.Although all the oil samples were characterized by a high con-tent of sesquiterpenes, a substantial chemical variability was ob-served. Indeed, a preliminary study on the leaf oil led to theidentification of azulene derivatives.[10] More recently, Ogunbinuet al. investigated the chemical composition of leaf and stembark oils from Nigeria.[11] The composition of leaf oil was domi-nated by sesquiterpene hydrocarbons: allo-aromadendrene

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(19.7%), (E)-β-caryophyllene (13.0%), as well as β-selinene,α-humulene and ar-curcumene (7.9–6.8% each) besidecaryophyllene oxide (14.4%). Stem bark oil was characterizedby the preeminence of other sesquiterpene hydrocarbonssuch as α-copaene, β-selinene, viridiflorene, α-guaiene, allo-aromadendrene and δ-cadinene (8.7-7.0% each) besideα-muurolol (8.7%). Finally, another sesquiterpene hydrocarbonzingiberene (26.7%) was the main compound identified inthe bark oil of P. longifolia var. pendula from Vietnam accompa-nied by (E)-β-caryophyllene (11.1%) and β-bisabolene (7.9%)whereas (E)-β-caryophyllene (30.0%), α-zingiberene (21.7%),aromadendrene (15.2%) and β-selinene (9.1%) were the majorcomponents of the leaf oil.[12,13]

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In continuation of our on-going work on the characterizationof aromatic plants of the Ivory Coast through the chemical com-position of their essential oils, we conducted this study on theleaf oil from P. longifolia.[14–16] In this paper we highlight thekey role of 13C nuclear magnetic resonance (NMR) (in combina-tion with column chromatography) in the identification of oxy-genated sesquiterpenes whose mass data are not compiled incommercial computerized mass spectrometry (MS) libraries.

Material and Methods

Plant Material

Leaves of P. longifolia were collected in Abidjan-Cocody, inJanuary 2011. Plant material was authenticated by Professor L.Aké Assi, from the Centre National Floristique (CNF, Abidjan,Ivory Coast).

Essential Oil Isolation and Fractionation

The leaves (7 × 1.4 kg) were submitted to hydrodistillation on aClevenger-type apparatus for 3 h. The oil (6 g) was decanted

Figure 1. Fractionation scheme

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and stored in a glass vial at 5 °C prior to analysis. A portion ofthe essential oil (EO) (4.502 g) was chromatographed on silicagel (ICN, 250–500μm, 135 g) and two fractions (F1, 4.060 g andF2, 0.428 g) were eluted with n-pentane and diethyl oxide,respectively (Figure 1). A part of the fraction F2 (0.375 g) waschromatographed once again on a silica gel column (Kieselgel,0.035–0.070μm, 20 g) and 12 fractions (F2.1–F2.12 = 25.9, 18.3,17.1, 25.2, 56.2, 43.3, 38.4, 30.1, 44.3, 44.7, 15.9 and 8.0mg) wereeluted with a gradient of solvents of increasing polarity (n-pen-tane/diethyl oxide 98/2 to 0/100).

Analytical Gas Chromatography

Analyses were carried out with a Clarus 500 Perkin Elmer (PerkinElmer, Courtaboeuf, France) apparatus equipped with twoflame ionisation detectors (FID), and two fused-silica capillarycolumns (50m×0.22mm i.d., film thickness 0.25μm), BP-1(polymethylsiloxane) and BP-20 (polyethylene glycol). The oventemperature was programmed from 60 °C to 220 °C at 2 °C/minand then held isothermal at 220 °C for 20min; injector tempera-ture: 250 °C; detector temperature: 250 °C; carrier gas, helium(0.8ml/min); split: 1/60; injected volume: 0.5μl. The response

of Polyalthia longifolia leaf oil

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13C NMR analysis of Polyalthia longifolia leaf oil

factor (RF) of each compound was calculated according to thecalculation method described by Tissot et al.[17] Methyloctanoate was used as an internal reference. Weight and per-centage of each compound were calculated using weight ofEO and reference, area and RF.

Gas Chromatography-MS Analysis

The essential oil was analysed with a Perkin Elmer TurboMass(Perkin Elmer) detector (quadrupole), directly coupled to aPerkin Elmer Autosystem XL (Perkin Elmer), equipped with afused-silica capillary column (60m×0.22mm i.d., film thickness0.25μm), Rtx-1 (polydimethylsiloxane). Carrier gas, helium at1ml/min; split, 1/80; injection volume, 0.2μl; injector tempera-ture, 250 °C; oven temperature programmed from 60 °C to 230 °Cat 2 °C/min and then held isothermal (45min); ion source temper-ature, 150 °C; energy ionization, 70 eV; electron ionization massspectra were acquired over the mass range 35–350Da.

NMR

All 13C NMR spectra were recorded on a Bruker AVANCE (Bruker,Wissembourg, France) 400 Fourier Transform spectrometer oper-ating at 100.623MHz for 13C, equipped with a 5-mm probe, indeuterated chloroform (CDCl3), with all chemical shifts referredto as internal tetramethylsilane (TMS). 13C NMR spectra were re-corded with the following parameters: pulse width (PW), 4μs(flip angle 45°); acquisition time, 2.7 s for 128 K data table witha spectral width (SW) of 24000Hz (240 ppm); digital resolution,0.183Hz/pt. The number of accumulated scans was 3000 forthe oil sample and fractions of chromatography (about 50mgof essential oil in 0.5ml of CDCl3).

Identification of Individual Components

Identification of individual components was based (i) on com-parison of their GC retention indices (RI) on apolar and polarcolumns, determined relative to the retention times of a seriesof n-alkanes with linear interpolation (‘Target Compounds’ soft-ware of Perkin-Elmer), with those of authentic compounds or lit-erature data,[18] (ii) on comparison of their mass spectral datawith those of reference compounds compiled in computerizedlibraries,[19–21] (iii) by 13C NMR spectroscopy, according to themethodology developed and computerized in our laboratories,using home-made software (see text).[22–25] Various compoundshave been identified by comparison of their 13C NMR chemicalshift values with those reported in the literature (see text).

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13C NMR Data of Some Components

2-Methylnonanal 8. 13C NMR data: δ: 204.25 C, 46.50 CH, 31.94CH2, 31.76 CH2, 29.67 CH2, 29.35 CH2, 27.00 CH2, 22.71 CH2,14.10 CH3, 13.50 CH3.

Zingiberenol I 48. (Fraction F2.8) 13C NMR data: δ: 134.32 CH,133.39 CH, 131.35 C, 124.66 CH, 67.39 C, 40.49 CH, 37.26 CH2,36.25 CH, 34.18 CH2, 29.66 CH3, 25.99 CH2, 25.74 CH3, 20.11CH2, 17.67 CH3, 15.73 CH3.

Zingiberenol II 51. (Fraction F2.12) 13C NMR data: δ: 134.57 CH,132.08 CH, 131.34 C, 124.67 CH, 69.85 C, 40.10 CH, 38.26 CH2,36.24 CH, 34.16 CH2, 28.37 CH3, 26.0 CH2, 25.74 CH3, 22.33 CH2,17.67 CH3, 15.70 CH3.


Caryophyllenol I 61. 13C NMR data: δ: 153.30 C, 136.95 C, 125.64CH, 109.91 CH2, 69.58 CH, 58.78 CH, 42.9 CH, 37.11 CH2, 36.63 CH2,33.77 C, 32.30 CH2, 30.18 CH3, 26.94 CH2, 22.16 CH3, 16.86 CH3.trans-β-Sesquiphellandrol 65 . 13C NMR data: δ: 145.19 C,

135.01 CH, 131.40 C, 126.49 CH, 124.60 CH, 113.11 CH2, 69.50CH, 36.01 CH, 35.55 CH, 34.10 CH2, 31.16 CH2, 25.97 CH2, 25.70CH3,17.67 CH3,16.08 CH3.

Preparation of Bornyl Formate 10

In a single-necked flask fitted with a condenser, 0.149 g(3.2mmol) of formic acid and 1.0 g (6.5mmol) of borneol in15ml of chloroform were introduced. The reaction mixture wasrefluxed for 5 h. The solvent was then removed on a rotary evap-orator. The reaction mixture (0.286 g) was chromatographed ona silica gel column (63–200μm). Bornyl formate (colourless oil,218mg, 1.2.10–3mol, 37.4%, GC purity: 95%) was eluted withpentane/diethyl oxide 98/2.NMR data of bornyl formate 10 (δ, ppm): 1H NMR: 8.11 (1H, s,

H-11), 5.01(1H, d, J = 9.98 Hz, H-1), 2.39 (1H, m, H-2b), 1.94 (1H, m,H-5a), 1.77 (1H, m, H-4a), 1.70 (1H, t, J = 4.48Hz, H-3), 1.32 (1H, m,H-5b), 1.25 (1H, m, H-4b), 1.03 (1H, dd, J = 9.98, 3.52Hz, H-2a), 0.91(3H, s, H-9), 0.89 (3H, s, H-8), 0.85 (3H, s, H-10). 13C NMR: 161.51(C-11), 79.98 (C-1), 48.83 (C-6), 47.91 (C-7), 44.90 (C-3), 36.72 (C-2),27.99 (C-4), 27.07 (C-5), 19.70 (C-9), 18.82 (C-8), 13.40 (C-10).

Results and DiscussionHydrodistillation of leaves from P. longifolia, collected inAbidjan-Cocody, allowed the isolation of an essential oil with apoor yield (0.05–0.07% w/w calculated on a fresh material basis).Analysis of the bulk sample was carried out by GC (in associationwith RI on two columns of different polarity), by GC-MS and by13C NMR according to a method developed by our group.[22–25]

According to the chromatogram profiles, it was evidencedthat P. longifolia leaf oil belonged to the family ofsesquiterpene-rich essential oils on the one hand and that co-elution of various components occurred on both capillary chro-matographic columns. Moreover, computer matching againstcommercial mass spectral libraries suggested the identificationof various major and minor components with very poor fits. Incontrast, the major components were unambiguously identifiedby 13C NMR using the computerized method and the NMR spec-tral library constructed in our lab. Unfortunately, 13C NMR doesnot allow the identification of minor components (less than0.3–0.4%) and after GC(RI) and GC-MS analysis most of the minorcomponents (probably oxygenated sesquiterpenes according totheir RI) remained unidentified.Identification of individual components of natural mixtures

(essential oils, resins and extracts) by MS and by 13C NMRexhibits analogies but also strong differences. Indeed, bothtechniques are based on the comparison of experimental data(mass fragments or 13C NMR chemical shifts) with those ofreference compounds compiled in computerized commercialor laboratory-made spectral data libraries. Every techniqueexhibits advantages and disadvantages. For instance, MS ismuch more sensitive than NMR although nowadays thedifference is reduced since ultra high-field spectrometers cameonto the market (proton spectra of small volatile compoundsmay be acquired at nanogram-scale).[26] Conversely, varioussesquiterpenes and diterpenes, usually found in essential oils,exhibit insufficiently differentiated mass spectra, as could be

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seen with spectra of α-cedrene and α-funebrene forinstance (two tricyclic sesquiterpene hydrocarbons that differonly by the stereochemistry of the ring junction) or incontrast with the spectra of epoxyfarnesatrienols andbisepoxyfarnesatrienes.[22,27] Moreover, frequently these com-pounds have a very close RI and therefore their identificationby GC-MS in combination with RIs remains a difficult task.Identification of individual components of a natural mixtureby GC-MS is also difficult if not impossible for compounds thatco-elute on the GC columns at our disposal and for heat sensi-tive compounds that rearrange on the injector or on the GCcolumn during the analysis.[16] The worst occurs when the MSdata of an essential oil component are not compiled in thecomputerized libraries that simultaneously contain spectra ofother compounds exhibiting similar fragmentations. As aconsequence, although GC-MS still remains a very efficientmethod for the analysis of volatiles, misidentification mayoccur during the analysis of a complex essential oil using thecombination of GC-MS and RIs, when the analyst neglect torespect some rules during the data treatment.

In contrast, the 13C NMR spectrum of a molecule may beconsidered as its fingerprint. In other words, two compounds,including sesquiterpenes, diterpenes, triterpenes and so one,exhibit always enough chemical shift values of their carbons suffi-ciently differentiated to allow their identification. Therefore, takinginto account various parameters (the number of observed signals,the number of overlapped signals, the difference of chemical shiftmeasured in the mixture and in the reference spectra) theidentification of an individual component of a complex mixtureis possible without individualization of the compound.[22–25]

The quality of the libraries is obviously crucial and anothermajor difference between MS and 13C NMR occurs at this level.More precisely, introducing one more compound in a computer-ized MS library requires that the mass spectrum of thatcompound should be recorded before being introduced in theMS library. Indeed, using MS data reported in the literature isnot an alternative way, as all the fragments, as well as their

Figure 2. Identification of individual components of an essential oil using 13

tion. Left: library constructed with 13C NMR spectra recorded in our laborato


respective intensities, are not systematically reported in theexperimental part. The major fragments that are in generalreported do not allow identification of sesquiterpenes and diter-penes that often exhibit insufficiently differentiated mass spec-tra. In contrast, the 13C NMR chemical shifts of every newcompound isolated from a natural mixture or synthesized arecompletely reported. Taking into account that nowadays highfield spectrometers allow the record of spectra in dilutesolutions, and therefore the difference of chemical shiftmeasured in the mixture and in the reference spectra is verythin, the chemical shift values reported in the literature may bedirectly introduced in the 13C NMR spectral data library.

Therefore, identification of the minor components of P.longifolia leaf oil was conducted by combination of chromato-graphic and spectroscopic techniques: GC(RI), GC-MS and 13CNMR, followed by fractionation of the bulk sample on a silicagel chromatographic column (CC) and combined analysis of allthe fractions by GC(RI) and 13C NMR. Concerning NMR, theexperimental procedure may be summarized as follow: the 13CNMR chemical shift values of the experimental spectra arematched against the spectra of reference compounds compiledinto two computerized libraries, the first one built with data com-ing from spectra recorded in our laboratory, the second one builtwith 13C NMR data reported in the literature (Figure 2).

In total, 70 compounds (11 monoterpenes, 53 sesquiter-penes, two acyclic compounds, three fatty acids and a diter-pene acid), accounting for 92.2% of the EO, were identified(Table 1). The composition of the EO was largely dominatedby sesquiterpene hydrocarbons (83.0%). (E)-β-Caryophyllene18 (27.5%), α-zingiberene 29 (11.9%), allo-aromadendrene 24(14.1%) and α-humulene 22 (8.3%) were the major components.Other sesquiterpene hydrocarbons usually produced by aromaticplants, such as α-selinene 30 (2.8%), β-selinene 28 (2.5%), trans-β-bergamotene 27 (1.9%), trans-α-bergamotene 20 (1.7%),α-copaene 15 (1.3%) and δ-cadinene 36 (1.2%) were identified.

Monoterpene hydrocarbons (2.6%) were represented byα-pinene 1 (1.6%) and (E)-β-ocimene 5 (0.6%) beside camphene

C nuclear magnetic resonance (NMR) spectroscopy without individualiza-ry; right: library constructed with 13C NMR data reported in the literature


Table 1. Components of Polyalthia longifolia leaf oil

No. Components° RI lit ɵ RIa RIp RF g/100 g Identification

1 α-pinene 936 a 930 1024 0.765 1.6 RI, MS, 13C NMR2 camphene 950 a 943 1071 0.765 0.6 RI, MS3 myrcene 987 a 980 1163 0.765 0.3 RI, MS4 limonene 1025 a 1021 1202 0.765 0.1 RI, MS5 (E)-β-ocimene 1041 a 1036 1252 0.765 0.6 RI, MS, 13C NMR6 2-nonanone 1074 a 1071 1389 0.889 tr RI, MS, 13C NMR7 linalool 1086 a 1085 1544 0.869 0.1 RI, MS, 13C NMR8 2-methylnonanal † 1157 1436 0.852 tr RI, MS, 13C NMR9 α-terpineol 1175 a 1173 1692 0.869 tr RI, 13C NMR10 bornyl formate + 1212 1576 0.985 0.1 RI, MS, 13C NMR11 thymol 1267 a 1268 2199 0.846 0.1 RI, MS, 13C NMR12 bornyl acetate 1270 a 1270 1579 0.958 0.1 RI, MS, 13C NMR13 geranyl acetate 1362 a 1360 1754 0.959 tr RI, 13C NMR14 α-ylangene 1376 a 1370 1482 0.787 0.1 RI, MS15 α-copaene 1379 a 1376 1494 0.787 1.3 RI, MS, 13C NMR16 isocaryophyllene 1409 a 1409 1574 0.752 0.3 RI, MS17 4,8-α-epoxycaryophyllane 1417 b 1410 1564 0.821 0.2 RI, MS, 13C NMR18 (E)-β-caryophyllenea 1421 a 1420 1600 0.752 27.5 RI, MS, 13C NMR19 4,8-β-epoxycaryophyllanea 1425 b 1420 1580 0.821 0.3 RI, 13C NMR20 trans-α-bergamotene 1434 a 1433 1587 0.752 1.7 RI, MS, 13C NMR21 (E)-β-farnesene 1446 a 1447 1666 0.752 0.8 RI, MS, 13C NMR22 α-humulene 1455 a 1452 1670 0.752 8.3 RI, MS, 13C NMR23 allo-aromadendrene 1462 a 1460 1647 0.752 14.1 RI, MS, 13C NMR24 ar-curcumenea 1473 a 1471 1769 0.708 4.3 RI, 13C NMR25 γ-muurolenea 1474 a 1471 1688 0.752 0.8 RI, 13C NMR26 β-humulenea 1474 a 1471 1675 0.752 0.7 RI, 13C NMR27 trans-β-bergamotene 1480 a 1479 1684 0.752 1.9 RI, MS, 13C NMR28 β-selinene 1486 a 1482 1718 0.752 2.5 RI, MS, 13C NMR29 α-zingiberene 1489 a 1488 1723 0.752 11.9 RI, MS, 13C NMR30 α-selinene 1494 a 1492 1719 0.752 2.8 RI, MS, 13C NMR31 sesquicineole 1507 a 1503 1741 0.821 0.1 RI, MS, 13C NMR32 γ-cadinene 1510 b 1505 1756 0.752 0.6 RI, MS, 13C NMR33 calamenene# 1512 b 1508 1828 0.762 0.4 RI, MS34 5,8-cyclocaryophyllan-4-ol 1514 a 1513 1949 0.821 0.2 RI, 13C NMR35 β-sesquiphellandrenea 1516 a 1515 1773 0.752 0.7 RI, 13C NMR36 δ-cadinenea 1520 a 1515 1756 0.752 1.1 RI, 13C NMR37 caryolan-8-ola 1566 a 1560 2044 0.821 0.5 RI, 13C NMR38 palustrola 1569 a 1560 1922 0.821 0.1 RI, 13C NMR39 4-formyl-5-nor-β-caryophyllene † 1564 1994 0.831 tr RI, 13C NMR40 caryolan-4-ola 1576 a 1570 2088 0.821 0.1 RI, 13C NMR41 caryophyllene oxidea 1578 a 1570 1976 0.831 2.3 RI, MS, 13C NMR42 5,11-epoxycadin-1(10)-enea 1578 c 1575 1929 0.831 tr RI, 13C NMR43 globulola 1589 a 1575 2067 0.821 0.1 RI, 13C NMR44 viridiflorol 1592 a 1582 2076 0.821 0.1 RI, MS, 13C NMR45 humulene oxide I 1593 a 1583 2009 0.831 0.1 RI, 13C NMR46 humulene oxide IIa 1597 a 1594 2032 0.831 0.6 RI, 13C NMR47 ledola 1600 a 1594 2022 0.821 0.4 RI, 13C NMR48 zingiberenol Ia † 1599 2107 0.821 0.6 RI, 13C NMR49 neo-intermedeola 1602 e 1599 2132 0.821 tr RI, 13C NMR50 muurola-4,10(14)-dien-1β-ol † 1611 2159 0.831 0.1 RI, 13C NMR51 zingiberenol IIa † 1615 2190 0.821 0.1 RI, 13C NMR52 caryophylla-4(14),8(15)-dien-5α-ola 1623 d 1615 2309 0.831 tr RI, 13C NMR53 caryophyllenol IIa ͋ 1615 2373 0.831 0.1 RI, 13C NMR54 1-epi-cubenola 1623 a 1615 2058 0.821 0.1 RI, 13C NMR55 τ-cadinola 1633 a 1626 2179 0.821 tr RI, 13C NMR56 τ-muurolola 1633 a 1626 2199 0.821 tr RI, 13C NMR57 torreyol 1632b 1629 2211 0.821 tr RI 13C NMR

(Continues)


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Table 1. (Continued)

No. Components° RI lit ɵ RIa RIp RF g/100 g Identification

58 cubenol 1636b 1631 2052 0.821 tr RI, MS, 13C NMR59 selin-11-en-4α-ola 1641 e 1638 2265 0.821 tr RI, 13C NMR60 α-cadinola 1643 a 1638 2245 0.821 0.3 RI, 13C NMR61 caryophyllenol Ia † 1638 2341 0.831 tr RI, 13C NMR62 β-bisabolol 1659 a 1655 2138 0.821 0.1 RI, 13C NMR63 α-bisabolol 1673 a 1666 2230 0.821 0.1 RI, 13C NMR64 bisabola-2,10-dien-1-ol † 1668 2296 0.821 0.1 RI 13C NMR65 trans-β-sesquiphellandrol † 1675 2362 0.831 tr RI, MS, 13C NMR66 phytone † 1718 2173 0.783 tr RI, MS, 13C NMR67 oleic acid 13C NMR68 linoleic acid 13C NMR69 linolenic acid 13C NMR70 kovaleic acid 13C NMR

Total 92.2

°Order of elution and percentages are given on apolar column, those with an asterisk * excepted, percentage on polar column. RIa,RIp = Retention indices on apolar (BP1) and polar (BP-20) column, respectively. RI lit ɵ : retention indices from literature, a ,[33] b ,[29]

c ,[36] d ,[45] e ,[46] + RIs of pure compounds, see text, † : RI not available in the literature, ͋: values reported on the literature varieddrastically from paper to paper. # correct isomer not specified. RI (italic) = retention indices in agreement with those measured inthe fraction of CC. 13C NMR (italic) = compounds identified by NMR in the fractions of CC.


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2, myrcene 3 and limonene 4. All of these hydrocarbons havebeen identified by comparison of their RIs on two columnsof different polarity and their MS and 13C NMR data with thoseof reference compounds compiled in commercial libraries (MS)or laboratory-built library (13C NMR).

Oxygenated monoterpenes (0.5%) were really minor compo-nents (tr-0.1% each). Linalool 7, thymol 11 and bornyl acetate12 were identified by the three techniques. Curiously the pres-ence of α-terpineol 9 and geranyl acetate 13 was only ensuredby NMR in the fractions of CC. Indeed, α-terpineol and geranyl ac-etate, although very common components of EOs, do not have agood fit in GC-MS analysis. They were probably overlapped withan unidentified component. In contrast, they were unambigu-ously identified by 13C NMR in the fraction of CC, F2.10 (1.2%)and F2.4 (4.0%), respectively. For component 10 (RIs = 1212/1576), a bornyl ester was suggested by MS and the characteristicsignals of the bornane skeleton (belonging to a compound thataccounted for 14.2%) were observed in the 13C NMR spectrumof fraction F2.1. According to its RI, the component is probablybornyl formate. Therefore, this compound was prepared byformylation of borneol and the chromatographic (RIs) and spec-troscopic data (MS, 13C NMR) of the unidentified compoundfitted perfectly with those of the genuine compound.

In parallel, two oxygenated acyclic non terpene compoundshave been identified at the trace level. 2-Nonanone 6 and2-methylnonanal 8 have been identified by GC(RI) and GC-MS inthe EO. Their identification was confirmed by 13C NMR infractions of CC (6=5.1% in F2.4; 8=7.4% in fraction F2.1). 13CNMR data of 2-methylnonanal 8 were not reported in the litera-ture. They have been assigned by comparison with NMR data of2-methyldecanal.[28]

In total, 19 sesquiterpene hydrocarbons have been identified,most of them including the main components cited in theprevious paragraph, by GC(RI) and by GC-MS and/or 13C NMR.RIs of these components fitted perfectly with those containedin our home-made data library and with those reported byJoulain and Konig.[18] Identification of minor sesquiterpene


hydrocarbons by GC(RI) and GC-MS was successful for mostcompounds, however, 13C NMR was revealed to be usefulfor the identification of the correct stereoisomer (trans-α-bergamotene 20, (E)-β-farnesene 21, trans-β-bergamotene 27),and crucial for the identification of compounds which co-elutedon the apolar column used to carry out GC-MS analysis(ar-curcumene 24, γ-muurolene 25 and β-humulene 26,β-sesquiphellandrene 35 and δ-cadinene 36). Other minor ses-quiterpene hydrocarbons were α-ylangene 14, isocaryophyllene16, γ-cadinene 32 and calamenene 33.

Oxygenated sesquiterpenes were also very minor compo-nents that accounted for 2.4% in total. Identification of variouscomponents by GC-MS was uncertain. Indeed, for a given GCpeak, several compounds were suggested with a similar scoreand it was hazardous to choose in spite of the help of RI, whenavailable. 13C NMR played a key role in the identification of thesecomponents, by matching the chemical shift of the spectra ofthe fractions of chromatography with those of referencecompounds whose NMR data are compiled in two laboratory-constructed libraries, the first one with spectra recorded in ourlab, the second one with data reported in the literature. Theiridentification is reported below.

NMR data of various oxygenated sesquiterpenes were presentin our library constructed with spectra recorded in ourlaboratory and all these components were easily identified infractions of CC, and then their content in the EO was evaluatedusing RI: sesquicineole 31, palustrol 38, caryophyllene oxide41, globulol 43, viridiflorol 44, ledol 47, humulene oxide II46, neo-intermedeol 49, caryophylla-4(14),8(15)-dien-5α-ol 52,caryophyllenol II 53, 1-epi-cubenol 54, τ-cadinol 55, τ-muurolol56, cubenol 58, selin-11-en-4α-ol 59, α-cadinol 60, β-bisabolol62 and α-bisabolol 63. It should be pointed out that variouscompounds were not identified in the EO, neither by MS norby 13C NMR. For instance caryolan-8-ol 37 (at the trace level inthe EO) was overlapped with palustrol 38 on the apolar column;caryolan-4-ol 40 was overlapped with caryophyllene oxide 41,etc. In parallel, 13C NMR is a powerful tool for differentiation of



stereoisomers and diastereoisomers. This point is perfectly illus-trated by the identification of α-bisabolol 63 which possessesidentical mass spectrum and RI on apolar and polar columnsvery close to that of its epimer epi-α-bisabolol. Therefore, it isquite difficult to identify the correct α-bisabolol diastereoisomerby GC-MS and GC(RI) when only one isomer is present in the EO.

Otherwise, various oxygenated sesquiterpene componentshave been identified by comparison of their chemical shiftsmeasured on the spectra of CC fractions with those reported inthe literature. Several of these compounds were suggested byGC-MS analysis. For others, no proposal was made duringGC-MS analysis or the proposal was ruled out by comparisonwith 13C NMR analysis of the fractions of CC.

Figure 3. Molecular structure of oxygenated sesquiterpenes identifiedin Polyalthia longifolia leaf oil. 17=4,8-α-epoxycaryophyllane, 19=4,8-β-epoxycaryophyllane, 34=5,8-cyclocaryophyllan-4-ol, 37= caryolan-8-ol, 39=4-formyl-5-nor-β-caryophyllene, 40= caryolan-4-ol, 42=5,11-epoxycadin-1(10)-ene, 48=zingiberenol I, 50=muurola-4,10(14)-dien-1β-ol, 51=zingiberenol II, 61= caryophyllenol I, 64=bisabola-2,10-dien-1-ol, 65= trans-β-sesquiphellandrol, 66=phytone. The absolutestereochemistry has not been established. The drawn structure is intendedto better illustrate the cis-trans relationship of the substituents

Components Suggested by MS and Confirmed by NMR

4,8-α-epoxycaryophyllane 17 and 4,8-β-epoxycaryophyllane 19.Both isomers have similar mass spectra and a very close RI(RIa/RIp = 1410/1564 and 1420/1580) and the β- isomer isoverlapped with (E)-β-caryophyllene 18 on the apolar columnused for GC-MS analysis. They accounted for 3.4% and 19.1%,respectively, in fraction F2.1 and they have been identified bycomparison of the chemical shifts in the 13C NMR spectrum ofthat fraction with those reported by Weyerstahl et al., who iso-lated both isomers from Brazilian Cangerana oil.[29] RIs fittedwith those reported on CP Sil5 CB column (1417 and 1425).

trans-β-Sesquiphellandrol 65 (bisabola-1,3(15), 10-trien-4-ol,7.3% in fraction F2.7, RI = 1675/2362). Its structure wassuggested by MS but the stereochemistry of the hydroxyl groupversus the side chain was not determined as both isomers havea similar mass spectra. Both sesquiphellandrol stereoisomershave been isolated from the EO of Zingiber officinale and theirstructural elucidation was based on chemical transformationand 1H NMR spectral data.[30] In contrast, 13C NMR data havenot been reported. Nevertheless, the 15 signals ofβ-sesquiphellandrol have been extracted from the spectrum offraction F2.7 and they were assigned by comparison with NMRdata of bisabola-1,10-diene-3,4-diol and those of cis and transp-mentha-1(7),2-dien-6-ol (NMR data of both isomers in ourspectral data library).[31] Therefore, the compound present inP. longifolia leaf oil is trans-β-sesquiphellandrol.

Phytone 66 (5.2% in fraction F2.4) was also suggested byGC-MS and confirmed by comparison of the carbon chemicalshifts with those reported by Ladépêche.[32]

37

Components not Identified by MS and Identified by NMR(Figure 3)

5,8-cyclocaryophyllan-4-ol 34 was present in the fraction F2.7(RIa/RIp = 1513/1949, 8.7%). Identification was carried out inagreement with the 13C NMR chemical shifts reported byWeyerstahl et al., who isolated this compound from BrazilianCangerana essential oil. The RI on an apolar column fitted withthe values (RIa = 1518 and 1514) was first reported byWeyerstahl et al. and subsequently by Hochmuth.[29,33]

Caryolan-8-ol 37 and caryolan-4-ol 40. Both isomers aretricyclic sesquiterpenols. Caryolan-8-ol 37 (synonym: caryolan-1-ol) was the main component (RIa/RIp = 1560/2044; 24.7%) offraction F2.8. 13C NMR chemical shifts fitted with those reported


by Tiliacos[34] and RI on the apolar column was in agreementwith that mentioned by Weyerstahl et al.[29] (RIa = 1566).Caryolan-4-ol 40 (syn = isocaryolan-8-ol) was the majorcomponent of fraction F2.11. (48.0%, RIa/RIp = 1570/2088). 13CNMR chemical shifts and the RI on an apolar column fitted withthose reported by Weyerstahl et al. (RIa = 1566).[29]

4-formyl-5-nor-β-caryophyllene 39 (4-formyl-8-methylene-4,11,11-trimethylbicyclo [6.2.0]decane) accounted for 5.7% inthe fraction F2.1, RIa/RIp = 1564/1994). 13C NMR chemical shiftswere assigned according to Abraham et al., who obtained thiscompound by biotransformation of (E)-β-caryophyllene withChaetomium cochliodes.[35]

5,11-epoxycadin-1(10)-ene 42 represented 3.7% of thefraction F2.4. 13C NMR chemical shifts and RI (RIa = 1575) werefitted with the data reported by Weyerstahl et al. (RIa = 1578),who isolated the compound from Brazilian Vassoura oil(Baccharis dracunculifolia).[36] It should be mentioned that 42was overlapped with globulol 43 on the apolar column.Humulene oxide I 45 (2,3-epoxy-α-humulene) accounted for

4.4% in fraction F2.5. Its RI on the apolar column (RIa = 1583) isclose to that measured by Srivastava et al. in the leaf oil ofSyzygium aromaticum (RIa = 1589).[37] 13C NMR chemical shiftsfitted with those reported by Khomenko et al. who obtainedthe compound by epoxidation of humulene.[38]

Zingiberenols I and II, 48 and 51. The major components offractions of CC F2.7 (57.0%) and F2.10 (17.7%) remainedunidentified after GC(RI) and GC-MS analysis. However, RI values


7


378

(1599/2107 and 1615/2190) and the similarity of the massspectra suggested the presence of oxygenated sesquiterpenestereoisomers. Moreover, the 15 signals belonging to each mol-ecule were extracted from the NMR spectra of fractions F2.7 andF2.10. Computer matching against the 13C NMR data libraryconstructed with literature data allowed the identification ofzingiberenols I and II.[39]

Zingiberenol exists in the form of four diastereoisomers(Figure 3): zingiberenol I or cis-4-(6-methyl-hept-5-en-2-yl)-1-methyl-cyclohex-2-en-1-ol (cis stereochemistry of the carbonchain and the methyl group) (two diastereoisomers) andzingiberenol II or trans-4-(6-methyl-hept-5-en-2-yl)-1-methyl-cyclohex-2-en-1-ol (two diastereoisomers). The four diastereoiso-mers have been obtained by synthesis.[39] The reported 13C NMRchemical shifts concerned the mixture of both epimers of cis andtrans isomers. For instance, the 13C NMR spectrum ofzingiberenols I contained 21 signals, as well as the 13C NMRspectrum of zingiberenols II. Therefore, six out of 21 signalsbelong solely to one epimer, six other signals belong solely tothe second epimer and the remaining nine signals belong toboth epimers. Polyalthia longifolia leaf oil contained only oneof both epimers of zingiberenol I and zingiberenol II (Figure 3).However, the exact stereochemistry of carbon C7 (relativeto C4 and C1) of these four diastereoisomers is not determinedto date.

Muurola-4,10(14)-dien-1β-ol 50 accounted for 4.5% in fractionF2.5 (RIa/Rip = 1611/2159). It was identified by comparison of13C NMR chemical shifts with those reported by Weyerstahlet al., who found this sesquiterpene alcohol in the EO of Aglaiaodorata from Vietnam.[40]

Torreyol 57 also known, inter alia, as δ-cadinol or cedrenolaccounted for 6.7% in fraction F2.10. The structure of thatcompound has been the subject of controversy. It was defini-tively established by stereoselective total synthesis and thechemical shifts belonging to this compound in F2.10 fit withthose reported by the authors.[41] Chemical shift assignmenthas been done using extensive 2D NMR (NOESY and Lanthanideshift reagent).[42] Moreover, the RI on apolar column (RIa = 1629)is in agreement with those reported, RI = 1631[43] and RI = 632.[29]

Caryophyllenol I 61, (caryophylla-3,8(15)-dien-5α-ol). FractionF2.8 of CC contained caryolan-8-ol 37 as major component(24.7%), followed by selin-11-en-4α-ol 59 (18.3%) and anothercompound with RIa/RIp = 1638/2341, 18.3%. The 15 chemicalshifts belonging to this compound were close to those ofcaryophyllenol II, a compound whose NMR data are compiledin our lab-constructed 13C NMR library. Therefore, we suspectedthe occurrence of the caryophyllenol epimer at the level of thefunctionalized carbon C5. 13C NMR chemical shifts ofcaryophyllenol I have been partially reported by Groweiss andKashman.[44] The five signals of functionalized carbons fittedperfectly with those reported. The complete 13C NMR data ofthat compound are described in the experimental part.

Bisabola-2,10-dien-1-ol 64 accounted for 17.9% in the fractionF2.6. 13C NMR chemical shifts fitted perfectly with those reportedby Sy and Brown, who isolated this compound from a solventextract of Alpinia densibracteata.[31]

Fatty and diterpene acids. The last fraction F2.12 of CC, elutedwith diethyl oxide, contained only acids. Three fatty acids, oleicacid (C18:1 Δ9), linoleic acid (C18:2 Δ9,12) and linolenic acid(C18:3 Δ9,12,15), were identified by comparison of their 13Cchemical shifts with those of pure compounds compiled in ourNMR spectral data library. The unassigned signals suggested


that the fourth acid was not a fatty acid but a diterpene acid.Indeed, the 13C chemical shifts fitted with those reported forkovalenic acid by Hara et al.,[3] who isolated this compound froma solvent extract of P. longifolia. In order to avoid esterification ofthe acids followed by GC, quantification of the four acids wasconducted by NMR, taking into account the mean intensity ofthe non overlapped, perfectly resolved signals of the protonatedcarbons of every compound. The four acids contributed equallyto the CC fraction. Owing that fraction F2.12 accounted for only2.2% of fraction F2 (weighted mass), it could be estimated thatevery acid was present at a trace level in the EO.

ConclusionIvoirian P. longifolia produced sesquiperpene-rich leaf oil,whose composition was dominated by (E)-β-caryophyllene,α-zingiberene, allo-aromadendrene and α-humulene. This EOpresents similarities with that of Vietnamese P. longifolia var.pendula.[12,13] Indeed, both oil samples exhibited high contentsof (E)-β-caryophyllene and α-zingiberene. They differed overallby the content of aromadendrene (Vietnamese oil) orallo-aromadendrene (Ivoirian sample) and also by the lack ofα-humulene in the Vietnamese oil. Ivoirian P. longifolia alsopresents similarities with Nigerian oil, which also containedamong the major compounds, allo-aromadendrene, (E)-β-caryophyllene and α-humulene.[11] However, the latter isdistinguished by the presence of caryophyllene oxide at anappreciable content and by the lack of α-zingiberene.

13C NMR spectroscopy combined with CC appeared crucial forthe identification of various minor components, particularlyoxygenated sesquiterpenes, whose MS data were not compiledin commercial or lab-constructed libraries. These EOcomponents have been identified from the 13C NMR spectrumof every fractions of CC by computer matching against two spec-tral data libraries, the first one constructed with referencespectra recorded in the laboratory, and the second oneconstructed with spectra of pure compounds reported in theliterature. Using the same procedure, fatty acids and a diterpeneacid have been identified without acido-basic extraction noresterification of the acids. It should also be pointed out that P.longifolia leaf oil contained only one epimer of zingiberenolI and II. Finally, the 13C NMR data of various compounds(bornyl formate, caryophyllenol I and trans-β-sesquiphellandrol)have been reported.

Acknowledgements

The authors wish to thank Professor L. Aké Assi for hisvaluable help in the identification of the plant and the Ministèrede l’Enseignement Supérieur de Côte d’Ivoire for providing aresearch grant to Z. A. Ouattara. MS spectra have beenrecorded on the equipment of the CPN Laboratory of theUniversity of Corsica.

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