UNIVERSITE DE YAOUNDE I THE UNIVERSITY OF YAOUNDE I

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CENTRE DE RECHERCHE ET DE POSTGRADUATE SCHOOLS OF
FORMATION DOCTORALE EN SCIENCES, SCIENCE, TECHNOLOGY AND
TECHNOLOGIE ET GEOSCIENCES GEOSCIENCES
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UNITE DE RECHERCHE ET DE DOCTORAL RESEARCH UNIT IN
FORMATION DOCTORALE EN CHIMIE ET CHEMISTRY AND APPLICATIONS
APPLICATIONS ******
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DEPARTEMENT DE CHIMIE ORGANIQUE

LABORATOIRE DES SUBSTANCES NATURELLES VEGETALES ET FONGIQUES

Chemical study guided by LC-MS and the

By :

WOUAMBA NJONTE Steven Collins

Supervision:

KOUAM FOGUE Siméon

Year 2020
 ABSTRACT
The work carried out in this thesis in the field of natural products and overlaps between
Organic Chemistry and Biology and deals with the Liquid Chromatography-Mass
Spectrometry (LC-MS) and antileishmanial activity guided investigation of Vernonia
guineensis Benth (Asteraceae) and, the standardization and preformulation of an improved
phytodrug from its active extracts.
To achieve this, Thus, the roots and aerial parts of the plant were extracted through
maceration and infusion to yield 06 crude extracts which were further subjected to LC-MS
analysis and antileishmanial assay. Extracts with promising chemical profile and/or
bioactivity were subjected to various liquid phase chromatographic techniques such as
Column Chromatography on silica gel and Sephadex LH-20 (CC), analytic and preparative
Thin Layer Chromatography (TLC) and to yield 34 compounds sorted as follows:
- Three ceramides: vernoguinamide (73) (a new derivative), elasticamide (74) and (2S, 3S,
4R) -2-N - [(2′R) -hydroxyhexacosanoyl]-4-hydroxysphinganine (75), all described for the
first time from the Asteraceae family.
- Four anthraquinones: physcion (76), erythroglaucin (77), revandchinone-3 (78) and emodin
(79), all described for the first time from the genus Vernonia.
- Three flavonoids: vernoguinoflavone (80) (a new derivative), quercetin (11) and luteolin
(14), all described for the first time from the species V. guineensis.
- Two glycerol ester derivatives: bisarachidicester (81) (a new derivative) and 2,3-
dihydroxypropyl heptacosanoate (82), all described for the first time from the Asteraceae
family.
- Two sesquiterpenes: vernopicrin (52) and vernomelitensine (53).
- Six steroids: vernoguinoside A (42) and vernoguinoside (44), β-sitosterol 3-O-β-D-glucoside
(83), stigmasterol 3-O-β-D-glucoside (84), stigmastérol (85) and β-sitostérol (86) .
- Six triterpenoids: hop-17(21)-en-3b-yl acetate (87) (isolated for the first time from the
Asteraceae family), lupeol (33), betulinic acid (88), β-amyrin (36), oleanolic acid (37) and
ursolic acid (38).
- Two carotenoids: 13-cis-β-carotène (89) and β-carotène (90), all described for the first time
from the genus Vernonia.
- Three fatty acids: tricosanoic acid (91), tetracosanoic acid (92), and pentacosanoic acid (93).
- Three alcohol derivatives: docosan-1-ol (94), triacontan-1-ol (95) and heptatriacontan-1-ol
(96).

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 The structures of these compounds were determined by interpretation of their 1D (1H,
13
C, DEPT) and 2D (HMQC, HMBC, 1H-1H COSY) NMR data and in some cases by
comparison of their data with those reported in the literature In order to confirm their
established structures, chemical transformations (methanolysis, epoxidation and acetylation)
were carried out on some isolated compounds.

The study of the aerial parts was orchestrated using an anti-leishmanial bioguided
methodology. However, other biological tests targeting the effect of extracts, fractions and
compounds on associated pathologies and the toxicity threshold were also evaluated. This
includes cytotoxic, antioxidant, antibacterial, and acute toxicity activity. The antileishmanial
activity was evaluated in vitro on the promastigote form of Leishmania donovani (1S
(MHOM/SD/62/1S) using the rezazurin colorimetric method. The cytotoxicity was evaluated
in vitro on the epithelial Vero cells line of monkey kidney by MTS spectrophotometric
method. The antioxidant properties of the extracts, fractions and isolated compounds were
evaluated by the methods of scavenging the radicals DPPH and ABTS, then by reduction of
ferric ions (FRAP) and by complexometric chelation of ferrous ions. Enteric infections,
constituting a risk factor for mortality associated with leishmaniasis, antimicrobial activity
against several strains of enteric bacteria such as Escherichia coli (ATCC25922), Salmonella
enterica (NR4294), Shigella flexineri (NR518), Salmonella Muenchen, Salmonella
typhimurium and Salmonella typhi (ATCC 19430) was evaluated by determining the
minimum inhibitory concentrations (MIC) and bactericidal (CMB) by the microdilution
method. The acute toxicity test was carried out over a period of 14 days, with 2 groups of 6
non-pregnant albino female rats of the Wistar strain according to the method described by
OECD line 438. From the results obtained, it emerges that the various crude extracts, fractions
and compounds tested present relatively high activities on all the pathologies targeted with a
low threshold of toxicity. Indeed, the ranges of antileishmanial activities (evaluation of the
IC50) were between [45.77―0.33] µg/mL, [13.45―0.39] µg/mL, and [46.27―0.65] µg/mL
respectively for the extracts fractions and compounds. Compared to other extracts, the extract
most active against Leishmania donovani was the CH2Cl2/EtOH/H2O extract (10:9:1), with an
IC50 = 0.33±0.07 µg/mL, and a selectivity index of 48 in the cytotoxicity assay. This extract,
showing promising antileishmanial activity compared to the reference molecule, amphotericin
B (IC50: 0.33±0.21 µg/mL), was subjected to sequential fractionation in order to identify the
active ingredients. The most promising fractions (IC50: 0.39±0.08 and 0.78±0.21 µg/mL) led
to the detection of 10 compounds by UPLC/DAD/HR (ESI)MS, among which 6 compounds

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were isolated, namely vernoguinoflavone (80, IC50 = 8.26±0.13 µg/mL), quercetin (11, IC50 =
3.22 ± 0.16µg / mL), luteolin (14, IC50 = 15.20 ± 0.02µg / mL), betulinic acid (89, IC50 = 6.21
± 0.07µg / mL), vernopicrin (52, IC50 = 0.68 ± 0.11µg / mL) and ursolic acid (38, IC50 =
0.94±0.11 µg/mL). However, the extract obtained by maceration in a MeOH/DCM mixture
(1:1) gives the best extraction yield and the best content of Vernopicrin, the most active
compound. This gave us to conclude on a synergistic action of the compounds resulting from
the active fractions. Concerning the antioxidant potential, the samples tested showed good
activities compared to ascorbic acid (IC50: 2.03―1.28 µg/mL), used in this test as a positive
control. Indeed, the ranges of antioxidant activity (evaluation of the IC50) were between
[250.50―4.08] µg/mL, [259.90―30.03] µg/mL, and [23.23―1.75] µg/mL respectively for
the extracts, fractions and compounds against [2.03―1.28] µg/mL for the positive control.
Concerning, the antibacterial activity, the various crude extracts from the aerial parts
demonstrated moderate activity with MIC values of between [500―1000] µg/mL. Certain
isolated compounds exhibited very good antibacterial activities on the strains tested (MIC:
3.2―100 μg/mL) exhibiting in some cases a bactericidal effect depending on the sensitive
germ. As for the roots of V. guineensis, the crude extract did not show good antileishmanial
activity (IC50> 100 µg/mL) but demonstrated moderate antimicrobial activity with MIC
values between 500 µg/mL and 2000 µg/mL. The ethyl acetate soluble fraction was the most
active (MIC: 62.4―500 µg/mL). From this fraction, the isolated compounds were tested
giving moderate activities (MIC: 31.2―125 µg/mL).
The extract obtained by aqueous infusion, which demonstrated good activity in all the
tests carried out, was found to be non-toxic at the maximum dose of 5000 mg/kg administered
to the rats, thus making the infusion of the aerial parts of V. guineensis a good candidate who
allowed the formulation of phytodrugs against visceral leishmaniasis (ointments and thermal
lotions) and associated pathology (instant infusion tea).

Key words: Vernonia guineensis, vernoguinamide, bisarachidicester, vernoguinoflavone,

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Chemical structures of isolated compounds

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