anti-infective and antiproliferative potential of african

Anti-Infective and Antiproliferative Potential of African Medicinal PlantsGuest Editors: Victor Kuete, Namrita Lall, and Thomas Efferth

Evidence-Based Complementary and Alternative Medicine

Anti-Infective and Antiproliferative Potentialof African Medicinal Plants

Evidence-Based Complementary and Alternative Medicine

Anti-Infective and Antiproliferative Potentialof African Medicinal Plants

Guest Editors: Victor Kuete, Namrita Lall,and Thomas Efferth

Copyright © 2012 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Evidence-Based Complementary and Alternative Medicine.” All articles are open access articlesdistributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited.

Editorial Board

Shrikant Anant, USAVassya Bankova, BulgariaWinfried Banzer, GermanyVernon Barnes, USADebra L. Barton, USAJairo Kenupp Bastos, BrazilDavid Baxter, New ZealandAlvin J. Beitz, USAPaolo Bellavite, ItalyFrancesca Borrelli, ItalyArndt Bussing, GermanyLeigh F. Callahan, USARaffaele Capasso, ItalyIl-Moo Chang, Republic of KoreaYunfei Chen, ChinaKevin W. Chen, USAJuei-Tang Cheng, TaiwanJen-Hwey Chiu, TaiwanJae Youl Cho, Republic of KoreaWilliam C. Cho, Hong KongShuang-En Chuang, TaiwanEdwin L. Cooper, USAVincenzo De Feo, ItalyAlexandra Deters, GermanyNobuaki Egashira, JapanPeter Fisher, UKJoel J. Gagnier, CanadaMichael Goldstein, USAS.-H. Hong, Republic of KoreaMarkus Horneber, GermanyChing Liang Hsieh, TaiwanBenny Tan Kwong Huat, SingaporeRoman Huber, GermanyAngelo Antonio Izzo, ItalyStefanie Joos, GermanyZ. Kain, USAJong Yeol Kim, Republic of KoreaCheorl-Ho Kim, Republic of Korea

Youn Chul Kim, Republic of KoreaYoshiyuki Kimura, JapanToshiaki Kogure, JapanAlfred Langler, GermanyLixing Lao, USAJang-Hern Lee, Republic of KoreaMyeong Soo Lee, Republic of KoreaTat leang Lee, SingaporeChristian Lehmann, CanadaPing-Chung Leung, Hong KongXiu-Min Li, USAChun Guang Li, AustraliaSabina Lim, Republic of KoreaGerhard Litscher, AustriaI.-Min Liu, TaiwanKe Liu, ChinaIrene Lund, SwedenGail Mahady, USAFrancesco Marotta, ItalyVirginia S. Martino, ArgentinaJames H. McAuley, AustraliaAndreas Michalsen, GermanyDavid Mischoulon, USAMark A. Moyad, USAStephen Myers, AustraliaVitaly Napadow, USAIsabella Neri, ItalyMartin Offenbacher, GermanyKi-Wan Oh, Republic of KoreaY. Ohta, JapanOlumayokun A. Olajide, UKThomas Ostermann, GermanyBhushan Patwardhan, IndiaBerit Smestad Paulsen, NorwayRichard Pietras, USAKhalid Rahman, UKCheppail Ramachandran, USA

Cesar R. Ramos-Remus, MexicoKe Ren, USAJose Luis Rıos, SpainPaolo Roberti di Sarsina, ItalyBashar Saad, Palestinian AuthorityAndreas Sandner-Kiesling, AustriaAdair Roberto Soares Santos, BrazilG. Schmeda-Hirschmann, ChileAndrew Scholey, AustraliaDana Seidlova-Wuttke, GermanySenthamil R. Selvan, USARonald Sherman, USAKan Shimpo, JapanVenil N. Sumantran, IndiaTakashi Takahashi, JapanToku Takahashi, USAJoanna Thompson-Coon, UKMei Tian, USAK. V. Trinh, CanadaAlfredo Vannacci, ItalySøren Ventegodt, DenmarkCarlo Ventura, ItalyWagner Vilegas, BrazilPradeep Visen, CanadaAristo Vojdani, USADietlind Wahner-Roedler, USAShu-Ming Wang, USAKenji Watanabe, JapanWolfgang Weidenhammer, GermanyJenny M. Wilkinson, AustraliaHaruki Yamada, JapanNobuo Yamaguchi, JapanHitoshi Yamashita, JapanKen Yasukawa, JapanE. Yesilada, TurkeyBoli Zhang, ChinaHong Zhang, ChinaRuixin Zhang, USA

Contents

Anti-Infective and Antiproliferative Potential of African Medicinal Plants, Victor Kuete, Namrita Lall,and Thomas EfferthVolume 2012, Article ID 535219, 2 pages

Antimicrobial Constituents of Artemisia afra Jacq. ex Willd. against Periodontal Pathogens,Garland More, Namrita Lall, Ahmed Hussein, and Thilivhali Emmanuel TshikalangeVolume 2012, Article ID 252758, 7 pages

Antimicrobial and Anti-Inflammatory Activities of Pterygota macrocarpa and Cola gigantea(Sterculiaceae), Christian Agyare, George Asumeng Koffuor, Vivian Etsiapa Boamah, Francis Adu,Kwesi Boadu Mensah, and Louis Adu-AmoahVolume 2012, Article ID 902394, 9 pages

Antimicrobial Effects of a Lipophilic Fraction and Kaurenoic Acid Isolated from the Root Bark Extractsof Annona senegalensis, Theophine Chinwuba Okoye, Peter Achunike Akah, Charles Ogbonnaya Okoli,Adaobi Chioma Ezike, Edwin Ogechukwu Omeje, and Uchenna Estella OdohVolume 2012, Article ID 831327, 10 pages

Evaluation of the Acetone and Aqueous Extracts of Mature Stem Bark of Sclerocarya birrea forAntioxidant and Antimicrobial Properties, Nicoline F. Tanih and Roland N. NdipVolume 2012, Article ID 834156, 7 pages

Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa) Cell Line,Danielle Berrington and Namrita LallVolume 2012, Article ID 564927, 11 pages

Synergistic Antimycobacterial Actions of Knowltonia vesicatoria (L.f) Sims, Antoinette Labuschagne,Ahmed A. Hussein, Benjamın Rodrıguez, and Namrita LallVolume 2012, Article ID 808979, 9 pages

Melanogenesis and Antityrosinase Activity of Selected South African Plants, Manyatja Brenda Mapunya,Roumiana Vassileva Nikolova, and Namrita LallVolume 2012, Article ID 374017, 6 pages

Antibacterial Activities of Selected Cameroonian Plants and Their Synergistic Effects with Antibioticsagainst Bacteria Expressing MDR Phenotypes, Stephen T. Lacmata, Victor Kuete, Jean P. Dzoyem,Simplice B. Tankeo, Gerald Ngo Teke, Jules R. Kuiate, and Jean-Marie PagesVolume 2012, Article ID 623723, 11 pages

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 535219, 2 pagesdoi:10.1155/2012/535219

Editorial

Anti-Infective and Antiproliferative Potential of AfricanMedicinal Plants

Victor Kuete,1 Namrita Lall,2 and Thomas Efferth3

1 Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon2 Department of Plant Science, Faculty of Agricultural and Biological Science, Pretoria 0002, South Africa3 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger weg 5, 55128 Mainz,Germany

Correspondence should be addressed to Victor Kuete, [email protected]

Received 20 May 2012; Accepted 20 May 2012

Copyright © 2012 Victor Kuete et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The importance of traditional medicine as a source ofprimary health care was first officially recognized by theWorld Health Organization (WHO) in the primary HealthCare Declaration of Alma Ata (1978) and has been globallyaddressed since 1976 by the Traditional Medicine Pro-gramme of the WHO. In Africa, traditional healers andremedies made from plants play an important role in thehealth of millions of people. Scientific evidence of their phar-macological potential is being provided continuously. Forthis special issue, we have invited investigators to contributeoriginal research articles as well as review articles that willprovide evidences on the basis of traditional knowledge ofthe use of African medicinal plants used in the treatment ofailments.

Six papers of this special issue focused on antimicrobialand two on antiproliferative properties of African medicinalplants. One paper of the issue reports the antibacterialproperties of the methanol extracts of some Cameroonianmedicinal plants and the effect of their associations withcurrently used antibiotics on multidrug-resistant (MDR)Gram-negative bacteria overexpressing active efflux pumps.Three extracts Garcinialucida, Garcinia kola, and Picral-ima nitida showed significant activities against such bac-teria and are suggested as possible alternative in chem-otherapy involving MDR bacterial species. Another paperhighlights the importance of Annona senegalensis andit active constituent, kaurenoic acid as a potential antibac-terial drugs against Bacillus subtilis, Pseudomonas aeruginosa,and Staphylococcus aureus. Another paper provides evidenceon the inhibitory potential of the acetone and aqueous

extracts of mature stem bark of Sclerocarya birrea on apanel of bacteria and fungi such Streptococcus pyogenes,Plesiomonas shigelloides, Aeromonas hydrophila, Salmonellatyphimurium, Cryptococcus neoformans, Candida glabrata,Trichosporon mucoides, and Candida krusei. There is alsoa paper which demonstrates that extracts from SouthAfrican medicinal plants Euclea natalensis A.DC., Knowltoniavesicatoria (L.f) Sims and Pelargonium sidoides DC cansuccessfully be combined with isoniazid (INH) to improveit antimycobacterial activity. This paper also demonstratesthat stigmasta-5,23-dien-3-ol isolated from K. vesicatoria isa bioactive compound againstM. tuberculosis. Another paperreports the antimicrobial activity of the ethanol extractof Artemisia afra and identified some of its componentsacacetin, scopoletin and betulinic acid as bioactive com-pounds against Gram-positive bacteria (Actinomyces naes-lundii, Actinomyces israelii and Streptococcus mutans), Gram-negative bacteria (Prevotella intermedia, Porphyromonas gin-givalis, and Aggregatibacter actinomycetemcomitans) and ayeast, Candida albicans. One of these six papers providesinformation on the possible use of Pterygota macrocarpa andCola gigantea in the fight of microbial infections involving E.coli, P. aeruginosa, S. aureus, B. subtilis, and C. albicans.

Two papers of this special issue were related to cancer.Paper one provides information on the ethanol leaf extractsof Harpephyllum caffrum as an antityrosinase agent for der-matological disorders such as age spots and melasoma. Theother paper reports the anticancer activities of African spices,Origanum vulgare, Rosmarinus officinalis, Lavandula spica,Laurus nobilis, Thymus vulgaris, Lavandula-x-intermedia,

2 Evidence-Based Complementary and Alternative Medicine

Petroselinum crispum, Foeniculum vulgare, and Capsicumannuum L. (Paprika). It identifies L. nobilis and R. officinalisas strong antiproliferative agents against the human cervicalcancer cell line, HeLa.

Victor KueteNamrita Lall

Thomas Efferth


Research Article

Antimicrobial Constituents of Artemisia afra Jacq. ex Willd.against Periodontal Pathogens

Garland More,1 Namrita Lall,1 Ahmed Hussein,1, 2 and Thilivhali Emmanuel Tshikalange1

1 Department of Plant Science, University of Pretoria, Pretoria 0002, South Africa2 Chemitsry Deparment, Universirty of Western Cape, Private Bag X17, Bellvile 7535, South Africa

Correspondence should be addressed to Namrita Lall, [email protected] Thilivhali Emmanuel Tshikalange, [email protected]

Received 23 January 2012; Accepted 6 March 2012

Academic Editor: Victor Kuete

Copyright © 2012 Garland More et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The phytochemical investigation of an ethanol extract of Artemisia afra led to the isolation of six known compounds, acacetin(1), 12α,4α-dihydroxybishopsolicepolide (2), scopoletin (3), α-amyrin (4), phytol (5), and a pentacyclic triterpenoid betulinicacid (6). The compounds were evaluated for antimicrobial activity against Gram positive (Actinomyces naeslundii, Actinomycesisraelii, and Streptococcus mutans), Gram negative bacteria (Prevotella intermedia, Porphyromonas gingivalis and Aggregatibacteractinomycetemcomitans previously known as Actinobacillus actinomycetemcomitans), and Candida albicans. The crude extract of A.afra inhibited the growth of all tested microbial species at concentration range of 1.6 mg/mL to 25 mg/mL. The compounds 1–6also showed activity range at 1.0 mg/mL to 0.25 mg/mL. Three best compounds (scopoletin, betulinic acid, and acacetin) whichshowed good antimicrobial activity were selected for further studies. Cytotoxicity of extract and compounds was determinedusing the XTT cell proliferation kit. The antioxidant activity of the extract and compounds was done using the DPPH scavengingmethod. The extract showed good antioxidant activity with an IC50 value of 22.2 μg/mL. Scopoletin had a strong transformation ofthe DPPH radical into its reduced form, with an IC50 value of 1.24 μg/mL which was significant to that of vitamin C (1.22 μg/mL).Acacetin and betulinic acid exhibited a decreased scavenging activity with the IC50 of 2.39 and 2.42 μg/mL, respectively. The extractand compounds showed moderate toxicity on McCoy fibroblast cell line and scopoletin was relatively nontoxic with an IC50

value of 132.5 μg/mL. Acacetin and betulinic acid also showed a smooth trend of non-toxic effects with IC50 values of 35.44 and30.96 μg/mL. The obtained results in this study confirm the use of A. afra in the treatment of microbial infections.

1. Introduction

Periodontal disease is a chronic, multifactorial disease of thetissue supporting teeth [1]. It is characterised by local infec-tion and inflammation in teeth supporting tissue leadingto connective tissue destruction and alveolar bone loss [2].The etiology of periodontitis is the oral bacteria [3, 4]. Ifleft untreated, periodontitis can have medical consequencessuch as weight loss, chronic pain, sore or loss teeth, swollengums, tooth decay, breakage of the maxillary or mandibularbones, and renal, coronary, and hepatic diseases [5–7].The population of periodontal bacteria begins to increaseas an anaerobic environment is produced due to oxygenscavenging activity of the early subgingival colonizers [4].These bacterial populations lead to biofilm formation whichconsists of microcolonies, extracellular layers, fluid channels,

and communication systems [8]. Biofilms may consist ofmore than 700 different microbial species which leave symbi-otically and incubate each other [9]. The biofilm formationand associated disease can be prevented by daily toothbrushing and chemotherapeutic agents such as chlorhexidine(CHX), fluorides biguanide antiseptics, quaternary ammo-nium-antiseptics and phenol derivatives [10, 11]. Althoughthese chemotherapeutic agents are effective they can causeside effects, such as gastrointestinal irritation, tooth staining,and gum irritation [11].

Dental treatment usually is expensive and not so eas-ily accessible, especially in developing countries; thereforehumans have turned to the use of natural traditional reme-dies to prevent oral ailments [12–15]. Artemisia afra (Africanwormwood, family Asteraceae) is widely distributed alongthe eastern parts of Africa. It grows in thick, bushy areas,


usually with tall stems up to 2 m high but sometimes as lowas 0.6 m. A. afra is a common species in South Africa witha wide distribution from the Cederberg Mountains in theCape, northwards to tropical East Africa and stretching as farnorth as Ethiopia [16, 17]. Worldwide there are about 500species of Artemisia, mainly from the northern hemisphere.Many of the other Artemisia species are aromatic perennialsand are used medicinally [18]. In southern Africa it isused to treat coughs, colds, diabetes, malaria, sore throat,asthma, headache, dental care, gout and intestinal worms[19]. In vitro studies done have revealed that A. afra is apotential antidepressant, cardiovascular, spasmolytic effects,antioxidant, and antimycobacteria [18, 20, 21]. Furthermore,the extracts of this plant species have shown activity againstTrypanosoma brucei brucei [22].

The rationale of this study was to determine the antimi-crobial, antioxidant, and cytotoxicity of Artemisia afra andisolated compounds against oral microorganisms which areresponsible for dental caries, gingivitis, and periodontitis.

2. Materials and Methods

2.1. Plant Material. Artemisia afra was collected at the SouthAfrican National Botanical Institute (SANBI), Pretoria.Voucher specimen was prepared and identified at the H. G.W. J. Schwelcherdt Herbarium (PRU), University of Pretoria.

2.2. Preparation of Extracts. Fresh plant material was soakedin 96% ethanol and homogenized into fine mesh. The extractwas then filtered through the Whatman No.1 filter paper. Thefiltrates were evaporated to dryness in a (BUCHI) Rotavaporunder reduced pressure of 40◦C.

2.3. Antimicrobial Assay

2.3.1. Microbial Strains. The microorganisms used in thisstudy include Actinomyces naeslundii (ATCC 19039), Actino-myces israelii (ATCC 10049), Aggregatibacter actinomycetem-comitans (ATCC 33384), Candida albicans (Med I), Por-phyromonas gingivalis (ATCC 33277), Prevotella intermedia(ATCC 25611), and Streptococcus mutans (ATCC 25175).Bacteria were grown in the Casein-peptone Soy Agar me-dium (CASO) (Merck SA (Pty) Ltd.) under anaerobic con-ditions in a jar with anaerocult A (Merck SA (Pty) Ltd.), at37◦C for 48 hours. Sabouraud Dextrose Agar medium (SDA)(Merck SA (Pty) Ltd.) was used for the culturing of Candidaalbicans and incubated at 37◦C for 24 hours under aerobicconditions. Subculturing was done once weekly.

2.4. Determination of Minimum Inhibitory Concentration(MIC) and Minimum Microbicidal Concentration (MMC).The microdilution technique using 96-well microplates [23]was used to obtain the MIC and MMC values of the crudeextract against microorganisms under study. The extractwas serially diluted in the 96-well plate with 48 hours oldmicroorganisms (5 × 106 CFU/mL) grown at 37◦C andthe final concentration of extract and positive control(CHX) ranged from 25.0 mg/mL to 0.8 mg/mL. Microbial

growth was indicated by adding 40 μL of (0.2 mg/mL) p-iodonitrotetrazolium violet (INT) (Sigma-Aldrich, SouthAfrica) to microplate wells and incubated at 37◦C for 48hours. MIC was defined as the lowest concentration thatinhibited the colour change of INT. The MMC was deter-mined by adding 50 μL of the suspensions from the wells,which did not show any growth after incubation duringMIC assays, to 150 μL of fresh broth. These suspensions werereincubated at 37◦C for 48 hours. The MMC was determinedas the lowest concentration of extract which inhibited 100%growth of microorganisms [24].

2.5. Antioxidant Activity. The free radical scavenging activitywas measured using 1, 1 diphenyl-2-picryl-hydraxyl (DPPH)assay [25] with slight modifications. The ethanol extractof A. afra and Vitamin C (positive control) 1000 μg/mL(20 μL) was added in the first three wells of a 96-well platecontaining 200 μL of distilled water to make up finalconcentration of 100 μg/mL and the remaining wells werefilled with 110 μL of distilled water. The first raws contain-ing the extract/compounds were serially diluted to wellswhich contain 110 μL of distilled water, and later, 90 μL ofmethanolic solution of DPPH (90 mM) was added to all thewells. The final concentrations of the extract/compoundsranged from 100 to 0.8 μg/mL. The plates were incubatedat 37◦C for 30 min and the absorbance was measured at517 nm using the ELISA plate reader. The percent radicalscavenging activity by A. afra was determined by comparisonwith ethanol (blank). The inhibition ratio was calculated asfollows: % DPPH radical-scavenging = (AC-AS)/AC × 100,where AC is absorbance of the control solution (containingonly DPPH solution), and AS is the absorbance of thesample in DPPH solution. The percentage of DPPH radical-scavenging was plotted against the plant extract/compoundsconcentrations (μg/mL) to determine the concentration ofextract/compound required to scavenge DPPH by 50%(EC50).

2.6. Determination of Cytotoxicity

2.6.1. Preparation of Extract and Compounds. Extract andcompounds were dissolved in dimethyl sulfoxide (DMSO)and stored at −20◦C. All tested compounds were diluted tothe final concentration with RPMI 1640 and control cultureswere diluted with 0.1% DMSO.

2.6.2. Cell Culture. McCoy cells were maintained in mono-layer culture at 37◦C and 5% CO2 with 10% PBS Medium,10 μg/mL of penicillin, 10 μg/mL, streptomycin, 40 μg/mLgentamycin, and 0.25 μg/mL fungizone.

2.6.3. Cell Proliferation Assay. A microtiter plate with McCoycells was used for testing all the ethanol extracts for cytotoxi-city following the method of [26]. Cytotoxicity was measuredby the XTT (sodium 3′-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acidhydrate) method using a cell proliferation kit II (RocheDiagnostics GmbH). Hundred microlitres of McCoy cells

Evidence-Based Complementary and Alternative Medicine 3

HO

HO

HO

HO

HOHO

HO

OH

OHO

O

O

O

OMe

OAc

MeO COOH

COOH

Phytol

12, 4-dihydroxbishopsolicepolide

1

2

3

4

5

6

Figure 1: Chemical structures of compounds isolated from the aerial part of Artemisia afra.

(1 × 105 mL) was seeded onto a microtiter plate and incu-bated for 24 h to allow the cells to attach to the bottom ofthe plate. Dilution series were made of the extract and com-pound and the various concentrations (400 to 3.1 μg/mL)were added to the microtitre plate and incubated for 48 h.The XTT reagents were added to a final concentration of0.3 mg/mL and the cells were incubated for 1-2 hours. Thepositive drug control (Zearalenone) at concentrations rangeof (10 μg/mL to 0.6 μg/mL) was included in the assay. Afterincubation the absorbance of the colour was spectropho-tometrically quantified using an ELISA plate reader, whichmeasured the optical density at 490 nm with a referencewavelength of 690 nm. The assay was carried out in triplicate.

2.6.4. Statistical Analysis. Statistical analysis was conveyed asmeans ± SD using GraphPad Prism 4.0 with a significantdifference of (P < 0.05).

3. Isolation and Identification of Compounds

The antibacterial compounds present in A. afra extract weredetermined by the direct bioautography method of chro-matograms using S. mutans [27]. The extract was spottedonto a TLC plate and developed using hexane: ethyl acetateat different ratios (1 : 1, 3 : 7, and 7 : 3). The plates werethoroughly dried and then the chromatograms were sprayed

with a dense culture of S. mutans, incubated overnightat 37◦C. The plates were further sprayed with 0.2 mg/mLof p-iodonitrotetrazolium (INT) (Sigma). Clear zones ofinhibition indicated compounds which inhibited bacterialgrowth. The isolation of compounds was performed usingcolumn chromatography. Fractionation was preceded byusing silica gel 60 (70–230 mesh) and Sephadex LH 20. Thinlayer chromatography (TLC) was performed on aluminumsheets coated with silica gel 60 F254 (Merck) and UV light wasused to detect compounds. TLC plates were further sprayedwith vanillin/sulphuric acid reagent. H1-NMR and 13C-NMRspectra were obtained by using Nuclear magnetic resonance(NMR) Germin 200 AT 199, 50 Hz, respectively.

The Isolation of A. afra was started with 200 g of ethanolextract on to a 100 mm diameter column. The column wasfilled with 2 kg silica gel, eluted with a mixture of hexane:ethyl acetate of increasing polarity (100 : 0 to 0 : 100). Fortyfractions were obtained and combined to make up 12 (I-XII)main fractions according to similarities of compounds asdetermined by TLC plate. A Sephadex column was conductedon the fraction X using 100% MeOH and it yielded 25subfractions which were combined in to three subfractions(L, M, N). Fraction N was fractionated using MeOH andyielded a pure compound 1. Fraction M was isolated usinga gradient of solvents DCM: Hex, 9 : 1 increasing polarity to3% to obtain a florescent blue compound 3.


Table 1: Mean MIC and MMC (mg/mL) results of Artemisia afra on oral microorganisms.

Plant extracts

Microorganisms tested

MIC (μg/mL) MBC (μg/mL)

Gram +ve Gram –ve Yeast Gram +ve Gram –ve Yeast

A.n A.i A.a P.i P.g C.a A.n A.i A.a P.i P.g C.a

A. afra 3.1 1.6 25.0 6.3 6.3 6.3 1.6 6.3 >1.0 1.25 25.0 >25.0

1 1.0 0.25 >1.0 1.0 1.0 >1.0 1.0 0.5 >1.0 >1.0 >1.0 >1.0

2 0.5 0.5 >1.0 1.0 >1.0 >1.0 1.0 0.5 >1.0 1.0 >1.0 >1.0

3 1.0 0.25 >1.0 0.5 >1.0 >1.0 >1.0 0.5 >1.0 1.0 >1.0 >1.0

4 1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0

5 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0 >1.0

6 0.25 1.0 >1.0 1.0 1.0 >1.0 0.5 1.0 >1.0 >1.0 >1.0 >1.0

Chlorhexidine 1.6 6.3 1.6 6.3 1.6 6.3 1.6 6.3 1.6 6.3 1.6 1.25

A.n: Actinomyces naeslundii; A.i: Actinomyces israelii; A.a: Actinobacillus actinomycetemcomitans; P.i: Prevotella intermedia; P.g: Porphyromonas gingivalis; C.a:Candida albicans.

0102030405060

100

708090

Vitamin C

50 25 12.5 6.25 3.125 1.5625 0.78125

Inh

ibit

ion

(%)

Concentration (µg/mL)

(a)

100 50 25 12.5 6.25 3.125 1.5625 0.78125

Inh

ibit

ion

(%)

Scopoletin

0

20

40

60


80100

(b)

Acacetin


Inh

ibit

ion

(%)

100 50 25 12.5 6.25 3.125 1.5625 0.781250

102030405060

(c)


Inh

ibit

ion

(%)

100 50 25 12.5 6.25 3.125 1.5625 0.78125

10

30

0

2025

5

15

Betulinic acid

(d)

Figure 2: The DPPH inhibitory activities of the isolated compounds and vitamin C.

Fraction VII was chromatographed using DCM: MeOH(95 : 5), and 20 fractions were obtained and combined to3 subfractions. Fraction G was fractionated using DCM:MeOH, increasing polarity on a Sephadex column andtwo fractions were obtained of which one was a purecompound 2, the second fraction was again fractionatedusing DCM: MeOH (95 : 5) and compound 6 was obtained.Compound 5 was isolated using Hex: EtoAc, at a ratio of9 : 1, using silica gel column. A succession of a blue-colouredcompound was observed on a TLC plate after application ofvanillin/sulphuric acid. Subfraction H from fraction VII waschromatographed using a sephadex column with ethanol asits mobile phase and it yielded a white precipitate of a purecompound 4.

4. Results and Discussion

The TLC profile gave a clear antibacterial activity of theextract and guide to isolate ideal compounds. In all solventsystems tested on TLC, both polar and nonpolar bendsdemonstrated activity by inhibiting the growth of S. mutans.Isolation from A. afra extract yielded six compounds(Figure 1), one known flavone Acacetin (1), sequiterpe-ne12α,4α-dihydroxybishopsolicepolide (2), a diterpene Phy-tol (5) [28, 29], and two pentacyclic triterpenes (4). A thor-ough revision of literature indicated that the data for penta-cyclic triterpene matched with those of α-Amyrin, a widelyspread triterpene in nature [30, 31]. The presence of the sig-nal at 3.0 of H-17 confirms the structure of Betulinic acid (6).


020406080

100120140160

3.063 6.125 25 50 100 200 400

Concentration

12.5

Con

trol

(%)

Artemisia afra

(a) (b)

Scopoletin

0

50

100

150

200

250

3.063 6.125 25 50 100

Concentration

12.5

Con

trol

(%)

(c)

Betulinic acid

0

50

100

150

200

250

3.063 6.125 25 50 100

Concentration

12.5

Con

trol

(%)

(d)

Figure 3: The cytotoxicity effects of A. afra extract and three selected compounds on the growth of the McCoy fibroblast cells.

We have compound (3) of which the forgoing data is identicalwith the known compound Scopoletin. All these compoundswere characterized by their 1H-NMR and 13C-NMR spectra.

The strong antimicrobial activity demonstrated by theethanol extract of A. afra has provided us with more evidencethat needed further chemical investigation of bioactivecompounds present. The extract of A. afra showed goodinhibitory effects against all Gram positive bacteria. It canbe noted that the MIC and MMC values varied from1.6 to 25.0 mg/mL among Gram positive bacteria. C. albi-cans which is a thick grower fungus was inhibited at aconcentration of 6.3 mg/mL. However, the MMC valueinsignificant as compared to the results of the MIC. TheA. actinomycetemcomitans was the resistant Gram negativebacteria as compared to all other tested bacteria (Table 1).The lowest MIC and MMC value of isolated compoundswas recorded at 0.25 mg/mL of compounds (6), (3), and (1)against A. israelii and A. naeslundii. Of all microorganisms,A. actinomycetemcomitans and C. albicans showed to beresistant against all compounds tested (Table 1).

The antioxidant activity of three selected compounds(scopoletin, acacetin and betulinic acid) revealed that theyare effective antioxidant agents (Figure 2). Scopoletin had astrong transformation of the DPPH radical into its reducedform, with an IC50 value of 1.24 μg/mL which was in closerange to that of vitamin C (1.22 μg/mL). Acacetin and Betul-inic acid exhibited a slightly low DPPH scavenging activitywith the IC50 of 2.39 and 2.42 μg/mL, respectively.

The cytotoxicity effects of ethanol crude extract of A. afraand three selected compound on the growth of Fibroblastcells are shown in Figure 3 and Table 2. The extract as a

Table 2: The IC50 values of crude extract and three selected isolatedcompounds.

Extract/compounds IC50(μg/mL) STD

Betulinic Acid 30.96 ±1.95

Acacetin 35.44 ±2.14

Scopoletin 132.5 ±1.85

Artemisia afra 16.95 ±1.82

Actinomycin-D 0.003364 ±0.00002

mixture of different components showed to be nontoxic onlower concentrations of 6.12 and 3.06 μg/mL, with the cellviability of 120%. However, toxic effects were apparent athigher concentration range of 12.50 to 400 μg/mL, withthe cell viability of 60 to 20%. Acacetin and betulinicacid also showed a smooth trend of nontoxic effects atlower concentrations and toxic at higher concentrationswith IC50 values of 35.44 and 30.96 μg/mL respectively. Theeffect of acacetin on lung cancer (A549) cell proliferationwas observed to have a dose-dependent manner with anIC50 value of 9.46 μM [32]. Apoptotic activity of betulinicacid against murine melanoma B16 cell line was reportedand it was discovered that betulinic acid induces apoptoticeffects with an IC50 of 22.5 μg/mL [33]. Reports postulatesthat triterpenes with a carboxyl group at c-28 shows morecytotoxic activity against cancer cell lines [34–36] and induceapoptosis [37]. Unexpectedly, one out of three compoundstested, scopoletin was relatively nontoxic with an IC50 valueof 132.5 μg/mL.


Acknowledgment

This work supported by the National Research Foundation.

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Research Article

Antimicrobial and Anti-Inflammatory Activities ofPterygota macrocarpa and Cola gigantea (Sterculiaceae)

Christian Agyare,1 George Asumeng Koffuor,2 Vivian Etsiapa Boamah,1 Francis Adu,1

Kwesi Boadu Mensah,2 and Louis Adu-Amoah1

1 Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences,Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

2 Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences,Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

Correspondence should be addressed to Christian Agyare, [email protected]

Received 15 February 2012; Accepted 31 March 2012

Academic Editor: Namrita Lall

Copyright © 2012 Christian Agyare et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Pterygota macrocarpa and Cola gigantea are African medicinal plants used in traditional medicine for the treatment of sores,skin infections, and other inflammatory conditions including pains. This study therefore aims at investigating the antimicrobialproperties of ethanol leaf and stem bark extracts of P. macrocarpa and C. gigantea using the agar diffusion and the micro-dilutiontechniques and also determining the anti-inflammatory properties of the extracts of these plants in carrageenan-induced footedema in seven-day old chicks. The minimum inhibitory concentration of both ethanol leaf and bark extracts of P. macrocarpaagainst the test organisms was from 0.125 to 2.55 mg/mL and that of C. gigantea extracts was 0.125 to 2.75 mg/mL. Extracts withconcentration of 50 mg/mL were most active against the test organisms according to the agar diffusion method. All the extracts ofP. macrocarpa and C. gigantea at 30, 100, and 300 mg/kg body weight except ethanol leaf extract of C. gigantea exhibited significantanti-inflammatory effects (P ≤ 0.001).

1. Introduction

The search for newer antimicrobial agents from varioussources has become imperative because of the emergenceof resistance strains of microorganisms against orthodoxantibiotics especially difficulty to treat infections fromresistant strains of bacteria [1] and also the fact that thenumber of scientists who are developing new antibacterialagents has dwindled, even as bacteria evolve ever moreclever mechanisms of resistance to antibiotics [2]. Therecent search for new antibiotics includes various sourcessuch as the synthetic compounds, bioactive agents fromaquatic microorganisms, and natural products includingmedicinal plants. In Africa and other developing countries,it is estimated that 70 to 80% of people rely on traditionalhealers and herbal practitioners for their health needs [3, 4]and medicinal plants are the main source of remedies usedin this therapy. Some of these medicinal plants are used

for the management of several different disease conditionssuch as bacterial infections, parasitic infections, skin diseases,hypertension, pains, and inflammation such as rheumatoidarthritis [5–8].

Several medicinal plants including their isolated com-pounds have been found to exhibit biological activitiesrelated to their traditional uses, for example, geraniinand furosin isolated from Phyllanthus mellerianus (Kuntze)Exell. have been found to possess wound healing propertiesascribed to this plant as wound healing agent [9]. Cryptole-pine, an alkaloid from Cryptolepine sanguinolenta, has beenshown to possess antimicrobial and antiplasmodial activitieswhich have gone to confirm its medicinal uses as anti-infective and antimalarial agent [10, 11].

Pterygota macrocarpa K. Schum. belongs to the familySterculiaceae and is known in local Asante-Twi languageas kyereye in Ghana. It is a large tree that grows in dense


semideciduous forests usually distributed in West Africafrom Sierra Leone to Cameroun. The soaked leaves areused to treat stomachache, pains, and disorders of digestion.Leaf decoctions are used for the treatment of gonorrheaand other urinary tract infections [12–14]. Traditionally, thebark is used in the management of haemorrhoids, dropsy,swellings, edema, gout, leprosy, and pain [15]. The seeds ofP. macrocarpa have been found to contain phytate, oxalateand tannins [16].

Cola gigantea A. Chev. belongs to the family Sterculiaceaeand is commonly known as giant cola and local Asante-Twi name is watapuo in Ghana. It is a large tree indry semideciduous forests in West Africa and the WestIndies. The nuts (mostly called kola) are often used totreat whooping cough, asthma, malaria, and fever. Othertraditional uses include increasing the capacity for physicalexertion and for enduring fatigue without food, stimulatinga weak heart, and treating nervous debility, weakness, lackof emotion, nervous diarrhea, depression, despondency,brooding, anxiety, and sea sickness [12, 14, 15, 17]. Kolanut is the name of the mature fruits of the Cola species[18] and has a bitter flavour and high caffeine content[19, 20], and when the fruit is ingested, it acts as stimulantsand thus creates an ecstatic and euphoric state [20]. Thecaffeine present acts as a bronchodilator, expanding thebronchial air passages [21]. These fruits are also chewedin communities during traditional ceremonies and also areknown to reduce hunger pangs. The ethanol leaf extract of C.gigantea has been shown to be active against Candida albicansand phytochemical screening of the leaf extract indicatedthe presence of alkaloids, saponins, tannins, anthraquinones,and cardenolides [22]. The aim of this study is to investigatethe antimicrobial and anti-inflammatory activities of ethanolstem bark and leaf extracts of P. macrocarpa and C. gigantea.


2.1. Plant Material and Chemicals. Stem bark and leaves ofPterygota macrocarpa and Cola gigantea were collected inJuly, 2007, from the Bobiri Forest Reserves of the ForestryResearch Institute of Ghana (FORIG) near Kubease, AshantiRegion, Ghana, and identified and authenticated by Dr. A.Asase, Department of Botany, University of Ghana, Ghana.Unless stated otherwise, all the chemicals were purchasedfrom Sigma (Deisenhofen, Germany).

2.2. Preparation of Extracts. The plant materials were airdried and powdered, and 200 g each of the dried powderedmaterial of P. macrocarpa and C. gigantea was extracted,respectively, with 70% ethanol (1.5 L) using Soxhlet appa-ratus. The ethanol extracts obtained were evaporated todryness under reduced pressure and kept in a dessicator.The yields of the stem and leaf extracts of P. macrocarpawere 4.2 and 12.4% w/w, respectively. And the yields of C.gigantea were 3.6 and 16.5% w/w for stem bark and leafextracts, respectively. Various quantities of the ethanol leafextract (CGLE) and ethanol stem bark extract (CGBE) ofC. gigantea and ethanol leaf extract (PMLE) and ethanol

stem bark extract (PMBE) of P. macrocarpa were dissolvedin normal saline and methanol for acute anti-inflammatoryand antimicrobial determinations, respectively.

2.3. Preliminary Phytochemical Screening. Phytochemicalscreening was conducted on both leaf and stem bark ofP. macrocarpa and C. gigantea to ascertain the presence ofcarbohydrates, tannins, sapogenetic glycosides, flavonoids,steroids, and alkaloids [23, 24]. The tannins content wasdetermined according to the method of Glasl [25] usingpyrogallol (Merck, Darmstadt, Germany, purity 99.5%,HPLC) as reference compound.

2.4. Determination of Antimicrobial Activity

2.4.1. Agar Diffusion Method. The antimicrobial activitiesof the extracts (PMLE, CGLE, PMBE, and CGBE) andreference drugs (chloramphenicol and clotrimazole (Sigma,Deisenhofen, Germany)) were determined using the agardiffusion method [26]. Nutrient agar (Oxoid Limited, UnitedKingdom) and Sabouraud agar (Oxoid Limited, UnitedKingdom) media were used for both determinations ofantibacterial and antifungal activities, respectively. 0.1 mLof 18 h culture of the test organisms (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcusaureus ATCC 25923, Bacillus subtilis NCTC 10073, andclinical fungal agent, Candida albicans, were used to seednutrient agar and Sabouraud agar plates, respectively. In eachof these plates, 4 equidistant wells (10 mm) were cut outusing sterile cork borer and were filled with 200 µL each ofthe different concentrations of extracts and reference drugsand allowed to diffuse at room temperature for 1 h. The zonesof inhibition were measured after 24 h incubation at 37◦C(for bacteria) and after 72 h at 30◦C (for fungi). The activitiesof the methanol (solvent) alone were also determined.

2.5. Determination of Minimum Inhibitory Concentration(MIC) Using Microdilution Technique. The MICs of theextracts (PMLE, CGLE, PMBE, and CGBE) against thetest bacteria were determined using the microdilutiontechnique as described by Eloff [27] and modified byAgyare and Koffuor [26]. Test solutions (100 mg/mL) ofboth extracts were prepared, test solution (25–100 µL) wasserially diluted with distilled water to 100 µg/mL, and50 µL of an 18 h old culture of one of the test bacteriagrown in nutrient broth (Oxoid Limited, United King-dom) was added to each well in the microplates. Thecovered microplates were incubated at 37◦C for 24 h. Toindicate growth, 30 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, thiazolyl blue) dis-solved in distilled water was added to the microplate wellsand incubated at 37◦C for 30 min. C. albicans was cultivatedin Sabouraud dextrose broth (Oxoid Limited, United King-dom) and then incubated for 3 days at 30◦C. The MICsof PMLE, CGLE, PMBE, and CGBE against the test funguswere determined according to the guidelines described inthe National Committee for Clinical Laboratory Standards[28] for filamentous fungi. The minimum inhibitory con-centration of the each extract against the test organisms was


Table 1: Preliminary phytochemical screening of dried leaves and stem barks of C. gigantea (CG) and P. macrocarpa (PM). +: presence ofsecondary metabolite; −: absence of secondary metabolites.

Secondary metabolites Alkaloids Saponins Flavonoids Steroids Carbohydrates Sapogenetic glycosidesTannins(% w/w)

Plant material/part

CG leaf + + + + + − 1.57

CG stem bark + + − + + − 1.02

PM leaf + + + + + + 1.45

PM stem bark + + − + + + 1.14

detected as the minimum concentration of extracts wherethere was no microbial growth, that is, nonformation ofblue color after the addition MTT to the medium [29]. Theprevious experiment was repeated three times.

2.6. Determination of Acute Anti-Inflammatory Activity. Thecarrageenan-induced inflammatory model in seven-day-oldchicks [30] was employed and the responsiveness of thesechicks to anti-inflammatory drugs/extracts was determined.

2.7. Experimental Animals. 7-days-old cockerels Gallus gal-lus (100–120 g) (Strain: Shaver 579) purchased from DarkoFarms Company Limited, Kumasi, Ghana, were maintainedin the Animal House of the Department of Pharmacology,Kwame Nkrumah University of Science and Technology,Kumasi, Ghana. The chicks were housed in stainless steelcages and fed with normal commercial poultry diet (GAFCO,Tema, Ghana), given water ad libitum, and maintainedunder laboratory conditions (temperature 28–30◦C, relativehumidity 60–70%, and normal light-dark cycle). A daybefore the experiment, the chicks were brought to thelaboratory and habituated to experimenter handling and theapparatus to minimize the effect of stress and novelty. The 7-day-old chicks were used in experiment. All procedures andtechniques used in these studies were in accordance with theNational Institute of Health Guidelines for the Care and Useof Laboratory Animals (NIH, Department of Health Servicespublication no. 83-23, revised 1985). The protocols for thestudy were approved by the Departmental Ethics Committee.

2.8. Experimental Design. At the beginning of the experi-ment the chicks (7 days old) were randomly assigned toone of fifteen groups (n = 5). The initial foot volumesof the chicks were measured using plethysmometer (IITCLife Science Inc., CA, USA) after which 0.01 mL of 2%carrageenan was injected into the plantar of the right footto induce inflammation. The inflammation produced wasmeasured, the increase in foot volumes was calculated, andthose with an increase between 15 and 40% were selected andput into thirteen groups of five after which they were injectedintraperitoneally with either diclofenac (Sigma, purity 98%HPLC) (10, 30 and 100 mg/kg) or dexamethasone (Sigma,purity 98% HPLC) (0.25, 0.5, and 1.0 mg/kg) based onrecommended effective human doses per body weight andPMLE, CGLE (30, 100, and 300 mg/kg), or PMBE andCGBE (30, 100 and 300 mg/kg) given orally based on

preliminary investigation. One group did not receive anydrug (control). Foot volumes were measured again at hourlyinterval posttreatment for 4 h. The percentage change in footvolume after induction and treatment of inflammation wascalculated and recorded for analysis.

2.9. Statistical Analysis. GraphPad Prism Version 5.0 forWindows (GraphPad Software, San Diego, CA, USA) wasused for all statistical analyses. Data are presented as mean± SEM (N = 5) and analyzed by one-way ANOVA followedby Dunnett’s multiple comparison’s test. P < 0.05 wasconsidered statistically significant in all analyses. The graphswere plotted using Sigma Plot for Windows version 11.0(Systat Software Inc., Germany).

3. Results

3.1. Preliminary Phytochemical Screening. Both the leaf andstem bark of P. macrocarpa and C. gigantea were found tocontain tannins (with varying amounts), alkaloids, steroids,saponins, and carbohydrate while the leaves of the twoplants contain flavonoids. The stem bark and leaves of P.macrocarpa were found to contain sapogenetic glycosides(Table 1).

3.2. Antimicrobial Activity. The ethanol extracts (PMLE,PMBE, CGLE, and CGBE) were found to be active against thetest bacteria (E. coli, P. aeruginosa, S. aureus, B. subtilis) withvarying mean zones of inhibition and C. albicans was foundto be less susceptible to the extracts. With respect to theagar diffusion method, the extracts (PMLE, CGLE, PMBE,and CGBE) with concentrations of 50 mg/mL exhibited thehighest activity against the test organisms (Table 3). Theminimum inhibitory concentration ranges of P. macrocarpaextracts (PMLE and PMBE) against the test organisms werefrom 0.125 to 2.55 mg/mL and those of C. gigantea extracts(CGLE and CGBE) were from 0.125 to 2.75 mg/mL (Table 2).

3.3. Anti-Inflammatory Activity. All the animals injectedwith carrageenan exhibited acute inflammation which man-ifested as increased foot volume. The control group howevershowed increased inflammation till the fourth hour. All theextract-treated groups except CGLE- (F3,80 = 1.80, P =0.1545: Figure 3) treated group exhibited significant anti-inflammatory effects (PMLE: F3,80 = 47.52, P < 0.0001;PMBE: F3,80 = 35.58, P < 0.001; CGBE: F3,80 = 1.80,


Table 2: Minimum inhibitory concentrations (MICs) of ethanol leaf extract (CGLE) and ethanol stem bark extract (CGBE) of C. giganteaand ethanol leaf extract (PMLE) and ethanol stem bark extract (PMBE) of P. macrocarpa determined by microdilution method. Theexperiments were repeated three times. Reference antimicrobial agents: CPC: chloramphenicol; CTZ: clotrimazole; ND: Not determined.

Extract/MIC(mg/mL)

S. aureusATCC 25923

B. subtilisNCTC 10073

E. coliATCC 25922

P. aeruginosaATCC 27853

C. albicans

CGLE 0.250 0.125 0.175 1.55 1.55

CGBE 0.125 0.150 0.250 2.75 1.75

PMLE 0.150 0.125 1.250 1.85 1.75

PMBE 0.125 0.175 0.155 2.55 0.75

CPC 0.025 0.020 0.025 0.055 ND

CTZ ND ND ND ND 0.025

Table 3: Antimicrobial activity of ethanol leaf extract (CGLE) and ethanol stem bark extract (CGBE) of C. gigantea and ethanol leaf extract(PMLE) and ethanol stem bark extract (PMBE) of P. macrocarpa by agar diffusion method. Activity of the methanol (solvent) used to dissolvethe extracts was determined. Mean zones of inhibition (plus diameter of well) are mean (mm) of 3 independent experiments, mean ± SD,n = 4 replicates, and diameter of well/cup = 10 mm. Reference antimicrobial agents: CPC: chloramphenicol (1 mg/mL); CTZ: clotrimazole(1 mg/mL); ND: Not determined.

Mean zones of growth inhibition (mm)

Extract (mg/mL)

Test organisms

S. aureus B. subtilis E. coli P. aeruginosa C. albicans

ATCC 25923 NCTC 10073 ATCC 25922 ATCC 27853

CGLE

10 19.67± 0.58 16.80± 0.40 12.20± 0.17 12.24± 0.13 12.65± 0.58

25 24.60± 0.53 19.33± 0.58 16.53± 0.24 15.50± 0.58 16.47± 0.48

50 28.33± 0.58 22.30± 0.42 19.67± 0.56 17.50± 0.47 22.13± 0.23

CGBE

10 16.67± 0.58 13.33± 0.58 12.30± 0.58 0.0 13.67± 0.58

25 13.00± 0.00 17.80± 0.35 14.12± 0.42 0.0 18.90± 0.48

50 18.67± 0.58 19.50± 0.50 18.67± 0.58 13.47± 0.58 21.85± 0.23

PMLE

10 0.0 17.80± 0.35 0.0 0.0 12.80± 0.20

25 14.60± 0.58 18.50± 0.50 19.33± 0.58 0.0 14.00± 0.00

50 19.67± 0.58 22.53± 0.12 21.47± 0.50 16.30± 0.23 22.33± 0.58

PMBE

10 0.0 12.33± 0.58 11.33± 0.58 0.0 12.80± 0.20

25 14.50± 0.50 14.67± 0.58 12.67± 0.58 0.0 15.40± 0.36

50 17.13± 0.23 16.73± 0.58 15.83± 0.29 12.3± 1.20 18.60± 0.58

CPC 25.67± 0.38 31.21± 0.38 30.67± 0.85 22.33± 0.38 ND

CTZ ND ND ND ND 25.60± 0.61

Methanol 0.0 0.0 0.0 0.0 0.0

P = 0.1545: Figures 1, 2, and 4). Similar effects were observedafter treating the animals with diclofenac (F3,80 = 79.81,P < 0.0001) and dexamethasone (F3,80 = 90.49, P < 0.0001)used as positive controls (Figure 5).

4. Discussion

The present studies indicate the antimicrobial and anti-inflammatory properties of ethanol extracts of P. macrocarpa(PMLE and PMBE) and C. gigantea (CGLE and CGBE).These findings were similar to our previous work [27] with

ethanol extract of Funtumia elastica Preuss Stapf. (Apocy-naceae) which confirmed the plant as having both antimicro-bial and anti-inflammatory properties. The antibacterial andantifungal activities of the P. macrocarpa are being reportedfor the first time. The MIC against the test bacteria is fromthe 0.125 to 2.55 mg/mL and the fungal agent (C. albicans) isfrom 0.75 to 1.75 mg/mL. With respect to the agar diffusionmethod, concentration of extracts from both leaves and thestem bark of P. macrocarpa less than 25 mg/mL did notexhibit activity against S. aureus and P. aeruginosa and lessactivity against E. coli at concentration of 25 mg/mL but


80

100

120

140

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Posttreatment time (hour)

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in fo

ot v

olu

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Control

0 1 2 3 4

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30 mg/kg PMLE

100 mg/kg PMLE

100 mg/kg PMLE

300 mg/kg PMLE

300 mg/kg PMLE

470.3 ± 24.08 361.8 ± 10.5 ∗∗∗ 356.6 ± 8.016 ∗∗∗ 350.4 ± 15.24 ∗∗∗

Figure 1: Effect of ethanol leaf extract (PMLE) of P. macrocarpa (30–300 mg/kg) on carrageenan-induced paw oedema. Values are expressedas mean ± SEM (N = 5), significantly different from control. ∗∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Control is the untreated birds.

80

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olu

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AUC values

30 mg/kg PMBE 100 mg/kg PMBE 300 mg/kg PMBE

470.3 ± 24.08 363.5 ± 15.94 ∗∗ 363.2 ± 18.15 ∗∗ 343.7 ± 10.86 ∗∗∗

Figure 2: Effect of ethanol stem bark extract (PMBE) of P. macrocarpa (30–300 mg/kg) on carrageenan-induced paw oedema. Values areexpressed as mean ± SEM (N = 5), significantly different from control. ∗∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Control is the untreatedbirds.

the MICs for the previous test bacteria were comparable tothe MICs of the C. gigantea extracts. This observation goesto support the assumption that size of inhibition halos ofdifferent extracts cannot be used for the determination ofthe relative antimicrobial potency since a more diffusiblebut less active extract could give a bigger diameter than anondiffusible but more active extract [27, 31].

The antimicrobial activities exhibited by the ethanolextracts of leaves and stem bark of C. gigantea (CGLE andCGBE) are in line with previous antimicrobial works onthe leaves of different species of Cola [22, 32, 33] where

different extracts of cola were found to exhibit inhibitoryactivities against certain bacteria and fungi with respect tothe leaf extract. The MIC range of both the leaf and stembark extracts of C. gigantea against the test bacteria is from0.125 to 2.75 mg/mL and the mean zones of inhibition ofthe different concentration (10–50 mg/mL) extracts (crude)were almost the same as those of the reference antibacterialagent, chloramphenicol at concentration of 1000 µg/mL.However, with the agar diffusion technique, the leaf extractsof C. gigantea (10 to 50 mg/mL) showed more activity againstboth test bacteria and fungus compared to the extracts from


100

120

140

160

180


Ch

ange

in fo

ot v

olu

me

0 1 2 3 4

Control

30 mg/kg CGLE

100 mg/kg CGLE

300 mg/kg CGLE

Control

AUC values

30 mg/kg CGLE 100 mg/kg CGLE 300 mg/kg CGLE

470.3 ± 24.08 459 ± 460.7 ± 28.46 ns 504.2 ± 27.29 ns27.83 ns

Figure 3: Effects of ethanol leaf extract (CGLE) of C. gigantea (30–300 mg/kg) on carrageenan-induced paw oedema. Values are expressedas mean ± SEM (N = 5), significantly different from control. ∗P < 0.05. Control is the untreated birds.

80

100

120

140


Ch

ange

in fo

ot v

olu

me

0 1 2 3 4

Control

30 mg/kg CGBE

100 mg/kg CGBE

300 mg/kg CGBE

Control

AUC values

30 mg/kg CGLE 100 mg/kg CGLE 300 mg/kg CGLE

470.3 ± 24.08 373.3 ± 16.14 ∗∗ 366.6 ± 10.44 ∗∗∗ 365.5 ± 6.734 ∗∗∗

∗∗∗

Figure 4: Effects of Ethanol stem bark extract (CGBE) of C. gigantea (30–300 mg/kg) on carrageenan-induced paw oedema. Values areexpressed as mean ± SEM (N = 5), significantly different from control. ∗∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Control is the untreatedbirds.

the stem bark. P. aeruginosa was found to be generallyless susceptible to all extracts from P. macrocarpa and C.gigantean, respectively (Table 3), and this was not surprisingsince it has been found to be resistant to most orthodoxantibiotics [34]. The antimicrobial properties may justify theuse of these plants for the treatment of various bacterial andfungal infections such as gonorrhea and urethral infectionsand sores.

The study also establishes the anti-inflammatory activityof the ethanol extracts of the leaves and stem bark ofP. macrocarpa and C. gigantean, respectively. Carrageenan-induced oedema has been commonly used as an experi-mental animal model for acute inflammation and is estab-lished to be biphasic. The early phase (1 to 2 hours) ofthe carrageenan model is chiefly mediated by serotonin,histamine, and increased synthesis of prostaglandins in the


80

100

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140

Posttreatment time

Ch

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0 h 1 h 2 h 3 h 4 h

Control10 mg/kg diclofenac1 mg/kg dexamethasone

Control

AUC values

200 mg/kg diclofenac 1 mg/kg dexamethasone

470.3 ± 24.08 363.5 ± 15.94 ∗∗∗ 363.2 ± 18.15 ∗∗∗

Figure 5: Effects of diclofenac (10 mg/kg) and dexamethasone (3 mg/kg) on carrageenan-induced paw oedema. Values are expressed asmean ± SEM (N = 5), significantly different from control. ∗∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Control is the untreated birds.

damaged tissues. The late phase is sustained by prostaglandinrelease and mediated by bradykinin, leukotrienes, poly-morphonuclear cells, and prostaglandins produced by tis-sue macrophages [35]. The extracts (PMLE, PMPE, andCGBE) inhibited the inflammation induced with car-rageenan in both phases indicating the ability of theseextracts to inhibit the synthesis or release of inflammatorymediators such as histamine, serotonin, bradykinin, andleukotrienes.

In comparing the four extracts (PMLE, CGLE, PMBE,and CGBE), only CGLE had insignificant anti-inflammatoryactivity in the carrageenan-induced hind paw oedema.However the CGBE had significant activity. The reasonfor this observation may be due to different chemicalcomposition of the stem bark and leaves of the sameplant. Traditionally, the bark is used in the management ofhaemorrhoids: dropsy, swelling, edema, gout, leprosy, andpain [15], and hence these results may confirm the medicinaluses of the bark. The CGBE extract may have relativelyhigh amounts of the bioactive constituents in relation tothe leaves. Phytochemical screening of C. gigantea showedthe presence of alkaloids and this confirms the findings ofSonibare et al. [22]. Members of the cola family are closelyrelated to Theobroma family of South America which havethe methylxanthine alkaloids such as caffeine, theobromine,and theophylline as secondary metabolites. Caffeine is one ofthe alkaloids of genus cola and it is used as an analgesic andanti-inflammatory adjuvant [36, 37].

There was no significant difference in anti-inflammatoryactivity between the leaf and stem bark extracts (PMLEand PMBE) of P. macrocarpa. However, the leaves aretraditionally used as diuretics, antiflatulence, and a remedyfor stomach, bladder, and urinary problems [15].

Powdered dried leaves and stem bark of P. macrocarpaand C. gigantea showed the presence of tannins and withdifferent tannin contents (1.02–1.57% w/w). Amoo andAgunbiade [38] reported on the high tannin content ofthe seeds of P. macrocarpa. Similar results were found inthe samples of leaves and bark of P. macrocarpa. Tanninsform a class of polyphenolic compounds which can act asantioxidants, antiviral, antibacterial, antiparasitic, and anti-inflammatory activity [39–42]. During the inflammatorycascade, antioxidants act as scavengers for free radicalsprotecting cells against oxidants which are mostly reactiveoxygen and nitrogen species [43–45]. The aforementionedfindings may justify the traditional medicinal uses of theP. macrocarpa and C. gigantea and hence there is the needto perform bioactivity fractionation of the active extractsto isolate the compounds that may be responsible for theantimicrobial and anti-inflammatory properties.

5. Conclusion

The minimum inhibitory concentration ranges of bothethanol leaf and bark extracts of P. macrocarpa against thetest organisms were from 0.125 to 2.55 mg/mL and thoseof C. gigantea extracts were from 0.125 to 2.75 mg/mL.Extracts (10, 25, and 50 mg/mL) of P. macrocarpa and C.gigantea exhibited antimicrobial activity with concentrationsof 50 mg/mL showing the highest zones of inhibition againstthe test organisms. All the extracts of P. macrocarpa andC. gigantea at 30, 100, and 300 mg/kg except leaf extract ofC. gigantea exhibited significant anti-inflammatory activity.The aformentioned activities may confirm the ethnobotan-ical uses of these two plants as antimicrobial and anti-inflammatory agents. It is recommended that the bioactive


extracts should be fractionated and the active compoundsresponsible for the previous pharmacological propertiesshould be isolated.

Conflict of Interests

The authors have no conflict of interest to declare.

Acknowledgments

The authors wish to express their gratitude to Mr. ThomasAnsah of the Department of Pharmacology, KNUST, Kumasi,Ghana, for his technical assistance and Miss Angela Mensahfor working on the project with them in the laboratory.

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Research Article

Antimicrobial Effects of a Lipophilic Fraction andKaurenoic Acid Isolated from the Root Bark Extracts ofAnnona senegalensis

Theophine Chinwuba Okoye,1 Peter Achunike Akah,1 Charles Ogbonnaya Okoli,1

Adaobi Chioma Ezike,1 Edwin Ogechukwu Omeje,2 and Uchenna Estella Odoh3

1 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Enugu State,Nsukka 410001, Nigeria

2 Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Nigeria,Enugu State, Nsukka 410001, Nigeria

3 Department of Pharmacognosy and Environmental Medicine, Faculty of Pharmaceutical Sciences, University of Nigeria,Enugu State, Nsukka 410001, Nigeria

Correspondence should be addressed to Theophine Chinwuba Okoye, [email protected]

Received 5 January 2012; Revised 20 February 2012; Accepted 20 February 2012


Copyright © 2012 Theophine Chinwuba Okoye et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Root bark preparation of Annona senegalensis Pers. (Annonaceae) is used in Nigerian ethnomedicine for treatment of infectiousdiseases. Extraction of the A. senegalensis powdered root bark with methanol-methylene chloride (1 : 1) mixture yielded themethanol-methylene extract (MME) which was fractionated to obtain the ethyl acetate fraction (EF). The EF on furtherfractionation gave two active subfractions, F1 and F2. The F1 yielded a lipophilic oily liquid while F2 on purification,precipitated white crystalline compound, AS2. F1 was analyzed using GC-MS, while AS2 was characterized by protonNMR and X-ray crystallography. Antibacterial and antifungal studies were performed using agar-well-diffusion method with0.5 McFarland standard and MICs calculated. GC-MS gave 6 major constituents: kaur-16-en-19-oic acid; 1-dodecanol; 1-naphthalenemethanol; 6,6-dimethyl-bicyclo[3.1.1]hept-2-ene-2-ethanol; 3,3-dimethyl-2-(3-methylbuta-1,3-dienyl)cyclohexane-1-methanol; 3-hydroxyandrostan-17-carboxylic acid. AS2 was found to be kaur-16-en-19-oic acid. The MICs of EF, F1, and AS2against B. subtilis were 180, 60, and 30 µg/mL, respectively. AS2 exhibited activity against S. aureus with an MIC of 150 µg/mL, whileF1 was active against P. aeruginosa with an MIC of 40 µg/mL. However, the extracts and AS2 exhibited no effects against Candidaalbicans and Aspergillus niger. Therefore, kaurenoic acid and the lipophilic fraction from A. senegalensis root bark exhibited potentantibacterial activity.

1. Introduction

The increase in the incidence of new and reemerging infec-tious diseases caused by organisms with high resistance ratesto standard antimicrobial agents has been a very challengingand global health burden. The indiscriminate and wide-spread antimicrobial use continues to cause significant in-crease in drug-resistant and multidrug-resistant bacteria[1, 2]. Medicinal plants have long been used in traditionalmedicine for treatment of various ailments including infec-tious diseases and many potent phytochemicals or second-ary metabolites possessing antimicrobial effects have been

isolated from plants [3, 4]. These constituents could serve asveritable lead compounds in the science of drug discovery,development, and research. An example is the startlingdiscovery of penicillin from a microscopic plant in 1928 thatlead to the synthesis of its derivatives such as penicillin G[5]. It is quite pertinent to note that since the discovery ofnalidixic acid in 1962, which led to the synthesis of morepotent fluoroquinolones and derivatives [6], there has notbeen the introduction of any major pharmacological class ofantibacterial agents. Hence, this is posing a great challenge toresearchers in the area of drug discovery and developmentof anti-infective agents and has equally lend credence to


the intensified research going on in the area of naturalproducts for the isolation of potent compounds that couldserve as lead in the discovery of new antibacterial agents [7].Screening of medicinal plants and other natural productshas led to the isolation of clinically active antibacterialagents [8]. Interestingly, many plant extracts have shown topossess antimicrobial effects and are being used in traditionalmedicine [9, 10]. Annona senegalensis Pers. (Annonaceae) isamong the medicinal plants that have been documented topossess antibacterial effects [11–13]. Also the ethnomedic-inal uses of the plant in the treatment of wounds andinfectious diseases such as diarrhea [14, 15] periodontal andother oral infections [16] had been reported. Furthermore,the anticonvulsant, sedative, and muscle relaxant [17, 18] aswell as anti-inflammatory [19] effects of the root bark extractand fractions of A. senegalensis have been reported.

Therefore, the objective of this study was to ascertain theantimicrobial effects of the root bark extracts and fractionsof A. senegalensis and to isolate and characterize the activephytochemical(s) responsible for these effects using proton-NMR and X-ray crystallography.


2.1. Plant Materials. Fresh roots of A. senegalensis were col-lected from Enugu-Ezike, Enugu State, Nigeria in the monthof June, 2007 and authenticated by a taxonomist, Mr. A.O. Ozioko, of the International Centre for Ethnomedicineand Drug Development (InterCEDD), Aku Road, Nsukka,Enugu State, Nigeria. A voucher specimen was depositedat the InterCEDD herbarium (specimen number: BDCP/INTERCEED–64).

2.2. Test Organisms. Clinical strains of Escherichia coli, Bacil-lus subtilis, Pseudomonas aeruginosa, Salmonella paratyphiand Staphylococcus aureus, Aspergillus, niger and Candidaalbicans, obtained from the Medical Laboratory Departmentof Bishop Shanahan Memorial Hospital, Nsukka, EnuguState, Nigeria and preserved in the Microbiology Unit of theDepartment of Pharmaceutics and Pharmaceutical Micro-biology, University of Nigeria, Nsukka, were used. Theseclinical strains were isolated from designated biological fluidsor sources as shown in Table 1.

2.3. Animals. Adult albino mice (18–30 g; n = 14) bred in theLaboratory Animal Facility of the Department of Pharmacol-ogy and Toxicology, University of Nigeria, Nsukka, were usedin the studies. The animals were maintained under standardlaboratory conditions and had free access to standard pellets(Guinea Feeds, Nigeria Plc) and water. On transfer to thework area, animals were allowed two weeks of acclimati-zation before the commencement of the experiments. Allanimal experiments were conducted in compliance with theNational Institute of Health Guidelines for Care and Use ofLaboratory Animals (Publication no. 85–23, revised 1985)and approval of the University Ethical Committee on the useof laboratory animals.

Table 1: Biological sources of clinical strains of the test organisms.

S/No Clinical strainBiological

source

1 Bacillus subtilis Wound

2 Escherichia coli Stool

3Pseudomonas

aeruginosaWound

4 Salmonella paratyphi Stool

5 Staphylococcus aureus Nasal discharge

6 Aspergillus niger Spoiled food

7 Candida albicansHigh vaginalswab (HVS)

2.4. Preparation and Extraction of Plant Materials. The rootbarks were peeled off, cut into pieces, and dried under shade.The dried root-bark was then pulverized into coarse powderusing a hammer mill. The powdered material (2.95 kg) wasextracted with a mixture of methanol: methylene chloride(1 : 1) using Soxhlet extractor to obtain the methanol:methylene chloride extract or the crude extract (MME).This was concentrated at reduced pressure using a rotaryevaporator to obtain a yield of 375 g (12.71% w/w).

2.5. Solvent-Guided Fractionation of MME and Bioactivity-Guided Studies. The methanol-methylene chloride extract(MME; 250 g) was subjected to solvent-guided fractionationin a silica gel (70-220 mesh, Merck Germany) column, suc-cessively eluted with n-hexane, ethyl acetate, and methanol inorder of increasing polarity. The fractions were concentratedunder reduced pressure in a rotary evaporator (below 40◦C)to obtain the hexane fraction (HF; 115 g; 46.0% w/w),ethyl acetate fraction (EF; 61 g; 24.4% w/w), and methanolfraction (MF; 69.5 g; 27.8% w/w). Bioactivity-guided studieson the extract and fractions using agar well diffusionmethod showed that EF had a potent antibacterial activitywith a relatively higher inhibition zone diameter (IZD)than the HF and MF. Subsequently, the ethyl acetate (EF)soluble fraction was subjected to column chromatographicseparation. The EF (50 g) was separated in a dry-packedsilica gel (70–220 mesh, Merck Germany) column of width4 cm and length 40 cm. The extract was mixed with thesilica gel and loaded on top of the prepacked column.The column was successively eluted with gradient mixturesof n-hexane and ethyl acetate (1 : 0, 9 : 1, 8 : 2, 7 : 3, 6 : 4,5 : 5, 4 : 6, 3 : 7, and 2 : 8) and the fractions collected in500 mL volume. The fractions were subsequently pooled andconcentrated to afford eight broad fractions, F1–F8, basedon the similarity of constituents visualized on silica gel pre-coated TLC plates (Uniplate-Analtech Co., USA), developedwith mixtures of n-hexane and ethyl acetate accordingly.Fraction F1 gave an oily liquid or lipophilic fraction, while F2(9–12; 2000 mL) when concentrated yielded white crystals.However, the crystals yielded by F2 were harvested andpurified by repeated washing with n-hexane and dried toobtain A. senegalensis crystals (AS2) (2.8 g; 5.6% w/w) whichwas stored in a refrigerator for activity studies.


2.6. Phytochemical Tests. The preliminary phytochemicalanalysis of methanol-methylene chloride extract (MME),ethyl acetate fraction (EF), hexane fraction (HF), methanolfraction (MF), and ethyl acetate subfraction isolated com-pound, AS2 were performed using standard phytochemicalprocedures as described by Harborne [20] and Trease andEvans [21]. Briefly, frothing test for saponins, Salkowski testfor terpenoids, Liebermann-Burchard tests for steroids, ferricchloride test for tannins, Keller-Killiani test for cardiac glyco-sides, Dragendorff ’s and Mayer’s test for alkaloids, Fehling’stest for reducing sugars, xanthoproteic test for proteins,iodine test for carbohydrates or starch, and ammonia testfor detection of flavonoids were performed for qualitativeidentification of the phytoconstituents present in [22]. Allreagents for the preliminary phytochemical analysis werefreshly prepared.

2.7. Identification and Characterization of AS2. Structuralelucidation of the pure crystals, AS2, was performed usingproton NMR and X-ray crystallography, since the com-pound is in crystal form. The melting point of AS2 wasalso determined using an analog melting point apparatus(Electrothermal, Cat. no. IA 6304, England). The identitywas established by comparison of the spectral data and X-raycrystallography of previously published compounds [23].

2.8. Acute Toxicity and Lethality (LD50) Test of AS2. The oralacute toxicity and lethality test (LD50) of the AS2 was per-formed in mice using the method described by Lorke [24].Briefly, the test was performed in two stages. In stage one,animals received oral administration of one of 10, 100, and1000 mg/kg (n = 3) of AS2 and observed for 24 h for numberof deaths. Since no death occurred in any of the groups in thefirst stage of the test, in stage two of the test, 1600, 2900, and5000 mg/kg doses of the AS2 were administered to a freshbatch of animals (n = 1) and was also monitored for 24 h.Since death occurred at the maximum dose (5000 mg/kg),the LD50 was estimated as the product of the square root ofthe dose that recorded death and the dose that recorded nodeath preceding it (in this case, 2900 mg/kg dose). We haveestimated and reported the acute toxicities and lethality testsof MME, EF, HF, and MF in a separate study [18].

2.9. GCMS Analysis of the Lipophilic Fraction (F1). The gaschromatography mass spectrometry (GCMS) of F1, thelipophilic subfraction, of the EF fractions was analyzed usingGCMS-QP2010 PLUS (SHIMADZU, JAPAN) in order tocharacterize the lipophilic components. Sample of the F1 wassuspended in 1 mL of ethyl acetate (Merck, Germany) and1 : l of this solution was analyzed by the gas chromatographycoupled with mass spectrometry equipped with a fusedsilica capillary column DB-5 (30 m × 0.25 mm × 0.25 m).The electron impact technique (70 eV) was used with theinjector temperature at 240◦C and that of the detector at230◦C. The carrier gas was helium at the working rate of1.7 mL/min. The column temperature was initially 60◦C andthen was gradually increased at the rate of 3 ◦C/min up to240◦C. For detection of the oil components, we used a flame

ionization detector set up at 230◦C. The identification of thecomponents of the lipophilic fraction was effected throughcomparison of substance mass spectrum with the database ofthe GC/MS (NIST 62.lib), the literature, and retention index[25, 26].

2.10. Antimicrobial Assay. Each of the extract (MME), frac-tions (HF, EF, and MF), F1 and AS2 was dissolved in dimethylsulfoxide (DMSO) to obtain 100 mg/mL concentration.Subsequently, the concentration was diluted to obtain 50, 25,12.5, and 6.25 mg/mL for the determination of the minimuminhibitory concentration (MIC) at the dose levels. Agar welldiffusion method as described in [27, 28] was employedfor the assay. The test organisms, the clinical isolates, wereprepared with a 0.5 McFarland standard and subcultured at37◦C and maintained on nutrient agar media for bacteriaand sabouraud agar media for fungi (Aspergillus niger andCandida albicans). Petriplates containing 20 mL of respectivemedium were seeded with selected microbial strains andincubated at 37◦C for 24 hours. Standard antimicrobialagents used as positive controls were gentamycin (Lek,Slovakia) and ciprofloxacin (Medreich, India). After 24 hoursthe inhibition zone diameters (IZD) were recorded and themean calculated. The minimum inhibitory concentrations(MICs) were then determined at various dilutions byextrapolation from the graphs of IZD squared (IZD2) againstlogarithm of the concentration.

2.11. Statistical Analysis. Data obtained were analysed bySPSS (Version 14) using One-Way Analysis of Variance(ANOVA) with Dunnet its test for multiple comparisonswith the control. Values are in mean ± SEM and wereconsidered significant at P < 0.05.

3. Results

3.1. Phytochemical Tests. Phytochemical tests of methanol-methylene chloride extract (MME) gave positive reactionswith phytochemical reagents with respect to alkaloids, car-bohydrates, flavonoids, fats and oils, glycosides, reducingsugars, resins, steroids, saponins, and terpenoids. The ethylacetate (EF) gave positive reactions for alkaloids, flavonoids,resins and terpenoids, while AS2 gave a strong positive reac-tion with Salkowski test. The phytoconstituents of hexanefraction (HF) and methanol fraction (MF) were also shown(Table 2).

3.2. Acute Toxicity and Lethality (LD50) Test of AS2. Themedium lethal dose (LD50) of the AS2 was found to be3800 mg/kg in mice indicating the good level of safety.

3.3. Identification and Characterization of AS2. The AS2 wasshown to be a white crystalline and odourless compound.The results of the proton NMR and X-ray crystallography,when compared with the spectral data of known compounds,established the identity of AS2 to be kaur-16-en-19-oic acidor kaurenoic acid, a diterpenoid, with the chemical structure


Table 2: Phytochemical constituents of extract and fractions.

Constituent MME EF HF MF AS2

Carbohydrate + − − + −Alkaloid + + + + −Reducing sugar + − − + −Glycoside + − − + −Saponins + − − + −Tannins − − − − −Flavonoids + + − − −Resin + + + + −Fats and oils + + + − −Steroids + − + + −Terpenoids + + + + +

Acidiccompounds

+ + − − −+= present; − = absent.

and X-ray crystallograph as shown (Figures 1, 2 and 3). Themelting point of AS2 was found to be 170–172◦C.

3.4. GCMS Analysis of the Lipophilic Fraction (F1). TheGCMS analysis of F1 revealed the presence of the fol-lowing 6 major constituents which include kaur-16-en-19-oic acid, 1-dodecanol, 1-naphthalenemethanol, 6,6-dimethyl-bicyclo [3.1.1]hept-2-ene-2-ethanol, 3,3-dimethyl-2-(3-methylbuta-1,3-dienyl)cyclohexan-1-methanol, and 3-hydroxyandrostan-17-carboxylic acid (Table 3). The chemi-cal structures of these constituents are shown in Figure 4.

3.5. Antimicrobial Assay. The extract, fractions, and AS2exhibited significant antibacterial activity and are devoidof any antifungal activity. The results of the inhibitionzone diameter (IZD) and MICs of the extract and fractionsrevealed activity against gram-positive and gram-negativeorganisms (Table 4). The order of potency against the variousbacteria isolates with respect to their MICs by the extractand fractions was B. subtilis (AS2 > F1 > HF ≡ MF > EF> MME), S. aureus (AS2 > MME), and P. aeruginosa (F1> MME) (Table 5). The MME exhibited an MIC of 0.370,8.75, 1.08, and 0.07 mg/mL against the clinical isolates of B.subtilis, S. aureus, P. aeruginosa, and S. typhi, respectively. Theextracts and isolate showed no antibacterial activity againstE. coli (Table 4). The F1 gave an MIC of 0.06 and 0.04 mg/mLagainst B. subtilis and P. aeruginosa, respectively. AS2 haspotency against S. aureus and B. subtilis with an MIC of0.15 and 0.03 mg/mL, respectively, whereas EF offered anMIC of 0.18 mg/mL against B. subtilis (Table 5). However, theantifungal test for MME, EF, HF, MF, F1, and AS2 against A.niger and C. albicans showed no activity.

4. Discussion

The antimicrobial effects of the methanol-methylene chlo-ride extract (MME), ethyl acetate fraction (EF), hexane

Figure 1: X-ray crystallograph of AS2.

HO

O Kaur-16-en-19-oic acid

Figure 2: Chemical structure of AS2.

fraction (HF), methanol fraction (MF), the lipophilic sub-fraction (F1), and the isolated compound, AS2 exhibitedappreciable antibacterial effects but are devoid of antifungalactivity. However, AS2 exhibited the lowest MIC value of30 µg/mL against B. subtilis and therefore exhibited the mostpotent activity when compared to the extracts and fractionof the root bark of A. senegalensis. The results of the proton


(3) Comment

(6) Site

(8) Author

Parameter Value Parameter Value

(1) Data file name H:/AS2-NIG/3/fid(2) Title AS2-NIG

(4) Origin Bruker BioSpin GmbH

(7) Spectrometer Spect

(9) Solvent CDCI3(10) Temperature 298.2(11) Pulse sequence zg30(12) Experiment 1D(13) Number of scans 8

(14) Receiver gain 362(15) Relaxation delay 1.0000(16) Pulse width 10.2500(17) Acquisition time 3.9846(18) Acquisition date 2010-10-03T10:48:06

(22) Lowest frequency −1640.8(23) Nucleus 1H(24) Acquired size 32768(25) Spectral size 65536

1400

1300

1200

1100

1000

900

800

700

600

500

400

300

200

100

0

−100

7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1

F1 (ppm)

2.65

2.06

2.02

1.99

1.91

1.88

1.86

1.84

1.63

1.61

1.59

1.55

1.52

1.49

1.47

1.25

1.15

1.10

1.08

1.06

1.02

1.01

0.96

4.81

4.75

7.22

AS2-NIG

2010-10-03T10:48:06(19) Modification date(20) Spectrometer frequercy 400.15(21) Spectral width 8223.7

(5) Owner Damaris

Figure 3: Spectral data analysis of AS2.

NMR and X-ray crystallography identified and characterizedAS2 to be a diterpene known as kaur-16-en-19-oic acid orkaurenoic acid (KA). The melting point of AS2 was found tobe 170–172◦C which was of a comparable range with that ofkaurenoic acid from a different source already reported [29].In a separate study, the antibacterial effects of the essentialoil from A. senegalensis have been reported [12] and moreimportantly the antibacterial activity of kaurenoic acid fromthe root extract of another plant, Viguiera arenaria, has beendocumented [30]. The extracts, F1 and the KA, exhibitedbetter antibacterial effects against gram-positive organismssuch as B. subtilis and S. aureus, than the gram-negative rodssuch as P. aeruginosa, S. paratyphi, and E. coli used in thestudy.

Therefore, the antibacterial effects of MME, EF, HF, MF,F1, and AS2 against organisms such as P. aeruginosa andS. aureus correlated with the ethnomedicinal use of theplant in wound healing, since P. aeruginosa and S. aureushad been implicated in the contamination of wounds andboils [31]. The antibacterial activity against P. aeruginosa isof interest because P. aeruginosa has been reported to beresistant to many antibacterial agents and identified asan opportunistic pathogen which causes complications inimmune-compromised patients [32]. The activity of rootbark extracts ofA. senegalensis against S. aureus has also beendocumented in a separate study [13]. Moreover, according toApak and Olila [13], the root bark extract of A. senegalensisexhibited no activity against E. coli which was consistent with


Table 3: Chemical constituents of lipophilic fraction (F1).

S/No Chemical nameMolecularformula

Molecularweight

Retention(KOVAT’s)

indexMS ions/fragments (m/e)

01 1-dodecanol C12H26O 186 145727, 41, 55, 70, 83, 97,111(112), 126, 140

026,6-dimethyl-bicyclo[3.1.1]hept-2-ene-2-

ethanolC11H18O 166 1290

27, 41 (43), 67, 79, 91, 105,122, 151.

03 Kaur-16-en-18-oic acid C21H32O2 316 2056

27, 41, 55, 67, 79, 91, 105,121, 133, 147, 159, 187,213, 241, 257, 273, 301,

316.

04 1-naphthalenemethanol C15H26O 222 168527, 41, 55, 69, 81, 109, 124,

191, 222.

053,3-dimethyl-2-(3-

methylbuta-1,3-dienyl)cyclohexan-1-methanol

C14H24O 208 159241, 55, 69, 81, 95, 109, 123,

139, 165, 177, 193.

063-hydroxyandrostan-17-

carboxylicacid

C20H32O3 320 237543, 67, 79, 93, 108, 121,135, 147, 161, 175, 194,215, 233, 248, 287, 302.

HO

(1)

OH

(2)

O(3)

−O

(4)

OH

(5)HO

O OH

(6)

Figure 4: Chemical structures of GCMS constituents of F1.


Ta

ble

4:In

hib

itio

nzo

ne

diam

eter

(IZ

D)

ofth

eex

trac

tan

dfr

acti

ons.

Org

anis

mM

ME

HF

MF

EF

F1A

S2C

IPG

EN

B.s

ubti

lis14.6

0±

0.12

13.3

0±

0.15

13.3

0±

0.06

14.0

0±

0.00

14.0

0±

0.23

16.0

0±

0.00

22.0

0±

0.12

26.0

0±

0.00

S.au

reus

14.2

0±

0.42

12.3

0±

0.35

+10.5

0±

0.17

10.3

3±

0.06

17.0

0±

0.00

28.0

0±

0.00

26.0

0±

0.00

P.ae

rugi

nosa

13.0

0±

0.00

++

+11.4

0±

0.31

+25.0

0±

0.00

22.0

0±

0.12

S.pa

raty

phi

12.6

0±

0.20

++

++

+25.0

0±

0.30

20.0

0±

0.00

E.c

oli

++

++

++

NA

NA

A.n

iger

++

++

++

NA

NA

C.a

lbic

ans

++

++

++

NA

NA

Val

ues

inm

m,a

rem

ean±

SEM

(AN

OV

A,D

un

net

its

post

hoc)

;n=

3;+

:no

grow

thor

no

acti

vity

;con

cen

trat

ion

ofA

S2=

50m

g/m

L;C

IP&

GE

N:4

0µ

g/m

Lw

hile

oth

ers

are

100

mg/

mL.

CIP

:cip

rofl

oxac

in,G

EN

:ge

nta

mic

in,N

A=

not

appl

icab

le.


Table 5: Minimum inhibitory concentration (MIC) of extract and fractions.

ORGANISM MME MF HF EF F1 AS2 CIP GEN

B. subtilis 370.00 150.00 140.00 180.00 60.00 30.00 0.28 0.02

S. aereus 8750.00 + + + + 150.00 1.18 0.23

P. aeruginosa 1080.00 + + + 40.00 + 3.60 0.79

S. paratyphi 70.00 + + + + + 0.39 1.77

MIC values are in µg/mL.

the lack of activity of MME, EF, HF, MF, F1, and AS2 againstE. coli identified in this study.

In addition to the antibacterial action of KA, thereis the possibility of the presence of other phytochemicalscontributing to the antibacterial activity of the extracts andfractions of A. senegalensis. This is more so since theF1, which consisted 6 major constituents, together withKA, exhibited potent antibacterial activity with an MIC of40 µg/mL against P. aeruginosa, whereas the kaurenoic acidalone did not show appreciable activity against P. aeruginosa.Hence, due to the presence of six major phytochemicals inthe GC-MS analysis of the F1, the observed antibacterialeffects could be attributed to the combined or single effects ofany of the compounds which include kaur-16-en-19-oic acid,1-dodecanol, 1-naphthalenemethanol, 6,6-dimethyl-bicyclo[3.1.1] hept-2-ene-2-ethanol, 3,3-dimethyl-2-(3-methylbu-ta-1,3-ienyl)cyclohexan-1-methanol, and 3-hydroxyandro-stan-17-carboxylic acid. The presence of kaurenoic acidamong these compounds is of practical interest as it wasalso established as the pure isolated compound, AS2. Thekaurenoic acid from the F1 gave the following mass ionson fragmentation with the GCMS analysis: 316 (molecu-lar ion), 301 (demethylation), and 273 (decarboxylation).Notably, steroids, organic acids, and alcohols dominated theremaining 5 compounds of the F1 and both organic acids aswell as alcohols are known to possess antibacterial activityespecially at higher concentrations [3]. However, F1 showeda strong indication and possibility of being a fixed oil, since itpossesses some characteristics attributable to fixed oils whichinclude odorless liquid at room temperature, pale yellowishin colour, permanent grease spot on filter paper whenheated in an oven and often contains mixture of organicacids as well as being an extraction product of n-hexane ormixture of n-hexane and ethyl acetate [33]. Medicinal plantswith preponderance of variety of secondary metabolites,such as tannins, terpenoids, essential oils, alkaloids, andflavonoids have been found in vitro, to possess antimicrobialproperties [3]. There is a recognizable loss of activity due tofractionation as the crude extract, MME, exhibited activityagainst all the organisms tested except E. coli, whereas thefractions were found to have lost activity to some of thetested organisms especially S. paratyphi. The presence ofditerpenoid compounds in the extracts and fractions of A.senegalensis really correlated well with the high presence ofresins in plants as diterpene acids occur well in plant resins.Some other published works have reported the isolation ofkaurenoic acid from the leaves of A. senegalensis [34, 35] andaerial parts of Espeletia semigloburata [36] which exhibited

antibacterial, anticancer, anti-inflammatory, and antipyreticeffects. The lack of activity against fungal organism, C.albicans, in the study was in consistent with other reportedwork on root bark extract of A. senegalensis which exhibitedlack of activity against C. albicans [16]. In other documentedstudies, terpenoids have shown to possess antibacterial [3, 37,38], antiviral [39, 40], and antiprotozoal effects [41]. Par-ticularly, antibacterial effects of diterpenoids isolated fromother plants have been reported [42, 43]. The results fromthis study tend to support the ethnomedicinal claim of rootbark of A. senegalensis in treatment of bacterial infectionsand wound healing particularly in the treatment urinarytract infections in veterinary animals [13]. Furthermore, KAcould equally serve as a veritable lead compound in thedevelopment of potent standard antibacterial agent.

5. Conclusion

Results of the study have indicated that a diterpenoid, kaur-16-en-19-oic acid or kaurenoic acid, has been identified asthe phytochemical constituent responsible for the antibacte-rial effects of root bark of Nigerian Annona senegalensis Pers.(Verbenaceae) and is devoid of appreciable antifungal effects.

Conflict of Interests

The authors declare that there is no conflict of interests.

Acknowledgments

The authors thankfully appreciate Professor Dr. MarkHamann of the Department of Pharmacognosy and Phyto-chemistry, University of Mississippi, USA, for performingthe proton NMR and the X-ray crystallography of AS2.Part of the financial support was from the Ministry ofEducation, Science and Technology Post Basic Programme,of the Federal Government of Nigeria for the Innovators ofTomorrow (IOT) Award Grant of the World Bank assistedStep-B project.

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Research Article

Evaluation of the Acetone and Aqueous Extracts ofMature Stem Bark of Sclerocarya birrea for Antioxidant andAntimicrobial Properties

Nicoline F. Tanih1 and Roland N. Ndip1, 2

1 Microbial Pathogenicity and Molecular Epidemiology Research Group, Department of Biochemistry and Microbiology,Faculty of Science and Agriculture, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa

2 Department of Microbiology and Parasitology, Faculty of Science, University of Buea, Box 63, Buea, Cameroon

Correspondence should be addressed to Roland N. Ndip, [email protected]

Received 9 January 2012; Revised 10 March 2012; Accepted 17 March 2012


Copyright © 2012 N. F. Tanih and R. N. Ndip. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

We assayed the antimicrobial activity of acetone and aqueous extracts of the stem bark of Sclerocarya birrea on some selected bac-teria and fungi species including; Streptococcus pyogenes, Plesiomonas shigelloides, Aeromonas hydrophila, Salmonella typhimurium,Cryptococcus neoformans, Candida glabrata, Trichosporon mucoides, and Candida krusei using both agar well diffusion and mini-mum inhibitory concentration (MIC) assays. Based on the levels of activity, the acetone extract was examined for total polyphenoliccontent, radical scavenging and antioxidant activities. Total phenols of the extract were determined spectrophotometrically. Theantioxidant activity was determined by the DPPH, ABTS and reducing power. All the bacteria and fungi species were susceptibleto the plant extracts. The acetone extract was the most active for the bacterial species with MIC (0.156–0.625 mg/mL) while theaqueous extract was the most active for the fungi species with MIC (0.3125–1.25 mg/mL). The polyphenolic compounds werefound as 27.2 mg/g tannic acid equivalent, 25.2 mg/g quercetin equivalent, 9.1 mg/g quercetin equivalent for phenols, flavonoidand flavonols respectively. The acetone extract exhibited a remarkable ability to scavenge radicals, strong reducing ability and apotential source of natural antioxidants. Both the acetone and aqueous extracts of S. birrea may provide a target for drug discovery.

1. Introduction

Awareness has increased recently on the possibility of health-related risk associated with oxidative stress [1, 2]. Oxidativestress initiated by highly reactive free radicals and oxygenspecies such as hydrogen peroxides, superoxide, lipid per-oxyl, hydroxyl, nitric oxide, and peroxynitrite is present inbiological systems from a wide variety of sources [3, 4]. Thefree radicals may oxidize nucleic acids, proteins, lipids, orDNA and cause degenerative diseases such as cancer, chronicinflammation, diabetes mellitus, atherosclerosis, myocardialinfarction, arthritis, anemia, asthma, and neurodegenerativediseases [2, 4]. Inflammation, free radical damage, andoxidative stress are often the byproduct of normal cellularprocesses and are implicated in almost all debilitating degen-erative conditions. Naturally, the human system has check-in

mechanisms to deal with oxidative damage and free radicalformation [2, 5]. These protective mechanisms maybe dis-rupted as a result of various pathological processes andthereby cause damage to the cells. Antioxidants have beenreported to have significantly remedied this destructive effect[1].

Synthetic antioxidants used as food additives have beenreported to be toxic both to humans and animals [5]. Pres-ently, one of the most common used synthetic antioxidant isbutylated hydroxytoluene (BHT) [2, 3]. This toxicity as wellas general consumer rejection has led to decreasing use ofsynthetic antioxidants and geometric growth in research intonaturally occurring products, particularly from medicinalplants, in search of alternative potent antioxidants. Muchwork has been done to find safe and potent natural anti-oxidants from various plant sources [4, 5].


Consequently, natural products such as plants and plantproducts have been the alternative target of natural antiox-idants for food and pharmaceutical products lately basedon their folkloric use in medicine since time immemorial.The presence of polyphenolic compounds such as flavonoids,phenols, flavonols, and proanthocyanidins in plants is asso-ciated with this antioxidant potential [4]. Synthetic antioxi-dants are generally compounds with phenolic structures ofvarious degrees of alkyl substitution, whereas antioxidantbioactivity of medicinal plants is partly attributed to phenoliccompounds [6]. Natural antioxidants that occur in medicinalplants act as freeradical scavengers and chain breakers, pro-oxidant metal ion complexes, and quenchers of singlet oxy-gen formation [7]. Antioxidant supplementation has beenobserved to be the most effective method to reduce oxidativestress. They retard the formation of toxic oxidation products,maintain nutritional quality, and increase shelf life [8].

Sclerocarya birrea is a medium-size-to-large deciduoustree with an erect trunk belonging to the family Anacar-diaceae [9]. It is widely used for the treatment of severalmorbidities including proctitis, dysentery, and diarrhoea inSouth Africa and Africa at large [9–11]. Bark decoctions areused by the Xhosa and Zulu people as enemas for diarrhoeaand the Vhavenda people for treating fevers, stomach ail-ments, and ulcers [9, 11]. Limited information exists onantioxidant activity of this plant. Mariod et al. [4] examinedthe antioxidant properties of methanolic extracts of differentplant parts including the stem bark of S. birrea but no find-ings are recorded for the antioxidant activity of the acetoneextract of the mature stem bark of S. birrea considering thatsuccessful isolation of biocompounds from plant materialis largely dependent on the type of solvent used in theextraction procedure as well as the plant part [10].

Microbial resistance to prevailing drugs remains an ever-growing challenge [12]. Bacterial species such as Aeromonashydrophila (A. hydrophila), Salmonella typhimurium (S. typh-imurium), and Plesiomonas shigelloides (P. shigelloides) areGram-negative organisms that cause gastrointestinal infec-tions while Streptococcus pyogenes (S. pyogenes) is a Gram-positive organism that causes respiratory infections; theyhave all been reported to be resistant to a number of antibi-otics [13, 14]. Furthermore, opportunistic fungi species likeCryptococcus neoformans (C. neoformans), Candida glabrata(C. glabrata), Trichosporon mucoides (T. mucoides), and Can-dida krusei (C. krusei), which have been significantly asso-ciated with immune-compromised individuals have beennoted to have an evolved resistance to drugs [10]. Thebiological activity of S. birrea has been described by severalresearchers but reports on the antimicrobial activity of theaqueous and acetone extracts of this plant on bacteria andfungi agents are limited to some species of microorganismsdespite the universal usage of this plant in traditional medi-cine [9–11, 15]. This study was therefore aimed at examiningthe polyphenolic content and antioxidative potential of theacetone extract of mature stem bark of S. birrea as wellas the bioactivity of the acetone and aqueous extracts onsome bacteria and fungi species of medical importance asa guide for the valorisation and authentication of its use incomplementary and alternative medicine.


2.1. Preparation of Plant Material. The stem bark of S. birreawas harvested from different trees at Nzhelele and trans-ported to the University of Fort Hare. Identification of theplant was carried out by botanists at the School of BiologicalSciences, University of Fort Hare, Alice with vouchersdeposited at the school’s herbarium (GEUFH01). The plantpart was washed with tap water, chopped into small pieces,and dried at 40◦C for one week in a hot air oven (Memment854, Western Germany). The dried plant material was pow-dered using a blender (ATO MSE mix, England). Driedpowdered plant material was further macerated in acetoneand water, respectively, and crude extract of the plant wasobtained as previously described [11].

2.2. Determination of Antimicrobial Activity of Mature StemBark of S. birrea. The antimicrobial activity of the maturestem bark of S. birrea was evaluated against four referencestrains of bacteria (Streptococcus pyogenes ATCC 49399, Ple-siomonas shigelloides ATCC 51903, Aeromonas hydrophilaATCC 35654 and Salmonella typhimurium ATCC 13311), andfungi (Cryptococcus neoformans ATCC 66031, Candida gla-brata ATCC 2001, Trichosporon mucoides ATCC 201382, andCandida krusei ATCC 14243), respectively. These organismswere selected based on their disease burden and increasingtrend of antibiotic resistance in the developing world [13, 14,16].

The agar well diffusion and broth microdilution methodswere used to determine the antibacterial and antifungalactivities of the acetone and aqueous extracts against thebacteria and opportunistic fungi [16]. Briefly, inocula of thebacteria and fungi species were prepared and adjusted to 0.5McFarland turbidity standards. This was plated on MuellerHinton (MH) agar for the bacterial species and potatodextrose agar for the fungi species. Inocula were spread uni-formly on the plate and allowed to dry for 15 minutes. Wells(10 mm in diameter) were punched into the agar using sterilestainless steel borer and filled with 100 µL of the extract at100 mg/mL. Ciprofloxacin (0.05 µg/mL) was used as positivecontrol for the bacteria, while amphotericin B was used forthe fungi species; 10% dimethyl sulfoxide (DMSO) wasincluded in all experiments as negative controls. The plateswere incubated at 37◦C for 1 to 6 days depending on theorganism (bacteria and fungi) after which diameters of zonesof inhibition were measured in millimetres. The experimentwas done in duplicates. Inhibitory activity of the plant wasindicated by a clear zone of no microbial growth around eachwell.

2.3. Determination of Minimum Inhibitory Concentration.The minimum inhibitory concentration (MIC) assay wasperformed in 96 well plates [11]. Extract to be tested wasprepared at a concentration of 10 mg/mL. Briefly, twofoldserial dilution of the extract was carried out in the test wellsin MH broth with concentration ranging from 0.156 to10.00 mg/mL. Twenty microliter of an overnight broth cul-ture of test organism was added to 180 µL of extract-con-taining medium. Our controls were prepared with culture


medium, bacterial suspension and broth only. Ciprofloxacinwas used as positive control for the bacteria species whileamphotericin B was used as positive control for the fungispecies. ELISA plate reader (Model 680, Biorad, Tokyo,Japan) was used to measure the absorbance of the platesbefore and after one to six days of incubation at 37◦C de-pending on the organism type. Absorbances were read at620 nm and compared to check for microbial growth. Thelowest concentration that inhibited the growth of the organ-ism was considered as the MIC of the extract.

2.4. Polyphenolic Compounds

2.4.1. Total Phenol. The phenolic content of the acetoneextract was determined spectrophotometrically by the FolinCiocalteu modified method [17]. Briefly, an aliquot of theextract (1 mL) was mixed with 5 mL of 10% Folin-Ciocalteureagent and 4 mL of Na2CO3 (75% w/v). This mixture wasvortexed for 15 s and incubated at 40◦C for 30 min for colourdevelopment. The absorbance of the samples was measuredat 765 nm (UV-VIS, Spectrophotometer Hewlett Packard,NJ, USA). The measurements were conducted in triplicateand the results reported as mean ± SD values. The result wasexpressed as mg/g tannic acid equivalent from the calibrationcurve.

2.4.2. Total Flavonoids. Total flavonoid was estimated usingthe method of OrdonEz [18]. This was based on theformation of a complex flavonoid-aluminium. A volume of0.5 mL of 2% AlCl3 ethanol solution was added to 0.5 mL ofextract solution. After one hour of incubation at room tem-perature, the absorbance was measured at 420 nm (UV-VISSpectrophotometer Hewlett, Packard, NJ, USA). All determi-nations were done in triplicate, and values were calculatedfrom calibration curve obtained from quercetin.

2.4.3. Total Flavonols. Determination of total flavonol con-tent was carried as previously described [19]. The reactionmixture consisted of 2.0 mL of the sample (acetone extract),2.0 mL of AlCl3 prepared in ethanol, and 3.0 mL of sodiumacetate (50 g/L) solution. The absorption at 440 nm was readafter 2.5 h at 20◦C. Total flavonoid content was calculated asquercetin (mg/g) equivalent from the calibration curve.

2.5. Determination of Reducing Power. The reducing powerof the acetone extract was evaluated according to the methodof Yen and Chen [20]. A volume of 1.0 mL of the extractprepared in distilled water and BHT and vitamin C (VIT C)(0–5.0 mg/mL) were mixed individually with the mixturecontaining 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and2.5 mL of potassium ferricyanide [K3Fe(CN)6] (1% w/v).The resulting mixture was incubated at 50◦C for 20 min,followed by the addition of 2.5 mL of trichloroacetic acid(10% w/v), which was then centrifuged at 3000 rpm for10 min. The upper layer of the solution (2.5 mL) was mixedwith 2.5 mL of distilled water and 0.5 mL of ferrous chloride(0.1%, w/v). The absorbance was measured at 700 nm againsta blank sample.

2.6. DPPH Radical Scavenging Activity. The determinationof scavenging activity of DPPH free radical in the acetoneextract solution was executed using the method of Liyana-Pathiranan and Shahidi [21]. A solution of 0.135 mM DPPHin methanol was prepared, and 1.0 mL of this solution wasmixed with 1.0 mL of extract prepared in methanol contain-ing 0.025–0.5 mg of the plant extract and standard drugsseparately (BHT and VITC). The reaction mixture was vor-texed thoroughly and left in the dark at room temperaturefor 30 min. The absorbance of the mixture was measuredspectrophotometrically at 517 nm as above. The ability of theplant extract to scavenge DPPH radical was calculated by theequation

DPPH radical scavenging activity

= {(Abs control− Abs sample)/(Abs control)

}× 100,(1)

where Abs control is the absorbance of DPPH radical +methanol Abs sample is the absorbance of DPPH radical +sample extract or standard.

2.7. ABTS Radical Scavenging Activity. This was done usingan antioxidant assay kit (Sigma, Germany, catalog no.CS0790). The principle of the antioxidant assay rests on theformation of a ferryl myoglobin radical from metmyoglobinand hydrogen peroxide, which oxidises the ABTS to producea radical cation. The trolox working solution was prepared bymixing the trolox standard and 2.67 mL and 1x assay buffer.The reconstituted solution (1.5 mM) was used to preparethe trolox standard curve. Briefly, concentrations of troloxstandard (0, 0.015, 0.045, 0.105, 0.21, and 0.42 nm) wereprepared using assay buffer. In the wells of the trolox curve,10 µL of a trolox standard and 20 µL of myoglobin workingsolution were mixed, while, in the wells of the test samples,10 µL of the test sample (acetone extract of S. birrea) and20 µL of myoglobin working solution were introduced andmixed. ABTS substrate working solution was added, themixture incubated at room temperature, and the reactionabrogated using a stop solution. Absorbance was read at405 nm.

2.8. Statistical Analysis. The experimental results were ex-pressed as mean± standard deviation (SD) of three replicatesand were subjected to paired Student’s t-test. Significantlevels were tested at P < 0.05.

3. Results

3.1. Antimicrobial Activity of Crude Extracts. The acetone andaqueous crude extracts of S. birrea at the different concentra-tions (100 mg/mL and 50 mg/mL) demonstrated antimicro-bial activity against all the microorganisms studied (Table 1).An inhibition zone diameter of ≥11 mm was chosen as abreakpoint for susceptibility [11]. S. pyogenes and P. shigel-loides were the most susceptible organisms to all extractswhile Salmonella typhimurium was the least susceptible withpartial zones of inhibition. A zone diameter of inhibition


Table 1: Zone of inhibition ± SD (mm) of the solvent extracts (mg/mL) of the stem bark of S. birrea against organisms.

OrganismAcetone Aqueous

100 50 100 50

S. pyogenes 27± 2.1 25± 0.7 22± 0.9 20± 0.8

A. hydrophila 20± 0.0 17± 1.2 20± 0.0 17± 1.6

P. shigelloides 26± 4.9 23± 2.1 26± 2.8 22± 0.7

S. typhimurium 16± 1.3 14± 2.3 18± 2.9 16± 0.7

C. neoformans 15± 2.1 14± 1.6 25± 0.7 19± 0.8

C. glabrata 17± 1.2 15± 2.3 20± 0.0 18± 1.6

T. mucoides 20± 4.1 18± 2.2 23± 2.9 21± 0.8

C. krusei 16± 1.5 14± 2.1 14± 2.8 12± 0.7

Table 2: MIC values in mg/mL of acetone and aqueous extracts of mature stem bark of S. birrea including the positive controls (ciprofloxacinand amphotericin B) against the selected microorganisms.

MicroorganismMinimum inhibitory concentration (mg/mL)

CiprofloxacinAcetone Aqueous

S. pyogenes 0.31250 0.62500 0.00396

A. hydrophila 0.15600 0.15600 0.01510

P. shigelloides 0.31250 1.25000 0.03120

S. typhimurium 0.62500 0.62500 0.15600

Amphotericin B

C. neoformans 0.31250 1.25000 0.06250

C. glabrata 0.31250 0.62500 0.02500

T. mucoides 0.31250 0.62500 0.02500

C. krusei 0.15600 0.31250 0.03125

of 27 ± 2.1 mm was recorded for S. pyogenes at 100 mg/mL.The acetone extract demonstrated good activity when com-pared to aqueous against all the bacterial species tested exceptfor Salmonella typhimurium where the aqueous extractshowed better activity at both concentrations (Table 1). Ci-profloxacin (0.025 mg/mL), the positive control, had a zonediameter of inhibition of 21–38 mm; the negative control(10% DMSO) showed no activity against our isolates. Thezones of inhibition of the extracts and antibiotic were com-pared; no statistically significant difference was observed(P > 0.05).

For the fungi isolates, the aqueous extract demonstrateda better activity when compared to the acetone extract withthe most susceptible organism being C. neoformans followedby T. mucoides with zone diameter of 25 ± 0.7 mm and 23 ±2.9 mm, respectively, at 100 mg/mL while the least activitywas reported for C. krusei. However, the acetone extractshowed a better activity against C. krusei when compared tothe aqueous extract at both concentrations.

3.2. MIC Determination. MIC of the extracts was deter-mined against the organisms with ciprofloxacin as the posi-tive control for the bacteria while amphotericin B was usedfor the fungi species. The acetone extract was the most activewith regard to all the bacterial species tested presenting withoverall smaller MIC values ranging from 0.156 to 0.625 mg/mL when compared to the aqueous extract, which had asMIC value 0.156 to 1.25 mg/mL. MIC value for the positive

control, ciprofloxacin, ranged from 0.00396 to 0.156 mg/mL.Of the bacterial species tested, Aeromonas hydrophila was themost sensitive (Table 2). For the fungi species, MIC valuesranged from 0.3125 to 1.25 mg/mL for the aqueous extract,which was less active when compared to the acetone extract(0.156 to 0.3125 mg/mL). The MIC of amphotericin Branged from 0.025 to 0.625 mg/mL. C. krusei was the mostsensitive of the fungi species tested (Table 2). However, therewas no statistically significant difference between the MIC ofthe extracts and that of the control antibiotic (P > 0.05).

3.3. Polyphenolic Compounds. Since the acetone extract wasmore active than the aqueous extract, it was assayed for poly-phenolic and antioxidant potential. The plant extract pos-sessed high phenol contents (27.2 mg/g tannic acid equiva-lent) followed by flavonoid (25.2 mg/g quercetin equivalent)and flavonols (9.1 mg/g quercetin equivalent). Phenolic com-pounds, especially flavonoids and phenols, have been shownto possess significant antioxidant activity.

3.4. Antioxidant Activity. The DPPH is a stable free radicalthat gives a strong absorption maximum at 517 nm emittinga purple colour. Absorbance decreases as a result of a colourchange from purple to yellow when DPPH radical is reducedby hydrogen from a free radical scavenging antioxidant toform the reduced DPPH-H. Figure 1 illustrates the DPPHradical scavenging activity of mature stem bark of S. birreacompared with VITC and BHT. The DPPH % inhibition


0

20

40

60

80

100

120

0 0.025 0.05 0.1 0.2 0.5

SampleVITCBHT

Concentration (mg/mL)

DP

PH

inh

ibit

ion

(%)

Figure 1: DPPH radical scavenging activity of the acetone bark ex-tract of S. birrea.

increased as the plant extract concentration increased (0,0.025, 0.05, 0.1, 0.2, and 0.5 mg/mL). As observed, the plantextract possessed tremendous DPPH radical scavengingactivity. Interestingly, the activity of the plant was verysimilar to that of VITC but higher than that of BHT.

In addition, antioxidant potency of the plant extract wasfurther evaluated using ABTS (2, 2′azino-bis (3-ethyebenz-thiazoline-6-sulfonic acid)). Figure 2 shows the radical scav-enging ability of the extract. As noted, an increase in theconcentration of the extract (0.015, 0.045, 0.105 0.21, and0.42 mg/mL) resulted in an increase of the absorbance. Ourplant extract demonstrated a better antioxidant activity whencompared to Trolox.

The plant extract was also evaluated for its ability toreduce iron (III) to iron (II) and compared with VITC andBHT standards (Figure 3). The reducing value of the extractwas significantly lower than that of BHT and vitamin C.Generally, as the concentration (plant extract, VITC, andBHT) increased from 0 to 0.5 mg/mL, the absorbance alsoincreased but at no point was the absorbance of plant extracthigher when compared to the reference drugs.

4. Discussion

Phytochemical compounds in plants are known to be bio-logically active aiding as antioxidants and antimicrobials [9].The problem of drug resistance seems an overwhelming chal-lenge. There has been an increasing trend towards usingmedicinal plants to treat various diseases, especially in devel-oping countries [22]. S. birrea has long been used in sub-Saharan Africa as a medicinal remedy for numerous ailments[9, 11] and it is believed to possess several therapeutic prop-erties. The effect of acetone and aqueous extracts of maturestem bark was tested on some microbes and the largestinhibitory zone diameter was recorded for the acetone extractagainst all the bacterial strains compared with the aqueousextract implying that acetone could be a better solvent forextraction of phytochemicals (Table 1). The efficiency of ace-tone in the extraction of phytochemicals has been reported

0

2

4

6

8

10

12

14

16

18

00.015 0.045 0.105 0.21 0.42

SampleTrolox

Concentration (µM)

Abs

orba

nce

(405

nm

)

Figure 2: ABTS radical scavenging activity of the acetone barkextract of S. birrea.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 0.025 0.05 0.1 0.2 0.5

SampleVITCBHT

Concentration (mg/mL)

Abs

orba

nce

(700

nm

)

Figure 3: Total ferric reducing potential of acetone bark extract ofS. birrea.

by some authors [9, 11]. The concentration 100 mg/mL dem-onstrated a better activity against the bacterial strains,though higher than 50 mg/mL which would be more bene-ficial as a prospective antimicrobial. Though S. pyogenes andP. shigelloides were the most susceptible of the bacteria eval-uated, other bacterial species were susceptible to both theacetone and aqueous extracts. Our results are consistent withthe findings of Eloff [9] who reported antimicrobial activityof both the bark and leaf extracts of S. birrea though notagainst the microorganisms we studied. Our results alsocorroborate the findings of Braca et al. [23] who documentedplant parts of S. birrea, namely, the leaves, roots, and barkused to treat several conditions including diarrhoea, hyper-tension, diabetes, dysentery, and inflammations to havesignificant antimicrobial effect. For the fungi species, theaqueous extract demonstrated a better antimicrobial activitycompared to the acetone extract with C. neoformans followedby T. mucoides being the most susceptible species. All fungi


species were susceptible to both acetone and aqueous extractsagain at a concentration of 100 mg/mL demonstrating betteractivity. This is in line with the finding of Masoko et al. [10]who found S. birrea to posses antifungal activity.

Interesting MIC values were observed for the differentsolvent extracts of S. birrea against the various bacterialspecies (Table 2). Ciprofloxacin used as the positive controlinhibited growth of all the bacterial species (MIC 0.00396–0.156 mg/mL). There was no statistically significant differ-ence between the MIC of extracts against bacterial speciesand that of ciprofloxacin (P > 0.05). The acetone extract wasvery active against all the tested pathogens except for Salmo-nella typhimurium where the aqueous extract showed a betteractivity. This is in line with the study of Eloff [9] who report-ed antibacterial activity of S. birrea leaves and bark extractagainst some Gram-positive and Gram-negative bacteria.

Also, there was good activity of the different solventextracts of S. birrea against the fungi species studied with nosignificant difference observed (P > 0.05). Amphotericin Bused as positive control had an MIC of 0.025–0.0625 mg/mL.The aqueous extract of S. birrea was very active against all thetested pathogens except for C. krusei where the acetoneextract showed a better activity. Our results are consistentwith the findings of Hamza et al. [24] who reported inhibi-tion of C. glabrata, C. krusei, and Cryptococcus neoformans bymethanolic (an organic solvent) extracts of S. birrea roots.Based on MIC values, the acetone extract of mature stembark of S. birrea would be a useful source for treating ail-ments caused by bacteria and fungi.

Polyphenols are the major plant compounds with high-level of antioxidant activity. Their ability to act as antioxidantrest on the fact that they are able to absorb, neutralize, andquench free radicals [7]. We evaluated the acetone extract ofmature stem bark of S. birrea for its polyphenolic (flavonoids,flavonols, and phenols) contents and antioxidant and anti-microbial activities of both acetone and aqueous extracts onsome selected microorganisms.

The plant extract possessed high polyphenolic contentwith phenols having the highest content followed by flavon-oid and flavonols. Our results are in line with the reportof Mariod et al. [4] who reported high total phenolic com-pounds in S. birrea bark although these authors did not iden-tify if they used mature or young stem bark of S. birrea andthe specific solvent used for extraction given that differentsolvents as well as plant parts may impact on the biologicalactivity differently [10].

The acetone extract demonstrated strong DPPH radicalscavenging activity and ABTS antioxidant activity (Figures 1and 2). Our plant demonstrated enormous activity, whichwas comparable to that of VITC a well-known standardantioxidant. These results are in line with the finding ofPretorius et al. [25] who reported that fruits harvested fromS. birrea are rich in vitamin C, hence it is not surprisingthat the stem bark is rich in vitamin C. Interestingly, theantioxidant activity of our extract was better than that ofBHT, a commercial antioxidant. However, Mariod et al. [4]reported antioxidant activity in the extract of S. birrea to berelative to that of BHA (butylated hydroxyanisole), a com-mercial antioxidant in foods. The scavenging activity of our

extract was better than the Trolox standard in the ABTS assay.The high polyphenolic (flavonoids, flavones and phenols)content in the extract might be associated to the high-level antioxidant found with the DPPH and ABTS assays.The reducing ability of the acetone extract of S. birrea wasdetermined by measuring the conversion of Fe+3 to Fe+2. Theobserved result showed that the extract possessed antioxidantactivity in a concentration-dependent manner. This effectmay suggest the ability of S. birrea to minimize oxidativedamage to some vital tissues in the body [26].

DPPH radical is a model of a stable lipophilic radical. Achain reaction in lipophilic radical is known to be initiated bylipid autoxidation [27]. The antioxidant reacts with DPPHradical, reducing the number of DPPH radical moleculesequal to the number of their available hydroxyl groups. Plantphenolics constitute one of the major groups of compoundsacting as primary antioxidant or free radical terminatorsand initiating oxidation processes [4, 28]. Synergism of thepolyphenolic compounds with each other in the plant extractmay contribute to its antioxidant activity [29]. The differ-ences in scavenging activity of the testing systems could bedue to the different mechanisms involved in the radical-anti-oxidant reaction. For example, the solubility of the extractsin different testing systems, substrate used, and quantizationmethod may influence the ability of herbs to quench differentradicals [30]. As a result, it may be difficult to compare anti-oxidant activity based on antioxidant assay because of thedifferent test system and the substrate to be protected [31].

In conclusion, polyphenolic compounds are present inthe acetone extract of S. birrea, which could serve as a poten-tial natural antioxidant. In addition, the extracts of this plantpossess significant antibacterial and antifungal potential,which could provide an affordable and effective platform fornewer drugs.

Acknowledgments

The authors are grateful to the Govan Mbeki Research andDevelopment Center, University of Fort Hare, Alice, SouthAfrica, and the National Research Foundation (NRF) (GrantReference CSUR 2008052900010), South Africa for financialsupport. Special thanks are due to Samie A and Njume C fortechnical assistance.

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Research Article

Anticancer Activity of Certain Herbs and Spices onthe Cervical Epithelial Carcinoma (HeLa) Cell Line

Danielle Berrington and Namrita Lall

Department of Plant Sciences, University of Pretoria, Pretoria 0002, South Africa

Correspondence should be addressed to Namrita Lall, [email protected]

Received 20 January 2012; Accepted 25 February 2012


Copyright © 2012 D. Berrington and N. Lall. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkeykidney (Vero) cell line and an adenocarcinoma cervical cancer (HeLa) cell line. The plants studied were Origanum vulgare L.(Oregano), Rosmarinus officinalis L. (Upright and ground cove rosemary), Lavandula spica L. (Lavender), Laurus nobilis L. (Bayleaf), Thymus vulgaris L. (Thyme), Lavandula x intermedia L. (Margaret Roberts Lavender), Petroselinum crispum Mill. (Curlyleaved parsley), Foeniculum vulgare Mill. (Fennel), and Capsicum annuum L. (Paprika). Antioxidant activity was determined usinga quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl) assay. The rosemary species exhibited effective radical scavenging capacitywith 50% inhibitory concentration (IC50) of 3.48 ± 0.218μg/mL and 10.84 ± 0.125μg/mL and vitamin C equivalents of 0.351 gand 1.09 g for McConnell’s Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) colorimetric assay. Only L. nobilis andO. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC50 of 34.46 ± 0.48μg/mL and126.3±1.00μg/mL on the HeLa cells and on the Vero cells 124.1 μg/mL± 18.26 and 163.8 μg/mL± 2.95 for L. nobilis and O. vulgare,respectively. Light (eosin and haematoxylin staining) and confocal microscopy (Hoechst 33342, acridine orange, and propidiumiodide staining) were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare.

1. Introduction

South Africa has a remarkable floral and cultural diversitywith an estimate of 30,000 species of higher plants, whichare mostly endemic to the region. A great number of thesespecies are used for traditional medicinal purposes whereZulu traditional healers use around 1032 different speciesfrom 147 various plant families [1]. It is estimated thataround 27 million South African citizens rely on thesetraditional medicines for their primary health care needs dueto the inaccessibility of western medicine to some regionsand the high cost [2]. Therefore, the demand for traditionalmedicines remains high in rural parts [1].

According to the World Health Organization (WHO)cancer is one of the leading causes of death worldwide, whichaccounted for 7.6 million deaths (around 13%) of the world’spopulation in 2008. They have furthermore estimated thatthe worldwide deaths are likely to rise to over 11 million

in 2030. The Cancer Association of South Africa (CANSA)has estimated that one in every six South African males andone in every seven South African females are diagnosed withcancer. One of the most prevalent types of cancer foundin South African females is cervical cancer which is alsothe most widespread cancer found in South African blackwomen and in poor-income countries.

Cervical cancer is caused by the Human Papilloma Virus(HPV) which forms warts in the throat and genital area.Although it is the most prevalent cancer in South Africanblack women it is also the most preventable cancer throughscreening and treatment of precancerous lesion and theuse of vaccines. Natural products are being tested for thetreatment of cancer as conventional cancer treatments suchas chemotherapy destroy cancerous as well as healthy cells.

In South Africa the use of plants for the treatment ofcancer is not common [3]. There is, however, a SouthernAfrican plant currently undergoing clinical trials for the


treatment of cancer. The plant is Combretum caffrum, morecommonly known as the “bush willow” which causes theshutdown of tumours and tumour necrosis [4]. Herbs andspices have been used for thousands of years as medicines.Through scientific research it is indicated that the bioactivecomponents present in herbs and spices can reduce therisk of cancer through their antimicrobial, antioxidant,and antitumorogenic activity and their ability to directlysuppress carcinogen bioactivation [5]. In this study commonherbs and spices were tested for their cytotoxic activity oncervical cancer (HeLa) cells. These plants were chosen asthey are readily available in South Africa and because theyare traditionally used all over Africa. This study will beused as a comparative study on future research which willfocus mainly on indigenous South African plants and theircytotoxic activity on cervical cancer cells.


2.1. Chemicals and Reagents. DPPH, ascorbic acid, Bouin’sfixative, haematoxylin, eosin, xylene, Hoechst 33342, propid-ium iodide, and N-propyl-gallate were of analytical gradeand supplied by Sigma Aldrich (St. Louis, MO, USA). Allcell lines, media, trypsin-EDTA, fetal bovine serum (FBS),phosphate buffer saline (PBS), antibiotics, and glycerol weresupplied by Highveld Biological (Pty) Ltd. (Modderfontein,Johannesburg, RSA).

2.2. Plant Material. All plant material was collected duringFebruary 2011 in Pretoria, Gauteng. The plants were identi-fied by the University of Pretoria Botanical Garden curator,Jason Sampson. The collected aerial parts of the plants wereshade-dried for two weeks.

2.3. Preparation of Acetone Extracts. The air-dried aerialparts of the plant were mechanically ground to produce a finepowder. The weighed samples were extracted with acetonefor 48 h and thereafter for 24 h using fresh solvent. The soluteof each plant was filtered with a Buchner funnel. Thereafterthe menstruum was evaporated to dryness using a vacuumrotary evaporator to give a dark green extract.

2.4. Antioxidant Activity—DPPH Assay. The method of duToit et al. [6] was followed to determine the radical scav-enging capacity (RSC) of the extracts. Slight modificationsto the method were made and are described briefly. Stocksolutions of vitamin C and the extracts were prepared atconcentrations of 2 mg/mL and 10 mg/mL, respectively. Toeach well in the top row of a 96-well plate, 200 μL ofdistilled water was added. To the rest of the wells 110 μL ofdistilled water was added as a medium. Twenty microlitres ofextract was added to the first top wells, in triplicate, followedby serial dilution with final concentrations ranging from3.9 μg/mL to 500 μg/mL for the extracts and 0.781 μg/mL to100 μg/mL for vitamin C [7]. Finally 90 μL of 0.04 M DPPHethanolic solution was added to each well, except for thenegative control where distilled water was added instead. Theplates were left in a dark room to develop for 30 minutes.

The RSC of the extracts was determined using a BIO-TEKPower-Wave XS multiplate reader at a wavelength of 515 nm,using KC junior software. The IC50 values of each extractwere calculated using GraphPad Prism 4 software. Lastly,the vitamin C equivalent for each extract was calculatedas follows: (IC50 of extract × 200 mg vitamin C)/IC50 ofvitamin C.

2.5. Cell Culture. The HeLa and Vero cell lines weremaintained in culture flasks containing Eagle’s MinimumEssentail Medium supplemented with 10% heat-inactivatedFBS and 1% antibiotics (100 U/mL penicillin, 100 μg/mLstreptomycin and 250 μg/mL fungizone). The cells weregrown at 37◦C in a humidified incubator set at 5% CO2. Cellswere subcultured after they formed a monolayer on the flask.The cells were detached by treating them with trypsin-EDTA(0.25% trypsin containing 0.01% EDTA) for 10 minutes andthen by adding complete medium to inhibit the reaction.

2.6. In Vitro Cytotoxicity Assay. Cytotoxicity was measuredby the XTT method using the Cell Proliferation Kit II. Themethod described by Zheng et al. [8] was used to performthe assay. Both HeLa and Vero cells were seeded (100 μL) ina 96-well microtitre plate (concentration 1 × 105 cells/mL).The plate was then incubated for 24 h at 37◦C and 5% CO2

to allow the cells to attach to the bottom of the wells. Theextracts were each prepared to a stock solution of 20 mg/mLand added to the microtitre plate. Serial dilutions were madeto range from a concentration of 400 μg/mL–1.563 μg/mLfor each extract. The microtitre plate was incubated for afurther 72 h. The control wells included vehicle-treated cellsexposed to 2% DMSO (sample concentration of 400 μg/mL)and the positive control Actinomycin D with concentrationsranging between 0.5 μg/mL and 0.002 μg/mL. After the 72 hincubation period, the XTT reagent (50 μL) was added toa final concentration of 0.3 mg/mL and the plate was thenfurther incubated for another 2 hours. After the incubationthe absorbance of the colour complex was read at 490 nmwith a reference wavelength set at 690 nm using a BIO-TEK Power-Wave XS multiwell plate reader. The assay wasperformed in triplicate to calculate an IC50 of the cellpopulation for each extract. The results were analysed usingthe GraphPad Prism 4 program.

2.7. Light Microscopy (Haematoxylin and Eosin Staining).The cancerous HeLa cells were exposed to 34.46 μg/mL(IC50) and 68.92 μg/mL (2IC50) of the Laurus nobilis extractand 126.3 μg/mL (IC50) and 252.6 μg/mL (2IC50) of theOriganum vulgare extract. The Vero cells were exposed toconcentrations of 124.1 μg/mL (IC50), 248.2 μg/mL (2IC50)and 34.46 μg/mL (HeLa IC50) for Laurus nobilis and con-centrations of 163.8 μg/mL (IC50), 327.6 μg/mL (2IC50),and 126.3 μg/mL (HeLa IC50) for Origanum vulgare. Theconcentrations were chosen due to their antiproliferativeactivity that was observed at these concentrations. Cellswere seeded at 100,000 cells per well in a 6-well plateon heat-sterilized cover slips and incubated for 24 h at37◦C at 5% CO2 for cell adherence. The cells were then


exposed to the above concentrations including a vehicle-treated control (2% DMSO), actinomycin D (0.05 μg/mL) aswell as cells propagated in growth medium. The cells wereincubated for a further 72 h at 37◦C. After 72 h the cellswere fixed with Bouin’s fixative for 30 min and stained bystandard haematoxylin and eosin procedures [9]. Briefly, thecells were rinsed in 70% ethanol for 20 min and thereafterstained in haematoxylin for a further 20 min. The cells wererinsed again and stained for 2 min in 1% eosin solution.After several rinsing procedures with different percentagesof ethanol, the cells were left in xylene for 10 min. Thecover slips were mounted to the microscope slides with theremaining resin and evaluated using a Nikon Stereo Lightmicroscope equipped with a 1.4 Apo oil lens under 400xmagnification.

2.8. Confocal Microscopy (Hoechst 33342 and PropidiumIodide Staining). A double fluorescent dye staining methodwas used according to Stander et al. [9]. Exponentiallygrowing cells were seeded at 100,000 cells per well in a6-well plate onto heat-sterilized cover slips and incubatedfor 24 h at 37◦C and 5% CO2 to allow the cells to attach.After the incubation period the medium was discarded andthe cells were exposed to the same concentrations as usedin Section 2.7 and incubated for a further 72 h. After 72 hthe treatment was inhibited by discarding the extract andwashing the cells with PBS. The cells were stained with0.5 mL of Hoechst 33342 solution (3.5 μg/mL in PBS) andincubated for 30 min at 37◦C incubator. After 30 min theHoechst 33342 solution was discarded and the cells werethen stained with 0.5 mL of propidium iodide (40 μg/mL inPBS) and incubated for a further 5 min at 37◦C. Thereafterthe cover slips were mounted on microscope slides withmounting fluid (90% glycerol, 4% N-propyl-gallate, 6%PBS) and observed under a confocal microscope (ZEISS,LSM 510 Meta confocal) using 1000x magnification.

Microscopy studies were done in triplicate so that theappearance of apoptosis or normal cell proliferation couldbe confirmed. An alternative is that blind sampling can bedone whereby microscope slides are numbered and viewedunder the microscope without knowing which treatment wasapplied and thereafter confirming which treatment was done.


3.1. Determination of Antioxidant Activity. All extractsshowed dose-dependent responses with concentrations rang-ing from 500 μg/mL to 3.90 μg/mL. The highest inhibi-tion was shown at 500 μg/mL and the least inhibition at3.90 μg/mL. The rosemary species showed the most effectiveRSC as recorded in Table 1. Bubonja-Sonje et al. [10]conducted a study on the extracts of Rosmarinus officinalisL. to test for their free radical scavenging activity ofDPPH. These extracts of rosemary were prepared from amethanol : water : acetic acid (90 : 9 : 1, v/v/v) solvent. Theantioxidant content was attributed to the fact that rosemarycontained a very rich source of polyphenolic compounds,more specifically phenolic diterpenes, such as Carnosol and

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9 10

Mea

sure

men

ts

Plant extract number

Vitamin C equivalents (g)

IC50 (µg/mL)

Figure 1: Comparison of the IC50 values and the Vitamin Cequivalents of the plant extracts.

Carnosic acid. Specifically carnosic acid was tested for itsantioxidant activity and was determined to have an IC50

value of 27.3±5.2μg/mL. In the present study the antioxidantactivity of the McConnell’s Blue and Tuscan Blue extractswere found to be better than that of the rosemary extractinvestigated by Bubonja-Sonje et al. [10]. The lavenderspecies showed minimal inhibition of DPPH for both forLavandula x intermedia and Lavandula spica. In a studyconducted by Gulcin et al. [11] aerial parts of the plantLavandula stoechas L. were used to determine the free radicalscavenging activity. This part of the plant contained oleanolicacid, ursolic acid, vergatic acid, β-sitosterol, α-amyrin, α-amyrin acetate, lupeol, erythrodiol, flavanoids, luteolin,acacetin, and vitexin. They found that the ethanol extracthad an IC50 of 60 μg/mL, which was notably much moreeffective than the IC50 values of Lavandula-x-intermedia andLavandula spica. As no previous studies have been reportedon the antioxidant content of Lavandula-x-intermedia andLavandula spica it is difficult to determine whether thecompounds present in Lavandula stoechas L. are also presentin the extracts used in the present study. The other plantmaterials had RSC ranging from good to relatively goodwhich can be seen in Table 1 and Figure 1 together withthe vitamin C equivalents for each extract. The vitamin Cequivalent is a measurement of the amount extract (g) thatwould be needed to have a RSC that is equivalent to theRSC of 200 mg vitamin C. The vitamin C equivalents werecalculated according to the following equation: (IC50 extract× 200 mg vitamin C)/IC50 vitamin C. The positive controlvitamin C had an IC50 of 1.98± 0.006μg/mL.

3.2. Cytotoxicity of Extracts. All plant materials were testedfor their anticancer activity using the XTT colorimetric assay.The plant extracts were prepared and screened for their invitro cytotoxic effects against HeLa and Vero cells. The assayis based on the ability of living cells to reduce the yellow-water soluble XTT into an insoluble formazan product [12].The IC50 values were used to determine the selectivity indexes


Table 1: A summary of the IC50 values of the plant extracts and their vitamin C equivalents.

Plant extractsIC50 values

(μg/mL) ± SDVitamin C equivalents for a

200 mg capsule (in g)

(1) Rosmarinus officinalis (McConnell’s Blue) 3.48± 0.22 0.351

(2) Rosmarinus officinalis (Tuscan Blue) 10.84± 0.12 1.09

(3) Lavandula-x-intermedia (Margaret Roberts) 138.5± 4.0 14.0

(4) Lavandula spica 125.5± 1.8 12.67

(5) Origanum vulgare 26.43± 1.58 2.67

(6) Petroselinum crispum 53.3± 2.63 5.38

(7) Laurus nobilis 30.85± 0.48 3.12

(8) Thymus vulgaris 36.13± 0.85 3.65

(9) Foeniculum vulgaris 38.36± 0.76 3.88

(10) Capsicum annuum (Paprika) 63.06± 1.91 6.27

(11) Vitamin Ca 1.98± 0.01 —aPositive control for antioxidant assay.

Table 2: Summary of the IC50 values and selectivity indexes of the plant extracts.

Plant extractsHeLa ICa

50

(μg/mL) ± SDVero IC50

(μg/mL) ± SDSIb

Rosmarinus officinalis (McConnell’s Blue) >100 12.03± 0.02 0.12

Rosmarinus officinalis (Tuscan Blue) 23.31± 0.055 23.64± 0.10 0.52

Lavandula-x-intermedia (Margaret Roberts) 182± 1.5 31.5± 0.14 0.17

Lavandulaspica 98.99± 1.28 26.3± 0.64 0.27

Origanumvulgare 126.3± 1.00 163.8± 2.95 1.30

Petroselinumcrispum 172.7± 0.25 105± 0.50 0.61

Laurusnobilis 34.46± 0.48 124.1± 18.8 3.61

Thymusvulgaris >200 138.4± 2.60 0.66

Foeniculumvulgare 129.7± 2.05 85.37± 5.26 0.66

Capsicum annuum (Paprika) >200 122.3± 3.76 0.58

Actinomycin Dc 0.002 0.027 13.5aFifty percent inhibitory concentration.

bSelectivity index.cPositive control for cytotoxicity assay.

(SI) of each extract which represents the overall activity. SIvalues were calculated as follows: (IC50 of Vero/IC50 of HeLa).The cytotoxicity and SI of the ten plant extracts are shown inTable 2.

Laurus nobilis and Rosmarinus officinalis (Tuscan Blue)strongly inhibited the proliferation of the HeLa cells as seenin Table 2. The lowest toxicity was observed from the Thymusvulgaris and Capsicum annuum (Paprika) extracts. All otherextracts had IC50 values ranging from 98.99± 1.28μg/mL to182 ± 0.15μg/mL. The IC50 value of Origanum vulgare wasnoted and was used in further mechanistic studies togetherwith Laurus nobilis. Actinomycin D, a known anticanceragent, had an IC50 value of 0.002 ± 0.0000395μg/mL. Thetoxicity on the Vero cells ranged from a high toxicity with anIC50 value of 12.03 ± 0.02μg/mL for Rosmarinus officinalis(McConnell’s Blue) to a low toxicity with an IC50 value of163.8 ± 2.92μg/mL for Origanum vulgare. It is interesting

to note that the two rosemary species exhibited varyingcytotoxicity against the HeLa cells. The anticarcinogenicactivity of rosemary is due to the major bioactive compoundssuch as rosmarinic acid, carnosic acid, and carnosol [13].Tuscan blue rosemary could possibly have a higher concen-tration of these bioactive compounds and therefore showshigher toxicity on the HeLa cells. In a similar study wherecompounds extracted from Rosmarinus officinalis were testedon various cancer cell lines, such as NCI-H82 (small lungcarcinoma), DU-145 (prostate carcinoma), Hep3D (livercarcinoma), K-562 (chronic myelois carcinoma), MCF-7,(breast adenocarcinoma), PC-3 (prostate adenocarcinoma)and MDA-MB-231 (breast adenocarcinoma) the IC50 valuesranged from 8.82 μg/mL to over 100 μg/mL [14]. The lowesttoxicity towards the HeLa cell line was seen in the Capsicumannuum and Thymus vulgaris extracts. In an article publishedby Cancer Research in Capsicum was reported to help in the


treatment of prostate cancer. Furthermore the essential oilof Thymus vulgaris was tested on head and neck squamouscell carcinoma (HNSCC) by Sertel et al. [15] and was foundto have a very low toxicity with an IC50 value of 369 μg/mL.The positive control actinomycin D showed an IC50 value of0.027± 0.00021μg/mL.

Extracts which showed SI values equal or greater thanone were chosen for further mechanistic studies. Fromthe SI values summarized in Table 2 it was noted thatOriganum vulgare and Laurus nobilis had SI values whichwere greater than one. When considering the affectivity ofthese extracts on HeLa cells it was noted that Laurus nobilishad the highest toxicity, whereas Origanum vulgare had amuch lower toxicity. When considering the toxicity of theseextracts towards the Vero cells, it is preferred that there isvery low toxicity as these cells are used as a comparisonto normal human cells. Origanum vulgare had the lowesttoxicity, whereas Laurus nobilis had a slightly higher toxicity.Both these extracts were chosen for further mechanisticstudies due to their SI values; however Origanum vulgareis not very effective against the HeLa cell line. However ina study conducted by Al-Kalaldeh et al. [16] the volatileoil extract of Origanum vulgare was very effective againsthuman breast adenocarcinoma cells (MCF7) with an IC50 of30.1 ± 1.14μg/mL. In the same study an ethanolic extract ofLaurus nobilis showed an IC50 of 24.49 μg/mL.

3.3. Light Microscopy. Laurus nobilis and Origanum vulgarewere qualitatively analyzed at their IC50 and 2IC50 valuesto investigate their influence on the HeLa cells and on theVero cells. The mechanism of action of both extracts wasdetermined by observing the change in cell morphologyafter the exposure of the cell lines to the extracts andcomparing them to vehicle-treated controls and mediumcontrols. In Figures 2(a) and 2(b) where the cells werepropagated in medium and in the presence of DMSO(vehicle-treated), respectively, normal proliferation seemedto take place and typical morphology was observed. InFigure 2(a) anaphase and interphase was observed whereas inFigure 2(b) metaphase and interphase was observed, whichare typical characteristics of the cell cycle. However in Figures2(c) and 2(d) normal cell proliferation was absent and signsof cell death and growth disorders started to appear. Inthese photographs the HeLa cells were exposed to the IC50

and 2IC50 values of Laurus nobilis extract. In both thesephotographs typical morphological features of apoptosisstarted to appear such as hypercondensed chromatin andthe degredation of DNA. The positive control actinomycinD also showed features of apoptosis, such as the presence ofapoptotic bodies (Figure 2(e)).

Observing the Vero cells in the absence and presence ofLaurus nobilis and actinomycin D (Figure 3) similar mor-phological changes were seen as in Figure 2. When the cellswere grown in medium only and in the presence of DMSOnormal cell proliferation took place such as metaphase(Figures 3(a) and 3(b)) and interphase (Figure 3(a)). Inthe photographs where the cells were exposed to the IC50

and 2IC50 values of Laurus nobilis changes in cellular

morphology started to take place. In both Figures 3(c) and3(d) cellular debris was observed which could be as a resultof lysis of apoptotic bodies. Furthermore the presence ofhypercondensed chromatin was seen in Figure 3(d). In thepresence of actinomycin D (Figure 3(f)) hypercondensedchromatin was also visible. An important observation wasmade in Figure 3(e) where the Vero cells were treated with theIC50 dose of Laurus nobilis on HeLa cells. In this photographnormal proliferation took place as seen by the presenceof metaphase. It was important to observe that the Verocells revealed less prominent features of cell death whenthey were treated at the IC50 of Laurus nobilis on HeLacells.

In the HeLa cell line when the cells were grown inmedium only and exposed to DMSO, normal cell cycle mor-phology was viewed. In Figure 4(a) anaphase was observedwhereas in Figure 4(b) cytokinesis was observed, which isthe last step in normal cell cycle before two daughter cellsare formed. In Figures 4(c) and 4(d) when cells were treatedat the IC50 and 2IC50 values of Origanum vulgare, apoptoticmorphological features were viewed such as hypercondensedchromatin and in the presence of actinomycin D apoptoticbodies were observed. Therefore when HeLa cells weretreated with the Origanum vulgare extract there was anincrease in morphological features which correlated withapoptosis.

In the Vero cell line, vehicle-treated controls (Figure5(b)) and medium-only controls (Figure 5(a)) were viableand still showed signs of proliferation as seen by thepresence of metaphase in both photographs. ActinomycinD (Figure 5(f)) as well as cells treated with 163.8 μg/mLand 327.6 μg/mL of the Origanum vulgare extract (Figures5(c) and 5(d)) showed prominent signs of cell death occur-ring through apoptosis such as hypercondensed chromatin.In Figure 5(e) where the Vero cells were treated with126.3 μg/mL of Origanum vulgare normal cell proliferationtook place where anaphase was observed. This concentrationis where 50% of the HeLa cells were inhibited by the extract,but showed less prominent and negligible signs of toxicity tothe noncancerous cell line.

3.4. Confocal Microscopy. Change in morphological featureswhen the cancerous (HeLa) and noncancerous (Vero) celllines were exposed to various concentrations of the acetoneextracts of Laurus nobilis and Origanum vulgare were con-firmed when visualizing the cells under confocal microscopy.The HeLa cells showed normal signs of cell proliferationwhen grown in medium only or when exposed to 2% DMSOas seen in Figures 6(a) and 6(b) respectively. Normal cellgrowth and proliferation was confirmed by the presenceof metaphase and anaphase. Actinomycin D (Figure 6(e))showed clear signs of cell death with the presence ofhypercondensed chromatin. Laurus nobilis treated HeLa cellsrevealed an increase in morphological features which werecharacteristic of apoptosis, which included the presence ofhypercondensed chromatin and reduced cytoplasm (Figures6(c) and 6(d)). The reduction in cytoplasm could havebeen due to the detachment of cells during the time ofincubation.


Anaphase

Interphase

(a)

Metaphase

Interphase

(b)

Hypercondensed chromatin

(c)

Condensed and fragmented DNA

(d)

Apoptotic

bodies

(e)

Figure 2: Haematoxylin and eosin staining of HeLa medium only control cells (a), vehicle-treated control cells (b), 34.46 μg/mL Laurusnobilis treated cells (c), 68.92 μg/mL Laurus nobilis treated cells (d), and 0.05 μg/mL actinomycin D (e), after 72 h of exposure (400xmagnification).

Interphase

Metaphase

(a)

Metaphase

(b)

Cellular debris

(c)


Cellular debris

(d)

Metaphase

(e)


(f)

Figure 3: Haematoxylin and eosin staining of Vero medium only control cells (a), vehicle-treated control cells (b), 124.1 μg/mL Laurusnobilis treated cells (c), 248.2 μg/mL Laurus nobilis treated cells (d), 34.46 μg/mL Laurus nobilis treated cells (e), and 0.05 μg/mL actinomycinD (f), after 72 h of exposure (400x magnification).


Anaphase

Interphase

(a)

Cytokinesis

(b)


(c)


(d)

Apoptotic bodies

(e)

Figure 4: Haematoxylin and eosin staining of HeLa medium only control cells (a), vehicle-treated control cells (b), 126.3 μg/mL Origanumvulgare treated cells (c), 252.6 μg/mL Origanum vulgare treated cells (d), and 0.05 μg/mL actinomycin D (e), after 72 h of exposure (400xmagnification).

Metaphase

(a)

Metaphase

(b)


(c)

Hypercondensed

chromatin

(d)

Anaphase

(e)

Hypercondensed

chromatin

(f)

Figure 5: Haematoxylin and eosin staining of Vero medium only control cells (a), vehicle-treated control cells (b), 163.8 μg/mL Origanumvulgare treated cells (c), 327.6 μg/mL Origanum vulgare treated cells (d), 126.3 μg/mL Origanum vulgare treated cells (e), and 0.05 μg/mLactinomycin D (f), after 72 h of exposure (400x magnification).


Metaphase

(a)

Interphase

(b)

Hypercondensedchromatin

(c)


Reduced cytoplasm

(d)


(e)

Figure 6: Hoechst 33342 and propidium iodide staining of HeLa medium only control cells (a), vehicle-treated control cells (b), 34.46 μg/mLLaurus nobilis treated cells (c), 68.92 μg/mL Laurus nobilis treated cells (d), and 0.05 μg/mL actinomycin D (e), after 72 h of exposure (1000xmagnification).

Metaphase

(a)

Anaphase

(b)

Hypercondensed

chromatin

(c)

Cellular debris

(d)

Metaphase

(e)

Apoptotic bodies

(f)

Figure 7: Hoechst 33342 and propidium iodide staining of Vero medium only control cells (a), vehicle-treated control cells (b), 124.1 μg/mLLaurus nobilis treated cells (c), 248.2 μg/mL Laurus nobilis treated cells (d), 34.46 μg/mL Laurus nobilis treated cells (e), and 0.05 μg/mLactinomycin D (f), after 72 h of exposure (1000x magnification).


Metaphase

(a)

Interphase

(b)

Reduced

cytoplasm andfragmented DNA

(c)


Cell membrane

blebbing

(d)

Hypercondensed

chromatin

(e)

Figure 8: Hoechst 33342 and propidium staining of HeLa medium only control cells (a), vehicle-treated control cells (b), 126.3 μg/mLOriganum vulgare treated cells (c), 252.6 μg/mL Origanum vulgare treated cells (d), and 0.05 μg/mL actinomycin D (e), after 72 h of exposure(1000x magnification).

Metaphase

(a)

Interphase

(b)


(c)

Hypercondensed

chromatin

Cell membraneblebbing

(d)

Metaphase

(e)

Apoptotic bodies

(f)

Figure 9: Hoechst 33342 and propidium iodide staining of Vero medium only control cells (a), vehicle-treated control cells (b), 163.8 μg/mLOriganum vulgare treated cells (c), 327.6 μg/mL Origanum vulgare treated cells (d), 126.3 μg/mL Origanum vulgare treated cells (e), and0.05 μg/mL actinomycin D (f), after 72 h of exposure (1000x magnification).


In the Vero cells normal cell cycle features such asmetaphase (Figure 7(a)) and anaphase (Figure 7(b)) wereobserved indicating that there were no growth deformities.However in Figures 7(c) and 7(d) changes in morphologystarted to appear such as the presence of hypercondensedchromatin and cellular debris. Furthermore in the pres-ence of actinomycin D apoptotic bodies started to appear(Figure 7(f)). However in Figure 7(e) normal cell cyclefeatures were viewed such as metaphase which indicates thatat the IC50 concentration of Laurus nobilis on HeLa cells therewere less prominent cellular features of cell death on Verocells.

In the presence and absence of Origanum vulgare onHeLa cells different morphological features were observed.In untreated cells or cells treated with 2% DMSO promi-nent features corresponding with normal cell growth andproliferation was observed, which included metaphase(Figure 8(a)) and interphase (Figure 8(b)). However in cellstreated with 126.3 μg/mL and 252.6 μg/mL of Origanumvulgare severe signs of cell death were viewed. In cellstreated with an IC50 concentration features such as reducedcytoplasm and fragmented DNA was seen (Figure 8(c)),whereas in cells treated at a 2IC50 concentration featuresappeared such as hypercondensed chromatin and cellularmembrane blebbing (Figure 8(d)). These features tend tobe characteristics of apoptotic cell death. Actinomycin D(Figure 8(e)) showed similar signs of apoptosis with theappearance of hypercondensed chromatin.

Vero cells showed normal cell proliferation when exposedto medium only or 2% DMSO treated cells. Cell cyclefeatures such as metaphase (Figure 9(a)) and interphase(Figure 9(b)) were observed. Cells exposed to IC50 and 2IC50

concentrations showed distinct signs of apoptosis such ashypercondensed chromatin (Figures 9(c) and 9(d)) andcellular membrane blebbing (Figure 9(d)). In Figure 9(e)normal cell proliferation was observed as seen by thepresence of metaphase. Actinomycin D also showed morpho-logical features which were characteristic of apoptosis such asapoptotic bodies (Figure 9(f)).

The two microscopy techniques were in agreementwith one another. The haematoxylin and eosin staining isbased on differentiating the nuclei from the cytoplasm. Thehaematoxylin stains the nuclei blue/purple whereas the eosinstains the cytoplasm red. The confocal microscopy is basedon differentiating intact cells from those that have lost theirmembrane integrity. The Hoechst 33342 penetrates intactcell membranes of viable cells and those undergoing apop-tosis and therefore stains the nucleus. Propidium iodide canonly stain the nucleus of cells that have lost their membraneintegrity and therefore stain the nucleus of cells undergoinglate apoptosis and necrosis [9]. In both cell lines the extractsshowed morphological changes including apoptotic bodies,hypercondensed chromatin, reduced cytoplasm, and cellulardebris which were indicative of possible apoptosis takingplace [17, 18]. In the HeLa cell line the common feature ofapoptosis occurring was hypercondensed chromatin whereasin the Vero cell line cellular debris as well as hypercondensedchromatin seemed to occur more often.

4. Conclusion

In this study common herbs and spices were chosen todetermine their anticancer activity. Herbs and spices werechosen due to their bioactive components which havethe ability to reduce the risk of cancer through theirantimicrobial, antioxidant, and antitumourogenic activity[19]. The rosemary species were found to have the highestantioxidant content which could be attributed to the highcontent of polyphenolic compounds. Therefore the rosemaryspecies possess potential chemopreventive properties. Afterperforming the XTT cytotoxicity assay it was hypothesizedthat Laurus nobilis, with an SI of 3.61, and Oregano vulgare,with an SI of 1.30, were the best candidates for furtherinvestigation. Both these extracts were further investigatedto determine their mechanism of cell death by observingthem under light microscopy and confocal microscopy. Fromthe microscopy images it was observed that in both celllines morphological changes did appear after exposure tovarious concentrations of the acetone extracts. Althoughthese microscopy images were in agreement with one anotherthat apoptosis was the possible mechanism of cell death,as seen by the characteristic features of hypercondensedchromatin (mostly in HeLa cells) and cellular debris (seenonly in Vero cells), they are not sufficient enough to definitelyconfirm that apoptosis is taking place and therefore moresensitive assays such as flow cytometry need to Be usedto confirm that apoptosis is taking place. However Laurusnobilis did show promising results as an anticancer agent dueto its relatively high toxicity on the HeLa cell line and at thissame concentration a low toxicity on the Vero cell line.

Acknowledgments

This study was supported by grants from the University ofPretoria and National Research Foundation of South Africa.The authors thank Alan Hall and the Microscopy Unit fromthe University of Pretoria for helping with the microscopy.

References

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Research Article

Synergistic Antimycobacterial Actions ofKnowltonia vesicatoria (L.f) Sims

Antoinette Labuschagne,1 Ahmed A. Hussein,1, 2 Benjamın Rodrıguez,3 and Namrita Lall1

1 Department of Plant Science, University of Pretoria, Gauteng Pretoria 0002, South Africa2 Department of Chemistry of Medicinal Plants, National Research Center, El-Tahrir Street, Dokki, Cairo 12311, Egypt3 Instituto de Quımica Organica, Consejo Superior de Investigaciones Cientıficas (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain




Copyright © 2012 Antoinette Labuschagne et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Euclea natalensis A.DC., Knowltonia vesicatoria (L.f) Sims, and Pelargonium sidoides DC. are South African plants traditionallyused to treat tuberculosis. Extracts from these plants were used in combination with isoniazid (INH) to investigate the possibilityof synergy with respect to antimycobacterial activity. The ethanol extract of K. vesicatoria was subjected to fractionation to identifythe active compounds. The activity of the Knowltonia extract remained superior to the fractions with a minimum inhibitoryconcentration (MIC) of 625.0 μg/mL against Mycobacterium smegmatis and an MIC of 50.00 μg/mL against M. tuberculosis.The K. vesicatoria extract was tested against two different drug-resistant strains of M. tuberculosis, which resulted in an MIC of50.00 μg/mL on both strains. The combination of K. vesicatoria with INH exhibited the best synergistic antimycobacterial activitywith a fractional inhibitory concentration index of 0.25 (a combined concentration of 6.28 μg/mL). A fifty percent inhibitoryconcentration of this combination against U937 cells was 121.0 μg/mL. Two compounds, stigmasta-5,23-dien-3-ol (1) and 5-(hydroxymethyl)furan-2(5H)-one (2), were isolated from K. vesicatoria as the first report of isolation for both compounds fromthis plant and the first report of antimycobacterial activity. Compound (1) was active against drug-sensitive M. tuberculosis withan MIC of 50.00 μg/mL.

1. Introduction

For the last 40 years there has been little progress in thetreatment of Tuberculosis (TB). The standard albeit datedtreatment regime is strict and lengthy (6–9 months) resultingin adverse side effects and inevitable patient noncompliance.It is no surprise that the emergence of multiple and exten-sively drug resistant strains of Mycobacterium tuberculosis(M. tb) is on the rise. The WHO recently reported that insome areas of the world, one in four people with TB becomesill with a form of the disease that can no longer be treatedwith standard drugs [1]. In addition, HIV/AIDS increasesthe risk for developing active TB and renders TB difficultto diagnose and treat. The TB-HIV/AIDS coinfection ratein South Africa is distressingly high, with an estimated73 percent of new TB patients coinfected with HIV [2].The search for new TB treatments that are effective againstresistant strains of M. tb and treatments which can augment

the potential of existing drugs against the disease is moreimportant today than at any other time in history. Withoutthe introduction of new treatments, TB patients will run outof options for effective drugs.

Plant products have received considerable attention aspotential anti-TB agents with a recent review emphasizingplant products as sources of antimycobacterial extracts andcompounds [3]. Most traditionally used plant therapies relyfor their effects on a variety of compounds and synergybetween these compounds, however, there are numerousbenefits for isolating and identifying active constituents fromthese bioactive plants. These benefits include characterisingtoxicity profiles, simpler determination of modes of action,and new activities of known compound which adds tothe wealth of information on phytochemicals. Combiningplant extracts and current TB drugs holds advantagessuch as decreased toxicity profiles, increased bioavailability


and activity, and reduced onset of microbial resistance.Testing isolated compounds from plant extracts, using wholeextracts alone, and in combination with other extracts andcurrent anti-TB drugs, covers a wider range for activity andpossible treatment therapies. In this study the synergisticantimycobacterial activity of three South African plants aswell as their cytotoxicity and the isolation, identification, andantimycobacterial activity of compounds obtained from theethanol (EtOH) extract of Knowltonia vesicatoria is reported.

In previous experiments, the EtOH extract of K. vesi-catoria (aerial parts) was found to be active against bothMycobacterium smegmatis and M. tuberculosis [4]. Knowlto-nia vesicatoria (Ranunculaceae) is relatively new to the fieldof tuberculosis research, and in general very little work hasbeen done on this specific plant, even though traditionaluses related to TB have been documented [5]. Recentlythe genus Knowltonia has been subsumed within the genusAnemone, and K. vesicatoria is now known as Anemonevesicatoria (L.f) Prantl [6]. To avoid confusion the nameKnowltonia vesicatoria has been retained throughout thepaper. One of the objectives of the study was to identify theantimycobacterial compounds present in the K. vesicatoriaextract and to eliminate the possibility of tannins as theantimycobacterial actives. In addition, extracts preparedfrom two well-known South African plants, Pelargoniumsidoides DC. (root, EtOH) and Euclea natalensis A.DC.(root, chloroform (CHCl3)) were included in the study toinvestigate the possibility of synergistic antimycobacterialaction when combined with each other, K. vesicatoria, andthe first-line antitubercular drug, isoniazid (INH).

Pelargonium species (Geraniaceae) are highly valued bytraditional healers for their curative properties and arewellknown for treatment of coughs, diarrhoea, and tuber-culosis. A review by Brendler and van Wyk [7] discusses allaspects of Pelargonium sidoides, including a complete andcomprehensive take on the anti-TB, antibacterial, antifungaland immunomodulating activity of this valuable plant.

The roots of Euclea species (Ebenaceae) are used insouthern African traditional medicinal preparations to treatchest complaints, chronic asthma, leprosy, and infections,among other ailments [8–11]. Euclea natalensis is a familiarplant to TB research and the antimycobacterial activityagainst M. tb by extracts and compounds of Euclea natalensisroots has been previously reported [12], including thesynergistic activity of this extract with INH [13].


2.1. Plant Material. Knowltonia vesicatoria syn Anemonevesicatoria was procured in the province of Gauteng, SouthAfrica. The stems and leaves (aerial parts) were collectedduring January. Previously, roots of P. sidoides were collectedfrom the Free State province, and roots from E. natalensiswere collected from KwaZulu-Natal province of South Africa.A voucher specimen (PRU 096449, P 092559 and N.L.22, resp.) for each plant was deposited and identified atthe H.G.W.J. Schweickerdt Herbarium (PRU), University ofPretoria, South Africa.

2.2. Microorganisms and Cell Lines. Mycobacterium smeg-matis (MC2 155) cultures, obtained from, American TypeCulture Collection (ATCC), Culture Collection (ATCC),MD, USA were kindly donated from the Medical ResearchCouncil (MRC) in Pretoria, South Africa. The cultures werekept on Middlebrook 7H11 agar (Sigma-Aldrich ChemicalCo., South Africa) and stored at approximately 8◦C, for nolonger than one month. Several aliquots of the preparedmycobacterial cultures in Middlebrook 7H9 broth (Sigma-Aldrich Chemical Co., South Africa) were frozen in cryovialsat −70◦C.

Three M. tb strains were used in the experimentalprocedures, which were carried out at the Medical ResearchCouncil (MRC), Pretoria, South Africa. H37Rv (ATCC27294), a drug-susceptible strain of M. tb, sensitive tothe first-line antituberculous drugs, INH, rifampicin (RIF),ethambutol (EMB), and streptomycin (STR), was obtainedfrom the ATCC. Mycobacterium tuberculosis were plated ontoslants of Lowenstein-Jensen (LJ) medium and allowed togrow for 3-4 weeks at 37◦C. Two clinical drug resistantstrains, 4388 (resistant to INH and EMB) and 5497 (resistantto RIF, INH, EMB, STR) were maintained in BACTEC 12Bmedium (7H12 medium containing 14C-labeled substrate,palmitic acid) at 37◦C until a growth index (GI) of 400 wasreached. Both resistant strains were obtained from an MRCproficiency test (round 14) from Belgium. These resistantstrains were only used to test the activity of the K. vesicatoria(EtOH) extract.

The histiocytic lymphoma cell line, U937, was obtainedfrom Highveld Biological (Pty) (Ltd.) (Sandringham, SouthAfrica) and maintained in complete RPMI 1640 medium(pH 7.2) (Sigma-Aldrich Chemical Co., South Africa),supplemented with 10% heat-inactivated foetal calf serum(FCS), 2 mM L-glutamine, and a 0.1% antimicrobial solution(pennicilin, streptomycin, and an antifungal. fungizone).

2.3. Extraction and Isolation. Prepared extracts of Eucleanatalensis (CHCl3) and Pelargonium sidoides (EtOH) werekindly donated by Professor N. Lall. The collected aerialparts of K. vesicatoria were allowed to air-dry in opensample bags away from direct sunlight. The dried plantmaterial (530.0 g) was extracted with 2.50 L of 100% EtOH.The solvent and plant material was placed on an electricshaker and vigorously shaken by hand twice a day forseven days. Each day the plant material were filtered, theextract concentrated under reduced pressure with a rotaryevaporator, and clean EtOH was added to the remainingplant material. A total yield of 148.4 g of dried extract wasobtained (28% of the total dried plant material). Sixty gramsof the K. vesicatoria EtOH extract was subjected to a solventpartitioning method for the removal of polyphenolics [14].The dried EtOH extract was dissolved in 1.5 L of a 9 : 1EtOH : dH2O solution which was then partitioned with anequal volume of hexane. The hexane layer was concentratedunder reduced pressure and stored for further testing of thenonpolar components (2.35 g). A portion of the 90% EtOHlayer was concentrated under reduced pressure and storedfor further testing (11.88 g). The remaining EtOH layer


(750 mL) was partitioned with equal volumes of CHCl3 :methanole (MeOH) (4 : 1) and water. The two layers werethen separated. The EtOH layer was concentrated underreduced pressure and stored (13.27 g). The chloroformiclayer was then washed with an equal volume (750 mL) of 1%w/v NaCl in water. The chloroformic layer was then driedunder reduced pressure, yielding 9.86 g of polyphenol-freeextract. All the layers were screened against M. smegmatis andthe active layers tested against M. tb.

The crude K. vesicatoria extract (28.00 g) was subjectedto silica column chromatography (CC, size 10 × 23 cm)using hexane: ethyl acetate (EtOAc) mixtures of increasingpolarity (0 to 100%) followed by MeOH. Fractions (1–26) of 500 mL were collected. Similar fractions were pooledtogether and dried, which resulted in nine main fractions.These nine fractions were subjected to a TLC bioautographicantibacterial assay using M. smegmatis. Fraction 3 (F3,404.0 mg) was subjected to silica gel CC (size 3× 25 cm)gradient elution from hexane to ethyl acetate (0 to 60%)on the basis of its inhibitory activity on M. smegmatis, toyield pure compound (1) (30.70 mg, Figure 2). Fraction 8(F8, 378.9 mg) from the first column contained very fewconstituents with one main compound and was chosen forfurther isolation with a silica gel CC (hexane-ethyl acetate,30 to 100%). Fifty subfractions were obtained and pooledtogether as two main fractions (F8.1 and F8.2). Fraction8.2 was further purified with a Sephadex column usingMeOH which resulted in the 3 : 2 isomer mixture of (2)and its enantiomer (3) (123.3 mg, Figure 2). The structuralidentity of the mixture was elucidated by physical (mp. [α]D)and spectroscopic (1H and 13C NMR) data and was alsosubjected to 2D COSY, HMQC, HMBC and NOESY spectra.The isomer mixture (30.00 mg) was subjected to columnchromatography (CC, 3× 30 cm) using 150 g of inactivatedsilica (1.00 mL d H2O for each 10.00 g of silica) eluted withhexane : EtOAc (40 to 60%). This procedure separated aportion (8.00 mg) of the main isomer (2) from the mixture.Isolation of minor isomer (3) was unsuccessful.

2.4. Antimycobacterial Activity of Solvent Partitioned Fractionson M. smegmatis Using Microplate Susceptibility Testing.Cryopreserved or freshly scraped colonies were suspended infresh Middlebrook 7H9 broth (Sigma-Aldrich Chemical Co.,South Africa) and incubated for 24 h at 37◦C. The overnightliquid culture was then transferred to a sterile test tubecontaining 20–25 glass beads (with a 2.00 mm diameter) andhomogenised by using a vortex mixer (Heidolph, Germany)for 5–10 min. The broth culture was then left still for 5–10 min to let larger clumps of mycobacteria settle. Thesupernatant was carefully decanted to a sterile flask andadjusted with Middlebrook 7H9 broth base to an opticaldensity of 0.2 (log phase) at 550 nm (Beckman DU-720UV spectrophotometer), yielding 1.26× 108 colony-formingunits per millilitre (CFU/mL). The microdilution test wasperformed in 96-well microtiter plates as described earlier[15, 16].

The plant extract and fractions were dissolved in 10%dimethyl sulfoxide (DMSO) to obtain a stock concentration

of 10 mg/mL. Ciprofloxacin (Sigma-Aldrich Chemical Co.,South Africa) at final concentrations ranging from 2.00 ×10−3 to 7.80 × 10−5 mg/mL served as the positive drugcontrol [17]. Serial twofold dilutions of each test sample weremade with Middlebrook 7H9 broth base (Sigma-AldrichChemical Co., South Africa) to yield volumes of 100 mL/wellwith final concentrations ranging from 2.50 to 0.08 mg/mL.Mycobacterium smegmatis (100 mL adjusted to an opticaldensity value of 0.2 to ensure the bacteria suspension wasat the start of the log phase and approximately 1.26 ×108 CFU/mL upon test commencement) was also added toeach well containing the samples and mixed thoroughly togive a final volume of 200 mL/well. The solvent control,DMSO at 12.50%, did not show inhibition on the growthof the bacteria. Tests were done in triplicate on two differentoccasions.

The plates were sealed with parafilm and incubatedat 37◦C for 24 h. The minimum inhibitory concentration(MIC) of samples was detected following addition (40 μL) of0.20 mg/mL p-iodonitrotetrazolium chloride (INT, Sigma-Aldrich Chemical Co., South Africa) to duplicates of eachsample triplicate and incubated at 37◦C for 30 min [16].Viable bacteria reduced the yellow dye to a pink colour.The MIC was defined as the lowest sample concentrationthat prevented this change and exhibited complete inhibitionof bacterial growth. The minimal bactericidal concentration(MBC) was determined by taking 50.0 μL aliquots from theremaining INT excluded wells to 150.0 μL of 7H9 broth in afresh 96-well plate. This new plate was sealed with parafilmand incubated for 48 h at 37◦C. The MBC was recorded asthe lowest concentration of sample which did not producethe pink colour change after the addition of 40.0 μL INT(0.20 mg/mL).

2.5. Antitubercular Rapid Radiometric Assay Using M. tuber-culosis. The radiometric respiratory techniques using theBACTEC 460 system (Becton Dickinson Diagnostic Instru-ment, Sparks, MD, USA) were used for susceptibility testingagainst all the M. tb strains as described previously [18].

Solutions of all the test samples were prepared in DMSOto obtain their respective concentrations. Previous resultsfor K. vesicatoria show an MIC of 50.00 μg/mL againstH37Rv [4], and was tested here at three concentrations(100.0, 50.00, and 25.00 μg/mL) in triplicate. This extractwas also tested against two drug-resistant strains of M. tbat the same concentrations. According to Lall et al. [12]the chloroform root extract of E. natalensis has an MICof 8.00 μg/mL against susceptible M. tb. This extract wastested again at concentrations ranging from 16.00, 8.00 to4.00 μg/mL in triplicate. The EtOH extract of P. sidoidesexhibited an MIC above 5000 μg/mL [19] and was testedat three concentrations (10000, 5000, and 2500 μg/mL) intriplicate. The hexane fraction (F2) and the tannin-freefraction (F4) obtained from the tannin clean-up partitioningwere tested in triplicate at three concentrations (100.0, 50.00,and 25.00 μg/mL) based on the antimycobacterial activityof these samples against M. smegmatis. Compounds (1)and (2) were tested in triplicate at four concentrations


Figure 1: Undifferentiated (A) and differentiated (B) U937 cells as viewed under a light microscope (100x magnification).

(200, 100, 50, and 25.00 μg/mL). All the samples weredissolved in 100% DMSO to obtain stock concentrations thatwere subsequently serially diluted twofold to give the threetest concentrations. None of the samples exceeded a finalDMSO concentration above 1.0% as control experimentsshowed that a final concentration of DMSO (1.0%) in themedium had no adverse effect on the growth of M. tb.The primary drug INH (Sigma-Aldrich Chemical Co., SouthAfrica), at a concentration of 0.02 mg/mL, served as thedrugcontrol in the bioassay.

2.6. Determining Synergistic Antimycobacterial Activity onM. tuberculosis Using the BACTEC Radiometric Assay. Theactivity of three different drug, and extract combinationswas evaluated at sub-MIC levels (below original MIC values)so that each component (extract or drug) was presentat concentrations corresponding to 1/2, 1/4, and 1/8 ofthe MIC. Analysis of the drug combination data wasachieved by calculating the fractional inhibitory concen-tration (FIC) index [20] with a general equation for usewith any number (n) of drugs in a combination as fol-lows: FIC = (MICa combination/MICa alone) + (MICb combination

+ MICb alone) +· · ·+ (MICn combination + MICn alone). Thesubscripts represent the different components in the drugcombination. The FIC was interpreted as follows: FIC ≤ 0.5,synergistic activity; FIC = 1, indifference/additive activity;FIC ≥ 2 or more, antagonistic activity [13, 21].

Three different combinations of plant extracts and INHwere tested for possible synergistic activity against the drugsusceptible M. tb strain. Combination 1 (C1) included theextracts of E. natalensis (CHCl3), K. vesicatoria (EtOH), andP. sidoides (EtOH) in a four-drug combination with the first-line drug INH. Combination 2 (C2) only included the threeplant extracts in a three-drug combination. Combination3 (C3) combined the K. vesicatoria (EtOH) extract andINH in a two-drug combination. The BACTEC radiometricmethod, as described before [13], was used to determine thesynergistic activity of these different amalgamations.

2.7. Cytotoxicity. All reagents were procured from HighveldBiological (Pty) (Ltd.) (Sandringham, South Africa) unless

indicated otherwise. The U937 cells were grown to a densityof 5 × 108 cells/mL, centrifuged, and washed with phosphatebuffered saline (PBS) solution. The concentration of cells wasadjusted to 1× 105 cells/mL in complete medium containinga final concentration of 0.10 μg/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Chemical Co., South Africa).Two hundred microlitres of the cell suspension were seededinto the inner wells of a 96-well tissue culture plate, whilethe outer wells received 200.0 μL of incomplete medium.The cells were incubated for 24 hours at 37◦C in an atmo-sphere of 5% CO2 to induce differentiation of monocytesto mature macrophages (Figure 1) [22, 23]. Cytotoxicitywas measured by the 2,3-bis(2-methoxy-4-nitro-5-sulfoph-enyl)-5-[(phenylamino)carbonyl]-2-H-tetrazolium hydrox-ide (XTT) method using the Cell Proliferation Kit II (RocheDiagnostics GmbH). Dilution series of E. natalensis, K.vesicatoria (EtOH crude and tannin-free), and P. sidoidesextracts were prepared at various concentrations (400.0 to3.125 μg/mL). Synergistic combinations were tested at MICand sub-MIC levels to maintain the correct ratios of eachextract as tested against M. tb, accordingly each combinationwas tested at different starting concentrations. Combination1 was made up to a combined concentration of 5058.2 μg/mLin the first well with a final concentration of 39.52 μg/mL.Combination 2 was made up to a combined concentrationof 5058.0 μg/mL in the first well with a final concentrationof 39.52 μg/mL and C3 with concentrations ranging from50.20 to 0.392 μg/mL. The pure compounds (INH, (1) and(2)) were made up to a stock concentration of 20.00 mg/mLand serially diluted to start with a concentration of 200.0 to1.562 μg/mL from the first wells to the last in the microtitreplates. The isomer mixture (compounds (2) and (3)) wastreated in the same way. These dilutions were added tothe inner wells of the microtiter plate and incubated for72 h. After 72 h, 50.0 μL of XTT reagent (1.0 mg/mL XTTwith 0.383 mg/mL PBS) was added to the wells and theplates were then incubated for 1-2 hours. The positivedrug, (Actinomycin D, Sigma), at a final concentrationrange of 5.0 × 10−2 to 3.9 × 10−4 μg/mL, was included.After incubation, the absorbance of the colour complexwas spectrophotometrically quantified using an ELISA platereader (PowerWave XS, Bio-Tek), which measures the OD


1

2

3

4

5

6

7

89

10

11

12

13

14

15

16

17

18

19

20

21

22

HO23

H24

25

26

27 28

29

30 31

Stigmasta-5, 23-dien-3-ol (1)

(a)

O

O

1

2

3

4

5

OH

5-(hydroxymethyl)furan-2(5H)-one (2)

(b)

O

O

OH

5-(hydroxymethyl)dihydrofuran-2(3H)-one (3)

(c)

Figure 2: Chemical structures of isolated compounds from Knowltonia vesicatoria.

at 450 nm with a reference wavelength of 690 nm. DMSO(0.04%) was added to serve as the control for cell survival.GraphPad Prism 4.03 software was used to statisticallyanalyse the 50% inhibitory concentration (IC50) values.


3.1. Identification of the Isolated Compounds

Compound (1): Stigmasta-5,23-dien-3-ol. (Figure 2) was ob-tained as clear crystals and was identified based on NMR data(1H and 13C) which were compared with those reported inliterature [24, 25].

Isomer mixture: 5-(hydroxymethyl)furan-2(5H)-one and5-(hydroxymethyl)dihydrofuran-2(3H)-one. (Figure 2) wasobtained as a yellow oil. This sample is a ≈ 3 : 2 mixtureof compounds (2) and (3), respectively, as was revealedby the integral of the 1H NMR spectrum and also bythe absence of HMBC and COSY correlations between thesignals corresponding to each one of the constituents.

Compound (2): 5-(hydroxymethyl)furan-2(5H)-one. (Fig-ure 2) [α]D-5.7 (c 0.16, CHCl2); 1H NMR (500 MHz,CDCl3): δ 7.47 (1H, dd, J3,2 = 5.8 Hz, J3,4 = 1.6 Hz, H-3),6.21 (1H, dd, J2,3 = 5.8 Hz, J2,4 = 2.1 Hz, H-2), 5.15 (1H,dddd, J4,2 = 2.1 Hz, J4,3 = 1.6 Hz, J4,5A = 3.9 Hz, J4,5B =5.1 Hz, H-4), 4.03 (1H, dd, J5A,5B = 12.2 Hz, J5A,4 = 3.9 Hz,

HA-5), 3.79 (1H, dd, J5A,5B = 12.2 Hz, J5b,4 = 5.1 Hz, HB-5).The absolute stereochemistry at the C-4 asymmetriccentre not determined. Since (2) was obtained as a yellowoily substance, the undefined stereochemistry at the C-4asymmetric centre is most likely the (S)- and not the(R)-form as the latter crystallises and the (S)-form is an oil[26].

3.2. Microdilution Assay Using M. smegmatis. Although stud-ies have shown that M. smegmatis could be more resistant todrug activity than M. tuberculosis [27–29], screening samplesagainst nonpathogenic M. smegmatis gives quick results inany laboratory fitted for microbial tests and provides a goodindication of which samples will also be active against M.tuberculosis. In most cases this strategy saves both timeand expensive reagents necessary to test numerous samplesagainst the pathogen, M. tb. In this case, the purpose ofscreening the partitioned fractions was to promptly establishif the tannin-free extract still exhibits the same activityas the tannin-containing extract and whether the tannin-concentrated fraction had higher antimycobacterial activitythan the whole extract. Results clearly indicate that this is notthe case (Table 1).

The tannin-free extract (F4) exhibited an MIC four-fold higher (2500 μg/mL) than that of the crude tannincontaining EtOH extract (625.0 μg/mL). This does notautomatically point to the conclusion that tannins are theresponsible components for Knowltonia’s antimycobacterial


Table 1: MIC and MBC values of the solvent-partitioned fractions against M. smegmatis compared to the crude EtOH extract of K.vesicatoria.

Sample MICa (μg/mL) MBCb (μg/mL)

K. vesicatoria (EtOH) 625.0 1250

F1 2500 >2500

F2 (hexane layer) 1250 2500

F3 >2500 >2500

F4 (tannin-free) 2500 >2500

F5 >2500 >2500

DMSO% 12.50 12.50

Ciprofloxacin 1.250 1.250aMinimum inhibitory concentration.

bMinimum bactericidal concentration.

activity. None of the partitioned fractions from K. vesicatoriaexhibited an MIC or MBC as low as that of the crude or“whole” EtOH extract. Fraction 1, which still contained all ofthe polyphenolic compounds but no nonpolar compounds,had the same activity profile as that of F4, with an MIC of2500 μg/mL and an MBC value of more than 2500 μg/mL.The fractions with concentrated levels of tannins, fractions3 (containing mostly polyphenols) and 5 (existing mainly ofNaCl and polyphenol residue), had no activity at the highestconcentration tested (2500 μg/mL) and were subsequentlynot included for the M. tb antimycobacterial test. Thehighest activity seen for the fractions was that of thenonpolar compounds (F2) with an MIC and MBC of 1250and 2500 μg/mL, respectively. Fraction 2 and the tannin-free fraction (F4) were tested against M. tb. The positivedrug control, ciprofloxacin, exhibited an MIC equal to itsMBC value of 1.250 μg/mL, which is comparable to itsmycobactericidal value found in the literature [30]. Thehighest percentage DMSO used for the samples in the assay(2.50%) did not inhibit M. smegmatis growth as evidentwith the MIC/MBC value of 12.50%. Taken as a whole,the results imply that either the active compounds weredamaged during the partitioning procedure or that synergywithin the K. vesicatoria extract itself forms the basis of itsantimycobacterial activity.

3.3. Antitubercular Rapid Radiometric Assay Using M. tuber-culosis. The antimycobacterial assays of the extracts againstM. tuberculosis using the BACTEC radiometric methodshowed that K. vesicatoria inhibited M. tuberculosis at anMIC of 50.0 μg/mL. Pelargonium sidoides and E. natalensisinhibited the bacteria at the 5000 μg/mL and 8.00 μg/mLagainst M. tuberculosis (Table 2). These results correspondvery well with previous MIC values obtained for all ofthe extracts [4, 11, 12, 18, 19]. The sensitive strain ofM. tb was not susceptible to either F2 (hexane layer) orF4 (tannin-free extract) obtained from the K. vesicatoriatannin clean-up procedure at the highest concentrationtested (100 μg/mL). The antituberculosis positive drug, INH,inhibited the growth of M. tuberculosis at 0.2 μg/mL duringall the independent assays.

The combination drug action showed that only C1 andC3 exhibited synergistic antimycobacterial activity. Althoughthe combined MIC of C1 (E. natalensis (CHCl3) + K.vesicatoria (EtOH) + P. sidoides (EtOH) + INH) wasreduced eightfold from 5058.2 to 632.30 μg/mL, the FIC (0.5)indicates that the combination had threshold synergisticactivity. This is mainly due to the high ratio of P. sidoides(5000 μg/mL equal to 98.8%) present in the combination.The same situation, where the P. sidoides extract overwhelmsthe other components in the amalgamation, is seen withC2 (E. natalensis (CHCl3) + K. vesicatoria (EtOH) + P.sidoides (EtOH)), which is slightly reflected by the FIC(1.5), indicating an additive effect of the combination. Thebest synergistic result with an FIC of 0.25 was seen forC3 (K. vesicatoria (EtOH) + INH) where the combinedMIC is reduced eightfold, from 50.20 to 6.275 μg/mL. Thissynergistic activity of C3 implies that if a patient were totake INH prescribed for the treatment of TB and combinedthis regime with the extract of K. vesicatoria, other possiblebenefits such as reduced length of INH treatment anddecreased toxicity due to lower intake concentrations ofK. vesicatoria could subsist. Testing the cytotoxicity of thiscombination is the next step to shed some light on thispossibility.

The isomer mixture exhibited no inhibitory activityagainst M. tb at the highest concentration tested (200.0 μg/mL). Since lactones are known to exhibit significantantibacterial properties, it was interesting to note that in thiscase the sterol was considerably more active than the lactonewith an MIC of 50.00 μg/mL compared to the lactone MICof 200.0 μg/mL. It has been reported that phytosterols haveanti-inflammatory, antibacterial, antifungal, antiulcerative,antioxidant, and antitumoral activities [31, 32]. Additionally,Saludes et al. [33] reported the antimycobacterial activity offive phytosterols isolated from Morinda citrifolia against M.tb. Three of these isolated sterols closely resemble the struc-ture of stigmasta-5,23-dien-3-ol, with the only differencesbeing the position and degree of saturation. Stigmasta-4-en-3-one and stigmasta-4,22-dien-3-one (used in combination)had an MIC lower than 2.00 μg/mL; stigmasterol had an MICof 32.00 μg/mL. Another phytosterol saringosterol, isolated


Table 2: Antimycobacterial activity against M. tuberculosis and cytotoxicity on U937 cells of test samples.

Samples MICa (μg/mL) FICb ΔGI4-3± SDc IC50 (μg/mL ± SD)d

V2e — — 22.0± 2.94 —

INH (0.2 μg/mL) 0.20 — −2.50± 2.12 >200.0

Actinomycin Df NTg — — 3.820± 0.258×10−3

K. vesicatoria EtOH 50.00h — −6.00± 0.58 41.25± 0.205

F4i >100.0 — 275.5± 45.3 64.77± 1.812

F2j >100.0 — 288.5± 65.1 NT

E. natalensis CHCl3 8.000 — −3.50± 0.88 12.22± 0.025

P. sidoides EtOH 5000 — 20.00± 1.85 43.54± 0.465

Compound (1) 50.00 — −9.00± 1.41 16.41± 0.135

Compound (2) 100.0k — — 44.70± 0.50

Isomer mixture (2) and (3) >100.0 — 117.0± 8.49 >200.0

C1 1.000/6.250/625.0/0.025l 0.50 −5.50± 3.53 100.3± 2.450

C2 4.000/25.00/2500m 1.50 17.00± 5.65 108.6± 0.89

C3 6.250/0.025n 0.25 −1.5± 0.707 121.7± 2.079aMinimum inhibitory concentration.

bFractional inhibitory concentration index.cΔGI value (mean ± standard deviation).dFifty percent inhibitory concentration.e10−2 inoculum control.f Cytotoxicity assay positive control.gNot tested.hMIC value for K. vesicatoria against both clinical drug-resistant M. tb strains: 4388 (resistant to INH and ETH; V2 ΔGI4-3 ± SD = 32.5 ± 4.95) and 5479(resistant to INH, EMB, STR, RIF; V2 ΔGI3-2 ± SD = 34.5±0.71) was found to be 50.00 μg/mL with ΔGI4-3 = 2.0±4.24 and ΔGI3-2 = 0.33±0.57, respectively.iFraction 4: tannin-free Knowltonia fraction.j Fraction 2: hexane Knowltonia fraction.kBACTEC MIC not determined, MIC determined via microplate assay.lEightfold reduction of respective MIC values for E. natalensis (CHCl3) + K. vesicatoria (EtOH) + P. sidoides (EtOH) + INH.mTwofold reduction of respective MIC values for E. natalensis (CHCl3) + K. vesicatoria (EtOH) + P. sidoides (EtOH).nEightfold reduction of respective MIC values for K. vesicatoria (EtOH) + INH.

from Lessonia nigrescens, (a brown algae) exhibited extremelylow toxicity compared to its MIC of 0.250 μg/mL [34].

Knowltonia vesicatoria inhibited both drug-resistantstrains of M. tuberculosis at an MIC of 50.0 μg/mL (Table 2).This activity indicates a mechanism of action different toINH, EMB, STR, and RIF. These drugs target cell wallsynthesis (INH, EMB), inhibit gene transcription (RIF), andinhibit protein synthesis (STR) [35]. The antimycobacterialmechanism of action of K. vesicatoria has to be investigated inorder to confirm this supposition as a different mechanism ofaction to INH could hold positive implications on preventingdrug resistance as well as targeting strains already resistantto INH with the use of a drug therapy that combines K.vesicatoria and INH.

3.4. Cytotoxicity. The XTT cytotoxicity assay is a rapid andcost-effective tool to help choose optimal candidates, thosesamples with low cytotoxicity and high antimycobacterialactivity, similar to a therapeutic dose, and to exclude anysamples too toxic to test at their antimycobacterial concen-tration for ensuing intracellular assays. The results obtained(Table 2) indicated that the cytotoxicity effects of the fourplant extracts on U937 cells demonstrated marginal toxicityexcept for E. natalensis, which showed high toxicity at afifty percent inhibitory concentration (IC50) of 12.22 μg/mL

against the macrophages. The tannin-free extract of K.vesicatoria (F4) had the highest IC0050 value (64.77 μg/mL)compared to the other plant extracts; the lowered toxicity ismost likely due to the lack of the protein binding polyphe-nols. Knowltonia vesicatoria and P. sidoides showed similartoxicity exhibiting IC50 values at 41.25 and 43.54 μg/mL,respectively (Table 2). With the synergistic combinations,the approximate IC50 values for combinations 1 and 2 werecalculated as 100.3 and 108.6 μg/mL, respectively. Synergisticcombination C3 had a resultant IC50 of 121.70 μg/mL. Thetoxicity profiles of the combinations were much better thanthe individual extracts indicating a synergistic action onlowering cytotoxicity.

The pure compounds, which included the antituberculardrug INH and the cytotoxic drug Actinomycin D, showedvery different cytotoxic profiles compared to the extractsand combinations. With the compounds isolated fromK. vesicatoria, compound (1) was more than twofold astoxic to the cells when compared to compound (2), withan IC50 of 16.41 and 44.70 μg/mL, respectively. The 3 : 2isomer mixture of 5-(hydroxymethyl)furan-2(5H)-one (2)and 5-(hydroxymethyl)dihydrofuran-2(3H)-one (3) was nottoxic to the macrophages even at the highest concentrationtested (200.0 μg/mL) with more than 50 percent of thecells still viable at this concentration. Isoniazid had asimilar effect on the cells, with 80 percent of the cells still


viable at 200.0 μg/mL, indicating an IC50 above this value.Actinomycin D, which served as the positive control in thecytotoxicity assay, exhibited an IC50 of 3.80 × 10−3 μg/mL.

Similar to a therapeutic index, it is necessary to comparethe difference between cytotoxicity and antimycobacterialactivity of the tested samples in order to choose the bestcandidates for possible treatment options. Of all the samplesINH had the broadest difference in toxicity when comparedto biological activity, followed by C3, E. natalensis, the isomermixture and the EtOH extract of K. vesicatoria. Pelargoniumsidoides had the narrowest therapeutic range followed by F4,C2, C1, and compounds (1) and (2).

4. Conclusion

This study adds to current literature by demonstrating thesynergistic antimycobacterial activity of the crude EtOHextract from the aerial parts of K. vesicatoria in its entiretyand in a drug combination with the first-line drug, isoniazid.Based on these findings we assume that a drug combinationof INH with K. vesicatoria could help prevent and combat-resistant strains; however, further studies will have to becarried out in order to demonstrate its efficacy.

This paper is the first to report on the isolationof stigmasta-5, 23-dien-3-ol, and 5-(hydroxymethyl)furan-2(5H)-one from K. vesicatoria; it is also the first report ofthe antimycobacterial activity of both these compounds.Subsequent and ongoing work includes investigating the pos-sible mechanism of action, intracellular antimycobacterialactivity, and measuring the immunomodulation of the mostactive candidates.

The various aspects of the challenges faced in TB drugdiscovery are applicable to other infectious agents. WithKnowltonia vesicatoria being reintroduced to the field ofmedicinal plant science, its extracts and isolated compoundscould also be applied to screening for activity against otherpathogens. It is the interplay of codeveloped methods on thenatural products chemical perspective that will improve thechances of treatment success.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

The authors are grateful to the staff at the Medical ResearchCouncil (Pretoria, South Africa) for kindly allowing themthe use of their facility and for donating the mycobacterialstrains, Ms. K. Le Roux and Ms. E. Mogapi (University ofPretoria) for maintaining the U937 cells, and the NationalResearch Foundation for financial support. This paper is partof the thesis submitted by the first author to the Departmentof Plant Science of University of Pretoria.

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Research Article

Melanogenesis and Antityrosinase Activity ofSelected South African Plants

Manyatja Brenda Mapunya,1, 2 Roumiana Vassileva Nikolova,1 and Namrita Lall2

1 Department of Biodiversity, School of Molecular and Life Sciences, University of Limpopo (UL), Private Bag X1106,Sovenga 0727, South Africa

2 Department of Plant Science, University of Pretoria, Pretoria 0002, South Africa




Copyright © 2012 Manyatja Brenda Mapunya et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Melanin is the pigment that is responsible for the colour of eyes, hair, and skin in humans. Tyrosinase is known to be the key enzymein melanin biosynthesis. Overactivity of this enzyme leads to dermatological disorders such as age spots, melanoma and sites ofactinic damage. Ten plants belonging to four families (Asphodelaceae, Anacardiaceae, Oleaceae, and Rutaceae) were investigatedfor their effect on tyrosinase using both L-tyrosine and L-DOPA as substrates. Ethanol leaf extracts (500 µg/mL) of Aloe ferox,Aloe aculeata, Aloe pretoriensis, and Aloe sessiliflora showed 60%, 31%, 17%, and 13% inhibition of tyrosinase activity respectively,when L-tyrosine was used as a substrate. Harpephyllum caffrum (leaves) at a concentration of 500 µg/mL had an inhibitory effectof 70% on tyrosinase when L-DOPA was used as a substrate. The IC50 of Harpephyllum caffrum (leaves and bark) were found to be51±0.002 and 40±0.035µg/mL, respectively. Following the results obtained from the tyrosinase assay, extracts from Harpephyllumcaffrum were selected for further testing on their effect on melanin production and their cytotoxicity on melanocytes in vitro.The IC50 of both extracts was found to be 6.25 µg/mL for melanocyte cells. Bark extract of Harpephyllum caffrum showed 26%reduction in melanin content of melanocyte cells at a concentration of 6.25 µg/mL. The leaf extract of this plant showed sometoxicity on melanocyte cells. Therefore, the bark extract of Harpephyllum caffrum could be considered as an antityrosinase agentfor dermatological disorders such as age spots and melasoma.

1. Introduction

Melanin is a pigment that occurs in humans, fungi, andplants [1]. It is responsible for the colour of eyes, hair,and skin in humans [2]. The pigment is secreted andproduced by the melanocytes cells, which are distributed inthe basal layer of the dermis, through a physiological processcalled melanogenesis [2–5]. It is formed through a series ofoxidative reactions involving the amino acid tyrosine in thepresence of the enzyme tyrosinase. There are two types ofmelanin pigments that can be produced by melanocyte cells,namely, eumelanin which is black or brown, and pheome-lanin which is red or yellow and alkali soluble [6, 7]. Thecolour of human hair and skin is determined by the type ordistribution and degree of melanin pigment. Each individual

of different racial group has more or less the same numberof melanocyte cells, thus the type of melanin produceddepends on the functioning of the melanocytes, for example,people with darker skin are genetically programmed toconstantly produce higher levels of melanin [2, 6, 7]. Themajor structural differences between dark and light skins interms of pigmentation are melanosome (organelles withinthe melanocyte cells) size and grouping. Melanosomes aresmaller and grouped in clumps in light skin, while they arelarger single organelle in dark skin [7, 8]. The role of melaninis to protect the skin against UV light damage by absorbingUV sunlight and removing reactive oxygen species [2, 3, 7].

The key enzyme that is responsible for melanin produc-tion is tyrosinase [9]. Hyperpigmentation of the skin occursdue to overactivity of tyrosinase enzyme and its underactivity


leads to hypopigmentation of hair. Overactivity of the en-zyme is associated with ageing while under-activity can oc-cur in any age group depending on a person’s heredity [10].Tyrosinase, also known as polyphenol oxidase, is a coppercontaining monooxygenase that catalyzes two distinct reac-tions involving molecular oxygen: hydroxylation of tyrosineto 3,4-dihydroxyphenylalanine (DOPA) by monophenolaseaction and oxidation of DOPA to DOPA-quinone by diphen-olase action [9, 11]. Quinones are highly reactive compoundsand can polymerize spontaneously to form high-molecular-weight compounds or brown pigments [12]. Apart fromanimals, tyrosinase is also widely distributed in plants and isa very important enzyme in controlling the quality of fruitsand vegetables. It catalyzes the oxidation of phenolic com-pounds to the corresponding quinones and is responsible forthe enzymatic browning of fruits and vegetables, which areof economic importance [2].

There is a variety of plants that are used traditionally forthe treatment of different skin problems. Poor skin penetra-tions and mutagenic effects of chemically derived com-pounds such as hydroquinone [9] used in cosmetics led tothe search for alternative herbal and pharmaceutical agentsto treat skin hyperpigmentation. Aloe species were selectedin this study because they are related species to Aloe verawhich is used in the markets for depigmentation purposes[13]. Other plants, selected in the present study (Table 1),are traditionally used in South Africa for skin-lighteningpurposes and/or for removing marks or pigments on the face[14]. Different parts of these plants are ground and used asfacial masks to remove spots and they are also used for skinlightening purposes. The aim of this study was to test theeffect of the selected plant extracts on tyrosinase enzyme andto identify which plant extracts can be used as possible skin-lightening agents.


2.1. Plant Material Collection and Extraction. Leaves andbark of selected plant species (Table 1) were collected fromthe Manie van der Schijff Botanical Garden of the Universityof Pretoria in July 2006. Some plant materials (Harpephyllumcaffrum and Calodendrum capensis both leaves and bark)were dried in shade while leaves of Aloe species were usedfresh. Traditionally paste of plant material mixed with wateris used for removing hyperpigmentation. However, due toethanol being a comparatively safer solvent (according to po-larity) and its polarity being not very different from that ofwater, and due to its antiseptic nature, this solvent was cho-sen for the preparation of extracts. Forty grams of each plantmaterial was ground with 200 mL absolute ethanol using aJannke & Kunkel grinder. Mixtures were left overnight andthen filtered through a Whatman filter paper (15 cm). Thesolvent was removed under a vacuum (BUCHI, Rotavapor,R-200) to yield dry extracts.

2.2. Chemicals and Reagents. Mushroom tyrosinase with theactivity of 6680 units/mg and Kojic acid (positive con-trol) were purchased from Sigma-Aldrich. Fetal calf serum

(FCS), trypsin, EDTA, L-glutamine, potassium phosphatebuffer (pH 6.5), penicillin/streptomycin/fungizone, and sod-ium pyruvate were purchased from Highveld Biological. TheCell Proliferation Kit II (XTT) (sodium 3-[1-(phenylam-inocarbonyl), 4-tetrazolium]-bis (4-methoxy-6-nitro) ben-zene sulfonic acid hydrate) labeling reagent) was purchasedfrom Roche Diagnostics.

2.3. Tyrosinase Enzyme Assay. The assay was performed us-ing relevant methods [9, 15]. Each powdered plant extractwas dissolved in dimethyl sulphoxide (DMSO) to a finalconcentration of 20 mg/mL. This extract stock solution wasthen diluted to 600 µg/mL in 50 mM potassium phosphatebuffer (pH 6.5). Serial dilutions were made to get eightconcentrations. Kojic acid was used as a control drug. In a 96-well plate, 70 µL of each extract serial dilution was combinedwith 30 µL of tyrosinase (333 Units/mL in phosphate buffer)in triplicates. After incubation at room temperature for 5minutes, 110 µL of substrate (2 mM L-tyrosine or L-DOPA)was added to each well. Final concentrations of the extractsamples ranged from 3.91 to 500 µg/mL. The final percentageof DMSO was 1% after the dilution. Optical densities ofthe reaction mixtures in the wells were then recorded at492 nm with the BIO-TEK Power Wave XS multi-well platereader. Final concentration of Kojic acid ranged from3.125 µg/mL to 400 µg/mL. Plant extracts which showedgood antityrosinase activity at a concentration of 60 µg/mLwere further investigated for their effect on melanin synthesisby melanocyte cells.

2.4. Melanocyte Cell Culture for the Investigation of Melanin

Inhibition by Plant Extracts

2.4.1. Preparation of Melanocyte Cell Culture. Mouse melan-ocyte cell line, B16-F10, was cultured in Dulbecco’s ModifiedEagle’s Medium (DMEM) containing 10% fetal bovine se-rum, 1.5 g/L NaHCO3, 2 mM L-glutamine, 10 µg/mL peni-cillin, 10 µg/mL streptomycin, and 0.25 µg/mL fungizone andincubated at 37◦C with 5% CO2 in a humidified atmosphere.Cells were subcultured in a ratio of 1 : 3 on every third orfourth day.

A cell suspension of 1×105 B16-F10 cells was prepared incomplete DMEM, supplemented with 10% FCS, and (10 mL)antibiotics (penicillin/streptomycin/fungizone). On day 0,B16-F10 cells in complete DMEM were dispensed into thewells of a 96-well plate (105 cells per well) and 24-well plate(104 cells per well). After an overnight incubation at 37◦Cin 5% CO2 and a humidified atmosphere, extract sampleswere added to the cells to the final concentration of 500,250, 125, 62.5, 31.25, 15.62, 7.81, and 3.91 µg/mL. Kojic acidwas used as a control drug. Final concentration of Kojicacid ranged from 400 to 3.125 µg/mL. Incubation at 37◦Cin 5% CO2 and a humidified atmosphere followed for threedays.

2.4.2. Effect of Plant Extracts on Melanin Synthesis. The effectof the plant extracts on melanin synthesis was determinedby washing the melanocyte cells in the 24-well plate with


Table 1: List of selected plants and their traditional uses.

Plants Common names Family name Medicinal use

Aloe aculeata Pole-Evans(Leaves)

Ngopane Asphodelaceae Used as a skin lightening [19]

Aloe arborescens Mill.(Leaves)

Ikalane/Umhlabana AsphodelaceaeLeaf extracts have shown to have significant woundhealing, antimicrobial, anti-ulcer and anticarcinogenicactivity [14]

Aloe ferox Mill. (Leaves) Ikhala/Inhlaba AsphodelaceaeSap in the leaves used traditionally as laxatives and can betaken for arthritis [14]

Aloe pretoriensis Pole-Evans(Leaves)

N/A Asphodelaceae Used as a skin lightening [19]

Aloe sessiliflora Pole-Evans(Leaves)

N/A AsphodelaceaeUsed traditionally to treat the uterus and believed topromote menstruation [14]

Aloe vera (L.) Burm.f.(Leaves)

N/A AsphodelaceaeThe gel from leaves is used as a remedy for minor burnsand scrapes and for sunburn [14]

Calodendrum capensisThumb. (Leaves)

Umbhaba Rutaceae Used as a facial mask [19]

Calodendrum capensisThumb. (Bark)

Umbhaba RutaceaeUsed traditionally in soaps and as a skin-lightener as whiteumemezi [14]

Harpephyllum caffrumBernh. (Leaves)

Umgwenya Anacardiaceae Used as a face mask [19]

Harpephyllum caffrumBernh. (Bark)

Umgwenya AnacardiaceaeAcne and eczema treatment, and is usually applied asfacial saunas and skin washes [14]

Sclerocarya birrea(A. Rich.) Hochst. (Nuts)

Morula AnacardiaceaeOil extracted from the kernels is Africa’s greatest skin careoil and as a skin-lightener (personal communication) [14]

Ximenia americana L.(Nuts)

Umthunduluka-obmvu OlacaceaeSeeds contain valuable oil that is used traditionally tosoothe leather and as cosmetic and skin ointment [14]

N/A Not available.

potassium phosphate buffered saline (PBS), and lysing with200 µL of sterile distilled water. Optical densities wererecorded at a wavelength of 405 nm. The effect of extractson melanin production was determined by comparing to thecontrol sample (medium with DMSO).

2.4.3. Toxicity Effect of Plant Extracts. The toxicity of theextracts on the B16-F10 cells was tested using the XTT cyto-toxicity assay. Fifty microliters of XTT reagent (1 mg/mLXTT with 0.383 mg/mL PMS) was added to the wells andincubated for one hour. The optical densities of the wellswere then measured at 450 nm (690 nm reference wave-length). By comparing to the control (DMEM with DMSO),cell survival was assessed.

2.5. Statistical Analysis. The results were analysed statisticallyusing one-way analysis of variance (ANOVA) and the leastsignificant differences (P < 0.01) were determined accordingto Duncan’s t-test.

3. Results

3.1. Effect of Plant Extracts on Tyrosinase Activity. Ethanolextracts from different parts of ten selected plants (Table 1)and Kojic acid (positive control) differed in their inhibitoryeffect on tyrosinase activity when using both L-tyrosine and

L-DOPA as substrates. Extracts from A. arborescens (leaves),A. vera (leaves), C. capensis (bark and leaves), and nut oilextract of S. birrea and X. americana did not inhibit thetyrosinase activity at tested concentrations (3.91 to 500 µg/mL), see Table 2. However, at a concentration of 500 µg/mLthe leaf extracts of A. aculeata, A. pretoriensis and A. sessil-iflora showed 31%, 17%, and 13% inhibition of tyrosinaseenzyme, respectively (Table 2). Leaf extract from Aloe feroxshowed inhibition of tyrosinase by 60%, 51% and 48%at 500 µg/mL, 250 µg/mL, and 125 µg/mL, respectively. Leafand bark extracts of H. caffrum showed significant (P <0.01) inhibition of the enzyme by 90% and 92% at 500 µg/mL, respectively, as compared to all other extracts tested(Table 2).

Plant extracts which exhibited inhibition of tyrosinase at500 µg/mL when using L-tyrosine as a substrate were furthertested for their effect on tyrosinase activity using L-DOPA asa substrate. Plants extract tested were H. caffrum (bark andleaves), A. aculeata, A. ferox, A. pretoriensis, and A. sessiliflora(leaves). A. pretoriensis and A. sessiliflora leaf extracts didnot show any inhibition of tyrosinase even at the highestconcentration tested (Table 2). Extracts of H. caffrum (barkand leaves) had stronger inhibitory effect on tyrosinase thanthe other plant extracts tested for tyrosinase activity usingL-DOPA as a substrate (Table 2). Kojic acid also had stronginhibitory effect on tyrosinase (88%, 83%, 74%, and 63% atconcentrations of 400, 200, 100, and 50 µg/mL, resp.) when


Table 2: Inhibitory activity of selected plants on tyrosinase whenboth tyrosine and L-DOPA are used.

Plant extracts [500µg/mL](%)

Tyrosine L-DOPA

Aloe aculeata Pole-Evans (Leaves) 31 —

Aloe arborescens Mill. (Leaves) — ∗

Aloe ferox Mill. (Leaves) 60 —

Aloe pretoriensis Pole-Evans (Leaves) 17 —

Aloe sessiliflora Pole-Evans (Leaves) 13 —

Aloe vera (L.) Burm.f. (Leaves) — ∗

Calodendrum capensis Thumb. (Leaves) — ∗

Calodendrum capensis Thumb. (Bark) — ∗

Harpephyllum caffrum Bernh. (Leaves) 90 70

Harpephyllum caffrum Bernh. (Bark) 92 60

Sclerocarya birrea (A. Rich.) Hochst. (Nuts) — ∗

Ximenia americana L. (Nuts) — ∗∗

not tested, —not active.

Table 3: The IC50 (concentrations at which half the tyrosinase acti-vity is inhibited) values of active plant extracts and positive control.

Plant extracts/ Positivecontrol

IC50 (Tyrosine)µg/mL

IC50 (L-DOPA)µg/mL

Harpephyllum caffrum(Leaves)

51± 0.002 125± 0.08

Harpephyllum caffrum(Bark)

40± 0.035 250± 0.12

Kojic acid 2.145± 0.082 26.66± 0.104

L-DOPA was used as a substrate. The IC50 of Harpephyllumcaffrum (leaves and bark) were found to be 51 ± 0.002 and40± 0.035µg/mL, respectively (Table 3).

3.2. Effect of Plant Extracts on Melanin Biosynthesis by MouseMelanocytes. Following the results obtained from tyrosinaseassay, extracts from H. caffrum (leaves and bark) were select-ed for further testing on their effect on melanin productionand their cytotoxicity on melanocytes in vitro since theyhad an inhibitory effect on tyrosinase when using both L-tyrosine and L-DOPA as substrates. The leaf extract fromAloe arborescens was selected to test its potential to promotemelanin production since it had no inhibitory effect on thetyrosinase. The bark extract of H. caffrum showed 26%reduction in melanin content of melanocyte cells at a concen-tration of 6.25 µg/mL (Figure 1(a)) while the leaf extract of A.arborescens showed 23% reduction in melanin content at thesame concentration. The leaf extract of H. caffrum showedtoxicity to melanocyte cells at most concentrations tested(Figure 1(b)).

3.3. Toxicity Effect of Plant Extracts. Extracts from H. caffrum(bark) and A. arborescens both showed low toxicity effect onmelanocyte cells (Figures 1(a) and 1(c)) at all concentrationstested with cell viability above 80%, and 70%. However,

leaf extracts of H. caffrum showed toxicity to melanocytescells (Figure 1(b)) at a concentration of 100 µg/mL. Kojicacid showed reduction in melanin production by melanocytecells with 69% and 61% at 3.12 µg/mL and 25.0 µg/mL, re-spectively (Figure 1(d)), and was not toxic to melanocytecells at all concentrations tested with cell viability of above80%.

4. Discussion

It is reported that Aloe vera’s derived compounds are used inskin-lightening agents [16] while from our results Aloe veradid not show any inhibition of tyrosinase. Active compoundsagainst tyrosinase from Aloe vera were isolated from the sapof the leaves [16, 17]. The aloes used in this study lacked thesap which may be due to the seasonal variation, even thoughthe time and season of plant’s collection in other studies wasnot specified.

Aloe arborescens had no inhibitory activity on tyrosinasebut when tested on melanocyte cells showed a reductionof melanin production by 23% instead of an increase. Thiscan be due to the fact that melanin biosynthesis is a multi-step pathway [1, 10] and thus the extracts may act on otherenzymes in the pathway rather than directly on tyrosinase.Leaf extracts of Aloe pretoriensis and Aloe sessiliflora had aninhibitory effect on tyrosinase when L-tyrosine was used as asubstrate but they did not show any activity on tyrosinasewhen L-DOPA was used as a substrate. This shows thatthey may act on monophenolase activity of tyrosinase byinhibiting conversion of tyrosine to L-DOPA [9, 11, 15].

The bark extract of Harpephyllum caffrum had the high-est inhibitory effect on tyrosinase and the highest reductionof melanin production by melanocytes cells as compared toall other plant extracts tested, except for Kojic acid. Thisextract and the Kojic acid were not toxic on melanocytescells as compared to the leaf extract of Harpephyllum caffrum.The results from this study show that Harpephyllum caffrum(bark) has the potential to serve as the source of chemicalconstituents for antpigmentation treatments.

Kojic acid (positive control) had the highest inhibitoryeffect on tyrosinase as compared to extracts of Harpephyllumcaffrum. It also resulted in a higher reduction of melaninproduction by melanocyte cells (38% at 6.25 µg/mL) as com-pared to the bark extract of Harpephyllum caffrum (26% at6.25 µg/mL). Kojic acid is reported to cause skin irritationwhen applied topically [18] but did not show any toxicity onthe melanocyte cells in concentrations tested (400 µg/mL to3.125 µg/mL) in this study.

5. Conclusions

From the present study, it can be concluded that scientificvalidation of the plant extracts used traditionally for treat-ment of skin, age-spots, dark marks, skin-lightening, andso forth is necessary in order to investigate their potentialas skin-lightening agents. Bark extract of Harpephyllum caf-frum, which exhibited good antityrosinase activity, inhibitedmelanin production in cell cultures, and did not show a toxic


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effect, will require further investigations in clinical studies inorder to determine its potential as a tyrosinase inhibitor.

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Research Article

Antibacterial Activities of Selected CameroonianPlants and Their Synergistic Effects with Antibiotics againstBacteria Expressing MDR Phenotypes

Stephen T. Lacmata,1 Victor Kuete,1 Jean P. Dzoyem,1 Simplice B. Tankeo,1

Gerald Ngo Teke,1 Jules R. Kuiate,1 and Jean-Marie Pages2

1 Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon2 Transporteurs Membranaires, Chimioresistance et Drug Design, UMR-MD1, IFR 88, Universite de la Mediterranee,Aix-Marseille II, Marseille, France

Correspondence should be addressed to Victor Kuete, [email protected] and Jules R. Kuiate, [email protected]

Received 20 October 2011; Accepted 13 December 2011

Academic Editor: Namrita Lall

Copyright © 2012 Stephen T. Lacmata et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The present work was designed to assess the antibacterial properties of the methanol extracts of some Cameroonian medicinalplants and the effect of their associations with currently used antibiotics on multidrug resistant (MDR) Gram-negative bacteriaoverexpressing active efflux pumps. The antibacterial activities of twelve methanol extracts of medicinal plants were evaluatedusing broth microdilution. The results of this test showed that three extracts Garcinia lucida with the minimal inhibitoryconcentrations (MIC) varying from 128 to 512 μg/mL, Garcinia kola (MIC of 256 to 1024 μg/mL), and Picralima nitida (MICof 128 to 1024 μg/mL) were active on all the twenty-nine studied bacteria including MDR phenotypes. The association ofphenylalanine arginine β-naphthylamide (PAβN or efflux pumps inhibitor) to different extracts did not modify their activities.At the concentration of MIC/2 and MIC/5, the extracts of P. nitida and G. kola improved the antibacterial activities of somecommonly used antibiotics suggesting their synergistic effects with the tested antibiotics. The results of this study suggest that thetested plant extracts and mostly those from P. nitida, G. lucida and G. kola could be used alone or in association with commonantibiotics in the fight of bacterial infections involving MDR strains.

1. Introduction

Bacterial infections are responsible for 90% of infectionsfound in health care services. The emergence of MDRbacterial strains appears as the major cause of treatmentfailure [1]. Among the known mechanisms of resistances,active efflux via resistance-nodulation-cell division (RND)pumps is one of the most occurring system in Gram-negativebacterial strains [2]. Efflux pumps are transport proteinsinvolved in the extrusion of toxic substrates (including virtu-ally all classes of clinically relevant antibiotics). The presentwork was therefore designed to investigate the antibacterialpotential against MDR bacteria expressing active effluxthough RND pumps. Medicinal plants of Cameroon used inthis study include the fruits of Citrus medica L. (Rutaceae),the bulbs of Allium sativum L. (Liliaceae) and Allium cepa

L. (Liliaceae), the seeds of Carica papaya Linn (Caricaceae),Cola acuminata (P. Beauv.) Schott and Endl. (Sterculiaceae),Buchholzia coriacea Engl. (Capparidaceae), Garcinia kolaHeckel (Guttifeare), and Garcinia lucida Vesque (Guttifeare),the seeds and fruits of Picralima nitida; the potential of theextract from the above plant extracts to increase the activityof some antibiotics on MDR bacteria was also investigated aswell as the role of bacterial efflux pumps in the resistance tothe tested plant extracts.

2. Material and Methods

2.1. Plant Materials and Extraction. The nine edible plantsused in this work were purchased from Dschang localmarket, west region of Cameroon in January 2010. Thecollected vegetal material were the fruits of Citrus medica,


the bulbs of Allium sativum and Allium cepa, the seeds ofCarica papaya, Cola acuminata, Buchholzia coriacea, Garciniakola, and Garcinia lucida, the seeds and fruits of Picralimanitida. The plants were identified by Mr. Tadjouteu Fulbert(Botanist) of the National Herbarium (Yaounde, Cameroon)where voucher specimens were deposited under a referencenumber (Table 1).

The fresh or powdered air-dried sample (1 kg) fromeach plant was extracted with methanol (MeOH) for 48 hat room temperature. The extract was then concentratedunder reduced pressure to give a residue that constituted thecrude extract. They were then kept under 4◦C until furtheruse.

2.2. Preliminary Phytochemical Investigations. The pres-ence of major secondary metabolite classes, namely, alka-loids, flavonoids, phenols, saponins, tannins, anthocyanins,anthraquinones, sterol, and triterpenes was determinedusing common phytochemical methods as described byHarborne [3].

2.3. Chemicals for Antimicrobial Assays. Ciprofloxacin (CIP),chloramphenicol (CHL), streptomycin (STR), tetracycline(TET), norfloxacin (NFX), cloxacillin (CLX), ampicillin(AMP), erythromycin (ERY), kanamycin (KAN), andcefepim (CEF) (Sigma-Aldrich, St Quentin Fallavier, France)were used as reference antibiotics. p-Iodonitrotetrazoliumchloride (INT) and phenylalanine arginine β-naphthylamide(PAβN) were used as microbial growth indicator and effluxpumps inhibitor (EPI), respectively.

2.4. Bacterial Strains and Culture Media. The studied mi-croorganisms include references (from the American TypeCulture Collection) and clinical (Laboratory collection)strains of Escherichia coli, Enterobacter aerogenes, Providenciastuartii, Pseudomonas aeruginosa, Klebsiella pneumonia, andEnterobacter cloacae (Table 2). They were maintained on agarslant at 4◦C and subcultured on a fresh appropriate agarplates 24 hrs prior to any antimicrobial test. Mueller HintonAgar was used for the activation of bacteria. The MuellerHinton Broth (MHB) was used for the MIC determinations.

2.5. Bacterial Susceptibility Determinations. The respectiveMICs of samples on the studied bacteria were determinedby using rapid INT colorimetric assay [4]. Briefly, the testsamples were first dissolved in DMSO/MHB. The solutionobtained was then added to MHB, and serially dilutedtwofold (in a 96-well microplate). One hundred microlitres(100 μL) of inoculum (1.5 × 106 CFU/mL) prepared inMHB was then added. The plates were covered with a sterileplate sealer, then agitated to mix the contents of the wellsusing a shaker and incubated at 37◦C for 18 hrs. The finalconcentration of DMSO was lower than 2.5% and doesnot affect the microbial growth. Wells containing MHB,100 μL of inoculums, and DMSO at a final concentration of2.5% served as a negative control. Ciprofloxacin was usedas reference antibiotic. The MICs of samples were detectedafter 18 hrs of incubation at 37◦C, following addition (40 μL)

of 0.2 mg/mL INT and incubation at 37◦C for 30 minutes[5]. Viable bacteria reduced the yellow dye to pink. MIC wasdefined as the lowest sample concentration that preventedthis change and exhibited complete inhibition of microbialgrowth.

Samples were tested alone and then, in the presenceof PAβN at 30 μg/mL final concentration. Two of the bestextracts, those from seeds of Garcinia kola and Picralimanitida fruits were also tested in association with antibioticsat MIC/2 and MIC/5. These concentrations were selectedfollowing a preliminary assay on one of the tested MDRbacteria, P. aeruginosa PA124 (see Supplemental MaterialS1 available online at doi:10.1155/2012/623723.). All assayswere performed in triplicate and repeated thrice. Fractionalinhibitory concentration (FIC) was calculated as the ratio ofMICAntibiotic in combination/MICAntibiotic alone and the interpreta-tion made as follows: synergistic (FIC ≤ 0.5), indifferent (0.5< FIC < 4), or antagonistic (FIC ≥ 4) [6]. (The FIC valuesare available in Supplemental Material S2).

3. Results

3.1. Phytochemical Composition of the Plant Extracts. Theresults of qualitative analysis showed that each plant con-tains various phytochemicals compounds such as alkaloids,anthocyanins, anthraquinons, flavonoids, phenols, saponins,tannins, and triterpenes as shown in Table 3.

3.2. Antibacterial Activity of the Plant Extracts. Extractswere tested for their antibacterial activities alone and incombination with PAβN on a panel of Gram-negativebacteria by the microdilution method. Results summarizedin Table 4 showed that the most active extracts were thosefrom Garcinia lucida (MIC ranged from 128 to 512 μg/mL),Garcinia kola (MIC from 128 to 1024 μg/mL), and the fruitsof Picralima nitida (MIC from 256 to 1024 μg/mL). Theantibacterial activities of these plant species were recordedagainst all the 29 studied microorganisms. Other extractsexhibited weak activities against a limited number of strainsstudied.

3.3. Role of Efflux Pumps in Susceptibility of Gram-NegativeBacteria to the Tested Plants Extracts. The various strains andMDR isolates were also tested for their susceptibility to theplants extracts, and reference antibiotic (ciprofloxacin) inthe presence of PAβN, an EPI. Preliminary tests showed thatPAβN did not have any antibacterial activity at 30 μg/mL.The association of the PAβN with the extracts reduced theMIC values of some of the extracts on some tested bacteria(Table 4). However, most of the studied extracts are not thesubstrates of the active efflux pumps.

3.4. Effects of the Association of Some Plants Extracts withAntibiotics. The strain P. aeruginosa PA124 was used tofind the appropriate subinhibitory concentration of theantibiotic-crude extract to be tested on other bacteria strains.The association of the extracts of P. nitida and G. kolareduced the MIC of ten antibiotics (CLX, AMP, ERY, KAN,


Ta

ble

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NC

Car

diov

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ases

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[7],

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gian

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ases

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[9]

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and

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[10]

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s,fr

uit

s,le

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nd

bark

Alk

aloi

ds,s

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trit

erp

enes

and

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1]

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tim

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[12]

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(Cap

pari

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Gas

troe

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met

han

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c[1

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]

Cit

rus

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(Ru

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6]

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tag

ain

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k,Tr

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t,E

c,Sa

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phys

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test

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infe

ctio

ns

[17]

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lkal

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(col

anin

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urg

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oun

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nce

rs[1

8,19

]

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ts,s

eeds

,an

dla

tex

kola

non

e,ko

lafl

avan

one,

and

garc

inia

flav

anon

e[2

0,21

]

An

tim

icro

bial

:se

eds

eth

anol

extr

act

agai

nst

Sa,

Sp,

Spn

,an

dH

i[2

2];

cyto

toxi

city

offr

uit

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met

han

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trac

t:w

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mia

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9]

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(Clu

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s

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ne,

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dsm

ethy

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[25]

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toto

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act:

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kac

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tyon

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and

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cells

and

pan

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lines

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r[2

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ine,

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[31]

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ycos

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nn

ins,

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des

and

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loid

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2]

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tim

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bial

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uit

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han

olan

ddi

chlo

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nst

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[31]

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otan

dst

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rk(a

queo

us

and

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anol

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ain

stSa

,Pa

,E

c,an

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ity

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oun

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Ds)

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tyof

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han

g;M

icro

orga

nis

ms

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Can

dida

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cans

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k:C

andi

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usei

;B

c:B

acill

usce

reus

;B

m:B

acill

usm

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m;B

s:B

acill

ussu

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s;B

st:B

acill

usst

earo

ther

mop

hilu

s;C

f:C

itro

bact

erfr

eund

ii;E

c:E

sche

rich

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li;H

i:H

aem

ophi

lus

influ

enza

e;K

p:K

lebs

iella

pneu

mon

iae;

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:Mor

gane

llam

orga

nii;

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Nei

sser

iago

norr

hoea

e;Pa

:Pse

udom

onas

aeru

gino

sa;P

f:P

lasm

odiu

mfa

lcip

arum

;Pm

:Pro

teus

mir

abili

s;P

v:P

rote

usvu

lgar

is;S

a:St

aphy

loco

ccus

aure

us;S

pn:S

trep

toco

ccus

pneu

mon

iae;

Sp:S

trep

toco

ccus

pneu

mon

iae;

St:

Salm

onel

laty

phi;

Tr:

Tric

hoph

yton

rubr

um;

Sf:

Stre

ptoc

occu

sfa

eeal

is;

Shf:

Shig

ella

flexn

eri;

Stm

:Sa

lmon

ella

typh

imur

ium

;Sp

:St

rept

ococ

cus

pneu

mon

ia).

bSc

reen

edac

tivi

ty:

sign

ifica

nt

(S:

CM

I<

100μ

g/m

L).

Mod

erat

e(M

:100

<C

MI≤

625μ

g/m

L).

Wea

k(W

:CM

I>

625μ

g/m

L).Q

:qu

alit

ativ

eac

tivi

tyba

sed

onth

ede

term

inat

ion

ofin

hib

itio

nzo

ne

[33]

.


Table 2: Bacterial strains and features.

Strains Features References

Escherichia coli

ATCC8739 and ATCC10536 Reference strains

AG100 Wild-type E. coli K-12 [31]

AG100A AG100 ΔacrAB::KANR [31, 34]

AG100ATETΔacrAB mutant AG100, owing acrF gene markedlyoverexpressed; TETR [31]

AG102 ΔacrAB mutant AG100 [35]

MC4100 Wild type E. coli

W3110 Wild type E. coli [36]

Enterobacter aerogenes

ATCC13048 Reference strains

EA-CM64CHLR resistant variant obtained from ATCC13048over-expressing the AcrAB pump

[37]

EA3Clinical MDR isolate; CHLR, NORR, OFXR, SPXR,MOXR, CFTR, ATMR, FEPR [38]

EA27Clinical MDR isolate exhibiting energy-dependentnorfloxacin and chloramphenicol efflux with KANR

and AMPR and NALR and STRR and TETR[38, 39]

EA289 KAN sensitive derivative of EA27 [40]

EA298 EA 289 tolC::KANR [40]

EA294 EA 289 ΔacrAB: ::KANR [40]

Enterobacter cloacae

ECCI69 Clinical isolatesLaboratory collection of UMR-MD1,University of Marseille, France

BM47 Clinical isolatesLaboratory collection of UMR-MD1,University of Marseille, France

BM67 Clinical isolatesLaboratory collection of UMR-MD1,University of Marseille, France

Klebsiella pneumoniae

ATCC12296 Reference strains

KP55 Clinical MDR isolate, TETR, AMPR, ATMR, and CEFR [41]

KP63 Clinical MDR isolate, TETR, CHLR, AMPR, and ATMR [41]

K24 AcrAB-TolCLaboratory collection of UMR-MD1,University of Marseille, France

K2 AcrAB-TolCLaboratory collection of UMR-MD1,University of Marseille, France

Providencia stuartii

NEA16 Clinical MDR isolate, AcrAB-TolC

ATCC29914 Clinical MDR isolate, AcrAB-TolC [42]

PS2636 Clinical MDR isolate, AcrAB-TolC

PS299645 Clinical MDR isolate, AcrAB-TolC

Pseudemonas aeruginosa

PA 01 Reference strains

PA 124 MDR clinical isolate [43]aAMP, ATMR, CEFR, CFTR, CHLR, FEPR, KANR, MOXR, STRR, and TETR. Resistance to ampicillin, aztreonam, cephalothin, cefadroxil, chloramphenicol,

cefepime, kanamycin, moxalactam, streptomycin, and tetracycline; MDR: multidrug resistant.

CHL, TET, FEP, STR, CIP, and NOR) at MIC/2 and/or MIC/5explaining the use of such concentrations. The associationsof the extracts of P. nitida fruits and G. kola with antibioticsdid not show any case of antagonism (FIC ≥ 4) meanwhileindifference was observed in some cases of the associations

of the extracts with FEP, CLX, and AMP (see Tables 5 and6, Supplemental Material S2). Many cases of synergy wereobserved in most of the strains with the associations G.kola/ERY against CM64, P. nitida/NOR against KP63, and P.nitida/ERY against PA124.


Ta

ble

3:E

xtra

ctio

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ects

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dph

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com

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acts

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nam

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sed

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Ph

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nn

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thra

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Ster

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Sapo

nin

s

Pic

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tida

Fru

its

13.5

6B

row

npa

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++

+−

++

+−

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eds

17.2

7B

row

npa

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++

++

+−

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itru

sm

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Bro

wn

past

e−

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−−

−−

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Alli

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rybu

lbs

18.9

9Ye

llow

pow

der

−−

−−

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esh

bulb

s4.

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row

npo

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r−

−−

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Seed

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row

npa

ste

+−

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acum

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Gar

cini

ako

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npa

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+−

++

+−

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npa

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lbs

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−+

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rybu

lbs

49.2

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row

npa

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−+

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−−

−−

−(+

):pr

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t;(−

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sen

t;∗ T

he

yiel

dw

asca

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late

das

the

rati

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the

mas

sof

the

obta

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met

han

olex

trac

t/m

ass

ofth

epl

ant

pow

der

orfr

esh

sam

ple.


Ta

ble

4:M

inim

alin

hib

itor

yco

nce

ntr

atio

n(μ

g/m

L)of

met

han

olex

trac

tsfr

omth

est

udi

edpl

ants

and

cipr

oflox

acin

.

Bac

teri

ast

rain

sP

lan

tsex

trac

tsa

and

MIC

(μg/

mL)

inth

eab

sen

cean

dpr

esen

ceof

PAβ

N(i

nbr

acke

t)C

AF

PN

FA

SB1

ASB

2B

CF

PN

SC

MF

GK

SG

LS

CP

SA

CB

1A

CB

2C

IPE

.col

iA

TC

C87

39—

1024

——

——

—51

251

2—

——

<0.

5A

TC

C10

536

—10

24—

——

1024

—51

251

210

24—

—64

W31

1010

24(1

024)

512

(512

)—

——

—(5

12)

—(1

024)

512

(256

)25

6(1

28)

——

—(1

024)

<0.

5(<

0.5)

MC

4100

—51

2—

——

1024

—51

225

6—

1024

1024

32A

G10

0A10

2451

2(1

28)

——

(512

)—

—(1

024)

—10

24(1

024)

256

(64)

1024

(102

4)—

—16

(8)

AG

100A

tet

1024

(102

4)10

24(5

12)

——

——

—25

6(2

56)

512

(512

)10

24(1

024)

1024

(102

4)—

32(8

)A

G10

2—

512

(128

)—

—(1

024)

—(1

024)

—(1

024)

—(1

024)

256

(64)

512

(256

)51

2(5

12)

——

32(1

6)A

G10

0—

512

——

1024

——

256

256

1024

—10

240.

5E

.aer

ogen

esA

TC

C13

048

—51

2—

——

——

512

256

——

—1

EA

294

—10

24—

——

——

512

256

—10

24—

64C

M64

1024

512

——

1024

—10

2425

625

6—

512

—32

EA

3—

512

——

——

—51

225

6—

——

32E

A29

8—

—(5

12)

——

——

—51

2(1

28)

256

(128

)—

——

1(<

0.5)

EA

2710

24(1

024)

512

(512

)—

——

——

256

(256

)25

6(2

56)

1024

(102

4)—

—1

(<0.

5)E

A28

9—

1024

(102

4)—

——

——

(102

4)51

2(5

12)

512

(256

)—

——

64(3

2)K

.pne

umon

iae

AT

CC

1129

610

24(1

024)

512

(256

)—

—(1

024)

—(1

024)

——

512

(512

)25

6(1

28)

—(5

12)

——

<0.

5(<

0.5)

KP

5551

2(5

12)

512

(256

)—

——

——

512

(512

)12

8(1

28)

1024

(102

4)10

24(1

024)

—(1

024)

32(4

)K

P63

—51

2—

——

——

512

512

——

—32

K2

—10

24—

——

——

512

(256

)25

6(1

28)

1024

(102

4)—

(512

)—

32(8

)K

2451

251

2—

——

——

512

256

1024

1024

—32

P.ae

rugi

nosa

PA01

—10

24(1

024)

——

——

—51

2(5

12)

512

(512

)—

——

32(4

)PA

124

—51

2—

——

——

1024

256

1024

——

128

P.st

uart

iiA

TC

C29

916

1024

(102

4)10

24(1

024)

——

——

—51

2(5

12)

256

(128

)10

24(1

024)

——

>64

(16)

NA

E16

1024

512

——

—10

24—

256

256

1024

1024

—64

PS2

636

—10

24—

——

——

128

128

——

—64

PS2

9964

510

24(1

024)

1024

(102

4)—

1024

(102

4)10

24(1

024)

1024

(102

4)25

6(2

56)

128

(128

)10

24(1

024)

——

<0.

5(<

0.5)

E.c

loac

aeB

M47

—25

6—

——

——

256

256

——

1024

64E

CC

I69

1024

512

——

1024

—10

2412

812

8—

——

128

BM

6710

2451

2—

——

——

256

256

1024

1024

—32

(—)

MIC

grea

ter

than

1024

μg/

mL;

a Ext

ract

from

CA

F:C

ola

acum

inat

afr

uit

;PN

F:P

icra

lima

niti

dafr

uit

s;A

SB1:

Alli

umsa

tivu

mdr

ybu

lbs;

ASB

2:A

llium

sati

vum

fres

hbu

lbs;

BC

F:B

uchh

olsi

aco

riac

eafr

uit

s;P

NS:

Pic

ralim

ani

tida

seed

s;C

MF:

Cit

rusm

edic

afr

uit

sju

ice;

GK

S:G

arci

nia

kola

seed

s;G

LS:

Gar

cini

alu

cida

seed

s;C

PS:

Car

ica

papa

yase

eds;

AC

B1:

Alli

umce

pafr

esh

bulb

s;A

CB

2:A

llium

cepa

dry

bulb

s;C

IP:c

ipro

flox

acin

.


Ta

ble

5:M

ICof

diff

eren

tan

tibi

otic

saf

ter

the

asso

ciat

ion

ofth

eex

trac

tof

Pic

ralim

ani

tida

fru

its

atM

IC/2

,MIC

/5ag

ain

stte

nM

DR

bact

eria

stra

ins.

An

tibi

otic

sE

xtra

ctco

nce

ntr

atio

nB

acte

rial

stra

ins,

MIC

(μg/

mL

)of

anti

biot

ics

inth

eab

sen

cean

dpr

esen

ceof

the

extr

act

AG

100

AG

100A

tet

AG

102

CM

64E

A3

EA

27E

A28

9K

P55

KP

63PA

124

CIP

0M

IC/2

MIC

/5

≤0.5

≤0.5

≤0.5

128

16(8

)S

32(4

)S

3216

(2)S

16(2

)S

≤0.5

≤0.5

≤0.5

256

64(4

)S

128(

2)S

≤0.5

≤0.5

≤0.5

6416

(4)S

32(2

)S

256

128(

2)S

256(

1)I

128

64(2

)S

64(2

)S

32 8(4)

S

8(4)

S

CH

L0

MIC

/2M

IC/5

42(

2)S

4(1)

I

>51

264

(>8)

S

128(>

4)S

128

16(8

)S

32(4

)S

512

64(8

)S

128(

4)S

512

64(8

)S

128(

4)S

648(

8)S

32(2

)S

512

64(8

)S

128(

4)S

3216

(2)S

16(2

)S

512

128(

4)S

256(

2)S

64 8(8)

S

32(2

)S

STR

0M

IC/2

MIC

/5

42(

2)S

2(2)

S

>51

225

6(>

2)S

512(>

1)

≤0.5

≤0.5

≤0.5

512

256(

2)S

512(

1)I

>51

264

(8)S

256(>

2)S

1616

(1)I

16(1

)I

1616

(1)I

16(1

)I

1616

(1)I

16(1

)I

128

128(

1)I

128(

1)I

>51

212

851

2

AM

P0

MIC

/2M

IC/5

3216

(2)S

16(2

)S

>51

251

2(1)

I

>51

2

256

64(4

)S

64(4

)S

512

512(

1)I

512(

1)I

>51

212

8(>

4)S

512(>

1)

6464

(1)I

64(1

)I

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

216

(>32

)S

16(>

32)S

TE

T0

MIC

/2M

IC/5

6416

(4)S

32(2

)S

256

128(

2)S

256(

1)I

81(

8)S

4(2)

S

128

32(4

)S

64(2

)S

512

64(8

)S

128(

4)S

82(

4)S

4(2)

S

328(

4)S

8(4)

S

84(

2)S

4(2)

S

168(

2)S

8(2)

S

8 2(4)

S

4(2)

S

CL

X0

MIC

/2M

IC/5

6432

(2)S

32(2

)S

>51

2>

512

>51

2

>51

212

8(>

4)S

256(>

2)S

>51

225

6>

512

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

251

2(>

1)>

512

KA

N0

MIC

/2M

IC/5

≤4 ≤4 ≤4

512

128(

4)S

128(

4)

1616

(1)I

16(1

)I

≤4 ≤4 ≤4

≤4 ≤4 ≤4

>51

251

2(>

2)S

>51

2

32≤4

(>8)

S

16(2

)I

328(

4)S

8(4)

S

512

128(

4)S

512(

1)I

128

64(2

)S

64(2

)S

ER

Y0

MIC

/2M

IC/5

6432

(2)S

64(1

)I

512

256(

2)S

256(

2)S

1616

(1)I

16(1

)I

256

32(8

)S

256(

1)I

328(

4)S

16(2

)S

84(

2)S

8(1)

I

128

128(

1)I

128(

1)I

6416

(4)S

32(2

)S

128

64(2

)S

128(

1)I

128

8(16

)S

8(16

)S

NO

R0

MIC

/2M

IC/5

3216

(2)S

32(1

)I

512

128(

4)S

128(

4)S

128

32(4

)S

64(2

)S

168(

2)S

16(1

)I

164(

4)S

16(1

)I

3216

(2)S

16(2

)S

6432

(2)S

32(2

)S

6432

(2)S

32(2

)S

644(

8)S

16(4

)S

128

32(4

)S

32(4

)S

FEP

0M

IC/2

MIC

/5

512

256(

2)S

512(

1)I

512

128(

4)S

512(

1)I

512

512(

1)I

512(

1)I

>51

225

6(>

4)S

>51

2

256

64(4

)S

128(

2)S

512

252(

2)S

512(

1)I

512

512(

1)I

512(

1)I

>51

2>

512

>51

2

512

256(

2)S

512(

1)I

512

512(

1)I

512(

1)I

():f

old

incr

ease

inM

ICva

lues

ofth

ean

tibi

otic

saf

ter

asso

ciat

ion

wit

hpl

ants

extr

act;

S:sy

ner

gy,I

:in

diff

eren

ce;A

MP

:am

pici

llin

;FE

P:c

efep

ime;

CH

L:ch

lora

mph

enic

ol;K

AN

:kan

amyc

in;N

OR

:nor

flox

acin

;ST

R:

stre

ptom

ycin

;TE

T:t

etra

cycl

ine;

CIP

:cip

rofl

oxac

in;C

LX

:clo

xaci

llin

;ER

Y:er

yth

rom

ycin

.


Ta

ble

6:M

ICof

diff

eren

tan

tibi

otic

saf

ter

the

asso

ciat

ion

ofth

eex

trac

tof

Gar

cini

ako

lase

eds

atM

IC/2

,MIC

/5ag

ain

stte

nM

DR

bact

eria

stra

ins.

An

tibi

otic

sE

xtra

ctco

nce

ntr

atio

nB

acte

rial

stra

ins,

MIC

(μg/

mL

)of

anti

biot

ics

inth

eab

sen

cean

dpr

esen

ceof

the

extr

act

AG

100

AG

100A

tet

AG

102

CM

64E

A3

EA

27K

P55

KP

63E

A28

9PA

124

CIP

0M

IC/2

MIC

/5

≤0.5

≤0.5

≤0.5

128

64(2

)S

64(2

)S

328(

4)S

8(4)

S

≤0.5

≤0.5

≤0.5

256

128(

2)S

128(

2)S

≤0.5

≤0.5

≤0.5

256

128(

2)S

256(

1)I

128

32(4

)S

128(

1)I

6432

(2)S

64(1

)I

32 <0.

516

(2)S

CH

L0

MIC

/2M

IC/5

44(

1)I

4(1)

I

>51

251

2(>

1)51

2(>

1)

128

16(8

)S

32(4

)S

512

256(

2)S

512(

1)I

512

512(

1)I

512(

1)I

648(

8)S

16(4

)S

3232

(1)I

32(1

)I

512

128(

4)S

256(

2)S

512

128(

4)S

256(

2)S

64 32(2

)S

32(2

)S

STR

0M

IC/2

MIC

/5

42(

2)S

2(2)

S

>51

2>

512

>51

2

<0.

5<

0.5

<0.

5

512

256(

2)S

512(

1)I

>51

2>

512

>51

2

168(

2)S

16(1

)I

168(

2)S

16(1

)I

128

128(

1)I

128(

1)I

168(

2)S

16(1

)I

>51

216

(>32

)S

128(>

4)S

AM

P0

MIC

/2M

IC/5

328(

4)S

32(1

)I

>51

2>

512

>51

2

256

128(

2)S

128(

2)S

>51

2>

512

>51

2

>51

2>

512

>51

2

6464

(1)I

64(1

)I

>51

2>

512

>51

2

>51

251

251

2

>51

2>

512

>51

2

>51

264

(>8)

S

256(>

2)S

TE

T0

MIC

/2M

IC/5

6432

(2)S

32(2

)S

256

128(

2)S

128(

2)S

82(

4)S

2(4)

S

128

64(2

)S

64(2

)S

512

256(

2)S

256(

2)S

82(

4)S

4(2)

S

82(

4)S

4(2)

S

164(

4)S

16(1

)I

328(

4)S

8(4)

S

8 4(2)

S

8(1)

I

CL

X0

MIC

/2M

IC/5

3216

(2)S

32(1

)I

>51

2>

512

>51

2

>51

251

251

2

>51

2>

512

>51

2

>51

2>

512

>51

2

128

32(4

)S

128(

1)I

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

212

8(>

4)S

>51

2

KA

N0

MIC

/2M

IC/5

≤4 ≤4 ≤4

512

32(1

6)S

256(

2)S

1616

(1)I

16(1

)I

≤4 ≤4 ≤4

≤4 ≤4 ≤4

>51

251

2>

512

324(

8)S

16(2

)S

512

256(

2)S

512(

1)I

32≤4

(>8)

S

32(1

)S

128

16(8

)S

64(2

)S

ER

Y0

MIC

/2M

IC/5

6416

(4)S

64(1

)I

512

32(1

6)S

512(

1)I

1616

(1)I

16(1

)I

256

16(1

6)S

32(8

)S

6416

(4)S

16(4

)S

84(

2)S

8(1)

I

6464

(1)I

64(1

)I

128

16(8

)S

32(4

)S

128

128(

1)I

128(

1)I

256

32(8

)S

256(

1)I

NO

R0

MIC

/2M

IC/5

328(

4)S

16(2

)S

512

128(

4)S

256(

2)S

128

64(2

)S

128(

1)I

164(

4)S

8(2)

S

168(

2)S

8(2)

S

328(

4)S

32(1

)I

128

32(4

)S

128(

1)I

648(

8)S

16(4

)S

6432

(2)S

32(2

)S

256

256(

1)I

256(

1)I

FEP

0M

IC/2

MIC

/5

512

512(

1)I

512(

1)I

>51

251

2(>

1)51

2(>

1)

>51

2>

512

>51

2

>51

225

6(>

2)S

256(>

2)S

>51

2>

512

>51

2

>51

2>

512

>51

2

>51

2>

512

>51

2

512

256(

2)S

512(

1)S

512

512(

1)I

512(

1)I

>51

251

2(>

1)51

2(>

1)

():f

old

incr

ease

inM

ICva

lues

ofth

ean

tibi

otic

saf

ter

asso

ciat

ion

wit

hpl

ants

extr

act;

S:sy

ner

gy;I

:in

diff

eren

ce;A

MP

:am

pici

llin

;FE

P:c

efep

ime;

CH

L:ch

lora

mph

enic

ol;K

AN

:kan

amyc

in;N

OR

:nor

flox

acin

;ST

R:

stre

ptom

ycin

;TE

T:t

etra

cycl

ine;

CIP

:cip

rofl

oxac

in;C

LX

:clo

xaci

llin

;ER

Y:er

yth

rom

ycin

.


4. Discussion

4.1. Antibacterial Activities and Chemicals Compositions of theTested Extracts. The phytochemical studies revealed the pres-ence of at least two classes of secondary metabolites in eachof the plant extracts. Several alkaloids, flavonoids, phenols,saponins, anthocyanins, anthraquinones, sterols, tannins,and triterpenes have been found active on pathogenicmicroorganisms [44, 45]. Some of these compounds werefound to be present in the plant species under this study, andthey could contribute to the observed antimicrobial activitiesof some plant extracts. The results of the phytochemicaltest on G. kola are in accordance with those obtained byOnayade et al., [46, 47]. Many compounds have been isolatedfrom G. kola, such as kolaflavone and 2-hydroxybiflavone[48–50] but their antimicrobials activities have not beenevaluated. However, Adegboye et al. [51] reported theactivity of G. kola on some streptomycin-sensitive Gram-positive bacteria strain. The present study therefore providesadditional information on the antibacterial potential of thisplant on MDR bacteria.

The previous phytochemical analyses on hexane extractfrom the seeds of G. lucida revealed several types ofcompounds [8, 23]. These include terpenoids, anthocyanins,flavonoids, and saponins derivatives. This report thereforeagrees well with the phytochemical data being reportedherein.

The results of the phytochemical analysis of the extractof fruits of P. nitida are similar to those obtained byKouitcheu [52]. Several alkaloids previously isolated fromthis plant include akuammicine, akuammine, akuammidine,picraphylline, picraline, and pseudoakuammigine [32, 53].Their antibacterial activities have not yet been demonstratedbut many alkaloids are known to be active on Gram-negativebacteria [33]. Differences were noted in the chemical compo-sition of the seeds and fruits of P. nitida, evidently explainingthe differences in the antibacterial activity of the two partsof this plant. In fact, the presence of tannins in the fruits maycontributes to its better activity compared to the seeds as theywere reported to inactivate the microbial adhesins, enzymes,transports proteins and cellular envelop [54].

Extracts from C. papaya, C. medica, B. coriacea, A. cepa,and C. acuminata showed weak activities against a limitednumber of strains. Nonetheless, the extracts from B. coriaceawere rather reported to have good antibacterial activities.Their weak activities as observed in the present paper couldtherefore be due to the multidrug resistance of the studiedbacteria.

4.2. Effects of the Association of Some Plants Extracts withAntibiotics. Three of the most active plants extracts (G. kola,G. lucida, and P. nitida) were associated with antibioticswith the aim to evaluate the possible synergistic effects oftheir associations. A preliminary study using P. aeruginosaPA124, one of the ten MDR bacteria used in this paper, wascarried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL,TET, FEP, STR, CIP, and NOR) to select the appropriatesub-inhibitory concentrations of the extract to be used. Theresults (see Supplemental Material S1) allowed the selection

of G. kola, G. lucida and their MIC/2 and MIC/5 as the sub-inhibitory concentrations. No antagonistic effect (FIC ≥ 4)was observed between extracts and antibiotics meanwhileindifference was observed in the case of CLX, FEP, AMP,which are β-lactams acting on the synthesis of the bacteriacell wall [55] (Tables 5 and 6, Supplemental Material S2).Many studies demonstrated that efflux is the mechanism ofresistance of bacteria for almost all antibiotic classes [56].It is well demonstrated that the efflux pumps reduce theintracellular concentration of antibiotics and consequentlytheir activities [57]. The MDR bacteria strains used inthis paper are known for their ability to overexpress activeefflux [58]. At MIC/2, synergistic effects were noted withthe association of NOR, CHL, TET (on 100% the studiedbacteria), ERY (on 80%), CIP (on 70%), and P. nitida extractmeanwhile G. kola extract also increased the activity ofNOR, TET (on 100%), ERY, and CIP (on 70%). Plant canbe considered as an efflux pumps inhibitor if a synergisticeffect with antibiotics is induced on more than 70% bacteriaexpressing active efflux pumps [6]. Therefore, the extractsfrom P. nitida and G. kola probably contain compounds thatcan acts as EPI. The results of the present paper corroboratewith those of Iwu et al. [7] reporting the existence ofsynergy effects between G. kola extract and gatifloxacin(G. kola/gatifloxacin in the proportions of 9/1, 8/2, 7/3,and 6/4) against Bacillus subtilis and the proportions of G.kola/gatifloxacin (at 9/1, 2/8, and 1/9) against Staphylococcusaureus.

The overall results of the present work provide baselineinformation for the possible use of the studied plants andmostly G. Lucida, G. Kola, and P. Nitida extracts in thetreatment of bacterial infections involving MDR phenotypes.In addition, the extracts of these plants could be used inassociation with common antibiotics to combat multidrugresistant pathogens.

Acknowledgments

The authors are thankful to the Cameroon National Herbar-ium (Yaounde) and University of Dschang Herbarium forplants identification.

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