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Journal of Ethnopharmacology 134 (2011) 803–812

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

ytotoxicity of some Cameroonian spices and selected medicinal plant extracts

ictor Kuetea,b, Benjamin Kruscheb, Mahmoud Younsb, Igor Voukenga, Aimé G. Fankama,implice Tankeoa, Stephen Lacmataa, Thomas Efferthb,∗

Department of Biochemistry, Faculty of Science, University of Dschang, CameroonDepartment of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany

r t i c l e i n f o

rticle history:eceived 22 October 2010eceived in revised form0 December 2010ccepted 21 January 2011vailable online 1 February 2011

eywords:ytotoxicityngiogenesisxtractspicesameroon

a b s t r a c t

Ethnopharmacological relevance: Several medicinal plants and spices are used traditionally to treat cancersin Cameroon.Aim: Methanol extracts from thirty-four spices and plants, with related ethnobotanical use were inves-tigated for their in vitro cytotoxicity on the human pancreatic cancer cell line MiaPaCa-2, leukemiaCCRF-CEM cells and their multidrug resistant (MDR) subline CEM/ADR5000, and the normal humanumbilical vein endothelial cells (HUVECs). In addition the anti-angiogenic properties of the most activeextracts were investigated.Material and methods: The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay was used for cytotoxic studies and the CAM-assay (chicken-chorioallantoic-membrane-assay) for anti-angiogenesis test.Results: The results of the cytotoxicity tests indicated that, when tested at 20 �g/ml, extracts from Xylopiaaethiopica, Echinops giganteus, Imperata cylindrica, Dorstenia psilirus and Piper capense were able to inhibitmore that 50% the proliferation of the three tested cancer cells (MiaPaCa-2, CEM/ADR5000 CCRF-CEM).

The lowest IC50 values of 6.86 �g/ml on MiaPaCa-2 and 3.91 �g/ml on CCRF-CEM cells were obtained withX. aethiopica, while the corresponding value of 6.56 �g/ml was obtained with P. capense on CEM/ADR5000cells. Against leukemia cells, no cross-resistance was observed with I. cylindrica, P. capense and Zinziberofficinalis. Extracts from D. psilirus and E. giganteus were able to inhibit angiogenesis by more than 50% inquail embryo.Conclusion: The overall results of the present study provide supportive data on the use of some Cameroo-

atme

nian plants for cancer tre

. Introduction

Pancreatic cancer is one of the most aggressive solid malig-ancies with high rate of resistance to most of the availablereatment modalities, such as chemotherapy and radiotherapyWente et al., 2008). Only 20% of pancreatic cancers are amenableo surgical resection at presentation and despite the medi-al advances made over the last 20 years, pancreatic cancerppear to have benefited the least in terms of survival (Garceat al., 2005). In the case of leukemia, MDR phenotypes haveecome a major concern to effective treatment. It is there-

ore important to develop new therapeutic strategies to treat

alignancies. It was reported that excessive angiogenesis is anmportant factor of the pathogenesis of many industrialized west-rn countries (Krenn and Paper, 2009). Therefore, compounds

∗ Corresponding author. Tel.: +49 6131 3925751; fax: +49 6131 3923752.E-mail address: efferth@uni-mainz.de (T. Efferth).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.01.035

nt.© 2011 Elsevier Ireland Ltd. All rights reserved.

with anti-angiogenic properties are of importance in the treat-ment and prevention of malignancies as well as other chronicdiseases (Paper, 1998; Carmeliet, 2003). Screenings of medic-inal plants used as anticancer drugs have provided modernmedicine with effective cytotoxic pharmaceuticals. More than60% of the approved anticancer drugs in United State of Amer-ica (from 1983 to1994) were from natural origin (Stévigny et al.,2005; Newman and Cragg, 2007). In the Cameroonian pharma-copoeia, there are still serious lacks of information on the useof large numbers of plants and spices traditionally employed inthe treatment of several ailments, including cancers. It belongsto the scientific community to fill this gap. However, it hasbeen recommended that ethnopharmacological usages such asimmune and skin disorders, inflammatory, infectious, parasiticand viral diseases should be taken into account when select-

ing plants used to treat cancer, since these reflect disease statesbearing relevance to cancer or cance-liker symptoms (Cordellet al., 1991; Popoca et al., 1998). Therefore, the present workwas designed to investigate the cytotoxicity of some plants

8 opharm

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04 V. Kuete et al. / Journal of Ethn

nd spices used locally in Cameroon to treat cancers, but notnventoried in the national pharmacopoeia. The study wasxtended to the search of anti-angiogenic properties of some ofhe most active extracts.

. Materials and methods

.1. Plant material and extraction

All medicinal plants used in the present work were collectedn Dschang, western region of Cameroon, between January andebruary 2009. The spices were obtained from Dschang local mar-et at the same period. The plants were identified at the Nationalerbarium (Yaounde, Cameroon) where voucher specimens wereeposited under the references numbers (Table 1). The air-driednd powdered plant material was soaked in methanol for 48 h, atoom temperature. The methanol extract was concentrated undereduced pressure to give the crude extract. This extract was thenonserved at 4 ◦C until further use.

.2. Cell lines and treatment

The human pancreatic cancer cell lines, MiaPaCa-2 (poorlyifferentiated), CCRF-CEM leukemia cells and their multidrugesistant subline, CEM/ADR5000 (moderately differentiated), werebtained from the American Type Culture Collection (Rockville,SA). Leukemia cells maintained in RPMI 1640 containing00 units/ml penicillin and 100 mg/ml streptomycin and sup-lemented with heat-inactivated 10% fetal bovine serum (FBS).iaPaCa-2 cells were maintained in DMEM containing 100 units/ml

enicillin,100 mg/ml streptomycin and 10% FBS (Invitrogen, Carls-ad, CA). Human umbilical vein endothelial cells (HUVECs) wereaintained in large vessel endothelial cell growth media. All cul-

ured cells were maintained in a humidified environment at 37 ◦Cith 5% CO2. Doxorubicin (Sigma–Aldrich, Schnelldorf, Germany)as used as a positive (cytotoxic) control. The concentration ofMSO was kept at or below 0.1% in all experiments.

.3. MTS cell growth inhibition assay

To assess cell proliferation, CellTiter 96® aqueous non-adioactive cell proliferation assay (Promega, Mannheim,ermany) was used according to the manufacturer’s instruc-

ions. The assay tests cellular viability and mitochondrial function.riefly, MiaPaCa-2 or HUVEC cells were grown in tissue cultureasks, and then harvested by treating the flasks with 0.025%rypsin and 0.25 mM EDTA for 5 min. Once detached, cells wereashed, counted and an aliquot (5 × 103 cells) was placed in eachell of a 96-well cell culture plate in a total volume of 100 �l.ells were allowed to attach overnight and then treated with plantxtracts. After 48 h, 20 �l MTS [3-(4,5-dimethylthiazol-2-yl)-5-3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]olution was added to each well and the plates were incubated at7 ◦C for 3 h. The absorbance of the product formazan, which isonsidered to be directly proportional to the number of living cellsn the culture, was measured at 490 nm and at 650 nm using a Pre-ision Microplate Reader (Molecular Devices, Sunnyvale, CA). Foreukemia cells, aliquot of 5 × 104 cells/ml (obtained from overnightuspension) were seeded in 96-well plates, and extracts weredded immediately. After 24 h incubation, plates were treatedith MTS solution as above mentioned. For the preliminary assays,

xtracts were tested at 20 �g/ml, and extracts with more than0% inhibition of the cell proliferation were further diluted twoold and tested for IC50 determinations. Doxorubicin was used asositive control. Each assay has been done at least three times,ith four replicate each. The viability was compared based on a

acology 134 (2011) 803–812

comparison with untreated cell and the inhibition percentage (%I)was determined using the formula:

%I =[

100(

1 − At

Ac

)]

where (At) is the absorbance of treated well and (Ac) is theabsorbance of control well. IC50 (on cancer cells) or EC50 (on HUVECcells) value is the concentration of sample required to inhibit 50%of the cell proliferation and was calculated from a calibration curveby a linear regression (Joshi et al., 2010) using Microsoft Excel. Theselective index was determined as the ratio EC50/IC50.

2.4. Angiogenesis test

2.4.1. Cultivation of quail eggsThe embryos were cultured according to the method described

by Wittmann et al. (1987). Briefly, fertilized quail eggs were incu-bated for 70 h at 38 ◦C and 80% relative humidity. After 70 h ofincubation the eggs were opened. For this purpose the eggs wereplaced in a vertical position to guarantee that the embryo floats inthe upper part of the egg. Afterwards, hole was cut in to the top ofthe egg and the complete content of the egg was transferred intoa Petri dish. By using this method, it could be guaranteed that thealbumin gets first into the Petri dish followed by the yolk with theembryo on top without exposing the embryo to shock-forces whichcould damage the vitelline membrane.

2.4.2. Chicken-chorioallantoic-membrane-assay (CAM-assay)Plant extracts with good cytotoxic activities on the tested can-

cer cell lines were tested for their anti-angiogenic effects. Themethod of D’Arcy and Howard (1967), with modifications accord-ing to Marchesan et al. (1998) was used. Briefly, the explantedembryo was placed in an incubator for 2 h at 38 ◦C to acclimatize tothe new ambience. Subsequently, the test substances were placedon the chorioallantoic membrane (CAM). Therefore, 2% agarosesolution was prepared and mixed 1:10 with the plant extractprior diluted in DMSO 0.1% final concentration. The final concen-tration of the substance was 20 �g/ml. Pellets with 0.1% DMSOserved as control. The agarose-pellets were then placed on thechorioallantoic membrane after they cooled down to room tem-perature. The Petri dishes with the quail embryos were placedin the incubator again and incubated at 38 ◦C and 80% relativehumidity for 24 h before documenting the effect of the appliedsubstances.

Imaging of the vascularized quail eggs was performed using adigital camera with 3×-magnification objective (Canon eos 500with a canon mp-e 65 2.8 macro objective). For illumination amercury-arc-lamp was used which provided a high fraction of blueand UV-light to obtain good contrast values between yolk andvessels. The pictured image section had a size of 5 mm × 5 mm.Following image acquisition, quantitative analysis was performedusing a software routine which was written in the ImageJ-macrolanguage, and the total small vessels number (or area) was thendetermined by the system. The percentage inhibition of vascular-ization was calculated using the formula:

%Inhibition of vascularization =(%vascularized area of treated sample%vascularized area of control sample

)× 100

2.5. Statistics

The one-way ANOVA at 95% confidence level was used for sta-tistical analysis.

V. Kuete et al. / Journal of Ethnopharmacology 134 (2011) 803–812 805

Table 1Medicinal plants used in the present study.

Family/plantspecies (Voucherspecimen)a

Traditional use Part usedtraditionally

Part used in thisstudy (extractionyield in %)b

Active compounds and previously screenedactivityc

AnnonaceaeMonodora

Myristica Dunal(2949/SFRCam)

Constipation, uterine hemorrhage,diuretic, fever (Udeala et al., 1980;Iwu et al., 1987; Okafor, 1987)spice

Kernels Kernels (17.23) Antimicrobial: volatile oil (Tatsadjieu et al., 2003)

Anti hypertensive activity; volatile oil (Kadouet al., 2007)

Xylopiaaethiopica (Dunal)A. Rich.16419/SRF-Cam

Wounds and skin infections, fever,tapeworm, stomach ache,dysentery, stomach ulcer (Irvine,1961; Thomas, 1965), spice.

Bark, leaves, roots,seeds

Seeds (15.89) Antimicrobial: volatile oil (Tatsadjieu et al., 2003)

Antioxidant: volatile oil (N’dri et al., 2009)Apiaceae

Apium graveolensL. (25582/HNC)

Bronchitis, asthma, liver andspleen diseases (Satyavati andRaina, 1976), spice

Whole plant, seeds Whole plant(19.84)

Mosquitocidal: sedanolide on mosquito larvae,Aedes aegyptii;Nematicidal: sedanolide, senkyunolide-N, andsenkyunolide-J on Panagrellus redivivus andsedanolide on Caenorhabditis elegans;Antifungal: sedanolide on Candida albicans andCandida parapsilasis (Momin and Nair, 2001)

Petroselinumcrispum (Mil.)Nym.ex A.W.Hill.(25583/HNC)

Laxative, duiretic, antiurolithiatic,antti-inflammatory, liver disease(Anonymous, 1983; Leung, 1980;Ashan et al., 1990; Al-Howirinyet al., 2003), spice

Whole plant Whole plant(13.38)

Anticancer: myristicin, apigenin (Zheng et al.,1992; Birt et al., 1997; Justesen et al., 1998);antioxidant, anti-inflammatory, antimutagenic:apigenin (Kuo et al., 1992; Lee et al., 1993; vanAcker et al., 1996).Antihypertensive (in the dog): apiin and luteolin(Occhiuto and Limardi, 1994).Antimicrobial: psoralen, bergapten (Lombaert etal., 2001; Kuete et al., 2007a)

ApocynaceaePicralima nitida

Stapf Th. and H.Dur. (1942/SRFK)

Malaria, male sexual impotence,dysmenorrhoea, gastrointestinaldisorder (Adjanohoun et al., 1996)

Bark, fruits Fruits (6.28)

CaesalpiniaceaeScorodophloeus

zenkeri Harms(44803/HNC)

Headache, cough, rheumatism;constipation (Ngono, 1999), spice.

Bark, seeds, woods Bark (8.71) Antimicrobial: 2,4,5,7-Tetrathiaoctane;2,4,5,6,8-pentathianonane;2,3,4,6,8-pentathianonane;2,3,5,6,8,10-hexathiaundecane;2,3,5-trithiahexane 5-oxide;2,4,5,7-tetrathiaoctane 2-oxide;2,3,5,7-tetrathiaoctane 3,3-dioxide;2,3,5-trithiahexane 3,3-dioxide (Kouokam et al.,2002)

CapparaceaePentadiplandra

brazzeana Baill.(42918/HNC)

Lumbago, hemorrhoids,stomachaches (El-Migirab et al.,1977), spice

Roots Roots (11.29)

Buchholziacoriacea Engl.(32124/SRF-Cam)

Earache, chest pains, kidney pains,washing small pox wounds,anthelmintic (Ajaiyeoba et al.,2001)

Bark, fruits Fruits (14.83) Antimicrobial and cytotoxic: lupeol andbeta-sitosterol (Ajaiyeoba et al., 2003)

CompositaeEchinops

giganteus var. lelyi(C. D. Adams) A.Rich.(23647/SRF-Cam)

Heart and gastric troubles (Tene etal., 2004), spice

Rhizomes Rhizomes (16.74) Antimicrobial: lupeol sitosteryl;ß-D-glucopyranoside (Kojima et al., 1990; Taneet al., 1995; Kuete et al., 2007c, 2008)

EbenaceaeDiospyros

canaliculata DeWildeman(9653/SRF/cam)

Gonorrhea and other bacterial andfungal infections, tuberculosis(Tangmouo et al., 2005; Kueteet al., 2009)

Bark Bark (22.07) Antimicrobial: diospyrone; plumbagin(Tangmouo et al., 2005; Kuete et al., 2009)

Diospyroscrassiflora Hiern(4924/SRFK)

Gonorrhea and other bacterial andfungal infections, tuberculosis(Tangmouo et al., 2005; Dzoyemet al., 2007; Kuete et al., 2009)

Bark Bark (18.66) Antimicrobial: crassiflorone; diospyrone;plumbagin (Tangmouo et al., 2005; Dzoyemet al., 2007; Kuete et al., 2009)

GnetaceaeGnetum

africanum Welw.(21165/HNC)

Treatment of enlarged spleen, sorethroats, deduction of pains ofchild-birth, antidotes to poison andsnake bite, septic wounds,antidiabetic (Smith, 1983; Ekop,2007)

Leaves, seeds Leaves (14.98)

806 V. Kuete et al. / Journal of Ethnopharmacology 134 (2011) 803–812

Table 1 (Continued)

Family/plantspecies (Voucherspecimen)a

Traditional use Part usedtraditionally

Part used in thisstudy (extractionyield in %)b

Active compounds and previously screenedactivityc

Gramineae (Poaceae)lmperata

cylindrica Beauv.var. koenigiiDurand et Schinz(30139/SRFK)

Diuretic and anti-inflammatoryagents (Nishimoto et al., 1968),scpice

Roots Roots (7.89) Cytoxic activity using brine shrimp assay:jaceidin; quercetagetin-3,5,6,3′-tetramethylether; �-Sitosterol-3-0-�-D-glucopyranosy1-6′ ′-tetradecanoate (Mohamed et al.,2009)Aggregation inhibitory activity: imperanene(Matsunaga et al., 1995)

GuttiferaeGarcinia lucida

Vesque(17974/SRF-Cam)

Gastric infections, antipoison(Nyemba et al., 1990)

Bark, fruits Fruits (23.89)

Garcinia kolaHeckel(27839/SRF-Cam)

Nervous alertness and induction ofinsomnia, purgative, woundhealing (Uko et al., 2001)

Roots, seeds, latex Seeds (22.19) Antimicrobial; kolanone kolaflavanone andgarciniaflavanone (Iwu, 1993; Hussain et al.,1982)

Psorospermumadamauense(9826/SRFCam)

Febrifugal, anti-poison, purgative,leprosy, skin diseases such asdermatitis, scabies and eczemas,subcutaneous wounds (Irvine,1961; Watt and Brayer-Brandwijk,1962; Uphof, 1968)

Bark, leaves, roots Bark (17.54) Antimicrobial: febrifuquinone, adamabianthrone(Tsaffack et al., 2009)

LabiataeThymus vulgaris

Linn. (25746/HNC)Antitussive action, infectiousdiseases, expectorant,antibroncholitic, antispasmodic,anthelmintic, carminative diureticproperties (Van Den Broucke et al.,1982; Sokovic et al., 2008;Imelouane et al., 2009)

Whole plant Whole plant(15.17)

Spasmolytic properties: thymol, carvacrol,cirsilineol, thymonin, 8-methoxycirsilineol,luteolin, apigenin (Van Den Broucke et al., 1982;Van Den Broucke and Lemli, 1982);Antimicobial: volatile oil; thymol (Sokovic et al.,2008; Imelouane et al., 2009); Antioxidant:5,4′-dihydroxy-6,7′ ,8,3′-tetramethoxyflavone;5,4′-dihydroxy-6,7,3′-trimethoxyflavone;5,4′-dihydroxy-7-methoxyflavone (Muira andNakatani, 1989)

LauraceaeCinnamomum

zeylanicum (Linn)Cor.(22309/SRFCam)

Stimulant, antiflatulent,antiemetic; antidiarrhoeal (Bakhru,1998), spice.

Bark, leaves Leaves (11.34) Antimicrobial: volatile oil (Ranasinghe et al.,2002); trans-cinnamaldehyde (Shahverdi et al.,2007)

Antioxidant: protocatechuic acid; cinnamtanninB-1; urolignoside; rutin;quercetin-3-O-˛-l-rhamnopyranoside(Jayaprakasha et al., 2006)

MoraceaeDorstenia elliptica

Bureau(44018/HNC)

Anti-snakebite, anti-infection andanti-rheumatic (Bouquet, 1969;Abegaz et al., 2000)

Twigs Twigs (5.55) Antimicrobial: psoralen; dorstenin; bergapten;O-[3-(2,2-dimethyl-3-oxo-2H-furan-5-yl)-3-hydroxybutyl]bergaptol;3-(3,3-dimethylallyl)-4,2′ ,4′-trihydroxychalcone(Kuete et al., 2007b; 2008)

Dorstenia maniivar manii Hook. f.(2135/HNC)

Rheumatism and stomachdisorders (Bouquet, 1969)

Leaves Leaves (9.71)

Dorstenia psilirusWelwitch(44839/HNC)

Arthralgia, cardiovasculardisorders, rheumatism, snakebites,headache, stomach disorders,diuretic, tonic, stimulant, analgesic(Ruppelt et al., 1991; Adjanohounet al., 1996; Ngadjui et al., 1998;Dimo et al., 2001), spice

Leaves, roots Roots (13.28) Antimicrobial; psoralen; 2-sitosterol glucosideanalgesic (Ngadjui et al., 1998; Kuete et al.,2007a)

MimosaceaeTetrapleura

tetraptera (Schum.and Thonn) Taub.(12117/SRFCam)

Tonic, purgative, emetic,convulsions, leprosy,anti-inflammatroy, rheumaticpains (Dalziel, 1948; Ojewole andAdesina, 1983; Oliver-Bever, 1983),spice

Bark, fruits Fruits (28.19) Antiparasitic: aridanin on Schistosoma mansoniand S. bovis (Adewunmi and Furu, 1989)

Anticonvulsant: volatile oil (Nwaiwu and Akah,1986)

Dichrostachysglomerata (Forssk.)Chiov.(15220/SRF-Cam)

Coughs in children, gynecologicaltroubles (Koudou et al., 1994),spice

Bark, fruits Fruits (8.06)

V. Kuete et al. / Journal of Ethnopharmacology 134 (2011) 803–812 807

Table 1 (Continued)

Family/plant species(Voucher specimen)a

Traditional use Part usedtraditionally

Part used in thisstudy (extractionyield in %)b

Active compounds and previously screenedactivityc

MyristicaceaePycnanthus

angolensis (Welw.)Ward(19536/SRFCam)

Stomach pain, chest pain, rhinitisproblems, malaria, toothache, fungalskin infections, worms, leprosy (Keayet al., 1964; Lok et al., 1983;Omobuwajo et al., 1992;Akendengué and Louis, 1994; Fortet al., 2000; Wabo et al., 2007;Abrantes et al., 2008)

Bark leaves, root,wood

Leaves (19.76) Antihelmintic and anticancer: dihydroguaiareticacid (Abrantes et al., 2008), Antihyperglycemic:pycnanthuquinones A and B (Luo et al., 1999; Fortet al., 2000; Gbolade and Adeyemi, 2008),Antimicrobial: pycnanthulignene Apycnanthulignene C 3,4-dimethoxy-3′ ,4′-methylenedioxy-7,7′-epoxylignan; genkwainin;pycnanthulignene C; 4,5-dimethoxy-3′ ,4′-methylenedioxy-2,7′-cycloligna-7,7′-diene;pycnanthulignene A; calycosin; biochanin A;prunetin (Nono et al., 2010; Kuete et al., 2010)

OlacaceaeOlax subscorpioidea

var. subscorpioideaOliv. (3528/SRFK)

Constipation, yellow fever, jaundice,venereal diseases, Guinea worm(Okoli et al., 2007)

Roots, seeds Seeds (21.72) Antibacterial and cytotoxic against Artemia salina:santalbic acid (Jones et al., 1995; Cantrell et al.,2003)

PeriplocaceaeMondia withei

(Hook.f.) Skeels(42920/HNC)

Aphrodisiacs, urinary tract infection,jaundice and headaches diarrhea(Adjanohoun et al., 1996; Noumiet al., 1998), spice

Whole plant, roots Roots (14.11)

PiperaceaePiper capense L.f.

(6018/SRFCam)Sleep inducing remedy, anthelmintic(Kokowaro, 1976; Van Wyk andGericke, 2000), spice

Roots, seeds Seeds (21.72) Antiplasmodial: kaousine; Z-antiepilepsirine(Kaou et al., 2010)

Piper guineense(Schum and Thonn)(6018/SRFC)

Respiratory infections, femaleinfertility, aphrodisiac (Noumi et al.,1998), spice

Leaves, fruits, seeds Seeds (19.27) Insecticidal:N-isobutyl-ll-(3,4-methylenedioxyphenyl)-2E,4E,10E-undecatrienamide;N-pyrrolidyl-12-(3,4-methylene-dioxyphenyl)-2E,4E,9E,11Z-dodecatetraenamide;N-isobutyl-13-(3,4-methylenedioxyphenyl)-2E,4E,12E-tridecatrienamide;N-isobutyl-2E,4E-decadienamide;N-isobutyl-2E,4E-dodecadienamide (Gbewonyoand Candy, 1992)Antimicrobial: volatile oil (Oyedeji et al., 2005)

SapotaceaeVitellaria paradoxa

C. F. Gaertn(2957/HNC)

Gastrointestinal infections, diarrhea,dysentery, worms, skins diseases,leprosy (Soladoye et al., 1989; Ferryet al., 1974)

Bark, leaves, fruits Bark (17.81)

RutaceaeFagara leprieuri

(Guill and Perr) Engl(37632/HNC)

Abdominal pain, asthma,appendicitis, toothache (Diniz et al.,2007), spice

Bark, seeds Seeds (21.06) Cytotoxic:3-hydroxy-1-methoxy-10-methyl-9-acridone;1-hydroxy-3-methoxy-10-methyl-9-acridone (4),1-hydroxy-2,3-dimethoxy-10-methyl-9-acridone(5),1,3-dihydroxy-2-methoxy-10-methyl-9-acridonewere found to be moderately active against lungcarcinoma cells (A549), colorectal adenocarcinomacells (DLD-1) and normal cells (WS1) (Ngoumfoet al., 2010)

Fagara macrophyllaEngl.(2716/SRFKcam)

Colds and stomach-ache, fever.Malaria (Adjanohoun et al., 1996)

Bark, seeds Seeds (18.29) Antitumour: nitidine chloride and6-methoxy-5,6-dihydronitidine (Wall et al., 1987);Molluscicidal and insecticidal: N-isobutylamides(Kubo et al., 1984)

ZingiberaceaeAframomum

citratum (Pereira) K.Schum. (37736/NHC)

Fever, intercostals pains, tonic,aphrodisiac (Tane et al., 2005), spice.

Leaves, fruits Fruits (6.17)

Aframomummelegueta (Roscoe) K.Schum. (39 065/HNC)

Constipation, fever, carminative(Dalziel, 1936)

Seeds Seeds (16.91) Antimicrobal: volatile oil (Oloke and Kolawole,1988)

Zinziber officinaleRoscoe(453125/HNC)

Infectious diseases, respiratory tractinfections, anticancer, indidigestion,diarrhea, nausea (Akoachere et al.,2002; Kato et al., 2006;Sakpakdeejaroen and Itharat, 2009)

Rhizomes Rhizomes (14.58) Enzymatic activity:2-(4-hydroxy-3-methoxyphenyl)ethanol and2-(4-hydroxy-3-methoxyphenyl)ethanoic acidinhibited the recombinant human aldose reductase(kato et al., 2006); cytotoxicity: 6-shogaol againsthuman A549, SK-OV-3, SK-MEL-2, and HCT15tumor cells (Kim et al., 2008).

a Plants were identified at the Cameroon National Herbarium (HNC).b The percentage of the methanol extract.c ( ): Data not available.

8 opharmacology 134 (2011) 803–812

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08 V. Kuete et al. / Journal of Ethn

. Results and discussion

In the US NCI plant screening program, a crude extract is gener-lly considered to have in vitro cytotoxic activity, if the IC50 valueollowing incubation between 48 and 72 h, is less than 20 �g/mlBoik, 2001). In the present work, thirty-four selected plant extractsere tested (at 20 �g/ml) for their cytotoxic effects, and the results

re summarized in Figs. 1–3 and Table 2. The results of Figs. 1–3ndicate that extracts from six of the 34 (17.6%) plants were ableo inhibit more that 50% the proliferation of the three tested can-er cells (MiaPaCa-2, CEM/ADR5000 and CCRF-CEM). Such extractsncluded Xylopia aethiopica, Echinops giganteus, Imperata cylindrica,orstenia psilirus and Piper capense, Zinziber officinale. However, 6

17.6%) extracts showed more than 50% inhibition of MiaPaCa-2ells (Fig. 1) and 10 (29.4%) on both CEM/ADR5000 (Fig. 2) or CCRF-EM (Fig. 3) cells. Taking in account the NCI criterion of anticancerctivities, extracts from X. aethiopica, E. giganteus, I. cylindrica, D.silirus, P. capense, Z. officinale, Olax subscorpioidea, Piper guineense,agara leprieuri and Aframomum melegueta could be considereds potential source of cytotoxic drug, as they showed IC50 valueselow 20 �g/ml on at least two cell lines (Table 2). The lowest IC50alues of 6.86 �g/ml on MiaPaCa-2 and 3.91 �g/ml on CCRF-CEMells were obtained with X. aethiopica, while the correspondingalue of 6.56 �g/ml was obtained with P. capense on CEM/ADR 5000ells (Table 2).

The development of resistance of cancer cells to cytotoxicgents appeared as one of the main cause of treatment break-own, leading to the recurrence of disease and death. Therefore,he susceptibility of MDR phenotypes to plant extracts is ofreat therapeutic interest. This explained the used of the humanesistant cell line CEM/ADR5000 which exerts the MDR pheno-ype of CCRF-CEM. When regarding the degree of resistance ofhe MDR phenotype (CEM/ADR5000), it was appeared that noross-resistance was observed with I. cylindrica, P. capense andinziber officinalis (Table 2), the values obtained being below 1.owever, higher degrees of resistance were also noted with dox-rubicin (>41.7) compared to the active extracts, suggesting thathey can also be of therapeutic importance in the treatmentf MDR phenotypes. This highlights the medicinal potencies ofome of the extract used traditionally in the treatment of can-ers. Besides, the most active extracts derived from commonlymployed spices, explaining their low toxicity on the HUVECells. However, all tested samples showed positive selectivityndexes (Table 2), though EC50 values of 21.0, 47.73 and 70.0 �g/ml

ere obtained, respectively, with A. melegueta, I. cylindrica and. guineense.

Anti-angiogenic compounds are gaining more and more interests a new approach in the prevention and treatment of cancer andnflammatory diseases (Krenn and Paper, 2009). The CAM assays a sensitive, easily feasible and cheap in vivo test for investiga-ions of the anti-angiogenic potential of individual compounds andlant extracts (Paper et al., 1995). The assay does not only provide

nformation on the efficacy of test samples in vivo but also on theiroxicity in vivo. Thus, for extracts used in food supplements theest system provides more information than in vitro assays suchs the inhibition of vascular endothelial growth factor (VEGF), ofEGF-catalyzed tube formation or of VEGF- or EGF-catalyzed cellroliferation as well as others (Krenn and Paper, 2009). Herein, thenti-angiogenic effects of X. aethiopica, D. psilirus, E. giganteus, Z.fficinale were investigated using fertilized quail eggs. The resultsre summarized in Fig. 4 and Table 2. These results indicated that

xtract from the tested plants were able to reduce at differentxtend the proliferation of blood vessels in quails embryo. How-ver, more than 50% inhibition was noted with D. psilirus and E.iganteus predicting that inhibition of angiogenesis could be one ofheir cytotoxic mechanism actions. Ta

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V. Kuete et al. / Journal of Ethnopharmacology 134 (2011) 803–812 809

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Fig. 1. Inhibitory percentage (%) of plant extracts at 20 �g/ml and doxorubicin at 10 �M on MiaPaCa-2 cell lines. No inhibition of the proliferation was noted with theextracts from Picralima nitida, Diospyros crassiflora, Thymus vulgaris, Dorstenia elliptica, Dorstenia manii and Pynanthus angolensis. Data with different superscript letters aresignificantly different (P < 0.05).

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ngolensis and Vitellaria paradoxa. Data with different superscript letters are signific

Few studies were focused on the anticancer activities of the mostctive plants reported herein. However, volatile oil from the fruitsf X. aethiopica was found to be cytotoxic to HepG-2 carcinoma cellsAsekun and Adeniyi, 2004). Compounds such as 6-shogaol isolated

ig. 3. Inhibitory percentage (%) of plant extracts at 20 �g/ml and doxorubicin at 10 �M oxtracts from Diospyros crassiflora and Vitellaria paradoxa. Data with different superscript

the three studied CEM/ADR5000 cancer cell lines. No inhibition of the proliferationlata, Diospyros crassiflora, Garcinia kola, Thymus vulgaris, Dorstenia manii, Pynanthusdifferent (P < 0.05).

from the rhizome of Zinziber officinale exhibited good cytotoxicityagainst human A549, SK-OV-3, SK-MEL-2, and HCT15 tumor cells(Kim et al., 2008) and the results reported in the present work arein consistence with these studies.

n CCRF/CEM cancer cell lines. No inhibition of the proliferation was noted with theletters are significantly different (P < 0.05).

810 V. Kuete et al. / Journal of Ethnopharmacology 134 (2011) 803–812

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The overall results of the present study provide supportive datan the use of some Cameroonian plants in the treatment of cancers.

cknowledgements

Authors are thankful to the National Herbarium of CameroonYaounde). V.K. is also very grateful to the German Academicxchange Service (DAAD) for the travel grant at the Johannes-utenberg University, Mainz, Germany.

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