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2017; 1 (2): 122–135
Open Chem., 2017; 15: 208–218
Research Article
Open Access
Nassifatou Koko Tittikpina*, Wouyo Atakpama, Hodabalo Pereki, Muhammad Jawad Nasim,
Wesam Ali, Stéphane Fontanay, Frédéric Nana, Chukwunonso ECC Ejike, Gilbert Kirsch,
Raphaël Emmanuel Duval, Patrick Chaimbault, Simplice D. Karou, Komlan Batawila,
Koffi Akpagana, Claus Jacob
‘Capiture’ plants with interesting biological
activities: a case to go
Journal xyz 2017; 1 (2): 122–135
The First Decade (1964-1972)
https://doi.org/10.1515/chem-2017-0024
Research Article
received March 31, 2017; accepted July 11, 2017.
were applied to the data. Those indices, in addition to a
bibliographic review, were then fed into a computer-aided
Abstract: TheMax
investigation
of natural
used in approach which predicted two interesting plants out of
Musterman,
Paulproducts
Placeholder
Traditional Medicine in Africa is complicated as modern the 43 species survey-recorded and their specific activities:
analytical and screening methods are often not available. Pterocarpus erinaceus sap against ringworm, Daniellia
Computer aided product identification from traditional oliveri sap against intertrigo and respectively their roots and
usage records (CAPITURE) may provide an interesting trunk barks against candidiasis. Subsequent laboratoryalternative and has been evaluated in the context of based studies have confirmed the predicted antimicrobial
an ethnobotanical survey on fungal diseases and their activities with MIC (128 µg/mL to 30 mg/mL) and without
on a normal human cell (MRC-5 cells).
traditional treatment
in Tchamba
District
(Togo).
53 any notable toxicity
Pharmacological
and
Mental
Self-transformation
in Ethic
Although
such
a
method
may not be flawless, it is able to
traditional healers
were
interviewed
and
their
knowledge
Comparison
provide
first
leads,
and
in
the face of limited resources, is
recorded. Several
indicators,
the
Use
Value
(UV),
Plant
Pharmakologische und mentale Selbstveränderung im
Part Value (PPV), Specific Use (SU) Value, Intraspecific an attractive alternative worth considering.
ethischen Vergleich
Use Value (IUV) and Informant Consensus Factor (ICF),
Keywords: Computer-assisted selection, ethnobotany,
https://doi.org/10.1515/xyz-2017-0010
fungal diseases, Tchamba; Togo
What Is So Different About
Neuroenhancement?
Was ist so anders am Neuroenhancement?
received February 9, 2013; accepted March 25, 2013; published online July 12, 2014
*Corresponding author: Nassifatou Koko Tittikpina, Faculty
of Pharmacy, Department of Bio-organic Chemistry, Building
Abstract: In the concept of the aesthetic formation of knowledge and its as soon
B 2.1., Room 1.13. Saarland State University, Campus D-66123
as possible
and success-oriented
application, insights and profits without the
Saarbrücken, Germany;
Laboratoire
de Botanique et Ecologie
reference
to
the
arguments
developed
1900. The main investigation also
Végétale, Université de Lomé, BP 1515, Lomé, Togo; Universitéaround
de
Lorraine, SRSMC,
UMR
7565,
54001
NANCY
Cedex.
France;
CNRS,
includes the period between the entry into force and the presentation in its current
Nature provides a treasure chest of natural products
SRSMC , UMR 7565,
ICPM,Their
1 boulevard
Arago
METZ
3. portrayal and narrative technique.
version.
function
as57078
part of
theCedex
literary
which often have amazing biological activities and have
France, E-mail: [email protected]
Wouyo Atakpama,
Hodabalo
Pereki,
Komlan
Batawila,
Koffi
been
used for
agricultural and medical applications for
Keywords: Function, transmission, investigation,
principal,
period
Akpagana: Laboratoire de Botanique et Ecologie Végétale,
centuries throughout the world. The lush vegetation in
Université de Lomé, BP 1515, Lomé, Togo
Western Africa is particularly rich in medical plants, and
Dedicated to Paul Placeholder
Muhammad Jawad Nasim, Wesam Ali, Claus Jacob: School of
the native tribes in countries such as Togo employ a vast
Pharmacy, Division of Bioorganic Chemistry, Building B 2.1.,
arsenal of plants and plant-derived products as part of
Room 1.13. University of Saarland, Campus D-66123 Saarbrücken,
Germany
their Traditional Medicine [1-6]. However, identifying
Stephane Fontanay, Raphael Emmanuel Duval: CNRS, UMR 7565,
which plants, plant products and substances contained
SRSMC, Vandoeuvre-lès-Nancy, F-54506 Nancy, France; Université de
therein are active and may be applied against certain
Lorraine, UMR 7565,
Faculté de Pharmacie,
F-54001, Nancy,
The SRSMC,
main investigation
also includes
the period between the entry into force and
diseases, and which may be toxic or less attractive
France; ABC Platform®,
Faculté de Pharmacie,
F-54001
Nancy, France
the presentation
in its current
version.
Their function as part of the literary porbecause of (traditionally) known side effects is far from
Frédéric Nana, Gilbert Kirsch, Patrick Chaimbault: Université de Lorraine,
trayal and narrative technique.
SRSMC, UMR 7565, 54001 NANCY Cedex. France; CNRS, SRSMC, UMR
trivial. Admittedly, modern analytical methods of sample
7565, ICPM, 1 boulevard Arago 57078 METZ Cedex 3. France
analysis and compound identification, such as automated
Chukwunonso ECC Ejike: Department of Medical Biochemistry,
chromatography coupled with mass spectrometry can
Faculty of Basic *Max
Medical
Sciences, Institute
Federal University
Musterman:
of Marine Ndufu-Alike,
Biology, National Taiwan Ocean University, 2 Pei-Ning
identify specific substances from crude materials within
Ikwo, PMB 1010Road
Abakaliki,
Ebonyi
State,
Nigeria
Keelung
20224,
Taiwan
(R.O.C), e-mail: [email protected]
hours.Ocean
At the
same time,
equally automated activity screens
Simplice D. Karou:
Ecole
Supérieure
des
Techniques
Biologiques
et
Paul Placeholder: Institute of Marine Biology, National Taiwan
University,
2 Pei-Ning
Alimentaires (ESTBA-UL),
Université
de
Lomé,
Lomé,
Togo
can
reveal
an
activity
or toxicity profile against target
Road Keelung 20224, Taiwan (R.O.C), e-mail: [email protected]
1 Introduction
1 Studies and Investigations
© ����
Mustermann
Placeholder,
published
by De Gruyter.
This
Open Access. ©Open
2017Access.
Nassifatou
Koko
Tittikpinaand
et al.,
published
by De Gruyter
Open.
Thiswork
workisis licensed under the Creative
licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives
�.� License.
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(micro-) organisms or cells within a day or two. Hence our
ability to screen for (new) medically active substances in
the laboratory today is rather advanced compared to the
cumbersome studies of the generation before us.
However, even the most advanced toolbox of analysis,
compound identification and robotic activity screens
cannot analyze the entire flora of the planet. Even those
powerful methods a priori require a certain narrowing
down of particularly interesting plants, and here often
rely heavily on the century-old knowledge of Traditional
Medicine. Indeed, whilst most developing countries, for
instance in Africa, Asia or South America can hardly afford
the kind of expensive methodology for (bio)analysis and
activity screens, those countries are exceptionally rich in
medical plants and traditional knowledge related to them.
The World Health Organization (WHO) has estimated that
80% of the population in the developing world still rely
traditional medicine to meet their healthcare needs [7].
In this situation, the famous question “Where to
begin?” may well be answered most efficiently by a brief
consultation of the Medicine (Wo)man. Let’s therefore
consider one country in Africa which stands for the
plight and opportunities of many others: Togo. In Togo,
economic hardship and an uneven access to healthcare
facilities and modern medication allow the spread of
infectious diseases, which in this country rank among the
top ten priority diseases [8,9]. Natural remedies based on
locally grown plants and traditional knowledge related to
them often represents the only source and resource to treat
such bacterial and fungal infections. Consequently, it is
imperative to investigate such plants further, and various
studies have been run in the country already to bring the
traditional therapeutic approach to the forefront [10-12].
Nonetheless, it is impossible to screen all plants of
Togo associated with one or more medical uses for all
(kind of) possible medical applications. In contrast, it may
also be risky to simply rely on one’s first impression and
gut feeling. Here, a more structured and ultimately also
more focused approach is warranted. We have therefore
developed a simple computer-aided “pre-selection
method” which (a) records hitherto unstructured and
orally passed on traditional knowledge in semi-structured
interviews, (b) extracts quantitative numerical values
from these testimonies, (c) uses these numerical values
in a computer-aided method to (d) rank, select and
hence identify the most promising plants or plant parts
as leads for further laboratory based investigations. This
method has several advances when compared to a simple
subjective “selection by experience” or literature survey.
On the one hand, it generates a record of traditional
uses which can be archived and preserved for future
Capiture, from field to laboratory 209
generations. On the other hand, it can be employed
rather successfully to narrow down the field of possible
plant candidates by applying an objective algorithm to an
ethnobotanical study, as we will demonstrate for plants
used to treat antimicrobial (antifungal) diseases in the
Tchamba prefecture of Togo.
2 Experimental procedure
2.1 Area of study: The Tchamba district of Togo
Togo is a country with a lush and also facet-rich vegetation,
and home to numerous known and suspected medical
plants (Figure 1) [13-16]. It is also ethnographically highly
diverse, and hence features a rich and diverse, often tribal
knowledge of traditional medicine, which is still the main
(re)source for the treatment of many diseases [9]. The
Tchamba District in Togo, which is located in the central
eastern part of the country, is unique as it is the only
district which brings together people from nine different
ethnic groups: Tchamba, Koussountou, Tem, Tem Fulani,
Kabyè, Ana-Ifè, Bassar, Lamba and Logba. We have
therefore selected this specific, narrow, local ethnical
melting pot of people, their cultures and traditions as it
(a) promises extensive yet also varied local traditional
medical knowledge and (b) can be surveyed efficiently
with its high concentration of traditional healers within
short travel distances. Furthermore, there has been no
previous investigation of traditional uses of plants against
infectious (fungal) diseases in this district, hence our
study will be unbiased and generate records and data
which in any case will be novel and original.
2.2 Ethnobotanical survey
In the Tchamba District of Togo (Figure 1), semi-structured
individual interviews on plants used for the treatment of
fungal diseases have been conducted with 53 traditional
healers (TH) in the main localities of the district in
September 2010. The questionnaire used, recorded
information on (a) personal data of the interviewee
(name, age, sex, level of education, ethnicity, living place,
specialty, knowledge origin) (b) the type of fungal diseases
treated, i.e. the name of the disease in the local language
and the symptoms, (c) the plant part(s) used, and its/
their mode of preparation and administration, as well as
(d) other diseases treated with the plant (s) mentioned by
the TH. The interviews were complemented by a brief field
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210 Nassifatou Koko Tittikpina et al.
Figure 1: Map of Togo showing the study area. A: Togo in West Africa. B: Togo and its different regions and districts. C: Tchamba District with
the localities prospected during the survey.
inspection of the plant species described and a collection
of small specimens of the mentioned plant species for
further, professional identification and authentication at
the Laboratory of Botany and Plants Ecology of University
of Lomé, with cross-reference to the website www.ipni.org
and for depositing voucher specimens at the Herbarium
of the University of Lomé. This field trip followed strict
rules in order to protect the rights of the TH with regard to
the information they provided and also to safeguard the
biodiversity of the area. Here, consent from the National
Association of Traditional Healers in Togo (CERMETRA)
was obtained prior to the survey and CERMETRA also
delegated two representatives who accompanied the
authors throughout the survey.
2.3 Turning testimonies into data
To convert structured interviews into quantitative,
numerical data, the information contained within the
questionnaire sheets collected was entered into Microsoft
Excel spreadsheets and analyzed. The analysis was based
on the computation of general and specific indices, some
of which have been described previously [11, 12, 17]. This
analysis was designed as robust yet also simple and
“doable” with access to limited resources. For instance,
Microsoft Excel spreadsheets, rather than expensive
statistic programs, were chosen as they are widely
available and enable simple calculations of parameters.
Besides Microsoft Excel, free software, such as Free Office,
Libre Office or WPS Office would also be suitable to conduct
such simple calculations.
The Informant Consensus Factor (ICF) was
determined to evaluate the consensus among traditional
healers as follows:
ICF =
Nur - nt
Nur -1
where Nur is the number of uses reported by respondents
for a particular disease and nt the number of plants species
reported to be used to treat this particular disease [12, 18].
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The Reported Use (RU) was defined as the total number
of uses reported for each plant and was subsequently used
to calculate the Use Value (UV) as indicator of the relative
importance of the species:
UV =
å U or RU
n
where ∑U (same as the RU, is the total number of citations
per species) and n the number of informants [11, 12, 18].
Not all parts of a given plant are equally used for
treatment; hence the Plant Part Value (PPV) provides
information on the parts of a plant most used to treat a
disease. The PPV is equal to the ratio between the total
number of reported uses for the plant’s part and the total
number of reported uses for that plant: RU plant part/ RU
.
plant
Similarly, not all plants are used to treat all (kinds
of) diseases, hence the Specific Use (SU) value was
introduced to reflect the number of times a plant part is
applied for a more specific use against a specific disease.
Finally, Intraspecific Use Value (IUV) was defined as
the ratio between the SU value of a plant part and the RU
for this plant: SU plant part/ RU plant. The IUV is probably the
most refined of the parameters discussed as it enables a
comparison between different parts of a plant used to treat
a specific type of disease [11, 17, 18].
2.4 Selection criteria to cap(i)ture plants of
interest
Equipped with the various parameters and values
calculated as described above, reliable selection criteria
has been developed to rank the 43 plants put forward
by the traditional healers. These criteria were crucial in
order to select a reasonable number of promising plants
and plant parts for further laboratory based assessment,
without, of course, ruling out any of the others (Figure 2).
After the RU and the UV for all plants were calculated, a
long-list of likely candidates was obtained. A cut-off based
on a RU (≥2) or a UV (≥ 0.0377) was applied to obtain a short
list with a reasonable number of entries. Subsequently, a
bibliographic review on the plants with the highest UV (≥
0.0377) or RU (≥2) was conducted. This resulted in a final
list of a small number of plants which were (a) frequently
used in the Tchamba District to treat (various) fungal
diseases, (b) were independently mentioned as medical
plants in the literature and (c) were still of sufficient
novelty to warrant further investigation.
Capiture, from field to laboratory 211
The first step is usually carried out after an
ethnobotanical survey: where researchers are able give
an overall global view on the plants used in a certain
community to treat certain diseases, and are able to
deduce the most important ones due to their high indices.
But researchers who would like to continue on a particular
plant, to confirm or infirm its traditional use and or study
its interesting chemistry, are impeded by questions they
ask themselves: which plant to choose? Which plant part
to focus on as a start? Which disease? And as a results
of these ambiguous questions, researchers end up making
selections of plants based on ‘good feeling’, or just working
on all the plants to find out the ‘most interesting ones’.
We are taking the example of this study to propose
a ‘more objective’ approach in the selection of the most
interesting plant(s)/plant part (s) on which to conduct
deeper research either in the pharmacology or natural
products chemistry. Subsequently after the first general
step selection , we are proposing two possible avenues to
proceed further, where the choice of avenue depends on
the particular situation at hand. We will introduce both
avenues here.
As part of the first avenue, one may initially define the
plant and then consider possible targets or target diseases
this plant may be effective against. The selection here is
based on the specific indices PPV, SU and IUV, which need
to be computed on the plants of the final list. The most
interesting plants will be the ones with the highest SU and
IUV.
The second avenue is more focused on the treatment
of a specific disease of interest, which is defined upfront,
and one may then hunt for the most promising plant or
plant part to treat this specific disease. It narrows down the
type of disease which may be treated (in our case different
kinds of fungal infections) and can rely on the ICF, SU and
IUV. As a first step, the ICF needs to be calculated for the
different types of fungal diseases reported by the TH. This,
places a focus on fungal diseases that have the highest
ICF. The next steps are to identify the plants parts used to
treat fungal diseases with the highest ICF and to compute
the SU and IUV on these plants parts. The most promising
plant parts that will come out from this computation will
also be the ones with the highest SU and IUV.
This method is preferably employed as an
ethnobiological recording and pre-screening exercise
before more eloquent and expensive analytical and
activity screening methods are unleashed.
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212 Nassifatou Koko Tittikpina et al.
In order to prepare suitable extracts for biological
activity studies, a preliminary assessment with different
mixtures of solvents and TLC analysis was performed
(TLC silica gel 60 F254, Merck Millipore, 1.05554.0001).
The best separation was obtained with a mixture of
methanol and dichloromethane MeOH-DCM (1:1, v/v) with
Pterocarpus erinaceus and MeOH (100%) with Daniellia
oliveri (solvents of a grade suitable for chemical synthesis
were used). Consequently, 3 Kg of powdered trunk barks
and roots, respectively, were saturated/soaked in these
solvents at room temperature for 48 h. The resulting
mixtures were filtered, the marc discarded and the eluates
evaporated under reduced pressure using a rotavapor to
obtain raw extracts. The raw extracts have further been
fractionated using liquid-liquid partition with solvents
of different polarities, ranging from 1 to 5: (1) petroleum
ether, (2) dichloromethane, (3) ethyl acetate, (4) 1-butanol
and finally (5) distilled water.
2.6 Antibacterial and antifungal assays
Figure 2: A schematic overview of the CAPITURE approach.
2.5 Collection of plant material and preparation of extracts
For the plants pre-selected by the algorithm described
above, an official letter of authorization from the ‘Direction
de la Protection des Végétaux’ was obtained to collect
specimens, and those were collected in the central region
of Togo (GPS coordinates: 09°11′689′′ North, 001°15′942′′
East) on the 19 June 2014 after a formal identification
by a botanist. A voucher specimen was deposited at the
Herbarium of the University of Lomé under the number:
TOGO 15076 for D. oliveri and TOGO 15077 for P. erinaceus.
After collection, trunk barks and roots were dried at 25°C
in the laboratory of Botany and Plants Ecology of the
University of Lomé at 25°C and milled. After milling, they
were sealed and brought to Europe by plane under the
official licence mentioned above. They were kept in a dry
and dark place until further use. Trunk barks (containing
sap) were collected in replacement of the fresh sap
because collection and biological testing of fresh sap as
used by traditional healers was not possible (the patient is
brought towards the tree, the trunk bark is cut and the sap
is applied directly onto the ringworm or intertrigo).
To assess the antimicrobial activity of the extracts,
selected strains of bacteria and fungi were employed, with
a particular focus on infections identified as part of the
CAPITURE method. Indeed, candidiasis is usually caused
by Candida albicans, Trycophyton rubrum is the pathogen
behind ringworm and in the case of intertrigo, the causes
could mainly be Candida albicans, Staphylococcus aureus
and Pseudomonas aeruginosa [19].
The activity assays therefore included Staphylococcus
aureus (ATCC 29213, ABC 1), Pseudomonas aeruginosa (ATCC
27853, ABC 4), as well as Candida albicans. All strains were
kindly provided by the ABC Platform® Bugs Bank. Activity
against Trycophyton rubrum was investigated previously
and there was no need to reproduce these studies.
Bacterial strains were grown on Mueller Hinton Agar
(MHA, Difco 225250) or Mueller Hinton Broth (MHB, Difco,
275730). C. albicans was grown on Sabourhaud agar (SA)
plates. The purity of the isolates was checked throughout
the study by examination of colony morphology and
employing the Gram staining procedure.
The antimicrobial activity on the raw extracts and their
fractions was estimated by employing the broth dilution
method to determine the Minimal Inhibitory Concentration
(MIC, the concentration that inhibits 100% of bacterial
growth) [20]. To determine the MIC, bacterial suspensions
were prepared by suspending one isolated colony from
MHA plates in 5 mL of MHB. After 24 h of growth, the
suspensions were diluted in distilled water to obtain a final
inoculum of 5×105 to 5×106 CFU (Colony Forming Units).
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Inoculum of C. albicans was prepared by suspending 5
isolated colonies from SA plates in 5 mL of distilled water.
This solution was diluted using Roswell Park Memorial
Institute medium without carbonates and with phosphates
(RPMI-1640, Sigma R6504) to obtain a suspension whose
concentration was equal to 0.5 McFarland units.
At this stage, twofold serial dilutions of extracts were
prepared in MHB for anti-bacterial activities and RPMI-1640
medium for anti-fungal activities, in 96-well plates (Greiner,
650161), starting from a stock solution of 120 mg/mL.
An equal volume of bacterial or fungal inoculum was
added to each well on the microtiter plate containing
0.05 mL of the serial extract. After incubation for 1824 h at 35°C for bacteria, 18-24 h for yeast, the MICs
were determined with a 96-wells plate reader at 540 nm
(Multiskan EX, Thermo Electron Corporation, France) as
the lowest concentration of compound whose absorbance
was comparable with the negative control wells (broth only
or broth with extract or compound, without inoculum).
Oxacillin, ticarcillin and Amphotericin B served as positive
controls and benchmark antibiotics and anti-fungal agents
for comparison and reference [21, 22]. Each condition was
repeated in 8 wells and results were expressed as means of
three independent experiments.
2.7 Cytotoxicity assay
In order to compare the - desired - activity of extracts
against microbes with their - rather undesired cytotoxicity against normal human cells, MRC-5 (ATCC
CCL-171) human lung fibroblasts were used as model. Cells
were grown in Minimum Essential Medium (MEM, 31095029, Life Technologies-Gibco®) supplemented with 10%
heat-inactivated fetal calf serum (CVFSV00-0U, Eurobio,
Courtaboeuf, France) and 2 mM L-glutamine (G7513100 mL, Sigma Aldrich), at 37°C in a 5% CO2 humidified
atmosphere. Cells were then plated at 104 cells per well in
96-well tissue culture plates (83.1835, Sarstedt, Germany)
and grown for 48 h at 37 °C in a 5% CO2 atmosphere.
Medium was discarded and replaced by fresh medium
containing increasing amounts of plant extract (range
1 µg/mL to 30 mg/mL) dissolved in dimethylsulfoxide
(DMSO). The final concentration of DMSO never exceeded
2 % of the final volume. Three different (negative) controls
were added: medium alone, cells in medium and extract
in medium. Each condition was repeated in eight wells.
After 24 h incubation, medium was discarded and cells
were washed with PBS.
The MTT assay was employed to quantify cytotoxic
impact [23]. 100 µL of medium containing 0.5 mg/mL MTT
Capiture, from field to laboratory 213
previously prepared in PBS were added to each well and
the plates were incubated for 4 h at 37°C. Then formazan
crystals were dissolved by the addition of 100 µL of SDS
(100 µg/mL), followed by incubation for 3 h at 37°C. Finally,
the absorbance was measured at 540 nm vs 690 nm using
a 96-wells plate reader (Multiskan EX, Thermo Electron
Corporation, France). Percentages of survival and the half
maximal inhibitory concentration (IC50) were calculated.
Experiments were repeated three times [21].
3 Results and Discussion
3.1 Identification of plants for focused
screening using the CAPITURE algorithm
Based on our interviews with 53 traditional healers in the
Tchamba District, 43 plant species belonging to 43 genera
and 27 botanical families have been identified in the
context of the treatment of fungal diseases. These plant
species form a long-list of plants that is provided in Table
1. For these 43 plants, the use value (UV) and the reported
use (RU) have been computed and compared. Eventually,
P. erinaceus (UV = 0.28), D. oliveri (UV = 0.11), F. virosa (UV
= 0.05) and P. pinnata (UV = 0.05) have yielded the highest
scores among the 43 species and, based on these values, it
appears that they are the preferred plants administered by
traditional healers to treat fungal diseases.
Since the UV values differ considerably between the
plants, we have been able to implement a fairly strict cutoff RU at 2 or UV at 0.04, which allows us to consider only
those plants with a RU ≥ 2 or an UV ≥ 0.04, yielding the
following short list of plants: Allium sativum, Anacardium
occidentale, Calotropis procera, Cochlospermum planchoni,
Quisqualis indica, Ricinus communis, Desmodium
gangeticum, Flueggea virosa, Daniellia oliveri, Pterocarpus
erinaceus, Xeroderris stuhlmannii, Milicia excels, Musa
sapientum, Piper guineense, Zea mays, Paullinia pinnata,
Nicotiana tabacu and Vitex doniana.
At this point, the bibliographic survey was performed
to complement the testimonies. To our great surprise, this
survey reveals that virtually none of plant species identified
have previously been studied in vivo for antifungal activity
(except P. erinaceus) [24]. Furthermore, the underlying
chemical composition and activity of the materials so far
is also not well documented. It also appears that none
of them, with the notable exceptions of P. erinaceus and
Ricinus communis, have been cited previously by other
ethnopharmacological studies [24, 25]. In contrast, plants
such as Allium sativum (the common garlic), Calotropis
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214 Nassifatou Koko Tittikpina et al.
Table 1: Long list of plants obtained after the survey.
Indices. ∑RU: sum of Reported Uses, UV: Use Value. Local names. Tch: Tchamba, Te: Tem, La: Lamba, K (Kounssountou), Lo: Logba, Ka:
Kabyè. Type of fungal and related diseases. R: Ringworm; I: Intertrigo; OC: Oral Candidiasis; SC: Sexual Candidiasis; F: Felon; Ony:
Onychomycosis. Plants parts. Bu: bulb; B: barks; Fr: fruits; FrS: fruits shell; R: roots; Rhi: rhizome; S: sap; Wp: whole plant; L: leaves; Se:
seeds; Tr: Trunk. Voucher number. NA: not appropriate.
Plant
Voucher specimen
Used part
Disease
∑ RU
UV
Allium sativum L. [cult.]
NA
Bu
R
2
0.04
Mangifera indica L. [cult.]
TOGO 01797
B
OC and SC
1
0.02
Anacardium occidentale L. [cult.]
TOGO 01768
B, Fr
OC, SC and I
2
0.04
Uvaria chamae P. Beauv.
TOGO 01950
R
SC
1
0.02
Anchomanes difformis (Blume) Engl.
TOGO 09515
Rhi
F
1
0.02
Cocos nucifera L. [cult.]
NA
FrS
F
1
0.02
Calotropis procera (Aiton) R.Br.
TOGO 02209
S
R
2
0.04
Tridax procumbens L.
TOGO 01151
Wp
OC and SC
1
0.02
Cochlospermum planchonii Hook.f.
TOGO 00466
B, R
SC and I
2
0.04
Quisqualis indica L.
TOGO 15085
R
OC and SC
2
0.04
Brysocarpus coccineus Thonn. ex Schumach.
TOGO 15081
R
OC and SC
1
0.02
Dioscorea alata L. [cult.]
TOGO 10414
Rhi
OC and SC
1
0.02
Dioscorea cayenensis Lam. [cult.]
TOGO 10424
Rhi
OC and SC
1
0.02
Bridelia ferruginea Benth.
TOGO 03089
L
R
1
0.02
Jatropha curcas L. [cult.]
TOGO 15092
Fr
OC
1
0.02
Ricinus communis L.
TOGO 03732
Fr, Se
OC, SC and R
2
0.04
Ficus virosa (Roxb. ex Willd.) Voigt
TOGO 03760
R, B
SC and Ony
4
0.08
Desmodium gangeticum (L.) DC
TOGO 05946
R
SC and R
2
0.04
Pterocarpus erinaceus Poir.
TOGO 15077
S, B, R
R, OC and SC
15
0.28
Cassia alata L.
TOGO 15094
L
R
1
0.02
Cassia occidentalis L.
TOGO 15088
Wp
Ony
1
0.02
Detarium microcarpum Guill. & Perr.
TOGO 00176
B
OC and SC
1
0.02
Daniellia oliveri (Rolfe) Hutch. & Dalziel
TOGO 15076
S, B
I, OC and SC
6
0.11
Khaya senegalensis (Desr.) A.Juss.
TOGO 01797
B
OC
1
0.02
Pseudocedrela kotschyi (Schweinf.) Harms
TOGO 12736
B
OC and SC
1
0.02
Xeroderris stuhlmannii (Taub.) Mendonça
& E.C. Sousa
TOGO 06769
Wp, B
R
2
0.04
Parkia biglobosa (Jacq.) R.Br. ex G.Don
TOGO 15084
Tr
F
1
0.02
Ficus thonningii Blume
TOGO 05199
L
R
1
0.02
Milicia excelsa (Welw.) C.C.Berg
TOGO 12751
Sa, B
F and SC
2
0.04
Musa sapientum L.
TOGO 15091
Fr
O and SC
3
0.06
Piper guineense (Schum and Thonn.)
TOGO 06862
Fr
F, OC and SC
2
0.04
Zea mays L. [cult.]
TOGO 11532
Fr
S and OC
2
0.04
Gardenia aqualla Stapf & Hutch
TOGO 07309
Tr
OC
1
0.02
Morinda lucida Benth
TOGO 07498
R
OC and SC
1
0.02
Citrus limon (L.) Burm.f. [cult.]
TOGO 15089
Fr, R, B
F, Ony, I, OC and SC
1
0.02
Citrus sinensis (L.) Osbeck
TOGO 15093
Fr
SC
1
0.02
Paullinia pinnata L.
TOGO 15082
R
Ony, F
4
0.08
Nicotiana tabacum L. [cult.]
TOGO O8500
L
R
2
0.04
Cola gigantea A. Chev
TOGO 08587
B
SC
1
0.02
Heliotropium indicum L.
TOGO 02508
L
OC
1
0.02
Vitex doniana Sweet
TOGO 09273
L, B
R
2
0.04
Aframomum melegueta [Roscoe] K. Schum
NA
Fr
OC
1
0.02
Unauthenticated
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procera and Annacardium occidentale have been studied
extensively before, yet those species do not score highly
in our own CAPITURE analysis [26-34]. Conducting this
review has helped identify a final list of plants as promising
candidates and as the most original plants to be analyzed
and studied further considering the feeble data of previous
biological or chemical studies on them: P. erinaceus, D.
oliveri, Ficus virosa and P. pinnata. The four plants are also
the most used ones by traditional healers as revealed by
the survey. To this final list, either the first or the second
avenues of the CAPITURE approach could be applied as
described in the methodology section.
Following the guidelines of the first option, the more
specific and informative PPV, SU and IUV parameters
were computed on the four species, identified as the most
promising candidates. The results obtained for the PPV
are summarized in Table 2.
The PPV parameter indicates which parts of a
particular plant are most frequently utilized by TH, hence
narrowing down the plant material to be studied. In the
case of P. erinaceus, the trunk bark (0.4) and the sap (0.53)
represent the parts most frequently employed by TH to
treat fungal infections. Similarly, in the case of D. oliveri,
the sap (0.71) and trunk bark (0.28) are most frequently
used, whilst for F. virosa and P. pinnata, the roots are the
only parts utilized. To address the question which fungal
infections to consider, the SU and the IUV parameters
were calculated. It becomes immediately apparent that
the sap of P. erinaceus is frequently used in the treatment
of ringworm and its roots to treat candidiasis, whilst the
sap of D. oliveri is utilized in the context of intertrigo and
its stems barks to treat candidiasis (Table 3).
Considering the second avenue of the CAPITURE
approach, we have calculated the Informant Consensus
Factor (ICF) as discussed in the previous section, on the
diseases identified by traditional healers: candidiasis
(oral and sexual), intertrigo, ringworm, onychomycosis
and felon (Table 4).
It has helped to identify ringworm (0.59), intertrigo
(0.57) and candidiasis (0.41) are the fungal diseases
with the highest ICF. This high value of ICF implies that
most of the TH agree on a set of plants from the short list,
which they use preferably to treat those three infections.
Consequently, we have computed the SU and IUV of those
plants, just focusing on their use in the treatment of the
three ailments. This has conducted to obtain the same
information as provided in Table 3.
To direct our in vitro biological screens, we have
combined both sides of the coin to see which parts of the
plants are used for the treatment of which specific fungal
infections. It seems that the sap of P. erinaceus is the most
215
Capiture, from field to laboratory Table 2: RU and PPV of most valuable plants parts.
Plant part
RU plant part
PPV
P. pinnata
Bark
Sap
Roots
Bark
Sap
Roots
6
8
1
2
5
4
0.40
0.53
0.07
0.28
0.71
1
F. virosa
Roots
4
1
P. erinaceus
D. oliveri
Table 3: SU and IUV as indicators of potential uses of the preselected plants.
P. erinaceus
D. oliveri
Plant
part
Sap
Roots
Specific reported
SU
use
Ringworm (8 times) 8
Candidiasis (1)
1
1
1
Sap
Barks
Intertrigo (5)
Candidiasis (1)
1
1
5
1
IUV
Table 4: The main Symptoms of the fungal diseases and the
Informant Consensus Factor (ICF).
Indices. Nur: number of uses; nt: number of plants species; ICF:
Informant Consensus Factor.
Disease
Nur
nt
ICF
Symptoms according to
traditional healers
Oral
candidiasis
31
25
0.20
Sexual
candidiasis
29
23
0.21
Itching on women genital
parts with sometimes
making urinating difficult
(sexual candidiasis), weird
white bands appearing
in children mouth (oral
candidiasis).
Ringworm
28
12
0.59
White circles appearing
on children heads with
sometimes a disappearing
of hair in the middle of the
circle.
Intertrigo
8
4
0.57
White weird things
appearing between the
toes associated with
wounds sometimes.
Onychomycosis 8
7
0.14
White or brown weird
things appearing on nails.
Felon
4
0
Inflammation of one’s
finger around the nail area
with sometimes white or
weird things, very painful,
forbidding to put one’s
hand in water.
4
Unauthenticated
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216 Nassifatou Koko Tittikpina et al.
attractive plant material employed against ringworm,
whilst the roots of this plant seem to be particularly effective
against candidiasis. The sap of D. oliveri appears to be
effective against intertrigo, and the trunk barks of this tree
bear promise in the treatment of candidiasis.
3.2 Screens for antimicrobial activity
We have therefore decided to follow this lead and to
investigate further the activity of extracts of D oliveri and
P. erinaceus against microbial infections: the trunk barks
of the first one and the roots of the second one against
Candida albicans, the trunk barks of the first one against
Staphylococcus aureus and Pseudomonas aeruginosa and
the trunk barks of the second one against Trycophyton
rubrum. As anticipated, the water extract of the roots of
P. erinaceus, the ethyl acetate fraction and water extracts
of the trunk barks of the other plant, are active against
Candida albicans with respective MIC values at 1.875
mg/mL, 30 mg/mL and 1.875 mg/mL. The activity of P.
erinaceus against Tricophyton rubrum was previously
tested with a total inhibition of fungal growth at 40 mg/
mL in vitro (97%) and in vivo (100%), values that were
superior to the ones obtained with Griseofulvine as
benchmark antifungal agent (92% at 40 mg/mL) [24]. D.
oliveri trunk barks were also tested against S. aureus and
P. aeruginosa (the other two germs involved in intertrigo).
As anticipated with the CAPITURE approach, activity
was observed on the two germs, with the methanolic raw
extract on S. aureus at 128 µg/mL and on P. aeruginosa
at 256 µg/mL. The following extracts derived from
a fractionation of the methanolic raw extract of the stems
barks of D. oliveri, were further been tested on the two
germs: petroleum ether, dichloromethane, ethyl acetate,
butanol and water fractions. Interestingly on S. aureus,
the MIC at 128 µg/mL obtained with the raw extract was
kept with almost all the fractions, except a non-significant
difference with the petroleum ether and dichloromethane
fractions where a MIC at 256 µg/mL was obtained. On P.
aeruginosa, no MIC has been observed with the non-polar
fractions, only with the butanol and water fractions with
also a conservation of the MIC at 256 µg/mL for the butanol
fraction and 128 µg/mL for the water fraction. The toxicity
of the active extracts and fractions of the two plants were
consequently tested on normal human cell lines, namely
MRC-5 cell lines. No toxicity has been observed at the
concentration at which the extracts or fractions were
active. To the best of our knowledge, no previous study
has reported the toxicity activities of these two plants on
MRC-5 cells. Besides, no previous study has also reported
the anti-bacterial activity of the two plants using the broth
dilution method as investigated in this study.
Eventually, it seems that the combination of a semiquantitative ethnopharmacological and literature survey
has provided one correct lead worth following up with
more detailed laboratory based studies. It should be
mentioned that there are some caveats associated with
such an approach and always room for improvement
and refinement. The specific indices PPV, IUV and SU,
for instance, have been first reported by Gomez-Beloz in
2002; who employed them to widen the knowledge about
specific pre-defined plants by interviewing 40 members
of the Winikina Warao community in Venezuela [11].
CAPITURE, on the other hand has explicitly avoided a
narrow focus on one community because of possible bias,
for instance based on a narrow tradition, superstition or
magic. Indeed, the results obtained do not disagree with
the information gathered from individual TH. Besides, in
CAPITURE, we did not compute the Disease Consensus
Index (DCI) indicative of specific plants used to treat a
single disease within a specific community [35]. In the
CAPITURE method, we have rather computed the ICF.
Indeed, the survey of TH did not focus on just one specific
disease but on fungal diseases in more general terms,
hence representing a broad disease category. Only towards
the final steps, the ICF has been employed to narrow down
this broad disease category to a more focused number of
specific fungal infections. Crucially, the latter were not
preset but defined by the TH themselves.
4 Conclusions
In summary, we have been able to use our method to
move on from a basic ethnopharmacological survey of 53
traditional healers in the Tchamba District of Togo to a
more structured, objective appraisal of existing knowledge
on the use of natural plant products against a spectrum of
common fungal infections. Whilst our approach still leaves
various questions unanswered (e.g. about plant species
eliminated from the list) and provides sufficient room for
improvement (e.g. by introducing further chemical and
environmental parameters), it nonetheless has allowed us
to narrow down the vast number of medical plants found
in Togo to a selected few which were also proven to be
highly active biologically (MIC as low as 128 µg/mL).
In future, we will expand this method to involve
more TH and to consider additional diseases. This way
we will not only identify additional leads for promising
medical plants, but we will also be able to record, store
and safeguard the century-old knowledge of TH which has
Unauthenticated
Download Date | 10/27/17 12:19 PM
been passed on through the generations and is always at
risk of “getting lost” in the mist of time. Importantly, this
kind of recording is structured and hence can capture tens
or even hundreds of testimonies, not in form of traditional
“stories from the forest” but as focused, comprehensive
yet down to the point questionnaires.
At the same time, we are investigating more closely
the few leads identified so far, especially in the context
of P. erinaceus and D. oliveri, bearing in mind that the
CAPITURE approach is only a pre-screen to be followed
by a full laboratory based analysis of the (active) chemical
ingredients found in the plants, an assessment of their
spectrum of biological activities and potential uses and a
full investigation of the underlying mode(s) of action. The
long-list with a total of 43 plants will always be around for
a reappraisal of plants, plant parts and infections they
may be active against should the need arise.
Eventually, the method described here not only serves
as an objective “selection tool” for promising plants based
on traditional knowledge which goes beyond the kind of
subjective inkling often used by researchers whilst sifting
through the woodlands or the literature associated with it.
It helps researchers save time and resources by allowing
them to directly work on the most interesting plants
(not all the plants recorded during an ethnobotanical
survey), as well as saving resources, which is crucial for
countries from the developing world. Equally it saves the
same resources and funding in the developed countries
because research could not be carried out on all the
plants of the world. Hence there is an apparent need for a
certain set of narrow(er) criteria. It also records, archives,
preserves and disseminates century-old knowledge held
by traditional healers which has been passed on orally
from generation to generation, which otherwise is bound
to be lost. Furthermore, the method demands a site visit,
personal contact with the TH and, above all, a professional
inspection of the plants employed, and eventually creates
an inventory of medical plants of the region, with voucher
specimen available for further studies. For all these
reasons, it is therefore certainly worth considering and
amending for future investigations.
Author contributions
NKT carried out the research, performed the different
experiments, elaborated the CAPITURE methodology and
wrote the manuscript. WA and HP helped run the research
(plants identification, treatment of the data) and mapped
the area. MJN and WA helped conceive some of the
Capiture, from field to laboratory 217
figures. SDK critically reviewed the methodology and the
manuscript. KB, KA, CJ, PC, RED and GK, supervised the
research. CECCE, FN, SF and all the other authors read,
improved the manuscript and approved its final version.
Acknowledgments: We are thankful to the traditional
healers of Tchamba District in Togo for sharing their
knowledge with us. Mr. Figui Nandji and Mr. Amoussou
Sadoukou from the CERMETRA (Centre de Recherche
en Medecine Traditionnelle et Appliquee) are also
acknowledged not only for the sharing but also for their
help during the survey. The leading members and all
the members of CERMETRA are warmly acknowledged.
We acknowledge the ‘Schlumberger Foundation’ for its
Faculty For The Future (FFTF) fellowship to NKT. Financial
support has also been provided by the Universities
involved, namely the University of Saarland, the University
of Lorraine and the University of Lome.
We also acknowledge financial support from ‘GradUS’
(University of Saarland, Germany), the French Ministry
of Further Education and Research (MRES), the French
National Scientific Research Centre (CNRS) and Region
Lorraine.
Conflict of interest: The authors declare no conflict of
interest.
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