Food Chemistry 149 (2014) 183–189

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Food Chemistry

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Volatile oil from striped African pepper (Xylopia parviflora, Annonaceae)possesses notable chemopreventive, anti-inflammatory andantimicrobial potential

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.10.093

⇑ Corresponding authors. Tel.: +39 0737404506 (F. Maggi). Tel.:+237 75004826;fax: +237 33451735 (L.A. Tapondjou).

E-mail addresses: filippo.maggi@unicam.it (F. Maggi), tapondjou2001@yahoo.fr(L.A. Tapondjou).

Verlaine Woguem a,b, Hervet P.D. Fogang a,b, Filippo Maggi c,⇑, Léon 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, Cameroonb Laboratory of Biochemistry of Medicinal Plants, Food Science and Nutrition, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroonc School of Pharmacy, University of Camerino, I-62032 Camerino, Italyd School of Biosciences and Biotechnology, University of Camerino, I-62032 Camerino, Italye School of Science and Technology, Chemistry Division, University of Camerino, I-62032 Camerino, Italy

a r t i c l e i n f o

Article history:Received 3 September 2013Received in revised form 18 October 2013Accepted 22 October 2013Available online 30 October 2013

Keywords:Xylopia parvifloraEssential oilCytotoxic activityAnti-inflammatoryAntimicrobial

a b s t r a c t

Fruits of Xylopia parviflora, well known as striped African pepper, are sold in the Cameroonian markets asa flavouring ingredient to make traditional soups. The essential oil hydrodistilled from fruits was ana-lysed for in vitro biological activities, namely cytotoxic, anti-inflammatory, antimicrobial and antioxidant,by MTT, nitric oxide inhibitory assay, agar disc diffusion method, and DPPH and ABTS assays. The essen-tial oil composition, analysed by GC and GC–MS, 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 stronginhibitory effect on tumour cells MDA-MB 231 and HCT116, with inhibition values close to those of cis-platin. A dose-dependent decrease in NO production was noted in RAW 264.7 macrophages treated withthe oil, revealing a promising anti-inflammatory potential. The essential oil showed a measurable antimi-crobial 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 nativepeople all over the world. Before agriculture, humans dependedon wild plants and animals for their daily needs. Nkui and Nahpoh are two traditional soups of the western region of Cameroonwhich contain many spices, among which are the fruits of Xylopiaparviflora (A. Rich.) Benth. (Annonaceae). X. parviflora, well knownas striped African pepper, is a small tree, 2–3 m high, distributed ineastern and central Africa in riparian sites. Its greenish fruits andseeds, normally boiled on a thread or on a stick of bamboo, arepounded and used in cooked foods or in the spicing of saucesand beverages (Jirovetz, Buchbauer, & Ngassoum, 1997; Karioti,Hadjipavlou-Litina, Mensah, Fleischer, & Skaltsa, 2004). As a tradi-tional remedy, the root decoction is taken by coastal peoples forstomach disorders. Other medicinal uses include treatment of

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 speciescontain bioactive components such as alkaloids (Harrigan, Bolzani,Gunatilaka, & Kingston, 1994; Johns, Lamberton, & Sioumis, 1968;Jossang, Lebceuf, Cave, & Pusset, 1991), acetogenins (Colman-Saizarbitoria, 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 thebiological activity of the volatile oil obtained from this spice.Therefore, as part of our continuous search for beneficial effectsof essential oils from Cameroonian spices (Fogang, Womeni,Piombo, Barouh, & Tapondjou, 2012; Fogang et al., 2012), we eval-uated the biological activities of the essential oil hydrodistilledfrom fruits of X. parviflora, i.e., antioxidant and antimicrobialcapacities, effects on the growth of tumour cell lines, andanti-inflammatory activity. Chemical investigation by GC-FID andGC–MS was undertaken to establish the composition–activityrelationship.

184 V. Woguem et al. / Food Chemistry 149 (2014) 183–189

2. Materials and methods

2.1. Plant material

Fruits of X. parviflora were collected in the Nde Division of theWestern Highlands of Cameroon. They were dried naturally onlaboratory benches at room temperature until constant weight.Identification was made by Mr. Nana Victor, taxonomist at theCameroon National Herbarium (Yaoundé), where a voucher speci-men was deposited (N. 6431/HNC/SRF).

2.2. Extraction of the essential oil

The dry fruits (100 g) of X. parviflora were separately groundand subjected to hydrodistillation with a Clevenger-type apparatususing 750 ml of deionised water for 4 h. The oil collected was driedover anhydrous sodium sulfate yielding 0.60% (w/w) of strongsmelling 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 pur-chased from Sigma–Aldrich (Milan, Italy). For retention indexdetermination, a mix of hydrocarbons ranging from n-octane (C8)to n-triacontane (C30) (Supelco, Bellefonte, PA) was used and rununder the experimental conditions reported below. All compoundswere of analytical standard grade. Analytical grade n-hexane sol-vent was purchased from Carlo Erba (Milan, Italy); it was distiledby a Vigreux column before use.

2.4. GC-FID and GC–MS analyses

For gas chromatographic separations, an Agilent 4890D instru-ment coupled to a flame 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 filmthickness; J & W Scientific, Folsom, CA), with the following temper-ature programme: 5 min at 60 �C, subsequently 4 �C/min up to220 �C, then 11 �C/min up to 280 �C, held for 15 min, for a totalrun of 65 min. Injector and transfer line temperatures were280 �C. Helium was used as the carrier gas, at a flow rate of1.4 ml/min; injection volume: 1 ll; split ratio, 1:34. A mixture ofaliphatic hydrocarbons (C8–C30) (Sigma, Milan, Italy) in n-hexane,was directly injected into the GC injector using the above temper-ature programme, in order to calculate the retention index of eachcompound. Oil samples were diluted 1:100 in n-hexane andinjected at a volume of 1 ll. Analysis was repeated 3 times. Datawere collected by using HP3398A GC Chemstation software (Hew-lett Packard, Rev. A.01.01). The relative amounts of essential oilcomponents, expressed as percentages, were obtained by FIDpeak-area normalisation by calculating the response factor (RF)of the FID for seven different classes of volatiles. Standardcompounds, 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 hydro-carbons; p-cymene for aromatic monoterpenes; 1,8-cineole andlinalool for oxygenated monoterpenes; (E)-caryophyllene anda-humulene for sesquiterpene hydrocarbons; caryophyllene oxidefor oxygenated sesquiterpenes; n-octanal for aldehydes andketones; 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 eachstandard a regression line was obtained by plotting the ratio ofinternal standard (mean value of n-octane and n-octadecane) toreference standard peak area versus the ratio of internal standardto reference standard concentration. Each dilution was analysedfive times. The correlation coefficients (r2) obtained for calibrationcurves of representative standards were all higher than 0.992. Thecorrection factor of each reference standard compound was calcu-lated as the slope of the linear regression equation. Whenevermore than one reference analyte for each class was considered,the correction factor was calculated by the mean of the slopes ofall representative compounds. Using the generalised responsefactor for compounds within the eight classes, the derived quanti-tative data may be considered as an approximation of the absolutequantification. For the calculation of volatile concentrations, ex-pressed in mg/g oil, the essential oils were injected three timesusing n-octane and n-octadecane as internal standards.

GC–MS analysis was performed on an Agilent 6890N gaschromatograph coupled to a 5973N mass spectrometer using anHP-5MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d.,0.1 lm film thickness; J & W Scientific) capillary column. The tem-perature programme was the same as above. Injector and transferline temperatures were 280 �C. Helium was used as the carrier gas,at a flow rate of 1 ml/min. Split ratio: 1:50; acquisition mass range:m/z 29–400. All mass spectra were acquired in electron-impact (EI)mode with an ionisation voltage of 70 eV. Oil samples were diluted1:100 in n-hexane and the volume injected was 2 ll. Data wereanalysed by using MSD ChemStation software (Agilent, VersionG1701DA D.01.00). Whenever possible volatile components wereidentified by co-injection with authentic standards (see Sec-tion 2.3). Otherwise, the peak assignment was carried out on thebasis of the standard of the International Organisation of theFlavour Industry (IOFI, http://www.iofi.org/) statement (Bicchi,Cagliero, & Rubiolo, 2011), i.e., by the interactive combination ofchromatographic linear retention indices that were consistent withthose reported in the literature (Adams, 2007; NIST 08, 2008) fornon-polar stationary phases, and MS data consisting of the com-puter matching with the WILEY275, NIST 08, ADAMS, and home-made (based on the analyses of reference oils and commerciallyavailable standards) libraries.

2.5. Cell culture

Human colon carcinoma cell line HCT116 was cultured inRPMI1640 medium with 2 mM L-glutamine, 100 IU/ml penicillin,100 lg/ml streptomycin, and supplemented with 10% heat-inacti-vated foetal bovine serum (HI-FBS) (PAA Laboratories GmbH, Pas-ching, Austria). Murine macrophage cell line RAW 264.7, humanbreast adenocarcinoma cell line MDA-MB 231, and human malig-nant melanoma cell line A375 were cultured in Dulbecco’s Modi-fied Eagle’s Medium (DMEM) with 2 mM L-glutamine, 100 IU/mlpenicillin, 100 lg/ml streptomycin, and supplemented with 10%HI-FBS. Cells were cultured in a humidified atmosphere at 37 �Cin 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 Quassintiet al. (2013). Briefly, cells were seeded at a density of 2 � 104

cells/ml. After 24 h, samples were exposed to different concentra-tions of essential oil (0.78–200 lg/ml). Authentic standards of themajor components a- and b-pinene, purchased fromSigma–Aldrich (Milan, Italy), were tested as well. The anticancerdrug cisplatin (0.01–50 lg/ml) was used as the positive control.Cells were incubated for 72 h in a humidified atmosphere of 5%

V. Woguem et al. / Food Chemistry 149 (2014) 183–189 185

CO2 at 37 �C. At the end of incubation, each well received 10 ll of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT) (5 mg/ml in phosphate-buffered saline, PBS) and the plateswere incubated for 4 h at 37 �C. The extent of MTT reduction wasmeasured spectrophotometrically at 540 nm using a Titertek Mul-tiscan microElisa (Labsystems, Helsinki, Finland). Experimentswere conducted in triplicate. Cytotoxicity was expressed as theconcentration 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 adensity of 5 � 105 cells/ml in the presence of essential oil(0.75–12 lg/ml). Cells were incubated with 1 lg/ml lipopolysac-charide (LPS), alone or co-incubated with essential oil and LPS(1 lg/ml) for 24 h. Aminoguanidine (200 lM) plus 1 lg/ml LPSserved as a control for the reduction of NO production (Kohet al., 2009). NO production was determined by measuring theamount of the primary stable reaction product nitrite with Griessreagent (Huygen, 1970) (1% sulfanilamide, 0.1% N-(1-naphthyl)eth-ylenediamine dihydrochloride, 5% H3PO4) by mixing 50 ll of cellculture supernatant with the same volume of reagents. After incu-bation for 10 min at room temperature, the absorption of theformed diazo dye was measured spectrophotometrically at540 nm using a Titertek Multiscan microElisa (Labsystems,Helsinki, Finland). The nitrite concentration was determined bycomparison with a sodium nitrite standard calibration curve in cul-ture medium (0–100 lM). The cell viability of the macrophageswas determined by MTT assay.

2.8. Antimicrobial activity

Microorganisms included in this study were Staphylococcusaureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonasaeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Candidaalbicans ATCC 24433. Antimicrobial activity of the essential oil andmajor components a- and b-pinene (Sigma, Milan, Italy) was as-sessed by disc diffusion test, by spotting 10 ll pure oil or standardcompound onto a paper disc and following the general guidelinesof the European Committee for Antimicrobial Susceptibility Testing(EUCAST, www.EUCAST.org), with previously described minormodifications, due to the nature of the substance tested (Quassintiet al., 2013). Activity was determined by measuring the diameter ofthe growth inhibition zone (inhibition zone diameter, IZD) visiblearound the paper disc (expressed in mm). Reported IZDs are inclu-sive of the paper disc diameter (6 mm). Therefore, a 6-mm IZDmeans no activity. Ten microlitres of each reference compound(a-pinene and b-pinene) per paper disc were used in the controlexperiments, while the known antimicrobials ciprofloxacin (5 lgper disc) and fluconazole (25 lg per disc) (NCCLS, 2004) were usedas a reference against bacteria and fungi, respectively.

2.9. Antioxidant activity

DPPH free radical-scavenging activity was evaluated on amicroplate analytical assay according to the previously describedprocedure by Srinivasan, Chandrasekar, Nanjan, and Suresh(2007). The stock solution was prepared by dissolving DPPH inmethanol and then stored at �20 �C until needed. The workingsolution was obtained by mixing stock solution with methanol toobtain an absorbance of 1 unit at 517 nm. Discoloration was mea-sured at 517 nm after incubation for 30 min in the dark. The freeradical-scavenging activity of each solution was then calculated

as percent inhibition according to the following equation:% inhibi-tion = 100 (A(blank) � A(sample))/A(blank)

Antioxidant activity of the essential oil was expressed as IC50,defined as the concentration of the test material required to causea 50% decrease in initial DPPH concentration. Trolox was used asreference. Results were expressed in lM Trolox equivalents(TE)/g of essential oil.

ABTS assay was performed following the procedure previouslydescribed (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 solu-tions of ABTS (7.4 mM) and potassium persulfate (2.6 mM) in equalquantities and allowing them to react for 12 h at room temperaturein the dark. The working solution was then obtained by mixingABTS+ solution with methanol to obtain a final solution withabsorbance of 1 unit at 734 nm measured with a Varian Cary 1spectrophotometer. Trolox was used as reference. Results wereexpressed in lM Trolox equivalents (TE)/g of essential oil. Thecapacity of free radical scavenging (IC50) was determined usingthe same previously used equation for the DPPH method. All dataof antioxidant activity were expressed as means ± standard devia-tions (SD) of triplicate measurements. The confidence limits wereset at p < 0.05. SD did not exceed 5% for the majority of the valuesobtained.

2.10. Statistical analysis

All assays were conducted at least three times with three differ-ent sample preparations. All data were expressed as mean ± stan-dard deviation (SD). Analysis of variance was performed usingInStat (GraphPad software, San Diego CA). A one-way ANOVAand unpaired Student’s t-test were used for these analyses, andp < 0.05 was considered to be statistically significant.

3. Results and discussion

3.1. Analysis of the essential oil

The chemical composition of the essential oil obtained from thefruits of X. parviflora is reported in Table 1. A total of sixty volatilecomponents, corresponding to 90.8% of the total composition, wereidentified in the essential oil. The major fraction was monoter-penes (70.7%) with hydrocarbons (50.0%) occurring in higher levelsthan oxygenated compounds (20.7%). The main components wereb-pinene (34.0%) and a-pinene (10.3%) among the former, andtrans-pinocarveol (5.0%) and myrtenol (4.6%) among the latter.These compounds are considered to be the main contributors tothe overall floral terpeny, smoky odour of the dried fruits of theWest African pepper tree (Tairu, Hofmann, & Schieberle, 1999).As a matter of fact, the high concentration of monoterpene hydro-carbons in X. parviflora is essential to give the pepper odour(Jirovetz et al., 1997). Minor contributions came from sesquiter-pene hydrocarbons (6.1%) and oxygenated sesquiterpenes(11.7%). Among these fractions none of the components reachedamounts higher than 2.2%.

Our results were consistent with those previously reported forthe volatile composition of fruits collected in a different region(Bayagam and Ngaoundere areas) of Cameroon (Jirovetz et al.,1997; Lamaty, Menut, Bessière, Amvam Zollo, & Fekam, 1989). Inthose cases, the major constituents were again b-pinene (40.0%and 22.6%, respectively) and a-pinene (14.0% and 12.9%, respec-tively), while trans-pinocarveol (2.7% and 2.6%, respectivley) andmyrtenol (2.2% and 1.7%, respectively) were slightly lower. Onthe other hand, among minor components, (E)-b-ocimene, whichwas quite abundant in those studies (5.4% and 4.1%, respectively),

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

N. Constituenta RFb Calc. LRIc Lit. LRId Essential oil IDf

ADAMS NIST08 %e mg/g

1 n-Hexanal 1.7 804 801 804 Trg 0.3 ± 0.0 RI,MS2 4-Hydroxy-4-methyl-2-pentanone 1.7 849 839 0.3 ± 0.1 3.4 ± 0.4 RI,MS3 Tricyclene 1.1 922 926 922 0.1 ± 0.0 0.7 ± 0.0 RI,MS4 a-Thujene 1.1 927 930 926 0.1 ± 0.0 1.0 ± 0.0 RI,MS5 a-Pinene 1.1 932 939 932 10.3 ± 0.0 104.4 ± 0.3 Std6 Camphene 1.1 947 954 947 3.3 ± 0.0 34.0 ± 0.1 Std7 Thuja-2,4(10)-diene 1.1 954 960 954 0.1 ± 0.0 0.9 ± 0.0 RI,MS8 Sabinene 1.1 973 975 971 Tr 0.3 ± 0.0 RI,MS9 b-Pinene 1.1 975 979 974 34.0 ± 0.1 346.1 ± 0.5 Std

10 Myrcene 1.1 993 990 991 0.1 ± 0.0 0.9 ± 0.0 Std11 p-Cymene 1.7 1028 1024 1028 1.3 ± 0.0 20.6 ± 0.3 Std12 Limonene 1.1 1031 1029 1028 0.6 ± 0.0 5.9 ± 0.0 Std13 1,8-Cineole 1.5 1034 1031 1034 1.7 ± 0.0 17.7 ± 0.2 Std14 (E)-b-Ocimene 1.1 1055 1050 1052 0.1 ± 0.0 0.9 ± 0.1 RI,MS15 Linalool 1.5 1103 1096 1100 0.9 ± 0.0 9.5 ± 0.4 Std16 a-Campholenal 1.5 1129 1126 1130 0.2 ± 0.0 1.9 ± 0.3 RI,MS17 trans-Pinocarveol 1.5 1140 1139 1141 5.0 ± 0.1 50.8 ± 0.6 Std18 Camphor 1.5 1146 1146 0.4 ± 0.0 3.9 ± 0.1 Std19 Pinocarvone 1.5 1165 1164 1165 0.4 ± 0.0 4.1 ± 0.3 RI,MS20 Borneol 1.5 1167 1169 1167 0.5 ± 0.1 4.9 ± 0.6 Std21 cis-Pinocamphone 1.5 1176 1175 Tr 0.2 ± 0.0 RI,MS22 Terpinen-4-ol 1.5 1179 1177 1179 0.8 ± 0.0 8.1 ± 0.3 Std23 cis-Pinocarveol 1.5 1188 1184 Tr 0.8 ± 0.0 RI,MS24 p-Cymen-8-ol 1.5 1190 1182 1189 0.1 ± 0.0 2.3 ± 0.2 RI,MS25 a-Terpineol 1.5 1192 1188 1189 1.0 ± 0.0 10.5 ± 0.1 Std26 Myrtenal 1.5 1195 1195 1195 2.5 ± 0.0 25.1 ± 0.4 Std27 Myrtenol 1.5 1197 1195 1196 4.6 ± 0.1 46.3 ± 0.8 RI,MS28 Verbenone 1.5 1211 1205 1211 0.5 ± 0.0 5.2 ± 0.1 Std29 trans-Carveol 1.5 1225 1216 1225 0.2 ± 0.1 1.6 ± 0.3 RI,MS30 Isobornyl acetate 1.5 1286 1285 1.0 ± 0.6 12.6 ± 1.2 Std31 d-Elemene 1.1 1336 1338 0.1 ± 0.0 1.5 ± 0.1 RI,MS32 a-Cubebene 1.1 1348 1348 0.3 ± 0.0 2.6 ± 0.1 RI,MS33 Cyclosativene 1.1 1360 1371 1360 0.5 ± 0.0 4.9 ± 0.9 RI,MS34 a-Copaene 1.1 1372 1376 0.5 ± 0.0 4.8 ± 0.2 RI,MS35 b-Cubebene 1.1 1387 1388 1385 0.3 ± 0.0 3.1 ± 0.1 RI,MS36 b-Elemene 1.1 1389 1390 1387 0.5 ± 0.0 4.7 ± 0.2 RI,MS37 (E)-Caryophyllene 1.1 1413 1419 1412 0.2 ± 0.0 2.2 ± 0.1 Std38 b-Copaene 1.1 1424 1432 1.1 ± 0.0 10.8 ± 0.1 RI,MS39 a-Humulene 1.1 1448 1454 1446 0.1 ± 0.0 1.3 ± 0.1 Std40 b-Selinene 1.1 1480 1490 1481 0.1 ± 0.0 0.8 ± 0.1 RI,MS41 trans-Muurola-4(14),5-Diene 1.1 1492 1493 1471 0.1 ± 0.0 1.3 ± 0.2 RI,MS42 a-Muurolene 1.1 1496 1500 1494 0.6 ± 0.0 6.0 ± 0.1 RI,MS43 trans-Calamenene 1.1 1519 1522 1.4 ± 0.1 14.6 ± 1.0 RI,MS44 d-Cadinene 1.1 1523 1523 1518 Tr 1.2 ± 0.2 RI,MS45 a-Calacorene 1.1 1539 1545 0.1 ± 0.0 1.1 ± 0.1 RI,MS46 Hedycaryol 1.3 1547 1548 1541 1.2 ± 0.0 11.8 ± 0.0 RI,MS47 b-Calacorene 1.1 1561 1565 0.1 ± 0.0 1.4 ± 0.0 RI,MS48 Spathulenol 1.3 1573 1578 1570 0.1 ± 0.0 1.3 ± 0.0 RI,MS49 Caryophyllene oxide 1.3 1576 1583 1573 2.1 ± 0.0 21.7 ± 0.0 Std50 Salvial-4(14)-en-1-one 1.3 1588 1594 0.3 ± 0.0 3.4 ± 0.0 RI,MS51 Humulene epoxide II 1.3 1602 1608 1602 0.8 ± 0.0 7.7 ± 0.1 RI,MS52 1,10-Di-epi-cubenol 1.3 1626 1619 1630 2.2 ± 1.0 26.7 ± 2.5 RI,MS53 Caryophylla-4(12),8(13)-dien-5-olh 1.3 1632 1640 1635 0.7 ± 1.0 1.3 ± 0.1 RI,MS54 Cubenol 1.3 1638 1646 0.8 ± 0.0 7.8 ± 0.1 RI,MS55 a-Muurolol 1.3 1644 1646 1644 2.1 ± 0.0 21.5 ± 0.1 RI,MS56 cis-Calamenen-10-ol 1.3 1658 1661 0.3 ± 0.0 3.0 ± 0.2 RI,MS57 trans-Calamenen-10-ol 1.3 1666 1669 0.2 ± 0.0 1.9 ± 0.0 RI,MS58 Eudesma-4(15),7-dien-1-b-ol 1.3 1682 1688 1.0 ± 0.0 10.4 ± 0.1 RI,MS59 Manoyl oxide 1.4 2004 2004 1.0 ± 0.0 9.9 ± 0.1 RI,MS60 Phyllocladene 1.4 2025 2017 0.2 ± 0.0 2.4 ± 0.0 RI,MS

Total identified (%) 90.8Oil yield (%) 0.6

Grouped compounds (%)Monoterpene hydrocarbons 50.0Oxygenated monoterpenes 20.7Sesquiterpene hydrocarbons 6.1Oxygenated sesquiterpenes 11.7Diterpenes 2.0

a Compounds are listed in order of their elution from an HP-5MS column. Their nomenclature was in accordance with Adams (2007).b 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 C8–C32 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 Identification 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 identified.

186 V. Woguem et al. / Food Chemistry 149 (2014) 183–189

Table 2In-vitro cytotoxic activity of essential oil from Xylopia parviflora.

Cell line (IC50 lg/ml)a

MDA-MB 231b A375c HCT116d

Essential oilXylopia parviflora 6.56 7.47 6.6395% C.I.e 6.02–7.14 6.89–8.10 6.01–7.31

Major componentsa-Pinene 31.0 44.8 63.195% C.I. 28.5–33.7 42.6–47.2 55.5–71.9b-Pinene 75.5 >200 57.295% C.I. 70.7–80.5 53.7–61.0

ReferenceCisplatin 2.07 0.15 2.6295% C.I. 1.69–2.22 0.11–0.20 2.41–2.86

a IC50 as the concentration of compound that affords a 50% reduction in cellgrowth (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., confidence interval.

V. Woguem et al. / Food Chemistry 149 (2014) 183–189 187

was very low in our sample (0.1%). These differences may be due tothe geographical origin of the samples.

3.2. Antiproliferative and anti-inflammatory activity

The essential oil of X. parviflora was tested in vitro for its poten-tial tumour cell growth-inhibitory effect on MDA-MB 231 humanbreast adenocarcinoma cell line, A375 human malignant mela-noma cell line, and HCT116 human colon carcinoma cell line, using

Fig. 1. Effects of Xylopia parviflora essential oil on NO production (a) and cell viability (b) bAG, aminoguanidine. Data are means ± SD of three independent experiments. ⁄p < 0.05 a

MTT assay. The results, collected in Table 2, show that essential oilexhibited a strong inhibitory effect against the human cancer cellsexamined. The highest activity was observed on MDA-MB 231 andHCT 116 cell lines, with IC50 values of 6.56 and 6.63 lg/ml, respec-tively, which are only three times weaker than those of the anti-cancer 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), confirm the potential of X. parviflora essential oil as apromising anticancer agent.

Very few of the compounds found in X. parviflora essential oilhave been tested for their anticancer properties. However, thecytotoxic effects of the commercially available major constituents,were found to be lower than that of the essential oil. b-Pinene wasslightly effective against MDA-MB 231 and HCT116 cell lines(Table 2). a-Pinene was more active on all tumour cells, showingmaximum inhibition on MDA-MB 231 cells (IC50 value of31.0 lg/ml). This compound has also been reported to exhibitapoptotic and antimetastatic activity on melanoma cells (Matsuoet al., 2011). This was partly supported by the IC50 value detectedon A375 cell line (31.0 lg/ml). The component trans-pinocarveolshowed little cytotoxic effect against MDA-MB 231 and some glio-blastoma (Nicoletti et al., 2012). The concentrations of b-pinene(34%), a-pinene (10.3%) and trans-pinocarveol (5%) in the oil donot justify completely the cytotoxic effect obtained for the wholeessential oil, indicating a possible synergism between the differentconstituents.

In order to evaluate potential inhibiting effects of X. parvifloraessential oil against stimulated macrophages, which could be a typ-ical indicator for anti-inflammatory activity, RAW 264.7 cells werestimulated by LPS; the effect of essential oil during a co-incubation

y LPS-stimulated RAW 264.7 macrophages. EO, essential oil; LPS, lipopolysaccaride;nd ⁄⁄p < 0.001 indicate significant differences compared to the LPS-treated group.

Table 3Activity on bacterial and yeast species of Xylopia parviflora essential oil and some purecompounds representing major constituents of the oil. Inhibition zone diameters areexpressed in mm ± SD.

Essential oil S. aureus E. faecalis E. coli P. aeruginosa C. albicans

Xylopia parviflora 14 ± 1 11 ± 2 11 ± 1 10 ± 1 12 ± 1

Major constituentsa-Pinene n.a.a n.a. n.a. n.a. 11 ± 0b-Pinene n.a. n.a. n.a. n.a. 9 ± 1

Positive controlCiprofloxacin 23 ± 1 n.r.b 33 ± 2 30 ± 2 n.a.Fluconazole n.a. n.a. n.a. n.a. 29 ± 1

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

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

Essential oil DPPH ABTS

TEACa

lmol TE/gIC50

b

lg/mlTEAClmol TE/g

IC50

lg/ml

X. parviflora 120 (±1.1) 590 (±1.5) 169 (±5.1) 188 (±4.2)

Main componentsb-Pinenea-Pinene 221 (±9.1) 322 (±13)trans-pinocarveol 204 (±5.9) 349 (±9)

ReferenceTrolox 17.9 (±0.2) 8.05 (±0.2)

a TEAC = Trolox equivalent (TE) antioxidant concentration.b IC50 = The concentration of compound that affords a 50% reduction in the assay.

188 V. Woguem et al. / Food Chemistry 149 (2014) 183–189

period of 24 h was determined by using NO production as a finalread-out parameter. Additionally, the influence of the essential oilon the viability of RAW 264.7 cells was investigated by MTT testafter a 24-h incubation time, to exclude non-specific toxicity(Fig. 1b). As shown in Fig. 1a, stimulation of macrophages withLPS resulted in a strong increase in NO production. A dose-depen-dent decrease in NO production was noted in cells treated with X.parviflora essential oil. The NO level in cells treated with 12 lg/mlof the essential oil was 37% of that of cells treated with LPS alone.

This effect is probably due to the presence of a-pinene, whichwas shown to decrease NO production in human chondrocytes(Neves et al., 2010). In addition, b-pinene and a-pinene are presentin several essential oils with anti-inflammatory activity (Miguel,2010). The high content of these compounds can be related tothe observed anti-inflammatory 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 activitiesof the oil and the two major constituents, namely a-pinene andb-pinene (together accounting for 40.3% of the oil), showed thatneither of the two were responsible for the activity observedagainst the bacterial species, whereas both of them inhibited thegrowth of C. albicans. Hence activity of X. parviflora oil in the caseof the yeast may be confidently ascribed to its content of a-pineneand b-pinene and associates well with the observed activity oneukaryotic human cell lines. This differential result is in agreementwith previous reports describing the antimicrobial properties ofextracts from other plants (Brusotti et al., 2013 and referencescited therein). Hence, other components occurring in the oil gavecontribution to the observed activity. Among them several com-pounds have shown moderate to good antibacterial activity, such

as 1,8-cineole (1.7% in X. parviflora 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 theessential oil and of its main components was tested with two dif-ferent methods, namely DPPH and ABTS+ assays. The essential oilshowed low antioxidant activity compared to the value reportedfor Trolox. The pure main compounds tested, i.e., a-pinene andtrans-pinocarveol, showed slightly higher radical-scavengingactivity towards DPPH than the oil, but no activity towards ABTS.b-Pinene did not show antiradical scavenging activity towardseither radical, even when tested at concentration above 4 mg/ml.

4. Conclusions

The high content of monoterpene hydrocarbons found in X.parviflora essential oil gives a typical pepper note and supportsthe use of the plant as an odorous spice in traditional Africancuisine. Interestingly, the biological activities herein reportedmay introduce new applications in the African pharmaceuticalmarket. The essential oil showed an important inhibitory activityagainst human tumour cells, being only three times lower thanthat of cisplatin; thus it is worthy of further investigation to under-stand the mode of action, especially on diet-related tumour cells,and to find an application for the African pharmaceutical market.The antimicrobial and anti-inflammatory activities of the oil alsohave the potential to be utilised.

Acknowledgments

The authors are grateful to the Italian Ministry of Education(MIUR) for the financial support (COOPERLINK 2011 Prot.CII113PPUC).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2013.10.093.

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