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The Metabolomic study of Calotropis procera Ait. from Burkina Faso, based on
chemical functional groups profiling using FTIR

Article in Journal of Complementary and Integrative Medicine · June 2020

DOI: 10.1515/jcim-2019-0134

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 DE GRUYTER Journal of Complementary and Integrative Medicine. 2020; 20190134

Prosper T. Kinda1 / Aminata P. Nacoulma1 / Samson Guenné1 / Moussa Compaoré1 /

Abdoulaye Djandé2 / Latifou Lagnika3 / Martin Kiendrébéogo1

The Metabolomic study of Calotropis procera Ait.

from Burkina Faso, based on chemical functional
groups profiling using FTIR
1 Laboratory of Applied Biochemistry and Chemistry ([Link].C.A), University Joseph KI-ZERBO, 03 P. O. Box: 7021, Oua-

gadougou 03, Kadiogo, Burkina Faso, E-mail: [Link]@[Link], natanac5@[Link], guesams@[Link],

mcompaore_3@[Link], martinkiendrebeogo@[Link]
2 Laboratoiry of Molecular Chemistry and Materials (LCMM), Organic Chemistry and Phtochemistry Team, University Joseph

KI-ZERBO, Ouagadougou, Burkina Faso, E-mail: djandeabdou@[Link]

3 Laboratory of Biochemistry and Bioactives Natural Substances, University of Abomey-calavi, 01 P. O Box: 348, Cotonou, Benin,

E-mail: llagnika@[Link]

Abstract:
Background: C. procera is an important wild medicinal plant used in different area of Burkina Faso for the
neuropsychiatric disorders treatment. It was reported to possess many pharmacological properties because of
its phytochemical diversity. This study was carried out to identify possible specific chemical characteristics form
C. procera leaves and root-bark samples, harvested in two regions of Burkina Faso, for a better selective use of
specimens in traditional medicine.
Methods: Plant materials (leaves and root-bark) were collected from five sites in each region. Samples pow-
ders and extracts were mixed with potassium bromide for the Fourier Transform Infrared Spectroscopy (FTIR)
analysis. A multivariate data analysis was performed to highlight differences in the FTIR spectral profile of
samples. Therefore, phytochemical contents such as phenolics, flavonoids and terpenoids were evaluated with
aqueous and methanolic extracts, using UV/visible light spectrophotometer method.
Results: Results of principal component analysis (PCA) showed a significant difference between leaves and
root-bark spectral profile, independently to the region of collection. These profiles possess characteristic signals
which could be exploited as biomarkers for plant organ discrimination. The phytochemical contents evaluation
showed that C. procera leaves contain more significant phenolics, and root-bark possess more terpenoid com-
pounds. This study of C. procera Ait. based on FTIR spectral characteristic and phytochemical content, suggest
that terpenoids, notably cardenolide-type could be a good biomarkers for C. procera samples characterization
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and to explain root-bark therapeutic potential.

Keywords: Burkina Faso, Calotropis procera, FTIR profile, neuropsychiatric, phytochemical
DOI: 10.1515/jcim-2019-0134
Received: June 13, 2019; Accepted: September 27, 2019

Introduction
Calotropis procera Ait. is a cultivable wild xerophytic shrub found across Africa, Asia and South America [1].
It is widely used in many countries for its therapeutic purposes. It has been recorded to possess numerous
pharmacological properties such as anticonvulsant, analgesic, anti-inflammatory, cytotoxic, antimicrobial and
hepatoprotective effects [2–4]. C. procera is also quoted to contain active compounds from terpenoid biogenesis
pathway such as cardenolides, triterpenes and steroids, phenolic pathway like flavonoids and tannins [4–6].
These compounds play an important role in traditional medicine and are responsible for plant pharmacolog-
ical and biological properties. C. procera is located in all regions of Burkina Faso, and its different organs are
widely used for diseases treatment such as neuropsychiatric disorders, liver diseases, drepanocytosis, malaria
and tumour [7–9]. However, the root-bark is the most valuable organs used by traditional healers for neuropsy-
chiatric diseases treatment in the Hauts-Bassins region of Burkina Faso [9]. This excessive use of root-bark does
not promote the sustainable management of this plant in Hauts-Bassins region. External factors like climate,
ground and altitude are known to influence plants phytochemical contents [10]. Metabolomics is the study of
Prosper T. Kinda is the corresponding author.
© 2020 Walter de Gruyter GmbH, Berlin/Boston.

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all metabolites in a biological matrix. It combines a simple extraction step followed by an analytical measure-
ment of the samples to get the metabolic fingerprint [11, 12]. Fourier transform infrared spectroscopy (FTIR) is
one of the most powerful tools to identify chemical functional groups present in compounds [13]. Then, it is the
more sensitive, rapid, selective and authenticate method to characterize and identify functional groups of phy-
toconstituents [14]. This analytical method was used to perform the metabolomic study of C. procera harvested
in Hauts-Bassins and Central regions of Burkina Faso. It was coupled with statistical analysis of multivariate
data methods. Then, Principal Component Analysis (PCA), a non-supervised analysis, was firstly performed
to observe global variability of data. Secondly, Orthogonal Projections to Latent Structures-Discriminant Anal-
ysis (OPLS-DA), a supervised analysis, was carried out to highlight responsible variables of discrimination.
This study aims to contribute to a better knowledge of the metabolic fingerprint of C. procera from Burkina Faso
regions, based on FTIR spectral profile and phytochemical content analysis, in order to explain its efficient use
in traditional medicine and to allow better control of the quality, efficiency and standardization of the plant
raw material.

Material and methods

Samples collection, powdering and extraction

Plant samples were collected from five (05) sites of the Central region (Donsin, Gonsé, Gampela, Saaba, Koubri)
and five (05) sites of the Hauts-Bassins region (Dinderesso, Samogan, Péni, Taga, Banfora). The collection sites
are represented in Figure 1. Ten (10) samples of leaves and ten (10) samples of root-bark of C. procera mature
trees were collected. All samples were harvested from August to October in the morning (from 6 am to 10 am).
Samples were dried under laboratory condition and powdered to obtain fine particles with homogenate size
(≤20 µm). Extraction was made according to two methods. Firstly, dry powder (25 g) of each sample was ex-
tracted by decoction with 500 mL of distilled water at 100 °C for 30 min. It was filtered with eight layers of
muslin cloth and centrifuged at 4000 rpm for 10 min. The supernatant was collected and lyophilized to dryness
to obtain aqueous extract. Secondly, Soxhlet method was used to obtain chloroform and methanol extracts.
Twenty-five grams (25 g) of dried powder was defatted with hexane under reflux using a Soxhlet apparatus for
8 h, followed by chloroformic extraction for 8 h after drying the marc. The same process was performed for the
methanolic extraction using the marc from chloroformic extraction. All extracts were kept at 4 °C until use.
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Figure 1: Localization of the collection sites.

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Metabolomic analysis

Sample preparation

For FTIR analysis, powder, aqueous, methanolic and chloroformic extracts from leaves and root-bark samples
collected in the two regions (Central, Hauts-Bassins) were used. Each sample was prepared with 98 mg of potas-
sium bromide and 2 mg of sample which were crushed to a fine powder. The powder was compressed under
a pressure of 10 tons with a press (Specac) to obtain a pellet. Each sample was prepared in three independent
pellets. A free pellet of potassium bromide (100 mg) was used for the base-line. The dataset contains sixty (60)
observations of C. procera powder, aqueous, methanolic and chloroformic extracts (n=10 × 2 × 3 pellets)

Infrared spectral recording and multivariate data analysis

The spectrum of each pellet was obtained using FTIR-ATR, Nicolet iS5 spectrometer (USA) in the range from
4000 to 600 cm−1 . SIMCA P+v (12, Sweden) software was used for multivariate data analysis (PCA and OPLS-
DA) according to previous methods [15, 16].

Quantitative determination of phytochemical compounds

For the phytochemical quantification, aqueous and methanolic extracts of sample from each collection site were
used. Data were expressed in mean ± SD with three independent samples. Tukey’s test was used to obtain the
p-value.

Total phenolic quantification

Total phenolics were determined using the Folin-Ciocalteu method as previously described by Guenné et al.,
[17] with a slight modification. Microtiter plate of 96-well was used. Each test well contained 25 μL of extract
solution (0.1 mg/mL in water), mixed with 125 μL of Folin-Ciocalteu reagent (0.2 N) 5 min later and 100 μL of
Na2 CO3 (75 g/L). A blank containing 25 μL of water, 125 μL of Folin-Ciocalteu reagent and 100 μL of Na2 CO3
was used. The plate was incubated for 2 h in the dark at room temperature, and absorbances were measured at
760 nm using a UV/visible light spectrophotometer (Epoch 251465, Biotek Instruments, USA). The experiments
were carried out in triplicate. A standard curve was plotted with gallic acid (y=0.005x + 0.0961; R2 =0.9995). The
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results were expressed as milligram gallic acid equivalents per gram of extract (mg GAE/g).

Total flavonoid quantification

Flavonoids content were estimated according to the Dowd method as adapted by Guenné et al., [17] with
some modification. In test wells of microplate, 100 μL of AlCl3 (2%) were mixed with 100 μL of extract so-
lution (1 mg/mL). After 10 min, the absorbances were measured at 415 nm against a blank contained 75 μL of
methanolic extract solution and 75 μL methanol. The experiments were carried out in triplicate and a quercetin
standard calibration curve (y=16.819x + 0.0898; R2 =0.9951) was used. The amounts of flavonoids in plant ex-
tracts were expressed as milligram quercetin equivalents per gram of extract (mg QE/g).

Total terpenoid quantification

Total terpenoid content was determined using the following procedure described by Chang et al. [18]. Briefly,
100 μL of each extract solution (10 mg/mL) were mixed with 150 μL of vanillin-glacial acetic acid solution and
500 μL of perchloric acid solution. The mixture solutions were heated for 45 min at 60 °C and then cooled in
an ice-water bath to the ambient temperature. Then, 2.25 mL of glacial acetic acid were added and absorbance
was measured at 548 nm, using a UV–visible light spectrophotometer (Epoch 251465, Biotek Instruments, USA).
An ursolic acid standard curve (y=0.2162x + 0.0559; R2 =0,999) was used. Results were expressed as milligram
ursolic acid equivalents per gram of extract (mgUAE/g).

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Results and discussion

FTIR spectra first derivative visual observation
The main characteristics signals of first derivative FTIR spectra were shown in Figure 2. Visual observation
revealed that leave profile was different to the root-bark profile (Figure 2A). At wavenumber 1043 cm−1 for
root-bark spectra and at 1372, 1510 cm−1 for leaves spectra were observed a characteristic signals. According to
the literature, signal at 1043 cm−1 corresponds to the S=O or C=S stretching and signals at 1372 and 1510 cm−1
were assigned to C-N of aromatic nitro group stretching frequency [17]. Many others characteristics signals
were observed at 974, 1117, 1221, 1455, 1680, 1754, 2850, 2939, 3640 cm−1 for both leaves and root-bark spectra,
at various intensities. These signals suggested the presence of functional groups such as alcohol C-O and O-
H vibrations respectively at 1117 and 3640 cm−1 , carboxylic C-O stretching at 1221 cm−1 . Wavenumbers 1680,
1754, 974 cm−1 correspond to conjugated aldehyde C=O, lactone or unsaturated carboxylic acid C=O and alkene
C=C vibrations, respectively [19, 20]. Alkane C-H vibrations were observed at 1455, 2850, 2939 cm−1 . The vi-
sual observation of plant parts spectra indicated that difference was not strong between leaves collected in the
two regions (Figure 2B). Therefore, a particular difference was observed at a wavenumber range from 2750 to
3000 cm−1 for root-bark samples collected from the Central region compared to the Hauts-Bassins region (Fig-
ure 2C). Wavenumbers 2830, 2900 cm−1 corresponds to aldehyde C-H vibration and 2945 cm−1 was assigned
to carboxylic acid O-H vibration. These characteristic signals of functional groups suggested the presence of
aldehyde containing compounds as biomarkers for root-bark collected from Central region and carboxylic acid
containing compounds for Hauts-Bassins.
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Figure 2: First derivative FT-IR of leaves and root-bark powders from Central and Hauts-Bassins regions. (A) Both leaves
and root-bark spectra, (B) leaves spectra, (C) root-bark spectra.

Multivariate data analysis of plant powder

PCA was performed on the corresponding FTIR spectra first derivative to have more information about spectra
variability. Using the score plots of the two first principal components, 81.68% of the variability in the dataset
of all samples was explained (PC1: 63.99%; PC2: 17.69%). These PCs clearly discriminate C. procera leaves pow-
der from root-bark powder. Root-bark samples were gathered in the positive side of PC1 axe, whereas leaves

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samples were found in the negative side of PC1 (Figure 3). This indicated a specific profile for each organ, inde-
pendently to the region or the site of collection. Elsewhere, in the score plot, the Hauts-Bassins region samples
were clustered in the positive side of PC2 and the Central region samples were found in the negative side (Fig-
ure 3). These results showed a discrimination of C. procera specimens from the Hauts-Bassins region compared
to those from the Central region, only according to the second principal component (PC2) axe. The multivari-
ate data analysis also revealed a homogeneous profile of root-bark samples according to the Hauts-Bassins
region specimens, located to the positive side of PC2. This discrimination was slightly observed in the leaves
samples, except for Samogan and Dinderesso specimens, which are similar to those from the Central region.
Likewise, PCA analysis split root-bark samples into two groups well discriminate between the Hauts-Bassins
region (Péni, Banfora, Samogan, Taga, Dinderesso) and the Central region (Donsin, Koubri, Saaba, Gampela,
Gonsé). The Hauts-Bassins region of Burkina Faso, belongs to the phytogeographical sector of south-Soudanien,
characterized by an average annual precipitations higher than 900 mm and an average temperatures oscillat-
ing around 26.8 °C. Elsewhere, the Central region belonging to the north-Soudanian area is characterized by
annual precipitations around 800 mm and an average temperatures around 28.1 °C [21, 22]. According to pre-
vious studies, climatic, ground and altitude impact on plants chemical composition [10]. Then, the difference
in environmental conditions observed between the Hauts-Bassins region and the Central region might explain
the variability correlated with samples phytochemical content and spectral profile.
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Figure 3: PCA analysis of C. procera specimens.

Multivariate data analysis of plant extracts

The unsupervised PCA analysis model permits to gather extracts into two well distinguish groups, according
to the polarity of the solvent (Figure 4). Polar extracts, both water’s and methanol’s, showed different spectral
profile compared to non-polar chloroform extracts. That indicated a difference between phytochemical content
extracted with polar solvents and those extracted with non-polar solvent. Then, to better understand the polar
extracts aggregation, a supervised OPLS-DA model was performed according to the extraction type and the
plant organ. Polar solvents scores permit to better distinguish leaves from root-bark than with PCA model.
Whereas, non-polar solvent extracts always showed high similarity in leaves and root-bark samples profile.
These results suggest that phytochemical content extracted with water and methanol in leaves samples were
both similar but were different to those extracted in root-bark samples. However, chloroform extracts contain
same chemical compounds from leaves and root-bark samples.

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Figure 4: PCA and OPLS DA analysis of leaves and root-bark aqueous, methanol and chloroform extracts.

The PCA loadings were also performed to correlate characteristic signals to spectra variability. The main dis-
criminant signals between samples in aqueous, methanol and chloroform extracts were identified and pre-
sented in Table 1. The examination of the loadings plot showed characteristic signals in various wavenumbers.
Wavenumbers 3650 and 1400 cm−1 were assigned to phenol hydroxyl (O-H) stretching and bending vibration,
respectively; 1750, 1680 cm−1 were assigned to the vibration of C=O stretching of carboxylic acid and aldehyde,
respectively. Wavenumbers at 1150, 1100 cm−1 corresponded to C-O stretching and 1045 cm−1 was assigned to
S=O or C=S stretching vibration. The signal at 1280 cm−1 was assigned to the aromatic C-O or C-N vibration;
1370, 1550 cm−1 corresponded to C-NO2 , N-O or N-H stretching; whereas 2850 cm−1 corresponded to the vi-
bration of C-H stretching [19, 20]. These functional groups are similar to those observed with powders profile,
and indicate the presence of aldehydes, carboxylic acids, esters, lactone, amides, amines, sulfoxide and aromatic
skeletal bands [14, 23, 24]. These characteristic signals belong to the main functional groups from terpenoids,
flavonoids, phenol acids and alkaloids [19, 20, 25, 26]. These results are coherent to those from Rani’s group
[27].

Table 1: Main discriminant peaks wavenumber and corresponding chemical functional groups.
Type of extract Wavenumber (cm−1 ) of signals Functional groups
From discrimination From discrimination
zones of leaves zones of root-bark
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spectra spectra
Aqueous 3650 3640 O-H free (stretching)
2850 1430 C-H (stretching, bending)
1690 1680 C=O aldehyde (stretching)
1370 C-NO2 (stretching)
1150 1160 C-O alcohol, C-N amine (stretching)
1045 S=O, C=S (stretching)

Methanolic 1080, 1100 1100 C-OH alcohol (stretching)

1400 O-H phenol (bending)
1530 1550 C-NO2 , N-H amide (stretching)
1740 C=O ester, lactone (stretching)

Chloroformic 1750 1750 C=O carboxylic acid, ester, lactone

1550 1550 C-NO2 , N-O nitro (stretching)
1120 1120 C-O secondary alcohol (stretching)
1280 1280 C-O aromatic ester, C-N aromatic amine
(stretching)

C. procera leaves, latex and root-bark are known to have an important content of cardenolides from terpenoids
biogenesis pathway. Most of these compounds exist in heterosidic form and their aglycone part possess alcohol,
aldehyde or carboxylic group, lactone group, aromatic nitrogen or sulphur functional group like in calotropin,
calotoxin, uscharin, voruscharin … [6] (Figure 5). These cardenolides structures contain an unsaturated cyclic
lactone which is characterized by the conjugated ketone signal at wavenumber 1690 cm−1 . This functional
group targeted C. procera “calotropin-type” cardenolides as biomarkers of leaves (Figure 6A). Then, character-

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 DE GRUYTER Kinda et al.

ized signals at wavenumber 1063 and 1340 cm−1 targeted C. procera “uscharin-type” cardenolides containing
sulphur and nitrogen as biomarkers for roots (Figure 6B and Figure 6C). These results were coherent with lit-
erature assessment about C. procera cardenolides found in plant organs [28]. The content of these compounds
were responsible of the variability observed between extracts FTIR spectra.

Figure 5: Cardenolide-type chemical structure.

Figure 6: Relative calotropin-type content at 1690 cm−1 (A) and Uscharin-type (B, C) content at 1063, 1340 cm−1 in C. pro-
cera organs. AUC: Area Under Curve.

Phytochemical contents
A quantitative analysis was performed with the aqueous and methanolic extracts to assess the variabil-
ity in phytochemical found in C. procera harvested in the Central and the Hauts-Bassins regions (Fig-
ure 7). The results showed that C. procera leaves contain phenolics from 66.40 ± 4,6 to 97.96 ± 5.20 mg-
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GAE/g and from 101.06 ± 13,70 to 140.95 ± 34,40 mgGAE/g for aqueous and methanolic extract, respectively.
Whereas, its root-bark contains phenolics from 38.75 ± 8.30 to 62.48 ± 1.50 mgGAE/g and from 71.94 ± 14.60
to 100.84 ± 28.60 mgGAE/g for aqueous and methanolic extract, respectively.

Figure 7: Phytochemical content of C. procera leaves and root-bark from Centre and Hauts-Bassins regions. Significant
difference **p<0.01, ***p<0.001.

Flavonoids were found in leaves from 5.10 ± 0.1 to 14.14 ± 0.80 mgQE/g for aqueous extract and from
9.65 ± 0.10 to 26.06 ± 1.30 mgQE/g for methanolic extract. In root-bark, flavonoids content was found from

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0.62 ± 0.50 to 20.52 ± 0.90 mgQE/g and from 0.39 ± 0.20 to 2.32 ± 0.30 mgQE/g in aqueous and methanolic
extract, respectively.
The leaves also contain terpenoids from 2.36 ± 1 to 27.03 ± 2.30 mgEUA/g and from 15.93 ± 3.1
to 75.13 ± 2.50 mgUAE/g for aqueous and methanolic extract, respectively. Whereas, root-bark contain
terpenoids from 17.78 ± 2.2 to 32.96 ± 1.00 mgUAE/g for aqueous extract and from 212.81 ± 3.60 to
348.95 ± 3.00 mgUAE/g for methanolic extract.
The data analysis shows that C. procera leaves contain more phenolics compounds (p<0.001) than root-bark,
independently to the region of collection or the type of extraction. Likewise, this analysis reveals an important
terpenoids content in root-bark extracts (p<0.001) compared to leaves extracts independently to the site of col-
lection. These observations are coherent with the results of PCA analysis, suggesting that cardenolides were
responsible of discriminant signals observed between leaves and root-bark spectra. Moreover, our results con-
firm that C. procera traditional uses and the efficacy of root-bark recipes are correlated with terpenoids content.
Indeed, in our previous study, 36.2% of all plant parts used in the treatment of neuropsychiatric disorders were
root samples. Specifically in C. procera parts used, root recipes represented 54.5% [9]. It has been reported that
terpenoids possess anti-inflamatory, anti-microbial, hepatoprotective, analgesic and sedative effects [29, 30],
then it is consistent to suggest that C. procera properties may be attributed to the presence of these compounds.
The specimens from Hauts-Bassins lightly showed best metabolites contents but data analysis did not reveal
significant difference (p>0.05) between phytochemical contents of C. procera specimens from the Central region
compared to the Hauts-Bassins region.
These quantitative values corroborate with the results of FTIR spectral evaluation. They reveal differences
of secondary metabolites contents between C. procera leaves and its root-bark.
Environmental conditions like climatic and ground influence plants phytochemical composition, partic-
ularly the secondary metabolites concentration [10]. Then, various compounds were stocked in the exposed
organs of plants in order to protect them against biotic and abiotic threats [31, 32]. Plant roots are the main
exposed organ because of its contact with ground. It receives directly aggression like dryness, flooding, in-
sects and micro-organism attacks. These observations might explained difference between leaves and root-bar
metabolites contents.

Conclusion
This metabolomic assessment of C. procera samples from the Central and the Hauts-Bassins regions of Burk-
ina Faso, permitted to reveal significant phytochemical variability between C. procera leaves and its root-bark
in both FTIR and quantitative evaluation, independently to the site of collection. Extraction with polar sol-
vents permitted to better distinguish leaves from root-bark phytochemical contents. Phenolic compounds were
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significantly high in leaves than in root-bark, whereas terpenoid compounds were significantly high in root-
bark. Similarly, multivariate data analyses suggest calotropin-type cardenolides as biomarkers for leaves and
uscharin-type cardenolides for roots. The FTIR spectral characteristics showed differences between regions for
the root-bark by the presence of aldehyde functional group in the Central region specimens and its oxidized
form, carboxylic acid in the Hauts-Bassins specimens. However, the quantitative phytochemical analysis did
not allow this discrimination between regions and suggest no significant difference between the specimens in
quantitative terms. Therefore, this metabolomic study coupled with the phytochemical assay allows the spec-
tral characterization of the powders and extracts of C. procera, and suggest that cardenolide-type could be used
as biomarkers for C. procera organs therapeutic potential. These methods could be used for plants raw material
standardization.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted
manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis,
and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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