Access to this full-text is provided by Wiley.
Content available from BioMed Research International
This content is subject to copyright. Terms and conditions apply.
Research Article
Beninese Medicinal Plants as a Source of
Antimycobacterial Agents: Bioguided Fractionation and In Vitro
Activity of Alkaloids Isolated from Holarrhena floribunda Used
in Traditional Treatment of Buruli Ulcer
Achille Yemoa,1Joachim Gbenou,2Dissou Affolabi,3
Mansourou Moudachirou,2André Bigot,1Séverin Anagonou,3Françoise Portaels,4
Anandi Martin,5and Joëlle Quetin-Leclercq6
1Unit´
e de Formation et de Recherche en Pharmacie, Facult´
e des Sciences de la Sant´
e (FSS), Universit´
ed’AbomeyCalavi(UAC),
04 BP 494 Cotonou, Benin
2LaboratoiredePharmacognosieetdesHuilesEssentielles(LAPHE),Facult
´
e des Sciences de la Sant´
e (FSS)
and Facult´
e des Sciences et Techniques (FAST), Universit´
e d’Abomey Calavi (UAC), 01 BP 188 Cotonou, Benin
3LaboratoiredeR
´
ef´
erence des Mycobact´
eries (LRM), Centre National Hospitalier de Pneumo-Phtisiologie (CNHPP),
01 BP 817 Cotonou, Benin
4Department of Biomedical Sciences, Institute of Tropical Medicine (IMT), Nationalestraat 155, 2000 Antwerpen, Belgium
5Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University,
K.L. Ledeganckstraat 35, 9000 Gent, Belgium
6Pharmacognosy Research Group, Louvain Drug Research Institute (LDRI), Universit´
eCatholiquedeLouvain(UCL),
B1 7203 Avenue E. Mounier 72, 1200 Bruxelles, Belgium
Correspondence should be addressed to Achille Yemoa; ayemoa@yahoo.fr
Received July ; Accepted August
Academic Editor: Parisa Farnia
Copyright © Achille Yemoa et al. is 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.
Buruli ulcer (BU) imposes a serious economic burden on aected households and on health systems that are involved in diagnosing
the disease and treating patients. Research is needed to nd cost-eective therapies for this costly disease. Plants have always been
an important source of new pharmacologically active molecules. Consequently we decided to undertake the study of plants used
in traditional treatment of BU in Benin and investigate their antimycobacterial activity as well as their chemical composition.
Extracts from forty-four () plant species were selected on account of reported traditional uses for the treatment of BU in Benin
and were assayed for antimycobacterial activities. Crude hydroethanolic extract from aerial parts of Holarrhena oribunda (G. Don)
T. Durand and Schinz was found to have signicant antimycobacterial activity against M.ulcerans (MIC = 𝜇g/mL). We describe
here the identication of four steroidal alkaloids from Mycobacterium ulcerans growth-inhibiting fractions of the alkaloidal extract
of the aerial parts of Holarrhena oribunda. Holadysamine was puried in sucient amount to allow the determination of its MCI
(= 𝜇g/mL). ese results give some support to the use of this plant in traditional medicine.
1. Introduction
Buruli ulcer (BU), caused by the environmental organism
Mycobacterium ulcerans and characterized by necrotizing
skinandbonelesions,posesimportantpublichealthissuesas
the third most common mycobacterial infection in humans
[]. e disease has become substantially more frequent over
the past decade, particularly around the Gulf of Guinea, and
has been detected or suspected in at least countries. Clinical
diagnosis of BU disease should be conrmed by PCR, as rec-
ommended by the World Health Organization (WHO), and
case patients should be treated with rifampin/streptomycin
Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 835767, 5 pages
http://dx.doi.org/10.1155/2015/835767
BioMed Research International
daily for weeks (therapy available since ), combined, if
necessary, with surgery.
In Benin, sociocultural believes and practices strongly
inuence the health-seeking behaviours of people aected by
BU. e rst recourse is oen traditional treatment. Most of
the components in the traditional treatment belong to the
plant kingdom []. Plants provide unlimited opportunities
for new drug leads because of the unmatched availability of
chemical diversity. Recently we carried out an ethnobotanical
survey involving seventeen traditional practitioners within
the Ouinhi community in the Zou Department (Benin). We
noted that about forty-nine dierent plants were used for the
traditional treatment of BU. Dierent parts of these plants
were included in various pharmaceutical forms for internal or
external use []. We realized a screening of plant extracts
used in traditional medicine to treat BU [,]. Results showed
that crude hydroethanolic extract of Holarrhena oribunda
was eective in inhibiting the growth of Mycobacter ium
ulcerans (MIC 𝜇g/mL) and worth further investigations.
Holarrhena oribunda (G.Don)T.DurandandSchinz
growsasashrubortreeuptomtall,withastem
diameter of up to cm. Its fragrant owers feature a white
corolla. Fruit is pale grey to dark brown with paired follicles,
each up to cm long. is species is known as four syn-
onyms: Rondeletia oribunda G. Don, Holarrhena africana A.
DC., Holarrhena wulfsbergii Stapf, Holarrhena ovata. In the
Republic of Benin, the common names of this plant are: Fon:
“kpakpatoun” []; Yoruba and Nago: “ako ire,” “ire Ibedji;”
Mina: “gaoti” [].Hoyeretal.[]inisolatedfromthe
bark of the trunk a steroidal alkaloid called holarrhesine, but
no activity was reported for this compound. ey also iso-
lated conessine from the bark of the root. A chemical study of
the leaves of H. oribunda achieved by Janot team in led
to the isolation of other new alkaloids including holaphylline
and holaphyllamine, while other alkaloids were isolated later.
Figure shows the structures of the chemical compounds
isolated from H. oribunda: holaphylline, holaphyllamine,
holamine, holaphyllinol, holaphyllidine, holadysamine, hol-
arrhesine, conessine, and progesterone [–]. Phytochemical
investigations on this plant have so far led to the identication
of a crude in vitro active alkaloid extract on M. tuberculosis
(MCI = . g/L) and other microorganisms [,].
Here we describe compounds identied from bioactive
fractions of the extract and evaluation of the inhibitory eect
of one of them on the growth of M. ulcerans.
2. Materials and Methods
2.1. Experimental
2.1.1. Plant Materials. Plant species was collected and iden-
tied by a botanist from the National Herbarium of Benin
and voucher specimens (Yemoa ) are deposited at the same
herbarium.
2.1.2. Preparation of Crude Hydroethanolic Extracts. Dried
plantsweregroundtoapowderwithapulverizator(National
Mixer Grinder Mx-N, Japan). g of powder was then
macerated h (at room temperature) in % ethanol in a
/ (w/v) ratio. e material was ltered through a Millipore
lterof.𝜇m (Acrodisc, USA). e ltrate was concentrated
under reduced pressure at less than ∘Cusingarotary
evaporator (Buchi Rotavapor R-/, Switzerland) to
obtain a crude residue.
2.1.3. Fractionation of Crude Extracts and Isolation of Active
Constituents. Crude hydroethanolic extract was fractionated
on silica gel (,–, mm Merck, Germany) by atmo-
spheric pressure liquid chromatography eluting with solvents
of increasing polarity, namely, hexane, dichloromethane,
ethyl acetate, and water, yielding fractions (F, F, F, and
F). Of these, fraction F (dichloromethane) was found
to cause growth inhibition of M. ulcerans and was, as a
result, selected for further work. is fraction was monitored
usingTLCplates(TLCsilicagel60F254S, Merck) and led
to characterize the presence of alkaloids (toluene : ethyl ace-
tate:diethylamine: [::], detection-Dragendor’s spray
reagent). We then performed a more specic extraction to
obtain an enriched alkaloid extract (Figure ).
We therefore proceeded to fractionation of this enriched
extract by Atmospheric Pressure Liquid Chromatography
(APLC) and Medium Pressure Liquid Chromatography
(MPLC) followed by gel ltration on Sephadex LH to
purify this enriched alkaloid fraction.
Atmospheric Pressure Liquid Chromatography (APLC). Alka-
loids enriched extract was repeatedly fractionated on silica
gel (,–, mm, Merck, Germany) eluting with
solvents of increasing polarity (mixture of CH2Cl2/MeOH
: to % MeOH followed by MeOH : H2O:and
MeOH : acetic acid : ). Fractions were monitored by TLC
(toluene : ethyl acetate : diethylamine : [ : : ], detection-
Dragendor’s spray reagent) and similar fractions were
combined and concentrated in vacuo. Prepuried fractions
obtained by APLC were rechromatographied by MPLC.
Medium Pressure Liquid Chromatography (MPLC). MPLC
wasperformedonglasscolumnspackedwithLiChroprepSi
(– 𝜇m) from Merck with a mobile phase composed of
CH2Cl2/MeOH : to % MeOH. Prepuried fractions
obtained were monitored by TLC (toluene : ethyl acetate : di-
ethylamine: [ : : ], detection-Dragendor ’s spray reagent)
and similar fractions were combined and concentrated in
vacuo followed by gel ltration on Sephadex LH (MeOH).
Five dierent puried fractions were obtained and analyzed
by high pressure liquid chromatography coupled to mass
spectroscopy (HPLC-MS or LC-MS) (ermo Scientic
Accela LC Systems, orbitrap).
LC-MS. High pressure liquid chromatography coupled to a
diode array detection and mass spectrometry with positive
electrospray ionization (HPLC-ESI-MSn)wasemployedto
rapidly separate and identify the constituents in these ve
puried fractions. e LC MS/MS system consisted in a
ermo Accela pump, autosampler, photodiode array detec-
tor, and ermo Scientic LTQ orbitrap XL mass spectrome-
ter.
BioMed Research International
N
O
HN
OH
O
O
HH
H
H
H
H
H
H
N
N
N
R1
R2
R1
R2
R1
R2
R3
R3
R4
CH3
R1=R2=H,
R1=R2=H,
Progesterone
Holadysamine: 3-methylamino pregn-5,16-dien-20-ol
holaphyllamine: 3𝛽-amino pregn-5-en-20-one
holamine: 3𝛼-amino pregn-5-en-20-one
holaphylline: 3𝛽-methylamino pregn-5-en-20-one
holaphyllinol: 3𝛽-methylamino pregn-5-en-20𝛽-ol
holaphyllidine: 3𝛽-methylamino pregn-5-en-20𝛼-ol
:
:
:
:
:
R1=H, R2=CH3–,
R1=H, R2=CH3–, R3=H, R4=OH,
R1=H, R2=CH3–, R3=OH, R4=H,
R1=R2=CH3–, R3=H: conessine
R1=H, R2=CH3–, R3=CH3C(CH3)CHCH2COO−: holarrhesine
F : Structures of chemical compounds isolated from H. oribunda.
Hexane phase
Aqueous solution CH2Cl2dried with Na2SO4
(total alkaloids)
Aqueous phase
(NaOH 10%)
Hexane (3 × 200 mL)
CH2Cl2(3 × 200 mL)
Hydroethanolic extract
(acidied with H2SO42%)
F : Scheme for preparation of alkaloids enriched extract.
Separation was performed using an analytical RP-C
Lichrospher- column ( × mm, particle size 𝜇m)
with a gradient using acetonitrile and , % triuoroacetic
acid (TFA) aqueous solution as the mobile phase. e gradi-
entusedstartsat%aqueoussolutionand%acetonitrile,
goingtoaplateauof%acetonitrileinmin.ese
conditionsareheldforminbeforereturningtotheinitial
conditions.egradientwaslinearandtheowratewas
. mL/min. e injection volume was 𝜇L; the column
temperature was ∘C.
High-resolution MS was measured with ESI source in the
positive mode. e following inlet conditions were applied:
capillary temperature ∘C, capillary voltage V, tube lens
V, sheath gas ow u.a, auxiliary gas ow u.a, and
sweep gas ow u.a. Data acquisition and processing were
performed with Xcalibur soware version ...
NMR. NMR spectra (1H, 13C, 1H-1HCOSY,and1H-13C
HSQC) were recorded using a Bruker-, MHz for 1H
and MHz for 13C. Chemical shis were expressed in ppm
(𝛿) using TMS (tetramethylsilane) (Aldrich Sigma, Germany)
as reference. For NMR analysis, the sample was dissolved
in 𝜇LofCDCl
3-MeOD ( : ) and transferred to a NMR
tube.
3. Results and Discussion
Our previous investigations allowed determining that the
hydroethanolic extract of H. oribunda inhibited growth
BioMed Research International
of M. ulcerans with a MIC value of 𝜇g/mL using the
resazurin microtiter assay (REMA) []. e CH2Cl2fraction
of this extract also inhibited growth of M. ulcerans with a
MIC value of 𝜇g/mL and contained alkaloids. Fraction-
ation of an enriched alkaloid extract (MIC = . 𝜇g/mL)
was undertaken as described in the Experimental Section,
allowing the identication of four dierent alkaloids in ve
dierent fractions and isolation of one of them with high
purity. e structure of the major compound was identied
as holadysamine (-methyl amino pregna,-dien--ol) by
MS and NMR []. LC-MS was used to identify alkaloids in
the other fractions, named compounds A, B, and C, and
gave molecular formula of, respectively, C22H37ON (MW:
.), C21H33ON (MW: .), and C23H37ON (MW:
.). According to bibliographic data on compounds
isolated from H. oribunda [,], they could correspond
to holaphyllinol (MW: .), holamine, or holaphyllamine
(MW: .), N,N-dimethyl holamine, N-dimethyl hola-
phyllamine or methyl holaphylline (MW: .).
HPLC-ESI-MS/MS allowed us to show that compound
A could correspond to holaphyllinol and B to holamine or
holaphyllamine while C seems to be a new compound. On
the basis of the analysis of spectroscopic data, we observed
the presence of a fragment at m/z ([M-H2O+H]+)and
thelossofawatermoleculeintheMS/MSfragmentations
ofCallowingustoproposeanalcoholicandnotaketonic
function on C (Figure ).
Further studies must be performed to conrm these
chemicalstructures.Itcouldnotbedonebecauseofthelack
of compound.
Fraction containing pure holadysamine was found to be
more active (MIC = 𝜇g/mL) than fractions containing A,
B, or C or the crude hydroethanolic extract ( 𝜇g/mL) but
less active than the reference rifampicin (MIC = 𝜇g/mL).
is low activity could not totally explain the use of this
plant in traditional treatment but as this plant is used in
mixtures with other plants; the various compounds present
in these plant extracts can act synergistically as it is the
case with the antibiotics ethambutol, clarithromycin, and
rifampicin []. Rastogi and Labrousse in their study showed
that the use of these antibiotics in combination results in
increased bactericidal eects compared to drugs used alone
or in combination with two of them.
4. Conclusions
Our results show that extracts of aerial parts of H. oribunda,
used by traditional healers to treat BU, exhibited signicant
in vitro antimycobacterial activities. Four active alkaloids
have been identied. Holadysamine the major compound
was found to be the most active one (MIC = 𝜇g/mL), but
this activity is lower than that of rifampicine. As synergy
may be found between dierent compounds, it would be
interesting to analyze the ecacy of the other alkaloids and
their combinations. Furthermore, as this plant is used in
association with other ones, it is also interesting to test plant
associations as used by traditional practitioners. Remedies
that would prove eective activity should be applied for
OH
N
Chemical formula: C23H37 NO
Exact mass: 343.28751
F : Proposed chemical structure for compound C.
toxicological and pharmacological studies. ere is also an
urgent need of standardization of traditional remedies based
on plants.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
Achille Yemoa was a doctoral student of the Wallonie
Bruxelles International (WBI). is research was nancially
supported by the WBI, the BURULICO Project no. INCO-
CT--, and CUD (Commission Universitaire pour
le D´
eveloppement). e authors gratefully thank the Bel-
gian National Fund for Scientic Research (FNRS) (FRFC
.. and CR) and the Special Fund for Research
(FSR). ey acknowledge the immense contribution of the
Ouinhi traditional practitioners to this study. ey also thank
Dr. M.-F. Herent for her help in LC-MS analysis.
References
[] D. S. Walsh, F. Portaels, and W. M. Meyers, “Buruli ulcer:
advances in understanding Mycobacterium ulcerans infection,”
Dermatologic Clinics,vol.,no.,pp.–,.
[] A.L.Y
´
emoa,J.D.Gb
´
enou, R. C. Johnson et al., “Identication et
´
etude phytochimique de plantes utilis´
ees dans le traitement tra-
ditionnel de l’ulc`
eredeBuruliauB
´
enin,” Ethnopharmacologia,
vol. , pp. –, .
[] A.Yemoa,J.Gbenou,D.Aolabietal.,“Buruliulcer:areview
of in vitro tests to screen natural products for activity against
Mycobacterium ulcerans,” Planta Medica,vol.,no.,pp.–
, .
[] E. A. Adjanohoun, V. Adjakidj`
e, M. R. Ahyi et al., “Contribu-
tion aux ´
etudes ethnobotaniques et oristiques en R´
epublique
Populaire du B´
enin,” M´
edecine Traditionnelle et Pharmacop´
ee,
l’Agence de Coop´
eration Culturelle et Technique, Paris, France,
.
[] G. A. Hoyer, A. Huth, I. Nitschke, and C. V. Szczepanski,
“Biologically active compounds from plants III Holarrhesine,
a new steroidal alkaloid from Holarrhena oribunda,” Planta
Medica, vol. , pp. –, .
[] M. M. Janot, A. Cave, and R. Goutarel, “Sterol alkaloids. III.
Togholamine, holaphyllamine, and holaphylline, new alkaloids
BioMed Research International
from the leaves of Holarrhena oribunda,” Bulletin de la Soci´
et´
e
Chimique de France, pp. –, .
[] R.Goutarel,A.Cave,L.Tan,andM.Leboeuf,“Steroidalkaloids.
XII. Synthesis of holaphyllamine and holamine,” Bulletin de la
Soci´
et´
eChimiquedeFrance, pp. –, .
[] M. Leboeuf, A. Cav´
e, and R. Goutarel, “Alcalo¨
ıdes st´
ero¨
ıdiques
LXXXII. Composition chimique des feuilles de l’ Holarrhena
oribunda Dur et Schinz. Isolement de la progest´
erone et de
quatre nouveaux alca lo¨
ıdes: m´
ethyl-holaphylline, holaphyllinol,
holaphyllidine et dihydroholaphyllamine,” Annales Pharmaceu-
tiques Franc¸aises,vol.,pp.–,.
[] S. Garg and K. K. Bhutani, “Chromatographic analysis of
Kutajarista—an ayurvedic polyherbal formulation,” Phytochem-
ical Analysis,vol.,no.,pp.–,.
[] S. Lambin and J. Bernard, “Action of some alkaloids on
Mycobacterium tuberculosis,” Comptes Rendus des S´
eances de la
Soci´
et´
edeBiologieetdeSesFiliales,vol.,pp.–,.
[] B. C. Adebayo-Tayo and A. A. Adegoke, “Phytochemical and
microbial screening of herbal remedies in Akwa Ibom State,
SouthSouthernNigeria,”Journal of Medicinal Plants Research,
vol. , pp. –, .
[] N. Rastogi and V. Labrousse, “Extracellular and intracellular
activities of clarithromycin used alone and in association
with ethambutol and rifampin against Mycobacterium avium
complex,” Antimicrobial Agents and Chemotherapy,vol.,no.
, pp. –, .
Available via license: CC BY
Content may be subject to copyright.
Content uploaded by Joëlle Quetin-Leclercq
Author content
All content in this area was uploaded by Joëlle Quetin-Leclercq on Nov 05, 2015
Content may be subject to copyright.
