Received Date: 18-Apr-2016

Revised Date: 21-Sep-2016

Accepted Date: 23-Sep-2016
Article Type: Full Paper
Accepted Article
Chemical diversity and antimicrobial activity of volatile compounds from

Zanthoxylum zanthoxyloides Lam. according to compound classes, plant

organs and Senegalese sample locations

by Yoro Tine a) b), Abdoulaye Diop c), William Diatta d), Jean-Marie Desjobert a)

Cheikh Saad Bouh Boye c), Jean Costa a), Alassane Wélé b) and Julien Paolini*a)
a
) Université de Corse, UMR CNRS 6134 SPE, Laboratoire de Chimie des Produits Naturels,

Campus Grimaldi, BP 52, F-20250 Corte, France (phone: +33-4-95450187; fax: +33-4-

95450180; e-mail: paolini@univ-corse.fr)

b
) Laboratoire de Chimie Organique et Thérapeutique, Faculté de Médecine, Pharmacie et

Odontologie, Université Cheikh Anta Diop, BP: 5005 Dakar-Fann, Sénégal

c
) Laboratoire de Bactériologie-virologie, Hopital Aristide Le Dantec, BP : 3001, Dakar,

Sénégal
d
) Laboratoire de Pharmacognosie et de Botanique, Faculté de Médecine, Pharmacie et

Odontologie, Université Cheikh Anta Diop, BP: 5005 Dakar-Fann, Sénégal

Abstract

The chemical diversity of Z. zanthoxyloides growing wild in Senegal was studied according

to volatile compound classes, plant organs and sample locations. The composition of fruit

essential oil was investigated using an original targeted approach based on the combination of

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 gas chromatography (GC) and liquid chromatography (LC) both coupled with mass

spectrometry (MS). The volatile composition of Z. zanthoxyloides fruits exhibited relative

Accepted Article
high amounts of hydrocarbon monoterpenes (24.3-55.8%) and non-terpenic oxygenated

compounds (34.5-63.1%). The main components were (E)-β-ocimene (12.1-39%), octyl

acetate (11.6-21.8%) and decanol (9.7-15.4%). The GC and GC-MS profiling of fruit

essential oils showed a chemical variability according to geographical locations of plant

material. The LC-MS/MS analysis of fruit oils allowed the detection of seven coumarins in

trace content. The chemical composition of fruit essential oils was compared with volatile

fractions of leaves and barks (root and trunk) from the same plant station. Hexadecanoic acid,

germacrene D and decanal were identified as the major constituents of leaves whereas the

barks (root and trunk) were dominated by pellitorine (85.8% and 57%, respectively), an

atypic linear compound with amide group. The fruit essential oil exhibited interesting

microbial activities against S. aureus and C. albicans, particularly the alcohol fraction of the

oil.

Keywords: Essential oils, Coumarins, Zanthoxylum zanthoxyloides, Antimicrobial activity,

GC-MS, LC-MS/MS.

Introduction. Plants of Zanthoxylum genus (Rutaceae family) are deciduous aromatic shrubs

and trees, comprises about 200 species native to warm temperate and subtropical region of

the world [1]. Thirty-five species have been reported in Africa, among them three taxa are

found in Senegal: Z. zanthoxyloides, Z. leprieurii and Z. rubescens [1]. Z. zanthoxyloides

(Lam.) Zepern and Timler is a medicinal plant widely used in ethnopharmacology throughout

Central and West Africa [1]. This shrub is traditionally used in abdominal and dental

problems, sickle-cell disease, leucoderma, asthma, and in fever and dyspepsia due to their

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 deodorant, disinfectant, and antiseptic properties [1-2]. Many studies have reported that plant

extracts exhibited good antimicrobial [3-13], insecticidal [14], anthelmintic [15],

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antiplasmodial [16-17], vasodilator [18], antisickling [19-21], cytotoxic [22-23],

antiinflammatory [24] and antioxydant activities [5][7].

Previous phytochemical studies on the secondary metabolites of Z. zanthoxyloides

showed the presence of terpenes [25-26], alkaloids (benzophenanthridines, furoquinolines,

aporphines) [26-28], flavonoids [29], amides [25-26][30] and coumarins [25-26][31-32] in

solvent extracts from various organs (roots, stem, bark, leaves and fruits). Moreover, some

papers have reported the chemical composition and biological activities of fruit essential oils

[33-40] from various geographical origins such as Benin and Cameroun. These studies

showed chemical variability according to the amounts of hydrocarbon monoterpenes (-

ocimene, -pinene) and oxygenated monoterpenes (citronellol, geraniol, geranial and

linalool). To the best of our knowledge, the present study is the first report on the chemical

composition and antibacterial activity of essential oils from Z. zanthoxyloides growing wild

in Senegal.

Coumarins and furanocoumarins are a wide group of plant secondary metabolites,

present in tissues of plants, belonging mainly to Apiaceae and Rutaceae families. Essential

oils from Citrus sp. present a high potential contribution to coumarin content in fragranced

products [41-44]. Many studies have also focused on the beneficial effects of this compound

class on human health [45-47]. However, furanocoumarins are also known to exhibit

phototoxic effects [48-49]. The European Cosmetics Directive 76/768/EEC have been

recently modified, introducing for the first time a concentration limit for the use of the

photosensitizing furocoumarins in cosmetics [50].

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 In order to explore the economic potential of Z. zanthoxyloides fruits, a targeted

metabolomic approach based on GC, GC-MS and LC/MS-MS analysis was used for the
Accepted Article
characterization of terpenes and coumarins in essential oils. The volatile compositions of

separated plant parts of Z. zanthoxyloides were also studied including fruit, leaf, bark (root

and trunk). Furthermore, the chemical variability of the essential oils were investigated

according to nineteen fruit samples from six Senegalese populations. The antimicrobial

activity of fruit essential oil and corresponding isolated fractions (hydrocarbons, esters,

carbonyls, alcohols) was evaluated against five strains including Escherichia coli ATCC

35218, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas

aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212 and Candida albicans ATCC

24433.

Results and Discussion. Comparison of volatile fraction obtained by hydrodistillation of

separated plant parts. In comparison with essential oil yield from fruits collected in

Kafountine (1%), hydrodistillation of the leaves, root and trunk barks of Z. zanthoxyloides

harvested on the same tree afforded drastically lower oil yields of 0.04%, 0.01% and 0.002%,

respectively (Table 1). As for other plants of Rutaceae family, this difference could let

suppose that fruits are the most aromatic organs relative to barks and leaves.

Combined analysis of the essential oils from separated plant organs led to the

identification of 79 components, accounting for 73.2 to 94.4% of the total oils (Table 1). 72

compounds were identified by comparing their Electronic Impact-mass spectra and their

retention indices with those of laboratory-made library. Seven constituents (with an asterisk

in Table 1) were identified by comparison of their EI-mass spectra and their apolar retention

indices with those of commercial libraries.

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 GC and GC-MS analysis of Z. zanthoxyloides fruit allowed identifying 39 compounds,

amounting to 94.4% of total oil composition. The volatile fraction of fruits displayed
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aliphatic oxygenated components (51.9%) and hydrocarbon monoterpene (36.5%) rich oil.

Indeed fruit essential oil exhibited (E)--ocimene (Table 1, Entry 13; 29.1%), octyl acetate

(Table 1, Entry 25; 21.3%) and decanol (Table 1, Entry 30; 14.4%) as main components.

The leaf essential oil was characterized by 53 volatile components, accounting for

73.2% of the total composition. In contrary to fruit essential oils, the leaf composition was

dominated by sesquiterpene hydrocarbons accounting to 28.4% of the total oil. The main

component of this fraction was germacrene D (Table 1, Entry 47; 7.6%). Moreover, non-

terpenic components were also reported as major compounds such as hexadecanoic acid

(Table 1, Entry 77; 14.8%) and decanal (Table 1, Entry 24; 5.4%). These results on leaf oil

composition differed from those reported in literature data. Indeed, the essential oil of plant

material collected in Benin [33] contains mainly (E)-β-ocimene (31.9%), α-pinene (26.5%)

and myrcene (30%), while the sample harvested in Ivory Coast [38] was dominated by

germacrene D (10.2%), myrcene (10%) and limonene (7.1%).

The bark of Z. zanthoxyloides showed atypic volatile composition characterized by only

one main component 75 present in root (85.8%) and trunk (57.0%), remained unknown using

GC and GC-MS methodology (comparison of retention indices and mass spectra of the

constituent with those reported in our own library and/or commercial data banks). Thus, this

compound was identified in bark as (2E,4E)-N-(2-methylpropyl)-2,4-decadienamide

(molecular formula C14H25NO, M= 223) by comparison of their EI-MS, 1H and 13

C-NMR

data (Fig. 2) with those previously described in literature [51-53]. This alkamide component,

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 also known as pellitorine, has been previously identified in solvent extracts of Z.

zanthoxyloides [25][30].
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Chemical variability of fruit essential oils according to geographical origins of samples. A

comparative study was conducted on 19 essential oil samples obtained by hydrodistillation of

Z. zanthoxyloides fruits collected from six distinct Senegalese populations (Fig. 1):

Kabrousse (four samples), Boucote (six samples), Kafountine (five samples), Palmarin (one

sample), Noflaye (two samples) and Keur massar (one sample). A fruit sample corresponds to

fruit collected on a tree feet.

The essential oil yields varied significantly according to geographical locations of plant

material, ranging from 0.91 to 1.73% (Table 2). The highest yields were observed for plant

samples from Boucotte (1.73%), Keur Massar (1.54%) and Noflaye (1.27%), while the

lowest were reported from Kabrousse (0.91%), Kafountine (0.98%) and Palmarin (0.98%).

These fruit oil yields were in accordance with those reported earlier by Fogand et al. (1.29%)

[37], Ngassoum et al. (1.1%) [34], Misra et al. (1%) [39] and Oulounde (0.65%) [36].

The analysis of the fruit essential oils by GC/FID and GC/MS allowed the identification

of 39 compounds accounting for 94.2 to 97.7% of the total compositions (Table 2). Oil

samples were dominated by monoterpene hydrocarbons (24.3-55.8%) such as (E)--ocimene

(Table 2, Entry 13; 12.1-39.0%), (Z)-β-ocimene (Table 2, Entry 12; 2.2-8.1%) and -pinene

(Table 2, Entry 3; 3.7-9.5%) associated with non-terpenic oxygenated compounds (34.5-

63.1%) as octyl acetate (Table 2, Entry 25; 11.6-21.8%), decanol (Table 2, Entry 30; 9.7-

15.4%) and octanol (Table 2, Entry 14; 3.5-14.5%). These results showed quantitative

chemical variability of essential oils according to geographical locations of Z. zanthoxyloides

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 populations (Table 2). For instance, the oil from the fruits collected from Kabrousse and

Boucote areas were found to have the highest amounts of (E)--ocimene (Table 2, Entry 13;
Accepted Article
39% and 37.9%, respectively) followed by Noflaye and Kafountine samples (Table 2, Entry

13; 29.1% and 28.6%, respectively), while samples from Palmarin and Keur massar areas

contained the lowest amounts of this component (Table 2, Entry 13; 6.1% and 12.1%,

respectively). It has been also found that the amount of octyl acetate varied between samples

collected from Palmarin, Keur massar and Kafountine (Table 2, Entry 25; 21.8%, 19.3% and

17.3%, respectively) and those of Boucotte, Kabrousse and Noflaye areas (Table 2, Entry 25;

12.3%, 11.6% and 10.5%, respectively).

The content of (E)-β-ocimene (41.5%) in oil sample from Benin [33] was close with

those reported in this work. Conversely, octyl acetate and decanol (in significant amounts in

Senegal samples) were absent in Benin sample which contained mainly oxygenated

monoterpenoids such as linalool (11.3%) and geranial (9.5%). Morover, Ngassoum et al. [34]

found that the fruit essential oil of Z. zanthoxyloides from Cameroon was dominated by

monoterpene hydrocarbons (55.4%), such as -pinene (38.2%) and (E)--ocimene (5.4%),

whereas two other studies [37][39] reported an essential oil composition rich in citronellol

and geraniol.

To synthesize the chemical composition data, Principal Component Analysis (PCA)

and Cluster Analysis (Dendrogram) were applied on the matrix linking essential oil

compositions and sample locations to identify possible relationships between the relative

percentages of volatile compounds and geographical origins. Figure 3 was obtained from the

correlation matrix calculated with the standardized matrix (See Supplementary Material). As

shown in Fig. 3, the principal factorial plane (constructed with axes 1 and 2) summarizes

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 60.59% of the entire variability of essential oils. The distribution of oil samples from Z.

zanthoxyloides fruit oils is reported in Fig. 3a. The distribution of variables (volatile
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components) is shown in Fig. 3b (19 variables). The plot established using PCA suggested

the existence of two main groups of essential oils (Fig. 3a). The first group (Group I),

including all oil samples from Senegal was characterized by highest amounts of -ocimene

and aliphatic oxygenated components such as octyl acetate, decanal, decanol and octanol.

The second group (Group II), represented by four oil samples from Cameroon (Cam1-4) and

one sample from Benin (Ben 1) was characterized by high contents of oxygenated

monoterpenes such as citronellol and geraniol and corresponding esters (citronellyl acetate

and geranyl acetate) or aldehydes (citronellal and geranial). Finally, one oil sample from

Benin (Ben 2) exhibited atypical chemical composition with high amount of -terpinene,

undecane and valencene. Statistical analysis using Cluster Analysis (CA) was also used to

show the correlation between chemotypes and geographical repartition of samples. The

dendrogram obtained by Canonical analysis CA reinforced the clustering observed using

PCA by grouping the 25 oil samples from Z. zanthoxyloides fruit into the same two main

clusters (Fig. 4).

Identification of coumarin components in fruit essential oils. Using targeted LC-MS/MS

methodology based on the screening of coumarin compounds, two simple coumarins (6,7-

dimethylesculetin, herniarin) and five furanocoumarins (psoralen, xanthotoxin, bergapten,

isopimpinellin, imperatorin) have been detected for the first time in the essential oil samples

of Z. zanthoxyloides fruits (Table 3 and Fig. 5). These components were identified by

comparison of retention times, MRM transition and mass spectra in fruit oils with those of

our own library (39 standard components of coumarins). The Multiple Reaction Monitoring

(MRM) transitions and optimized MS/MS parameters (declustering potential, entrance

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 potential, collision cell entrance potential, collison energy, collision cell exit potential) of

each coumarin from fruit oil were described in Table 3. Xanthotoxin was first isolated and
Accepted Article
characterized from an alcoholic extract of Z. zanthoxyloides fruits [31][32]. Thereafter,

Adesina (1986) reported the presence of three simple coumarins (scoparone, scopoletin,

umbelliferone) and five furocoumarins (imperatorin, xanthotoxin, bergapten, marmesin and

psoralen) in dichloromethane extract of dried fruits [25]. It should be noted that all coumarin

compounds identified in this study have been previously reported in essential oils of other

plants from Rutaceae family, especially Citrus and Ruta species [41-44].

Antibacterial activity of fruit essential oils and isolated compound fractions. The essential oil

from Kafountine (mixture of five oil samples) was subjected to flash chromatography and

afforded five fractions, which were then analyzed by GC and GC/MS to determine their

chemical composition (Table 4). Finally, they were tested for their antimicrobial properties.

Antimicrobial screening of the essential oil and its fractions were made by disk

diffusion method against five bacteria (E. coli ATCC 35218, E. coli ATCC 25922, S. aureus

ATCC 29213, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212) and one yeast strain (C.

albicans ATCC 24433). The results showed that the mean inhibition zones of the whole

essential oil and its fractions were less than those of positive controls, gentamycin

(30g/disc) and amphotericin B (10μg/disk), except for the fraction (F5) which had higher

inhibition zones than positive controls against S. aureus and C. albicans (Table 5). The

negative control with DMSO has not showed any inhibition zone against the tested

pathogens.

The total fruit oil and corresponding isolated fractions did not exhibit any antibacterial

activity against E. coli, P. aeruginosa and E. faecalis. The comparison between chemical

compositions of fractions F2 (decanal, octyl acetate and decyl acetate) and F3 (octyl acetate,

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 hexyl acetate, decyl acetate) indicated that carbonyl linear compound (decanal) has higher

antibacterial effect against S. aureus than ester components (octyl acetate, hexyl acetate,
Accepted Article
decyl acetate). In addition, hydrocarbon monoterpenes of fraction F1 (-ocimene

stereoisomers, -pinene) did not exhibited any antibacterial property against S. aureus.

Conversely, the fraction (F5) rich in linear alcohols (octanol and decanol) exhibited the

highest inhibitory activity on S. aureus as well as C. albicans. The oxygenated oil fraction

(F2-F4) showed medium antifungal activity against Candida albicans, while the hydrocarbon

monoterpenes (fraction F1) exhibited low biological effect.

Antibacterial activity of fruit essential from Z. zanthoxyloides has already been reported

[34][37]. Ngassoum et al. [34] have measured a medium activity against S. aureus, E.

faecalis and E. coli. The divergence between present results and those of Ngassoum et al. can

be related to the difference in the composition of the tested essential oils. Indeed, these two

essential oils depict significant differences in their α-pinene concentrations (4.5% vs 38.2%,

respectively). Moreover, this compound was known by his antibacterial activity [55]. Another

published paper [37] on antimicrobial activity of essential oil from Z. zanthoxyloides fruits

showed no relevant activity against E. coli ATCC 25922, P. aeruginosa ATCC 27853, E.

faecalis ATCC 29212 and C. albicans ATCC 24433. This essential oil consisted mainly of

citronellol (29.9%), geraniol (11.5%), citonellyl acetate (5.5%) and limonene (5.5%).

Conclusion. This study reported the chemical variability and the antibacterial activity of the

fruit oils of Z. zanthoxyloides from Senegal. According to the geographic origin of the

populations, the oil composition of the fruits exhibited quantitative variations in the contents

of hydrocarbon monoterpenes (-ocimene stereoisomers, -pinene) and aliphatic oxygenated

compounds (octyl acetate, decanal, decanol). The fruit essential oil contained also trace

amount of simple coumarins and furanocoumarins. The fruits exhibited higher essential oil

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 yield than the other parts of the plant. Moreover, the main volatile constituent of barks (root

and trunk) is pellitorine whereas leaf oils are dominated by hexadecanoic acid and
Accepted Article
germacrene D. Finally, fruit oil is effective for inhibition or S. aureus and C. albicans,

particularly the alcohol fraction with high amount of octanol and decanol. Like other plants

of Rutaceae family, Z. zanthoxyloides fruit oils could be used in cosmetic and/or food

products for their antimicrobial activity as well as in perfumery for their flavouring

properties.

Acknowledgement

We thank the Ministry of Higher Education and Scientific Research of Senegal,

Embassy of France in Senegal and the Territorial Authority of Corsica for their financial

support.

Experimental Part

Plant material. Nineteen samples of Z. Zanthoxyloides fruit pericarps were collected in

may 2015 from six localities of Senegal (Fig.1): Kabrousse, Boucote, Kafountine, Palmarin,

Noflaye, Keur massar. Each fruit sample is harvested on the same tree. Leaf, root bark and

trunk bark samples were collected on the same tree from one station (Kafountine).The plant

material was identified by Dr William Diatta from the Department of botanical and

pharmacognosy of University Cheikh Anta Diop of Dakar.

Essential Oil Isolation. Plant material were air-dried for 14 days at room temperature.

Samples were hydrodistilled (6 h) using a Clevenger-type apparatus according to the method

recommended in the European Pharmacopoeia [56]. The yields of essential oils (w/w,

calculated on dry weight basis) were given in the table 2.

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 GC and GC/MS Analysis. Analyses were carried out using a Perkin-Elmer Autosystem

XL GC apparatus (Walthon, MA, USA) equipped with dual flame ionisation detection (FID)
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system and fused-silica capillarycolumns, namely, Rtx-1 (polydimethylsiloxane) and Rtx-

wax (poly-ethyleneglycol) (60 m × 0.22 mm i.d; film thickness 0.25 μm). The oven

temperature was programmed from 60 to 230°C at 2°C/min and then held isothermally at

230°C for 35 min: hydrogen was employed as carrier gas (1 mL/min). The injector and

detector temperatures were maintained at 280°C, and samples were injected (0.2 μL of pure

oil) in the split mode (1:50). Retention indices (RI) of compounds were determined relative to

the reten-tion times of a series of n-alkanes (C5–C30) by linear interpolationusing the Van

den Dool and Kratz (1963) equation with the aidof software from Perkin-Elmer (Total Chrom

navigator). The relative percentages of the oil constituents were calculated from the GC peak

areas, without application of correction factors.

Samples were also analysed with a Perkin-Elmer Turbo mass detector (quadrupole)

coupled to a Perkin-ElmerAutosystem XL, equipped with fused-silica capillary columns Rtx-1

and Rtx-Wax. The oven temperature was programmed from 60 to 230°C at 2°C/min and then

held isothermally at 230°C (35 min): hydrogen was employed as carrier gas (1 mL/min). The

following chromatographic conditions were employed: injectionvolume, 0.2 μL of pure oil;

injector temperature, 280°C; split, 1:80; ion source temperature, 150°C; ionisation energy, 70

eV; MS (EI) acquired over the mass range, 35–350 Da; scan rate, 1 s.

Identification of the components was based on: (a) comparison of their GC retention

indices (RI) on non-polar and polar columns, determined from the retention times of a series

of n-alkanes with linear interpolation, with those of authentic compounds or literature data;

(b) on com-puter matching with commercial mass spectral libraries [57–59] and comparison

of spectra with those of our personal library; and (c) comparisonof RI, MS and NMR spectral

data of authentic compounds or literature data.

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 LC-MS/MS analysis. Solvents used for liquid chromatography were LC-MS grade

acetonitrile (ACN) obtained from Fisher Scientific (Illkirch, France). Deionized water was
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purified using a MilliQ water (Millipore, Bedford, MA, USA) purification system. Reference

coumarins (98 % purity determined by high-performance liquid chromatography (HPLC)

were purchased from Extrasynthese (Geney, France). Solutions of reference compounds were

prepared by dissolving the compounds in ACN at 0.1 mg/mL and then filtered through a 0.2-

m polytetrafloroethylene (PTFE) filter. Direct-infusion MS analyses were performed with

reference solutions at a concentration of 0.1 mg/L in volume ratio of ACN/H2O = 5:5.

The LC system consisted of a Flexar ultra high performance liquid chromatography

(UHPLC; Perkin-Elmer) with two Flexar FX-10 UHPLC pumps, a Flexar solvent manager, a

275-Flexar autosampler, and a Flexar LC PE200 column oven. UHPLC analyses were

performed on a 100 mm2.1 mm i.d. 3 m, LUNA 3U C18 column (Phenomenex) and the

column temperature was set at 30 °C. A volume of 10 L of sample was injected using an

injection loop of 15 L in full loop mode. The mobile phase consisted of MilliQ water

(solvent A) and ACN (solvent B) at a flow rate of 500 L/min. The column was equilibrated

(A:B; v/v) in 90:10 for 5 min, and elution was carried out with the following linear gradient

90:10 in 5 min, 80:20 in 5 min, 70:30 in 5 min, 60:40 in 5 min, 50:50 in 5 min, an increase

from 50 % B to 100 % in 5 min, and 100 % B for 7 min.

MS2 conditions was carried on AB Sciex 3200 QTRAP (Toronto, Canada) 3200

QTRAP linear triple quadrupole fitted with an Atmospheric Pressure Chemical Ionization

(APCI) ion sources operating in positive mode. Ions were detected with the utilization of the

multiple reaction monitoring mode (MRM). Multiple reactions monitoring transitions were

optimized for all analytes. The process of selection of ion pairs was followed by the

optimization of ionization conditions. High purity nitrogen was used as both nebulizer and

turbo gas. The APCI source was operated with following settings in positive mode: curtain

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 gas (CUR) 25 psi, nebulizer gas (GS1) 31 psi, heater gas (GS2) 65 psi, ion spray voltage (IS)

5000 V and temperature 450°C. The precursor ion [M+H]+ for each of the 39 analytes was
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determined in MS1 full scan tests and the product ions in MS/MS experiments were then

monitored for each transition in MRM mode. The directly infused (concentration: 0.1mg/L;

flow rate: 10 µL/min) respective standards in the MS/MS machine were used for optimizing

perfect conditions for maximum product ion formation, fragmentation and detection of each

analyte. Multiple EPI spectra were recorded in our MS spectral library for each compound.

Data acquisition was performed in the multiple reaction monitoring (MRM) mode, followed

by an EPI scan (MRM-EPI). EPI mass spectra were recorded in the range of m/z = 50–500 at

4000 Da/s. Prior to LC-MS/MS analysis, essential oil was diluted to 1/100 in acetonitrile and

analyzed under the same conditions as the standard.

NMR analysis. The structure elucidation of pelitorine was carried out by 1H and 13
C

NMR, DEPT, and 2D-NMR (HSQC). Spectra were measured in deuterated chloroform using

a Bruker Avance 400 Fourier Transform spectrometer (Wissembourg, France) operating at

400 MHz for 1H-NMR and at 100 MHz for 13C-NMR and equipped with a 5 mm probe. All

shifts were referred to the internal standard tetrame-thylsilane (TMS). NMR spectra were

recorded with the following parameters: pulse width, 4 μs (flip angle, 45°); acquisition time,

2.7 s for 128 K Data table with aspectral width of 25,000 Hz (250 ppm); CPD mode

decoupling; digital resolution, 0.183 Hz/pt. The number of accumulated scanswas 3000–5000

for each sample depending of the amount ofproduct. The 1H-NMR spectra were recorded

with the following parameters: flip angle, 30°; acquisition time, 2.56 s for 32,000 data table

with a spectral width of 7000 Hz (17.5 ppm). 2D-NMR sequences were recorded using

Bruker microprograms.

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 Essential-Oil Fractionation. A mixture of five oil samples from Kafountine (3.98 g)

was submitted to liquid chromatography on a normal phase silica column (40 g, 20–40 mm,
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Clarisep, Bonna Agela Technologies, Willington, USA) using an automatized Combi Flash

apparatus (Teledyne Isco, Inc. Lincoln, NE, USA) equipped with a fraction collector

monitored by an ultraviolet detector (230 nm, 250 nm). The apolar fraction was eluted with

hexane (n-C6H14) and the polar fractions were eluted with a mixture of diisopropyloxide

(iPr)2O/hexane with increasing polarity. The elution gradient percentage of (iPr)2O/nC6H14

(time) was: 0 mL/100 mL (5 min); 2 mL/100 mL (6 min); 3.5 mL/100 mL (6 min); 5 mL/100

mL (6 min); 5-10 mL/100 mL (6 min); 10-100 mL/100 mL (6 min) and 100 mL/100 mL (6

min). The oil fractionation of the essential oil led to one apolar fraction (F1) and four polar

fractions (F2, F3, F4, F5). All fractions were analyzed by GC and GC/MS.

Statistical Analysis. Data analyses were performed using principal component

analysis (PCA) and cluster analysis (CA). Both methods aim at reducing the multivariate

space in which objects (oil samples) are distributed, but are complementary in their ability to

present results. Indeed, PCA provides the data for plots in which both objects (oil samples)

and variables (oil components) are represented, while canonical analysis (CA) informs a

classification tree in which objects (sample locations) are gathered. The PCA was carried out

using the function PCA of the statistical XLSTAT software (Addinsoft, Paris). The

discriminate variables (volatile components) have been selected using function of the

statistical software. A dendrogram (tree) was produced by CA using Ward’s method of

hierarchical clustering, based on Euclidean distances between pairs of oil samples.

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 Microbial strains. The microorganisms used in the present investigation included

reference strains from the American Type Culture Collection (ATCC): Escherichia coli
Accepted Article
ATCC 35218, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213,

Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Candida

albicans ATCC 24433. All of the strains were grown on Mueller−Hinton agar (MHA) for the

bacteria and Saboureaud dextrose agar (SDA) with chloramphenicol for yeast.

Determination of antibacterial activity. Antibacterial activity of the essential oil and its

fractions were assayed using the agar disc diffusion method [60]. Inocula were prepared by

diluting overnight cultures in Mueller-Hinton Broth (MHB, Oxoid) medium to approximately

106 CFU/mL. Filter paper discs (Whatman disc, 6 mm diameter) were impregnated with 10l

of the essential oil and placed onto the inoculated Petri dishes containing Mueller-Hinton 2

Agar. In addition, reference disks without any oil and gentamicin (30g/disc), were used for

comparison. After incubation at 37 ± 1 °C for 18−24 h for bacteria and at 37 ± 1 °C for

24−48 h for yeast, the diameters of inhibition zones were measured (mm) and recorded as the

mean ± standard deviation (SD) (Table 5). Each test was performed in triplicate separate.

According to the width of the inhibition zone diameter expressed in mm, results were

appreciated as follows: not sensitive (−) for diameter equal to or below 8.0 mm, moderately

sensitive (+) for diameter between 8.0 and 14.0 mm, sensitive (++) for diameter between 14.0

and 20.0 mm and extremely sensitive (+++) for diameter equal to or longer than 20.0 mm.

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Table 1. Comparison of volatile composition of separated organs (fruit, leaf, bark) of Z.

zanthoxyloides from the same station (Kafoutine)

Different parts
a b c d
No Compounds Ir l Ir a Ir p
Fruits Leaves Root Trunk

1 3-Methyl-2-butenol* 751 748 1316 0.7 - - -

2 Nonane* 900 898 900 0.2 - - -

3 α-Pinene 936 931 1015 2.5 0.5 tr -

4 Camphene 950 943 1066 tr - - -

5 Sabinene 973 964 1120 tr - - -

6 β-Pinene 978 970 1108 tr - - -

7 Octanal 981 980 1290 tr 0.1 tr 0.2

8 Myrcene 987 979 1159 0.1 0.4 - -

9 Hexyl acetate 1006 997 1271 0.4 - - -

10 Limonene 1025 1020 1201 0.3 0.5 - -

11 1,8-Cineole 1024 1020 1211 0.2 0.3 - -

12 (Z)-β-Ocimene 1029 1024 1230 4.4 0.2 - -

13 (E)-β-Ocimene 1041 1034 1247 29.1 5.9 - -

14 Octanol 1063 1058 1531 4.5 0.2 - 0.4

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 15 Nonan-2-one 1074 1070 1388 0.1 - - -

16 Nonanal 1076 1083 1394 0.1 0.2 0.1 3.2

Accepted Article
17 Linalool 1086 1081 1544 2.4 0.6 tr 0.5

18 Undecane* 1100 1100 1100 0.6 - - -

19 allo-Ocimene 1126 1118 1370 0.1 - - -

trans-Limonene-1,2-
20 1130 1120 1456 0.1 - - -
epoxide

21 Nonanol* 1149 1156 1635 0.1 0.2 - 0.2

22 Terpinen-4-ol 1164 1161 1600 tr - - -

23 -Terpineol 1176 1179 1700 0.1 0.1 - -

24 Decanal 1180 1185 1498 6.2 5.4 0.1 0.8

25 Octyl acetate* 1994 1198 1478 21.3 0.7 - -

26 Citronnellol 1213 1214 1764 - - - 0.5

27 Geraniol 1235 1232 1844 - 0.4 - 1.3

28 (E)-2-Decenal 1240 1241 1652 0.1 - - 1.2

29 Geranial 1244 1247 1731 - - - 0.2

30 Decanol* 1259 1261 1765 14.4 1.2 - 0.1

31 (E-E)-2,4-decadienal 1290 1291 1820 - - 0.2 4.8

32 Myrcenyl acetate 1313 1326 1721 - 0.2 - -

34 -Elemene 1340 1337 1467 - 0.8 - -

33 2-Undecanal 1343 1726 - - 0.2 1.1

35 Geranyl acetate 1362 1361 1752 - 0.2 0.1 1.9

36 β-Elemene 1389 1388 1589 0.5 3.7 - 0.2

37 Dodecanal 1389 1389 1708 0.3 - - -

38 Decyl acetate 1390 1395 1674 3.7 1.1 - -

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 39 -Caryophyllene 1421 1424 1591 0.5 4.7 - 0.3

40 γ-Elemene 1429 1427 1638 - 0.7 - -

Accepted Article
41 Geranylacetone 1430 1428 1851 - 0.2 0.1 0.5

42 β-Copaene 1430 1444 1581 - 0.2 - -

43 α-Humulene 1455 1450 1660 0.3 2.5 - 0.2

44 Trans-β-Ionone 1468 1466 1936 - 0.3 - 0.1

45 γ-Muurolene 1474 1468 1681 - 0.2 - -

46 -Curcumene 1473 1473 1769 - - - 0.2

47 Germacrene D 1479 1476 1704 0.3 7.6 - 0.1

48 β-Selinene 1486 1483 1712 0.1 0.5 - 0.1

49 Valencene 1494 1489 1719 0.2 0.5 - -

50 α-Selinene 1494 1495 1720 - 1.8 - 0.2

51 (E-E)--Farnesene 1498 1498 1744 0.1 - - -

52 δ-Guaiene 1502 1498 1711 - 0.5 - -

53 Pentadecane 1500 1501 1500 0.4 - - -

54 -Bisabolene 1503 1504 1720 - - - 0.3

55 γ-Cadinene 1507 1511 1752 - 0.3 - -

56 -Cadinene 1534 1535 1743 - 0.7 - -

57 Elemol 1541 1536 2072 - 0.2 - -

58 cis-3-Hexenyl benzoate 1545 1554 2088 - 0.3 - -

59 (E)-Nerolidol 1553 1546 2037 - 0.6 - 0.3

60 Germacrene B 1552 1552 1828 tr 3.7 - 0.4

61 Epiglobulol 1565 2013 - 0.6 - -

62 Caryophyllene oxyde 1578 1576 1980 - 1.6 0.1 0.9

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 63 Globulol 1589 1575 2074 - 0.2 - -

64 Ledol 1600 1596 2029 - 1.0 - -

Accepted Article
65 Humulene epoxyde II 1602 1598 2044 - - - 0.2

66 -Cadinol 1633 1625 2169 - 0.9 - 0.2

67 α-Cadinol 1643 1645 2231 - 2.4 - -

68 (2Z, 6Z)-Farnesol 1694 1653 2269 - 0.3 - -

Eudesma-4(15),7-dien-
69 1671 1667 2347 - 0.7 - -
1-β-ol

70 -Bisabolol 1673 1674 2217 - - - 0.4

71 Germacrone 1684 1678 2216 - - - 0.3

72 (2E, 6E)-Farnesol* 1710 1701 2235 tr 0.4 - -

73 α-Cyperone 1741 1729 2358 - 0.5 - -

74 (E, E)-Farnesyl acetate 1822 1818 2260 tr 0.4 - -

75 Pellitorine° 1923 2811 - - 85.8 57

76 (E, E)-Farnesylacetone 1895 2366 - 0.4 - 0.1

77 Hexadecanoic acid 1962 2903 - 14.8 3.6 1.9

78 (E)-Phytol 2014 2105 2617 - 0.6 - -

79 Tricosane 2300 2300 2300 - - - 0.3

Hydrocarbon compounds 39,7 35,9 tr 2.3

Oxygenated compounds 54,7 37,3 90.3 78.3

Hydrocarbon monoterpenes 36,5 7,5 tr -

Oxygenated monoterpenes 2,8 2,3 0.4 9.8

Hydrocarbon sesquiterpenes 2 28,4 - 2

Oxygenated sesquiterpenes - 10,8 0.1 2.4

Aliphatic hydrocarbons 1,2 - - 0.3

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 Aliphatic oxygenated components 51,9 24,2 89.8 66.1

Total identified 94.4 73.2 90.3 80.6

Accepted Article
Yields (w/w vs dry material) 1 0.04 0.01 0.002
a
Order of elution is given on apolar column (Rtx-1).
b
Retention indices of literature on the apolar column (lr l) [55].
c
Retention indices on the apolar Rtx-1 column (Ir a).
d
Retention indices on the polar Rtx-Wax column (Ir p).
* Compound identified with commercial library [55].
° Identification reported in Table 3.
tr: trace (%<0,05%)

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ccepted Articl Table 2. Chemical variability of the essential oils from Z. zanthoxyloides fruits according to sample locations in Senegal

Chemical variability of the fruits essential oilse

a b c d
No Compounds Ir l Ir a Ir p
Kabrousse Boucote Kafountine Palmarin Noflaye Keur Massar

1 3-Methyl-2-butenol* 751 748 1316 0.3±0.3 0.3±0.2 0.4±0.2 0.4 0.5±0.1 0.5

2 Nonane* 900 898 900 0.1±0.1 0.1±0.0 0.2±0.1 0.1 0.2±0.0 0.1

3 α-Pinene 936 931 1015 7.2±3.1 7.8±2.4 5±2.3 3.7 6.1±2.9 9.5

4 Camphene 950 943 1066 0.1±0.0 0.1±0.0 0.1±0.0 0.1 0.1±0.0 0.1

5 Sabinene 973 964 1120 0.1±0.1 0.1±0.1 tr 0.1 5.1±7.1 -

6 β-Pinene 978 970 1108 0.1±0.1 0.2±0.1 0.1±0.0 0.1 0.3±0.1 0.2

7 Octanal 981 980 1290 1.4±0.8 0.9±0.2 0.7±0.4 0.9 1.6±0.0 1.8

8 Myrcene 987 979 1159 1±0.1 1.1±0.2 0.9±0.5 0.7 1±0.1 1

9 Hexyl acetate 1006 997 1271 0.3±0.1 0.4±0.1 0.4±0.2 0.6 0.2±0.1 0.5

10 Limonene 1025 1020 1201 0.3±0.1 0.4±0.1 0.4±0.2 0.2 0.3±0.1 0.3

11 1,8-Cineole 1024 1020 1211 0.2±0.1 0.3±0.1 0.3±0.1 0.2 0.3±0.1 0.3

12 (Z)-β-Ocimene 1029 1024 1230 7.9±0.7 8.1±1.0 5.3±0.9 3.2 4.6±0.6 2.2

13 (E)-β-Ocimene 1041 1034 1247 39±1.7 37.9±3.4 28.6±2.0 16.1 29.1±2.4 12.1

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ccepted Articl 14 Octanol 1063 1058 1531 4.4±1.4 4.7±2.1 3.5±2.4 14.5 6.2±1.3 6.8

15 Nonan-2-one 1074 1070 1388 0.1±0.1 0.1±0.0 0.2±0.1 0.1 0.1±0.0 0.1

16 Nonanal 1076 1083 1394 0.1±0.0 0.1±0.1 0.2±0.1 0.3 0.2±0.1 0.2

17 Linalool 1086 1081 1544 4.5±2.3 4.8±3.6 9.0±5.0 2.8 4.7±2.2 17

18 Undecane* 1100 1100 1100 0.2±0.1 0.2±0.1 0.4±0.2 0.3 0.3±0.0 0.3

19 allo-Ocimene 1126 1118 1370 0.2±0.0 0.1±0.1 0.1±0.0 0.1 0.1±0.0 0.1

20 trans-Limonene-1,2-epoxide 1130 1120 1456 0.1±0.1 0.1±0.1 0.1±0.1 0.1 0.2±0.1 -

21 Nonanol* 1149 1156 1635 0.1±0.0 0.3±0.2 0.2±0.1 0.8 0.3±0.2 0.2

22 Terpinen-4-ol 1164 1161 1600 tr Tr tr tr 1.2±1.7 -

23 -Terpineol 1176 1179 1700 tr 0.1±0.0 tr 0.1 0.3±0.2 0.2

24 Decanal 1180 1185 1498 5.5±0.1 3.7±0.5 5.3±0.6 5.4 6.6±3.0 7.6

25 Octyl acetate* 1994 1198 1478 11.6±1.0 12.3±2.7 17.3±2.7 21.8 10.5±4.7 19.3

30 Decanol* 1259 1261 1765 9.7±1.2 9.7±3.4 9.7±4.3 15.4 11.7±5.9 9.7

36 β-Elemene 1389 1388 1589 0.2±0.1 0.1±0.1 0.2±0.2 0.2 0.5±0.4 0.2

37 Dodecanal 1389 1389 1708 0.2±0.1 0.2±0.1 0.3±0.1 0.2 0.1±0.1 0.2

38 Decyl acetate 1390 1395 1674 2.1±0.9 1.9±0.4 3.0±1.0 2.7 1.8±0.9 2.6

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ccepted Articl 39 -Caryophyllene 1421 1424 1591 0.2±0.1 0.2±0.1 0.3±0.2 0.2 0.1±0.0 0.3

43 α-Humulene 1455 1450 1660 0.1±0.0 0.1±0.0 0.2±0.1 0.1 0.1±0.1 0.2

47 Germacrene D 1479 1476 1704 0.2±0.1 0.2±0.1 0.2±0.1 0.1 0.2±0.1 0.1

48 β-Selinene 1486 1483 1712 tr tr 0.1±0.0 0.1 0.1±0.1 -

49 Valencene 1494 1489 1719 0.1±0.0 0.1±0.1 0.2±0.1 0.1 0.1±0.1 -

51 (E-E)--Farnesene 1498 1498 1744 tr 0.3±0.3 0.7±0.6 2.3 0.1±0.0 1.3

53 Pentadecane 1500 1501 1500 0.1±0.1 0.2±0.1 0.3±0.1 0.5 0.5±0.1 0.4

60 Germacrene B 1552 1552 1828 0.1±0.1 0.1±0.1 tr - 0.1±0.1 -

72 (2E, 6E)-Farnesol* 1710 1701 2235 tr tr tr 0.1 0.1±0.0 0.2

74 (E, E)-Farnesyl acetate 1822 1818 2260 0.2±0.2 0.1±0.1 tr 0.1 0.2±0.1 0.3

Hydrocarbon compounds 56.9±5.1 57.2±4.2 44.3±3.7 28.3 48.7±1.5 28.4

Oxygenated compounds 40.8±5.1 39.8±4.9 49.9±5.5 66.5 46.2±0.7 67.5

Hydrocarbon monoterpenes 55.8±4.1 40.5±3.6 24.3 46.6±2.1 25.5

Oxygenated monoterpenes 4.8±2.3 5.3±3.7 9.5±5.0 3.2 5.3±2.3 17.5

Hydrocarbon sesquiterpenes 0.8±0.4 1±0.4 1.9±0.6 3.1 1.1±0.7 2.1

Oxygenated sesquiterpenes 0.2±0.1 0.1±0.1 Tr 0.2 0.3±0.1 0.5

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ccepted Articl Aliphatic hydrocarbon compounds 0.4±0.1 0.4±0.2 0.9±0.3 0.9 1.0±0.1 0.8

Aliphatic oxygenated compounds 35.8±3.1 34.5±7.0 41.3±9.3 63.1 40.7±3.0 49.5

Total identified 97.7±0.5 97.0±1.1 94.2±1.7 94.8 94.9±0.8 95.9

Yields (w/w vs dry material) 0.91±0.28 1.73±0.72 0.98±0.42 0.98 1.27±0.37 1.54
a
Order of elution is given on apolar column (Rtx-1).
b
Retention indices of literature on the apolar column (lr l) [55].
c
Retention indices on the apolar Rtx-1 column (Ir a).
d
Retention indices on the polar Rtx-Wax column (Ir p).
e
Localities of sampling.
* Components identified with commercial library [55].
tr: trace (%<0,05%)

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ccepted Articl
Table 3. Optimized MS/MS dectection parameters of seven coumarins of Z. zanthoxyloides fruit oils

Transition Optimized MS parameters

Analytes Tr/min MW/Da
Q1 mass/Da Q3 mass/Da DP/V EP/V CEP/V CE/V CXP/V

6,7-Dimethylesculetin 17.9 206.1 207.1 151.2 61 4.5 12 29 4

Herniarin 20.8 176.1 177.1 121.1 56 4 12 27 4

Psoralen 22.7 186.1 187.1 131.1 56 10.5 12 33 4

Xanthotoxin 23.2 216.1 217.1 202.1 71 12 14 61 4

Bergapten 24.7 216.1 217.1 202.0 61 8.5 14 27 4

Isopimpinellin 24.9 246.1 247.1 217.1 71 10.5 14 23 4

Imperatorin 31.5 270.2 271.2 203.1 51 5 14 17 4

DP=declustering potential, EP=entrance potential, CEP=collision cell entrance potential, CE=collison energy, CXP= collision cell exit

potential

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 Table 4. Major components of fractions obtained by liquid chromatography of collective

essential oil from Z. zanthoxyloides fruits (Kafoutine samples)

Accepted Article
Relative percentages of main components
Compounds
EO F1 F2 F3 F4 F5

α-Pinene 4.5 4.3 0.1 - - -

(Z)-β-Ocimene 6 15.5 0.1 - - -

(E)-β-Ocimene 29.7 70.1 0.6 - - -

Octanol 8.6 - - - 0.7 35.7

Linalool 6.3 - - - 82.6 0.3

Decanal 4.1 - 21.3 0.5 - -

Octyl acetate 16.1 - 57.1 90.5 1.3 -

Decanol 14 - - - 11.8 59.4

Decyl acetate 2.1 - 11.3 4.1 0.2 -

EO: Essential oil; F: fraction

Elution gradient (iPr)2O/nC6H14 of increasing polarity

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ccepted Articl Table 5. Antimicrobial activity of fruit essential oil from Z. zanthoxyloides and corresponding isolated fractions

Inhibition zone diameters (mm)

Bacterial and fungal strains

Samples
E. coli E. coli S. aureus E. faecalis P. aeruginosa C. albicans
ATCC 35218 ATCC 25922 ATCC 29213 ATCC 29212 ATCC 27853 ATCC 24433

EO R R 14.5±0.7 R R 17.5±0.7

F1 R R R R R 12.6±0.6

F2 R R 17.0±1.4 10.7±0.6 R 15.1±1.3

F3 R R R 11.1±0.2 R 20.5±0.7

F4 11.5±0.4 11.3±0.1 17.3±0.4 8.6±0.8 R 18.8±0.4

F5 R R 25.5±2.1 8.3±0.1 R 38.3±1.1

Gentamycin (30 μg/disk) 17 15 25 14 14 -

Amphotericin B (10 μg/disk) * - - - - - 22

EO: Essential oil; F: fraction; R: resitant strain; -:not tested

‘*’ Value reported in the literature [53]

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Accepted Article
Fig. 1. Sampling of Z. zanthoxyloides (fruits, leaves and stems, trunk) from Senegal.

19 samples of Z. Zanthoxyloides fruit pericarps: Kabrousse (12°21' N, 16°42' W; four

samples), Boucote (12°25'N, 16°44'W; six samples), Kafountine (12°56'N, 16°44'W; five

samples), Palmarin ( 14°1'N, 16°46'W; one sample), Noflaye (14°45' N, 17°12'W; two

samples), Keur massar (14°46'N, 17°17'W; one sample). Fruit samples were collected on the

same tree. Leaf, root bark and trunk bark samples harvested on the same tree of Kafountine

location.

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 Fig. 2. Chemical structure, Mass Spectra and NMR data of pellitorine

EI-MS 70 eV (rel. int.): 223 (27.1%); 152 (38.8%); 151 (100%); 96 (62.0%); 95 (22.4%); 81
Accepted Article
(62.7%); 69 (19.8%); 67 (21.2%); 57 (19.6%); 41 (25.6%).
13
C-NMR  (ppm): 166.66 C(1); 121.62 C(2); 141.49 C(3); 128.20 C(4); 143.40 C(5); 32.93

C(6); 28.48 C(7); 31.38 C(8); 22.48 C(9); 14.01 C(10); 46.99 C(1’); 28.61 C(2’); 20.13

C(3’); 20.13 C(4’).

1
H-NMR δ (ppm), J (Hz): 7.18 (1H, dd, J=14.97, 10.75 Hz, H-3); 6.03-6.14 (1H, m, H-5);

6.08-6.17 (1H, m, H-4); 5.77 (1H, d, J=14.97 Hz, H-2) ; 5.64 (br, 1H, N-H); 3.16 (2H, t,

J=6.4 Hz, H-1’); 2.12-2.18 (2H, m, H-6); 1.77-1.83 (1H, m, H-2’); 1.38-1.45 (2H, m, H-7);

1.25-1.35 (2H, m, H-8); 1.25-1.35 (2H, m, H-9); 0.94 (3H, d, J=8.05 Hz, H-3’); 0.94 (3H, d,

J=8.05 Hz, H-4’); 0.85 (3H, t, J=6.4 Hz, H-10).

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 Fig. 3. PCA of chemical composition of the Z. zanthoxyloides fruit oils from Senegal and
those described in literature data (see Supplemetary material).
Accepted Article
Senegal: Cabrousse (Cab 1-4), Boucote (Bou 1-6), Kafountine (Kaf 1-5), Palmarin (Pal 1),

Noflaye (Nof 1-2), Keur massar (Keu 1).

Cameroon: four samples (Cam 1-4) [34, 37, 39, 40]

Benin: two samples (Ben 1-2) [33, 36]

(a) Distribution of samples

10
Ben 2

4
F2 (19.69 %)

Group I : Group II :
2 19 fruits oils 4 fruit oils from Cameroon
Keur Kaf4
from Senegal Kaf2
Nof1 kaf5 Ben 1 Cam 2
0 Cab1 Kaf1
Palm Cab3 Cam 4
Bo1
Bo4 Nof2 Cab4 Cam 1
Cab2 Bo2
-2 Cam 3
Bo6 Kaf3
Bo5 Bo3

-4

-6
-10 -8 -6 -4 -2 0 2 4 6 8 10 12
F1 (40.90 %)

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 (b) distribution of variables
Accepted Article
1
-Terpinene Undecane
Valencene
0.75

0.5 Decanal

0.25
F2 (19.69 %)

Geranial
0
Citronellol
Octan-1-ol Isopulegol
α-Pinene Citronellyl acetate
-0.25 Linalol Geraniol
Cis-β-Ocimene Geranyl acetate Citronellal
Octyl acetate Decan-1-ol Limonene
-0.5 Trans-β-Ocimene

-0.75

-1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
F1 (40.90 %)

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 Fig 4. CA of chemical composition of A of the essential oils from Z. zanthoxyloides fruits.
Dendrogram of agnes (x = ch, metric = "euclidean", stand = TRUE, method = "ward")
Accepted Article
Coding numbers of sample locations corresponding to those of Figure 3.

Fig. 5. Chemical structures of coumarins identified in Z.. zanthoxyloides essential oil

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