Anda di halaman 1dari 7

Food Chemistry 149 (2014) 183189

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Volatile oil from striped African pepper (Xylopia parviora, Annonaceae)


possesses notable chemopreventive, anti-inammatory and
antimicrobial potential
Verlaine Woguem a,b, Hervet P.D. Fogang a,b, Filippo Maggi c,, Lon A. Tapondjou a,, Hilaire M. Womeni b,
Luana Quassinti c, Massimo Bramucci c, Luca A. Vitali c, Dezemona Petrelli d, Giulio Lupidi c,
Fabrizio Papa e, Sauro Vittori c, Luciano Barboni e
a

Laboratory of Environmental and Applied Chemistry, Faculty of Science, University of Dschang, P.O. Box 183, Dschang, Cameroon
Laboratory of Biochemistry of Medicinal Plants, Food Science and Nutrition, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon
c
School of Pharmacy, University of Camerino, I-62032 Camerino, Italy
d
School of Biosciences and Biotechnology, University of Camerino, I-62032 Camerino, Italy
e
School of Science and Technology, Chemistry Division, University of Camerino, I-62032 Camerino, Italy
b

a r t i c l e

i n f o

Article history:
Received 3 September 2013
Received in revised form 18 October 2013
Accepted 22 October 2013
Available online 30 October 2013
Keywords:
Xylopia parviora
Essential oil
Cytotoxic activity
Anti-inammatory
Antimicrobial

a b s t r a c t
Fruits of Xylopia parviora, well known as striped African pepper, are sold in the Cameroonian markets as
a avouring ingredient to make traditional soups. The essential oil hydrodistilled from fruits was analysed for in vitro biological activities, namely cytotoxic, anti-inammatory, antimicrobial and antioxidant,
by MTT, nitric oxide inhibitory assay, agar disc diffusion method, and DPPH and ABTS assays. The essential oil composition, analysed by GC and GCMS, was dominated by monoterpene hydrocarbons (50.0%)
responsible for the pepper odour, such as b-pinene (34.0%) and a-pinene (10.3%). The oil induced a strong
inhibitory effect on tumour cells MDA-MB 231 and HCT116, with inhibition values close to those of cisplatin. A dose-dependent decrease in NO production was noted in RAW 264.7 macrophages treated with
the oil, revealing a promising anti-inammatory potential. The essential oil showed a measurable antimicrobial activity against all the species tested, while the radical scavenging activity was low.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Wild vegetables have been used since ancient times by native
people all over the world. Before agriculture, humans depended
on wild plants and animals for their daily needs. Nkui and Nah
poh are two traditional soups of the western region of Cameroon
which contain many spices, among which are the fruits of Xylopia
parviora (A. Rich.) Benth. (Annonaceae). X. parviora, well known
as striped African pepper, is a small tree, 23 m high, distributed in
eastern and central Africa in riparian sites. Its greenish fruits and
seeds, normally boiled on a thread or on a stick of bamboo, are
pounded and used in cooked foods or in the spicing of sauces
and beverages (Jirovetz, Buchbauer, & Ngassoum, 1997; Karioti,
Hadjipavlou-Litina, Mensah, Fleischer, & Skaltsa, 2004). As a traditional remedy, the root decoction is taken by coastal peoples for
stomach disorders. Other medicinal uses include treatment of

Corresponding authors. Tel.: +39 0737404506 (F. Maggi). Tel.:+237 75004826;


fax: +237 33451735 (L.A. Tapondjou).
E-mail addresses: lippo.maggi@unicam.it (F. Maggi), tapondjou2001@yahoo.fr
(L.A. Tapondjou).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.10.093

infertility, insertion of root pieces into nostrils for headache relief,


the bark as analgesic and antispasmodic (Nishiyama et al., 2006),
and the leaves in the treatment of malaria fever. Xylopia species
contain bioactive components such as alkaloids (Harrigan, Bolzani,
Gunatilaka, & Kingston, 1994; Johns, Lamberton, & Sioumis, 1968;
Jossang, Lebceuf, Cave, & Pusset, 1991), acetogenins (ColmanSaizarbitoria, Gu, & McLaughlin, 1994), terpenes (Harrigan,
Gunatilaka, David, Chan, & John-son, 1994; Martins, Osshiro,
Roque, Marks, & Gottlieb, 1998) and essential oils (Brophy,
Goldsack, & Forster, 1998).
As far as we know, no investigations have been made on the
biological activity of the volatile oil obtained from this spice.
Therefore, as part of our continuous search for benecial effects
of essential oils from Cameroonian spices (Fogang, Womeni,
Piombo, Barouh, & Tapondjou, 2012; Fogang et al., 2012), we evaluated the biological activities of the essential oil hydrodistilled
from fruits of X. parviora, i.e., antioxidant and antimicrobial
capacities, effects on the growth of tumour cell lines, and
anti-inammatory activity. Chemical investigation by GC-FID and
GCMS was undertaken to establish the compositionactivity
relationship.

184

V. Woguem et al. / Food Chemistry 149 (2014) 183189

2. Materials and methods


2.1. Plant material
Fruits of X. parviora were collected in the Nde Division of the
Western Highlands of Cameroon. They were dried naturally on
laboratory benches at room temperature until constant weight.
Identication was made by Mr. Nana Victor, taxonomist at the
Cameroon National Herbarium (Yaound), where a voucher specimen was deposited (N. 6431/HNC/SRF).
2.2. Extraction of the essential oil
The dry fruits (100 g) of X. parviora were separately ground
and subjected to hydrodistillation with a Clevenger-type apparatus
using 750 ml of deionised water for 4 h. The oil collected was dried
over anhydrous sodium sulfate yielding 0.60% (w/w) of strong
smelling light yellow oil which was stored at 20 C until used.
2.3. Chemicals

a-Pinene, camphene, b-pinene, myrcene, p-cymene, limonene,


1,8-cineole, linalool, trans-pinocarveol, camphor, borneol,
terpinen-4-ol, a-terpineol, myrtenal, verbenone, isobornyl acetate,
(E)-caryophyllene, a-humulene, caryophyllene oxide were purchased from SigmaAldrich (Milan, Italy). For retention index
determination, a mix of hydrocarbons ranging from n-octane (C8)
to n-triacontane (C30) (Supelco, Bellefonte, PA) was used and run
under the experimental conditions reported below. All compounds
were of analytical standard grade. Analytical grade n-hexane solvent was purchased from Carlo Erba (Milan, Italy); it was distiled
by a Vigreux column before use.
2.4. GC-FID and GCMS analyses
For gas chromatographic separations, an Agilent 4890D instrument coupled to a ame ionisation detector (FID) was used.
Volatile components were separated on an HP-5 capillary column
(5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d., 0.17 lm lm
thickness; J & W Scientic, Folsom, CA), with the following temperature programme: 5 min at 60 C, subsequently 4 C/min up to
220 C, then 11 C/min up to 280 C, held for 15 min, for a total
run of 65 min. Injector and transfer line temperatures were
280 C. Helium was used as the carrier gas, at a ow rate of
1.4 ml/min; injection volume: 1 ll; split ratio, 1:34. A mixture of
aliphatic hydrocarbons (C8C30) (Sigma, Milan, Italy) in n-hexane,
was directly injected into the GC injector using the above temperature programme, in order to calculate the retention index of each
compound. Oil samples were diluted 1:100 in n-hexane and
injected at a volume of 1 ll. Analysis was repeated 3 times. Data
were collected by using HP3398A GC Chemstation software (Hewlett Packard, Rev. A.01.01). The relative amounts of essential oil
components, expressed as percentages, were obtained by FID
peak-area normalisation by calculating the response factor (RF)
of the FID for seven different classes of volatiles. Standard
compounds, each representing the chemical classes determined,
were selected among those available in the authors laboratory.
i.e., b-pinene, limonene and c-terpinene for monoterpene hydrocarbons; p-cymene for aromatic monoterpenes; 1,8-cineole and
linalool for oxygenated monoterpenes; (E)-caryophyllene and
a-humulene for sesquiterpene hydrocarbons; caryophyllene oxide
for oxygenated sesquiterpenes; n-octanal for aldehydes and
ketones; n-octane and n-octadecane for alkanes. Each standard,
diluted in n-hexane, was injected at four different concentrations
(0.04, 0.08, 0.16 and 0.40 mg/ml) using n-octane and n-octadecane

as internal standards at a concentration of 0.04 mg/ml. For each


standard a regression line was obtained by plotting the ratio of
internal standard (mean value of n-octane and n-octadecane) to
reference standard peak area versus the ratio of internal standard
to reference standard concentration. Each dilution was analysed
ve times. The correlation coefcients (r2) obtained for calibration
curves of representative standards were all higher than 0.992. The
correction factor of each reference standard compound was calculated as the slope of the linear regression equation. Whenever
more than one reference analyte for each class was considered,
the correction factor was calculated by the mean of the slopes of
all representative compounds. Using the generalised response
factor for compounds within the eight classes, the derived quantitative data may be considered as an approximation of the absolute
quantication. For the calculation of volatile concentrations, expressed in mg/g oil, the essential oils were injected three times
using n-octane and n-octadecane as internal standards.
GCMS analysis was performed on an Agilent 6890N gas
chromatograph coupled to a 5973N mass spectrometer using an
HP-5MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d.,
0.1 lm lm thickness; J & W Scientic) capillary column. The temperature programme was the same as above. Injector and transfer
line temperatures were 280 C. Helium was used as the carrier gas,
at a ow rate of 1 ml/min. Split ratio: 1:50; acquisition mass range:
m/z 29400. All mass spectra were acquired in electron-impact (EI)
mode with an ionisation voltage of 70 eV. Oil samples were diluted
1:100 in n-hexane and the volume injected was 2 ll. Data were
analysed by using MSD ChemStation software (Agilent, Version
G1701DA D.01.00). Whenever possible volatile components were
identied by co-injection with authentic standards (see Section 2.3). Otherwise, the peak assignment was carried out on the
basis of the standard of the International Organisation of the
Flavour Industry (IOFI, http://www.io.org/) statement (Bicchi,
Cagliero, & Rubiolo, 2011), i.e., by the interactive combination of
chromatographic linear retention indices that were consistent with
those reported in the literature (Adams, 2007; NIST 08, 2008) for
non-polar stationary phases, and MS data consisting of the computer matching with the WILEY275, NIST 08, ADAMS, and homemade (based on the analyses of reference oils and commercially
available standards) libraries.
2.5. Cell culture
Human colon carcinoma cell line HCT116 was cultured in
RPMI1640 medium with 2 mM L-glutamine, 100 IU/ml penicillin,
100 lg/ml streptomycin, and supplemented with 10% heat-inactivated foetal bovine serum (HI-FBS) (PAA Laboratories GmbH, Pasching, Austria). Murine macrophage cell line RAW 264.7, human
breast adenocarcinoma cell line MDA-MB 231, and human malignant melanoma cell line A375 were cultured in Dulbeccos Modied Eagles Medium (DMEM) with 2 mM L-glutamine, 100 IU/ml
penicillin, 100 lg/ml streptomycin, and supplemented with 10%
HI-FBS. Cells were cultured in a humidied atmosphere at 37 C
in the presence of 5% CO2.
2.6. MTT cytotoxicity assay
The MTT assay was used as a relative measure of cell viability.
Cell-viability assays were carried out as described by Quassinti
et al. (2013). Briey, cells were seeded at a density of 2  104
cells/ml. After 24 h, samples were exposed to different concentrations of essential oil (0.78200 lg/ml). Authentic standards of the
major components a- and b-pinene, purchased from
SigmaAldrich (Milan, Italy), were tested as well. The anticancer
drug cisplatin (0.0150 lg/ml) was used as the positive control.
Cells were incubated for 72 h in a humidied atmosphere of 5%

V. Woguem et al. / Food Chemistry 149 (2014) 183189

CO2 at 37 C. At the end of incubation, each well received 10 ll of


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide
(MTT) (5 mg/ml in phosphate-buffered saline, PBS) and the plates
were incubated for 4 h at 37 C. The extent of MTT reduction was
measured spectrophotometrically at 540 nm using a Titertek Multiscan microElisa (Labsystems, Helsinki, Finland). Experiments
were conducted in triplicate. Cytotoxicity was expressed as the
concentration of compound inhibiting cell growth by 50% (IC50).
The IC50 values were determined with GraphPad Prism 4 software
(GraphPad Software, San Diego, CA).
2.7. Nitric oxide inhibitory assay
RAW 264.7 macrophages were seeded in 96-well plates at a
density of 5  105 cells/ml in the presence of essential oil
(0.7512 lg/ml). Cells were incubated with 1 lg/ml lipopolysaccharide (LPS), alone or co-incubated with essential oil and LPS
(1 lg/ml) for 24 h. Aminoguanidine (200 lM) plus 1 lg/ml LPS
served as a control for the reduction of NO production (Koh
et al., 2009). NO production was determined by measuring the
amount of the primary stable reaction product nitrite with Griess
reagent (Huygen, 1970) (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, 5% H3PO4) by mixing 50 ll of cell
culture supernatant with the same volume of reagents. After incubation for 10 min at room temperature, the absorption of the
formed diazo dye was measured spectrophotometrically at
540 nm using a Titertek Multiscan microElisa (Labsystems,
Helsinki, Finland). The nitrite concentration was determined by
comparison with a sodium nitrite standard calibration curve in culture medium (0100 lM). The cell viability of the macrophages
was determined by MTT assay.
2.8. Antimicrobial activity
Microorganisms included in this study were Staphylococcus
aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Candida
albicans ATCC 24433. Antimicrobial activity of the essential oil and
major components a- and b-pinene (Sigma, Milan, Italy) was assessed by disc diffusion test, by spotting 10 ll pure oil or standard
compound onto a paper disc and following the general guidelines
of the European Committee for Antimicrobial Susceptibility Testing
(EUCAST, www.EUCAST.org), with previously described minor
modications, due to the nature of the substance tested (Quassinti
et al., 2013). Activity was determined by measuring the diameter of
the growth inhibition zone (inhibition zone diameter, IZD) visible
around the paper disc (expressed in mm). Reported IZDs are inclusive of the paper disc diameter (6 mm). Therefore, a 6-mm IZD
means no activity. Ten microlitres of each reference compound
(a-pinene and b-pinene) per paper disc were used in the control
experiments, while the known antimicrobials ciprooxacin (5 lg
per disc) and uconazole (25 lg per disc) (NCCLS, 2004) were used
as a reference against bacteria and fungi, respectively.
2.9. Antioxidant activity
DPPH free radical-scavenging activity was evaluated on a
microplate analytical assay according to the previously described
procedure by Srinivasan, Chandrasekar, Nanjan, and Suresh
(2007). The stock solution was prepared by dissolving DPPH in
methanol and then stored at 20 C until needed. The working
solution was obtained by mixing stock solution with methanol to
obtain an absorbance of 1 unit at 517 nm. Discoloration was measured at 517 nm after incubation for 30 min in the dark. The free
radical-scavenging activity of each solution was then calculated

185

as percent inhibition according to the following equation:% inhibition = 100 (A(blank) A(sample))/A(blank)
Antioxidant activity of the essential oil was expressed as IC50,
dened as the concentration of the test material required to cause
a 50% decrease in initial DPPH concentration. Trolox was used as
reference. Results were expressed in lM Trolox equivalents
(TE)/g of essential oil.
ABTS assay was performed following the procedure previously
described (Re et al., 1999), applied to a 96-well microplate assay
(Esparza-Rivera, Stone, Stuchnoff, Pilon-Smits, & Kendall, 2006).
The ABTS+ stock solution was prepared by mixing the two solutions of ABTS (7.4 mM) and potassium persulfate (2.6 mM) in equal
quantities and allowing them to react for 12 h at room temperature
in the dark. The working solution was then obtained by mixing
ABTS+ solution with methanol to obtain a nal solution with
absorbance of 1 unit at 734 nm measured with a Varian Cary 1
spectrophotometer. Trolox was used as reference. Results were
expressed in lM Trolox equivalents (TE)/g of essential oil. The
capacity of free radical scavenging (IC50) was determined using
the same previously used equation for the DPPH method. All data
of antioxidant activity were expressed as means standard deviations (SD) of triplicate measurements. The condence limits were
set at p < 0.05. SD did not exceed 5% for the majority of the values
obtained.
2.10. Statistical analysis
All assays were conducted at least three times with three different sample preparations. All data were expressed as mean standard deviation (SD). Analysis of variance was performed using
InStat (GraphPad software, San Diego CA). A one-way ANOVA
and unpaired Students t-test were used for these analyses, and
p < 0.05 was considered to be statistically signicant.
3. Results and discussion
3.1. Analysis of the essential oil
The chemical composition of the essential oil obtained from the
fruits of X. parviora is reported in Table 1. A total of sixty volatile
components, corresponding to 90.8% of the total composition, were
identied in the essential oil. The major fraction was monoterpenes (70.7%) with hydrocarbons (50.0%) occurring in higher levels
than oxygenated compounds (20.7%). The main components were
b-pinene (34.0%) and a-pinene (10.3%) among the former, and
trans-pinocarveol (5.0%) and myrtenol (4.6%) among the latter.
These compounds are considered to be the main contributors to
the overall oral terpeny, smoky odour of the dried fruits of the
West African pepper tree (Tairu, Hofmann, & Schieberle, 1999).
As a matter of fact, the high concentration of monoterpene hydrocarbons in X. parviora is essential to give the pepper odour
(Jirovetz et al., 1997). Minor contributions came from sesquiterpene hydrocarbons (6.1%) and oxygenated sesquiterpenes
(11.7%). Among these fractions none of the components reached
amounts higher than 2.2%.
Our results were consistent with those previously reported for
the volatile composition of fruits collected in a different region
(Bayagam and Ngaoundere areas) of Cameroon (Jirovetz et al.,
1997; Lamaty, Menut, Bessire, Amvam Zollo, & Fekam, 1989). In
those cases, the major constituents were again b-pinene (40.0%
and 22.6%, respectively) and a-pinene (14.0% and 12.9%, respectively), while trans-pinocarveol (2.7% and 2.6%, respectivley) and
myrtenol (2.2% and 1.7%, respectively) were slightly lower. On
the other hand, among minor components, (E)-b-ocimene, which
was quite abundant in those studies (5.4% and 4.1%, respectively),

186

V. Woguem et al. / Food Chemistry 149 (2014) 183189

Table 1
Chemical composition of the essential oil hydrodistilled from fruits of Xylopia parviora.
N.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

Constituenta

n-Hexanal
4-Hydroxy-4-methyl-2-pentanone
Tricyclene
a-Thujene
a-Pinene
Camphene
Thuja-2,4(10)-diene
Sabinene
b-Pinene
Myrcene
p-Cymene
Limonene
1,8-Cineole
(E)-b-Ocimene
Linalool
a-Campholenal
trans-Pinocarveol
Camphor
Pinocarvone
Borneol
cis-Pinocamphone
Terpinen-4-ol
cis-Pinocarveol
p-Cymen-8-ol
a-Terpineol
Myrtenal
Myrtenol
Verbenone
trans-Carveol
Isobornyl acetate
d-Elemene
a-Cubebene
Cyclosativene
a-Copaene
b-Cubebene
b-Elemene
(E)-Caryophyllene
b-Copaene
a-Humulene
b-Selinene
trans-Muurola-4(14),5-Diene
a-Muurolene
trans-Calamenene
d-Cadinene
a-Calacorene
Hedycaryol
b-Calacorene
Spathulenol
Caryophyllene oxide
Salvial-4(14)-en-1-one
Humulene epoxide II
1,10-Di-epi-cubenol
Caryophylla-4(12),8(13)-dien-5-olh
Cubenol
a-Muurolol
cis-Calamenen-10-ol
trans-Calamenen-10-ol
Eudesma-4(15),7-dien-1-b-ol
Manoyl oxide
Phyllocladene

RFb

1.7
1.7
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.7
1.1
1.5
1.1
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.3
1.1
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.4
1.4

Calc. LRIc

804
849
922
927
932
947
954
973
975
993
1028
1031
1034
1055
1103
1129
1140
1146
1165
1167
1176
1179
1188
1190
1192
1195
1197
1211
1225
1286
1336
1348
1360
1372
1387
1389
1413
1424
1448
1480
1492
1496
1519
1523
1539
1547
1561
1573
1576
1588
1602
1626
1632
1638
1644
1658
1666
1682
2004
2025

Lit. LRId

IDf

Essential oil

ADAMS

NIST08

%e

mg/g

801
839
926
930
939
954
960
975
979
990
1024
1029
1031
1050
1096
1126
1139
1146
1164
1169
1175
1177
1184
1182
1188
1195
1195
1205
1216
1285
1338
1348
1371
1376
1388
1390
1419
1432
1454
1490
1493
1500
1522
1523
1545
1548
1565
1578
1583
1594
1608
1619
1640
1646
1646
1661
1669
1688

804

Trg
0.3 0.1
0.1 0.0
0.1 0.0
10.3 0.0
3.3 0.0
0.1 0.0
Tr
34.0 0.1
0.1 0.0
1.3 0.0
0.6 0.0
1.7 0.0
0.1 0.0
0.9 0.0
0.2 0.0
5.0 0.1
0.4 0.0
0.4 0.0
0.5 0.1
Tr
0.8 0.0
Tr
0.1 0.0
1.0 0.0
2.5 0.0
4.6 0.1
0.5 0.0
0.2 0.1
1.0 0.6
0.1 0.0
0.3 0.0
0.5 0.0
0.5 0.0
0.3 0.0
0.5 0.0
0.2 0.0
1.1 0.0
0.1 0.0
0.1 0.0
0.1 0.0
0.6 0.0
1.4 0.1
Tr
0.1 0.0
1.2 0.0
0.1 0.0
0.1 0.0
2.1 0.0
0.3 0.0
0.8 0.0
2.2 1.0
0.7 1.0
0.8 0.0
2.1 0.0
0.3 0.0
0.2 0.0
1.0 0.0
1.0 0.0
0.2 0.0

0.3 0.0
3.4 0.4
0.7 0.0
1.0 0.0
104.4 0.3
34.0 0.1
0.9 0.0
0.3 0.0
346.1 0.5
0.9 0.0
20.6 0.3
5.9 0.0
17.7 0.2
0.9 0.1
9.5 0.4
1.9 0.3
50.8 0.6
3.9 0.1
4.1 0.3
4.9 0.6
0.2 0.0
8.1 0.3
0.8 0.0
2.3 0.2
10.5 0.1
25.1 0.4
46.3 0.8
5.2 0.1
1.6 0.3
12.6 1.2
1.5 0.1
2.6 0.1
4.9 0.9
4.8 0.2
3.1 0.1
4.7 0.2
2.2 0.1
10.8 0.1
1.3 0.1
0.8 0.1
1.3 0.2
6.0 0.1
14.6 1.0
1.2 0.2
1.1 0.1
11.8 0.0
1.4 0.0
1.3 0.0
21.7 0.0
3.4 0.0
7.7 0.1
26.7 2.5
1.3 0.1
7.8 0.1
21.5 0.1
3.0 0.2
1.9 0.0
10.4 0.1
9.9 0.1
2.4 0.0

922
926
932
947
954
971
974
991
1028
1028
1034
1052
1100
1130
1141
1165
1167
1179
1189
1189
1195
1196
1211
1225

1360
1385
1387
1412
1446
1481
1471
1494
1518
1541
1570
1573
1602
1630
1635
1644

2004
2017

Total identied (%)


Oil yield (%)

90.8
0.6

Grouped compounds (%)


Monoterpene hydrocarbons
Oxygenated monoterpenes
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Diterpenes

50.0
20.7
6.1
11.7
2.0

RI,MS
RI,MS
RI,MS
RI,MS
Std
Std
RI,MS
RI,MS
Std
Std
Std
Std
Std
RI,MS
Std
RI,MS
Std
Std
RI,MS
Std
RI,MS
Std
RI,MS
RI,MS
Std
Std
RI,MS
Std
RI,MS
Std
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
Std
RI,MS
Std
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
Std
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS

Compounds are listed in order of their elution from an HP-5MS column. Their nomenclature was in accordance with Adams (2007).
Relative response factor (RF) of FID detector for the main chemical groups occurring in the essential oil.
c
Linear retention index on HP-5MS column. Experimentally determined using homologous series of C8C32 alkanes.
d
Linear relative retention index taken from Adams (2007) and/or NIST08 (2008).
e
Percentage values are means of three determinations standard deviation.
f
Identication methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wiley, ADAMS and NIST08 MS database; RI, based on
comparison of RI with those reported in ADAMS and NIST08.
g
Tr, traces (mean value below 0.1%).
h
Correct isomer not identied.
b

V. Woguem et al. / Food Chemistry 149 (2014) 183189


Table 2
In-vitro cytotoxic activity of essential oil from Xylopia parviora.
Cell line (IC50 lg/ml)a
MDA-MB 231b

A375c

HCT116d

6.56
6.027.14

7.47
6.898.10

6.63
6.017.31

95% C.I.
b-Pinene
95% C.I.

31.0
28.533.7
75.5
70.780.5

44.8
42.647.2
>200

63.1
55.571.9
57.2
53.761.0

Reference
Cisplatin
95% C.I.

2.07
1.692.22

0.15
0.110.20

2.62
2.412.86

Essential oil
Xylopia parviora
95% C.I.e
Major components

a-Pinene

a
IC50 as the concentration of compound that affords a 50% reduction in cell
growth (after 72 h of incubation).
b
Human breast adenocarcinoma cell line.
c
Human malignant melanoma cell line.
d
Human colon carcinoma cell line.
e
C.I., condence interval.

was very low in our sample (0.1%). These differences may be due to
the geographical origin of the samples.
3.2. Antiproliferative and anti-inammatory activity
The essential oil of X. parviora was tested in vitro for its potential tumour cell growth-inhibitory effect on MDA-MB 231 human
breast adenocarcinoma cell line, A375 human malignant melanoma cell line, and HCT116 human colon carcinoma cell line, using

187

MTT assay. The results, collected in Table 2, show that essential oil
exhibited a strong inhibitory effect against the human cancer cells
examined. The highest activity was observed on MDA-MB 231 and
HCT 116 cell lines, with IC50 values of 6.56 and 6.63 lg/ml, respectively, which are only three times weaker than those of the anticancer drug cisplatin. Slightly lower was the activity on A375,
with IC50 value of 7.47 lg/ml. These low IC50 values (i.e.,
<20 lg/ml), as reported by the US NCI plant screening programme
(Boik, 2001), conrm the potential of X. parviora essential oil as a
promising anticancer agent.
Very few of the compounds found in X. parviora essential oil
have been tested for their anticancer properties. However, the
cytotoxic effects of the commercially available major constituents,
were found to be lower than that of the essential oil. b-Pinene was
slightly effective against MDA-MB 231 and HCT116 cell lines
(Table 2). a-Pinene was more active on all tumour cells, showing
maximum inhibition on MDA-MB 231 cells (IC50 value of
31.0 lg/ml). This compound has also been reported to exhibit
apoptotic and antimetastatic activity on melanoma cells (Matsuo
et al., 2011). This was partly supported by the IC50 value detected
on A375 cell line (31.0 lg/ml). The component trans-pinocarveol
showed little cytotoxic effect against MDA-MB 231 and some glioblastoma (Nicoletti et al., 2012). The concentrations of b-pinene
(34%), a-pinene (10.3%) and trans-pinocarveol (5%) in the oil do
not justify completely the cytotoxic effect obtained for the whole
essential oil, indicating a possible synergism between the different
constituents.
In order to evaluate potential inhibiting effects of X. parviora
essential oil against stimulated macrophages, which could be a typical indicator for anti-inammatory activity, RAW 264.7 cells were
stimulated by LPS; the effect of essential oil during a co-incubation

Fig. 1. Effects of Xylopia parviora essential oil on NO production (a) and cell viability (b) by LPS-stimulated RAW 264.7 macrophages. EO, essential oil; LPS, lipopolysaccaride;
AG, aminoguanidine. Data are means SD of three independent experiments. p < 0.05 and p < 0.001 indicate signicant differences compared to the LPS-treated group.

188

V. Woguem et al. / Food Chemistry 149 (2014) 183189

Table 3
Activity on bacterial and yeast species of Xylopia parviora essential oil and some pure
compounds representing major constituents of the oil. Inhibition zone diameters are
expressed in mm SD.

a
b

Essential oil

S. aureus

E. faecalis

E. coli

P. aeruginosa

C. albicans

Xylopia parviora

14 1

11 2

11 1

10 1

12 1

Major constituents
a-Pinene
b-Pinene

n.a.a
n.a.

n.a.
n.a.

n.a.
n.a.

n.a.
n.a.

11 0
91

Positive control
Ciprooxacin
Fluconazole

23 1
n.a.

n.r.b
n.a.

33 2
n.a.

30 2
n.a.

n.a.
29 1

n.a., not active. No inhibition zone around the disk was visible.
n.r., not recommended by the guidelines.

as 1,8-cineole (1.7% in X. parviora oil), a-terpineol (1.0%), linalool


(0.9%), verbenone (0.5%), borneol (0.5%) and camphor (0.4%)
(Koutsoudaki, Krsek, & Rodger, 2005; Santoyo et al., 2005;
Sivropoulou et al., 1997).
3.4. Antioxidant activity
As reported in Table 4, the radical-scavenging activity of the
essential oil and of its main components was tested with two different methods, namely DPPH and ABTS+ assays. The essential oil
showed low antioxidant activity compared to the value reported
for Trolox. The pure main compounds tested, i.e., a-pinene and
trans-pinocarveol, showed slightly higher radical-scavenging
activity towards DPPH than the oil, but no activity towards ABTS.
b-Pinene did not show antiradical scavenging activity towards
either radical, even when tested at concentration above 4 mg/ml.

Table 4
In-vitro radical-scavenging activities of essential oil from Xylopia parviora.
Essential oil

DPPH
TEACa

IC50b

lmol TE/g

lg/ml

TEAC
lmol TE/g

lg/ml

X. parviora

120 (1.1)

590 (1.5)

169 (5.1)

188 (4.2)

Main components
b-Pinene
a-Pinene
trans-pinocarveol

221 (9.1)
204 (5.9)

322 (13)
349 (9)

Reference
Trolox
a
b

4. Conclusions

ABTS

17.9 (0.2)

IC50

8.05 (0.2)

TEAC = Trolox equivalent (TE) antioxidant concentration.


IC50 = The concentration of compound that affords a 50% reduction in the assay.

period of 24 h was determined by using NO production as a nal


read-out parameter. Additionally, the inuence of the essential oil
on the viability of RAW 264.7 cells was investigated by MTT test
after a 24-h incubation time, to exclude non-specic toxicity
(Fig. 1b). As shown in Fig. 1a, stimulation of macrophages with
LPS resulted in a strong increase in NO production. A dose-dependent decrease in NO production was noted in cells treated with X.
parviora essential oil. The NO level in cells treated with 12 lg/ml
of the essential oil was 37% of that of cells treated with LPS alone.
This effect is probably due to the presence of a-pinene, which
was shown to decrease NO production in human chondrocytes
(Neves et al., 2010). In addition, b-pinene and a-pinene are present
in several essential oils with anti-inammatory activity (Miguel,
2010). The high content of these compounds can be related to
the observed anti-inammatory activity.
3.3. Antimicrobial activity
Results of antimicrobial activity tests are summarised in Table 3.
The essential oil was active against all the microbial species tested,
including the yeast C. albicans. The comparison between activities
of the oil and the two major constituents, namely a-pinene and
b-pinene (together accounting for 40.3% of the oil), showed that
neither of the two were responsible for the activity observed
against the bacterial species, whereas both of them inhibited the
growth of C. albicans. Hence activity of X. parviora oil in the case
of the yeast may be condently ascribed to its content of a-pinene
and b-pinene and associates well with the observed activity on
eukaryotic human cell lines. This differential result is in agreement
with previous reports describing the antimicrobial properties of
extracts from other plants (Brusotti et al., 2013 and references
cited therein). Hence, other components occurring in the oil gave
contribution to the observed activity. Among them several compounds have shown moderate to good antibacterial activity, such

The high content of monoterpene hydrocarbons found in X.


parviora essential oil gives a typical pepper note and supports
the use of the plant as an odorous spice in traditional African
cuisine. Interestingly, the biological activities herein reported
may introduce new applications in the African pharmaceutical
market. The essential oil showed an important inhibitory activity
against human tumour cells, being only three times lower than
that of cisplatin; thus it is worthy of further investigation to understand the mode of action, especially on diet-related tumour cells,
and to nd an application for the African pharmaceutical market.
The antimicrobial and anti-inammatory activities of the oil also
have the potential to be utilised.
Acknowledgments
The authors are grateful to the Italian Ministry of Education
(MIUR) for the nancial support (COOPERLINK 2011 Prot.
CII113PPUC).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2013.
10.093.
References
Adams, R. P. (2007). Identication of essential oil components by gas chromatography/
mass spectrometry. Carol Stream, Illinois: Allured Publishing Corporation.
Bicchi, C., Cagliero, C., & Rubiolo, P. (2011). New trends in the analysis of the volatile
fraction of matrices of vegetable origin: A short overview. A review. Flavour and
Fragrance Journal, 26, 321325.
Boik, J. (2001). Natural compounds in cancer therapy. MN, USA: Oregon Medicinal
Press.
Brophy, J. J., Goldsack, R. J., & Forster, P. I. (1998). The essential oils of the Australian
species of Xylopia (Annonaceae). Journal of Essential Oil Research, 10, 469472.
Brusotti, G., Ibrahim, M. F., Dentamaro, A., Gilardoni, G., Tosi, S., Grisoli, P., et al.
(2013). Chemical composition and antimicrobial activity of the volatile
fractions from leaves and owers of the wild Iraqi Kurdish plant Prangos
peucedanifolia Fenzl. Chemistry & Biodiversity, 10, 274280.
Colman-Saizarbitoria, T., Gu, Z. M., & McLaughlin, J. L. (1994). Two new bioactive
monotetrahydrofuran annonaceous acetogenins from the bark of Xylopia
aromatica. Journal of Natural Products, 57, 16611669.
Esparza-Rivera, J. R., Stone, M. B., Stuchnoff, C., Pilon-Smits, E., & Kendall, P. A.
(2006). Effects of ascorbic acid applied by two hydrocooling methods on
physical and chemical properties of green leaf stored at 5 C. Journal of Food
Science, 71, 270276.
Fogang, H. P. D., Tapondjou, L. A., Womeni, H. M., Quassinti, L., Bramucci, M., Vitali,
L., et al. (2012). Characterization and biological activity of essential oils from
fruits of Zanthoxylum xanthoxyloides Lam. and Z. leprieurii Guill. & Perr., two
culinary plants from Cameroon. Flavour and Fragrance Journal, 27, 171179.

V. Woguem et al. / Food Chemistry 149 (2014) 183189


Fogang, H. P. D., Womeni, H. M., Piombo, G., Barouh, N., & Tapondjou, L. A. (2012).
Bioefcacy of essential and vegetable oils of Zanthoxylum xanthoxyloides seeds
against Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). Journal of Food
Protection, 75, 547555.
Harrigan, G. G., Bolzani, V. da S., Gunatilaka, A. A. L., & Kingston, D. G. I (1994).
Kaurane and trachylobane diterpenes from Xylopia aethiopica. Phytochemistry,
36, 109113.
Harrigan, G. G., Gunatilaka, A. A. L., David, G. I., Chan, G. W., & John-son, R. K. (1994).
Isolation of bioactive and other oxoaporphine alkaloids from two annonaceous
plants, Xylopia aethiopica and Miliusa cf. banacea. Journal of Natural Products, 57,
6873.
Huygen, C. (1970). Reaction of nitrose dioxide with Griess-type reagents. Analytical
Chemistry, 42, 407409.
Jirovetz, L., Buchbauer, G., & Ngassoum, M. (1997). Investigation of the essential oils
from the dried fruits of Xylopia aethiopica (West African Peppertree) and
Xylopia parviora from Cameroon. Ernaehrung, 21, 324325.
Johns, S. R., Lamberton, J. A., & Sioumis, A. A. (1968). Alkaloids of Xylopia papuana.
Australian Journal of Chemistry, 21, 13831386.
Jossang, A., Lebceuf, M., Cave, A., & Pusset, J. (1991). Alkaloids of the Annonaceae. 96.
Dehydroxylopine and dehydrocorytenchine, novel isoquinoline alkaloids from
Xylopia vieillardi. Journal of Natural Products, 54, 466472.
Karioti, A., Hadjipavlou-Litina, D., Mensah, M. L. K., Fleischer, T. C., & Skaltsa, H.
(2004). Composition and antioxidant activity of the essential oils of Xylopia
aethiopica (Dun.) A. Rich. (Annonaceae) leaves, stem bark, root bark, and fresh
and dried fruits, growing in Ghana. Journal of Agricultural and Food Chemistry, 52,
80948098.
Koh, E. M., Kim, H. J., Kim, S., Choi, W. H., Choi, Y. H., Ryu, S. Y., et al. (2009).
Modulation of macrophage functions by compounds isolated from Zingiber
ofcinale. Planta Medica, 75, 148151.
Koutsoudaki, C., Krsek, M., & Rodger, A. (2005). Chemical composition and
antibacterial activity of the essential oil and the gum of Pistacia lentiscus var.
chia. Journal of Agricultural and Food Chemistry, 53, 76817685.
Lamaty, G., Menut, C., Bessire, J. M., Amvam Zollo, P. H., & Fekam (1989). The
essential oil of Xylopia parviora (A. Rich) Benth. from Cameroon. Journal of
Essential Oil Research, 1, 247248.
Martins, D., Osshiro, E., Roque, N. F., Marks, V., & Gottlieb, H. E. (1998). A
sesquiterpene dimer from Xylopia aromatica. Phytochemistry, 48, 677680.
Matsuo, A. L., Figueiredo, C. R., Arruda, D. C., Pereira, F. V., Borin Scutti, G. A.,
Massaoka, M. H., et al. (2011). Pinene isolated from Schinus terebinthifolius Raddi
(Anacardiaceae) induces apoptosis and confers antimetastatic protection in a
melanoma model. Biochemical and Biophysical Research Communications, 411,
449454.

189

NCCLS. (2004). Methods for antifungal disk diffusion susceptibility testing of yeasts:
Approved guideline, M44-A. Wayne, PA: NCCLS.
Miguel, M. G. (2010). Antioxidant and anti-inammatory activities of essential oils:
A short review. Molecules, 15, 92529287.
Neves, A., Rosa, S., Gonalves, J., Runo, A., Judas, F., Salgueiro, L., et al. (2010).
Screening of ve essential oils for identication of potential inhibitors of IL-1induced NF-jB activation and NO production in human chondrocytes:
Characterization of the inhibitory activity of a-pinene. Planta Medica, 76,
303308.
Nicoletti, M., Maggi, F., Papa, F., Vittori, S., Quassinti, L., Bramucci, M., et al. (2012). In
vitro biological activities of the essential oil from the resurrection plant
Myrothamnus moschatus (Baillon) Niedenzu endemic to Madagascar. Natural
Product Research, 26, 22912300.
Nishiyama, Y., Moriyasu, M., Ichimaru, M., Iwasa, K., Kato, A., Mathenge, S. G., et al.
(2006). Secondary and tertiary isoquinoline alkaloids from Xylopia parviora.
Phytochemistry, 67, 26712675.
NIST 08. (2008). Mass spectral library (NIST/EPA/NIH). Gaithersburg, USA: National
Institute of Standards and Technology.
Quassinti, L., Lupidi, G., Maggi, F., Sagratini, G., Papa, F., Vittori, S., et al. (2013).
Antioxidant and antiproliferative activity of Hypericum hircinum L. subsp. majus
(Aiton) N. Robson essential oil. Natural Product Research, 27, 862868.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology and Medicine, 26, 12311237.
Santoyo, S., Cavero, S., Jaime, L., Ibanez, E., Senorans, F. J., & Reglero, G. (2005).
Chemical composition and antimicrobial activity of Rosmarinus ofcinalis L.
essential oil obtained via supercritical uid extraction. Journal of Food Protection,
68, 790795.
Sivropoulou, A., Nikolaou, C., Papanikolaou, E., Kokkini, S., Lanaras, T., & Arsenakis,
M. (1997). Antimicrobial, cytotoxic, and antiviral activities of Salvia fructicosa
essential oil. Journal of Agricultural and Food Chemistry, 45, 31973201.
Srinivasan, R., Chandrasekar, M. J. N., Nanjan, M. J., & Suresh, B. (2007). Antioxidant
activity of Caesalpinia digyna root. Journal of Ethnopharmacology, 113, 284291.
Tairu, A. O., Hofmann, T., & Schieberle, P. (1999). Characterization of the key aroma
compounds in dried fruits of the West African peppertree Xylopia aethiopica
(Dunal) A. Rich (Annonaceae) using aroma extract dilution analysis. Journal of
Agricultural and Food Chemistry, 47, 32853287.