ISSN: 2320-5407 Int. J. Adv. Res.

12(11), 1421-1428

Journal Homepage: -www.journalijar.com

Article DOI:10.21474/IJAR01/19957
DOI URL: http://dx.doi.org/10.21474/IJAR01/19957

RESEARCH ARTICLE
PHYTOCHEMICAL STUDY AND EVALUATION OF THE ANTIOXIDANT ACTIVITIES OF LEAVES
FROM VERNONIAAMYGDALINA

Demel Axel Adou1,2, Kouassi Pierre Alain Konan2, Jean Brice Bredou2, Djele Alette Edwige Ziale2, Yapo
Severin Katou 1,2, Janat Akhanovna Mamyrbékova-Békro2 and Békro Yves-Alain2
1. UFR des Sciences et Technologies, Université Alassane Ouattara (UAO).
2. Laboratoire de Chimie Bio-Organique Et de Substances Naturelles, UFR des Sciences Fondamentales et
Appliquées, Université NanguiAbrogoua (UNA).
……………………………………………………………………………………………………....
Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History The aim of this study is to highlight the phytochemical composition
Received: 17 September 2024 and antioxidant potential of the ethereal extract of the leaves of
Final Accepted: 27 October 2024 Vernoniaamygdalina, a plant used in traditional medicine in Côte
Published: November 2024 d'Ivoire.Phytochemical studies using TLC and LC-MS revealed, in
addition to coumarins, flavonoids and phenolic acids. Determination of
Key words:-
Vernoniaamygdalina, Phytochemical the antioxidant potential by TLC and spectrophotometry using the
Screening, Antioxidant DPPH radical showed the antioxidant power of Vernoniaamygdalina
leaves with a CR50 of 0.01995 ± 0.2x10-5 % mg/mL.The many
pharmacological properties of V. amygdalina would therefore depend
on the coexistence of these phytocompounds.

Copyright, IJAR, 2024,. All rights reserved.

……………………………………………………………………………………………………....
Introduction:-
The end of the 20th century was marked by a number of major health crises (COVID-19, Ebola, Avian flu, etc.)
affecting human beings.This upsurge in pathologies could be linked to global upheavals (production methods, new
technologies, climate change, etc.) and oxidative stress (Akissi, 2021).Indeed, free radicals can have deleterious
effects on many molecules, including proteins, lipids, RNA, DNA and carbohydrates (Kostova et al., 2006; Lü et al.,
2010).Furthermore, oxidative stress-related diseases such as inflammatory conditions, cancers, diabetes, accelerated
ageing and cardiovascular disease are public health issues (Adou et al., 2022).In this way, a great deal of research
has been carried out to discover new bioactive compounds, particularly of natural origin, with diversified and unique
chemical structures to tackle this upsurge.Moreover, plants remain one of the most promising sources of molecules
in all areas of health, both for the treatment and prevention of certain pathologies.According to Newman and Cragg
(2020), around 75% of medicines for the period 1981-2019 are of natural origin.In this context,
Vernoniaamygdalina, a plant traditionally used in Côte d'Ivoire, was studied. The aim was to determine the
phytochemical composition and antioxidant potential of a selective extract of Vernoniaamygdalina leaves.

Materials and Methods:-

Plant material
The leaves of Vernoniaamygdalina were collected in Anyama (5° 29′ 40″ north, 4° 03′ 06″ west, a town in the south
of Côte d'Ivoire in the Autonomous District of Abidjan). The identification was confirmed by the late ASSY Jean of
the Centre National de Floristique (CNF) in Abidjan (5° 20′ 11″ Nord, 4° 01′ 36″ Ouest), in accordance with

Corresponding Author:-Demel Axel Adou

Address:-UFR des Sciences et Technologies, Université Alassane Ouattara (UAO). 1421
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existing herbaria. The various organs were dried under air conditioning (18°C) for two weeks, then ground using an
electric grinder (Moulinex) to obtain the powders used for analysis.

Methods:-
Extraction
Coumarin extraction was carried out according to the method described by Grinkevitch and Safronitch (1993), with
a few modifications. In a reflux heater, 30 g of plant powder were subjected to hydrolysis in HCl (2N, 100 mL) for 2
hr. After cooling, the pomace was dried in a fume hood for 48 h, then recovered in hexane (24 h).The pomace is
then extracted with MeOH (100 mL) in a Soxhlet for 2 h. After removal of the solvent, the extracted mass is
successively treated with KOH solutions at 0.5% (w/v) (90 mL) and 5% (w/v) (20 mL) for 1 h. The alkaline fraction
obtained is treated with ethyl ether (3 × 50 mL) to remove undesirable compounds.After settling and separation, a
5% (v/v) HCl solution is carefully added to the alkaline phase up to pH= 2. The reaction mass was then treated with
ethyl ether (3 × 100 mL). The diethyl ether fractions of the leaves were used for the rest of the study.

Phytochemical analysis by TLC

Thin-layer chromatographic (TLC) qualitative analysis of the ethereal extracts was carried out in accordance with
the operating methods referenced in the bibliography (Dekker et al., 2003; Newman et al., 2003; Békro et al., 2007;
Mamyrbékova-Békro et al., 2008; Cazes et al., 2010; N'Gaman et al., 2013).Deposits were made on resized
chromatoplates (silica gel 60 F254, aluminum support, 20 × 20 cm, Merck). After migration in the solvent system
toluene/chloroform/ethyl acetate (1/3/1/0.5), chromatograms were observed under UV at 254 and 365 nm, before
revelation with basic lead acetate, 5% (w/v) methanolic KOH solution and ammonia.

Phytochemical analysis by LC-MS

Analysis was carried out on an HPLC chain (Agilent 1260 Infinity) coupled to a mass spectrometer (Q-TOF-MS
Agilent 6530) equipped with an ESI source. A Sunfire® Waters C18 column with a length of 150 mm, a diameter of
2.1 mm and a particle diameter of 3.5 μm was used. The mobile phase consisted of a gradient of solvents A (H2O +
0.1% HCO2H) and B (ACN).A linear gradient was used for 41 min in the following proportions: 0-5% B (0-5 min),
5-95% B (5-15 min), 95-100% B (15-25 min), 100% B (25-30 min), 100-0% B (30-32 min) and 0% B (32-41 min).
Sample injection volume and flow rate are set at 5 μL and 250 μL/min respectively. Data analysis is performed
using Agilent MassHunter Workstation software.

Determination of antioxidant potential

The antioxidant power of ethereal coumarin extracts was tested following the method described by Takao et al .
(1994). Deposits weré made on resized chromatoplates (silica gel 60 F254, aluminum support, 20 × 20 cm, Merck).
After migration in the solvent system toluene / chloroform / ethyl acetate/ MeOH (1/3/1/0.5), chromatograms were
dried and then treated with a methanolic solution of the stable radical DPPH (2 mg/mL).After an incubation time of
30 min, the phytoconstituents of extracts with potential free radical scavenging activity are revealed as pale yellow
imprints on a violet background.The method of Blois (1958), adopted by Kabran et al. (2014), was used to measure
the antioxidant activity of coumarin extracts.DPPH is solubilized in absolute MeOH to obtain a solution with a
concentration of 0.3 mg/mL. Different concentration ranges (1; 0.5; 0.25; 0.1; 0.05; 0.025 and 0.01 mg/mL) of each
extract are prepared in the same solvent. 1 mL extract and 2.5 mL methanolic solution of DPPH.After shaking, the
tubes are placed in the dark for 30 min. The absorbance of the mixture is then measured at 517 nm against a blank
consisting of 1 mL pure MeOH and 2.5 mL DPPH solution. Ascorbic acid (vitamin C) is the positive reference
control. DPPH reduction percentages (%R) are calculated according to formula (1):

(1)
%R = (Ab – Ae) / Ab × 100

Ab: absorbance of blank

Ae: absorbance of sample

The concentration required to reduce 50% of the DPPH radical concentration (CR 50) was determined using
OriginPro 9.1 software (Etekpo et al., 2018; Tano et al., 2019).

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Statistical analysis
A statistical study based on one-way analysis of variance (ANOVA) was carried out using OriginPro 9.1 software.
The difference between means was considered significant at the 5% level. All experimental measurements were
performed in triplicate and results expressed as means plus or minus standard deviations (Mean ± SD).

Results and Discussion:-

Phytochemical screening by TLC
The chromatograms were interpreted with reference to the literature (Wagner and Bladt, 1996; Dekker, 2003; Cazes,
2010). In view of the results obtained, the appearance of blue, yellow, orange and fluorescent blue coloration reflects
the presence of coumarins in V. amygdalina leaf extracts (Table 1).In addition, the presence of flavonoids was
detected by Neu's reagent in leaf extract. Indeed, this specific reagent reveals flavonoids in blue, yellow, green or
orange (Wagner and Bladt, 1996). Dagnon et al. (2019) showed the presence of flavonoids in leaf extract.The results
obtained are partly in line with the work of certain authors. Indeed, E. Canh Pham et al, 2024 showed that V.
amygdalina leaves are known to be rich in phenolic acids, tannins and flavonoids.This entire study highlighted the
absence of coumarin in the crude extract. Oluwaseun et al (2017) confirmed the presence of flavonoids, alkaloids,
tannins and phenolic compounds in V. amygdalina organs.

Table 1:- Synopsis of the phytochemical composition of V. amygdalina.

Fraction Rf Color Phytocompounds
0,08 Yb1,c1,d1,e1,e2; Bfa2,b2; coumarinsa,b,c,d; 7-hydroxycoumarinor 6,7-
c2,d2
B dialcoxycoumarina,b/flavonoide
0,12 Bc2 coumarinc
0,25 Ya1,b1,c1,d1,e1; coumarina,b,c,d; 7-hydroxycoumarin or 6,7-
Bfa2,b2,c2,d2,e2 dialcoxycoumarina,b/ flavonoide
VA 0,43 Ya1; Bd2 Coumarina,d
0,52 Bd2 coumarind
0,58 Ora1; Gb1; Ra2,b2,c2,d2 NAc,d; 6-hydroxy-7-alcoxycoumarina,b
0,62 Orc1,d1; Brc2 coumarinc,d
0,92 Ba2 coumarina
Rf : retention factor ; B : blue ; Y : yellow ; Br :brown ; G : green ; Bf : blue-fluorescent ; Or : orange; a2 : 365 nm ;
b1 : NH3; b2 : NH3 at 365 nm: c1 : ((AcO2)2Pb); c2 : ((AcO2)2Pb) at 365 nm ; d1 : KOH; d2 : KOH at 365 nm; e2 : Neu
at 365 nm ; NA : not appreciated, VA: Vernonia amygdalina

LC-MS profile of the ethereal extract of V. amygdalina leaves

Positive-mode LC-MS analysis of the ethereal extract of V. amygdalina leaves revealed 20 peaks (Figure 1). Data on
maximum absorbances and the masses of the various fragment ions (Table 2) helped to highlight the co-presence of
coumarins, flavonoids and phenolic acids.

Coumarins areillustrated by peak 1, 5, 6, 7, 8, 10, 13, 14, 15, 16, 18 and 19. Peaks 1, 5, 7, 8, 10, 15, 16, 18 and 19
were attributed to auraptene, imperatorine, coumarin, aleuritine, 4,7,8-trihydroxy-6-methoxy-2-oxo-chroman-3-
carboxylic acid and a furanocoumarin derivative respectively.Auraptene is characterized by a molecular ion at m/z
299 [M+H]+ and daughter ions at m/z 163 [M+H-C10H16]+ (loss of 2 isoprenic units); 135 [M+H-C10H16-CO]+ and
117 [M+H-C10H16-CO-H2O]+.Peak 5 was identified as imperatorine, with a molecular ion at m/z 271 [M+H]+ and
daughter ions at m/z 201 [M+H-C5H10]+ (loss of 2-methylbut-2-ene); 163 (furanic ring breakage of a
furanocoumarin); 135 and 117.Analysis of peak 16 identified 4,7,8-trihydroxy-6-methoxy-2-oxochroman-3-
carboxylic acid. Indeed, this phytocompound is characterized by a molecular ion at m/z 271 [M+H] + and daughter
ions at m/z 227 [M+H-CO2]+; 209 [M+HCO2-H2O]+; 181 [M+H-CO2-H2O-CO]+; 164 [M+H-CO2-2H2O-CO]+; 139
and 107.These proposals are based on a similar pattern of fragmentation pathways and UV spectra. Peak 6 indicates
the presence of 3,4-dihydrocoumarin (m/z 149 [M+H]+) with daughter ions at m/z 121 [M+H-CO]+ and 105 [M+H-
CO2]+.In addition, different fragmentations of the molecular ion corresponding to peak 13, giving m/z 157 [M+H-
CO]+ , m/z 142 [M+H-CO2]+ and m/z 125 [M+H-CO3]+ suggest psoralen. Peak 14 showed under positive mode
ionization a molecular ion at m/z 400 [M+H]+, fragmentation of this ion showed daughter ions at m/z 352, a base

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peak at m/z 191 due to loss of the glycosyl unit and m/z 175 suggesting that this compound may be an isomer of
scopoline.

Figure 11:-Mass Spectrum of the ether extract of V.amygdalina leaves.

Table 2:-Synopsis of the LC-MS profile of the phytochemical composition of the ethereal extract of V. amygdalina
leaves.
MS
Mola (m/z) Molecula
Pi Rt(min MS/MS
λmax (nm) r Ion r Phytocompounds
c ) Ion
Mass [M+H] formula
+

226
163,137,
1 3,29 285 298 299 C19H22O3 Auraptene
135,117
315
205 283,190,
2 4,71 298 299 C16H10O6 Irolone
226 145,117
227
253,235,
3 8,60 260 270 271 C15H10O5 Apigenin
154,137,117,109
295
215 255,180,163,149,11
270 271 C16H14O4 Alpinetin
235 9
4 10,12
280 212 213 195,167 C13H8O3 Urolithin

201, 163, 135, 117,

227 270 271 C16H14O4 Imperatorine
103
5 11,33 280
330 CinnamicacidderivativeFragesi
180 181 163, 145, 103 C9H8O4
ne
235 370 371 353,269 C21H22O6
12,29
6 280 148 121,105 C9H8O2 3,4-dihydrocoumarin

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149

210 270 271 163,117,103 C16H14O4 Coumarin

7 13,26 240
241 242 NI
285
400,369,264,234,14
215 416 C21H20O9 Aleuritin
417 6
8 13,76 240
2,4,5-
265 198 197 179, 161, 119, 101 C9H8O5
trihydroxycinnamiqueacid
21024028 NI
9 14,37 266 267 138,107 C8H8O4
5
210
245
10 15,78 270 271 149, 135, 117, 103 C16H14O4 Furanocoumarinederivative
270

21025031
11 17,40 315 316 153 Dihydroxybenzoichexosideacid
5
250
12 18,01 280 281 NI
315
341 342 325,311,281,268 dimethoxylflavanon
13 18,77 215285
186 187 157,142,125 C11H6O3 Psoralene
250
285 400 401 352,191,175 Scopolineisomer
14 19,93 310
21024534
270 271 163, 135, 117, 103 C16H14O4 furanocoumarinderivative
5
4,7,8-trihydroxy-6-methoxy-2-
210 270 271 163, 135, 117, 103 C11H10O8 oxo-chroman-3-
15 22,05
260 carboxyliqueacid
120 121 NI
210
16 24,03 250 270 271 149,135,117,103 C16H14O4 furanocoumarinderivative
270
17 25,29 642 643 NI
210 596 597 C26H44O15 NI
18 25,90 250
270 271 149, 135, 117, 103 C16H14O4 furanocoumariederivative
270
28,53 210 656 657 NI
19
28,53 255 270 271 201,135,117,103 C16H14O4 Imperatorine
210 610 611 466,303,177,122 C27H30O16 Rutine
20 29,39 250
568 569 NI
270
NI : Not Identified

Five (5) flavonoids were identified in the ethereal extract of V. amygdalina. These were irolone (peak 2); apigenin
(peak 3); alpinetin (peak 4); dimethoxyflavanone (peak 13) and rutin (peak 20). Indeed, irolone (peak 2) is
characterized by a molecular ion at m/z 299 [M+H]+ and daughter ions at m/z 283 [M+H-H2O]+; 190 [M+H-H2O-
C6H6O]+; 145 [M+H-H2O-C6H6O-CH4O2]+ and 117 [M+H-H2O-C6H6O-CH4O2-CO]+.Apigenin (peak 3) and rutin
(peak 20) were revealed by molecular ions at m/z 271 [M+H] + and 611 [M+H]+ respectively, and the fragmentation
of these ions is similar to that obtained in Table 2. The fragmentation of peak 4 suggests alpinetine at m/z
271([M+H]+). The fragment at m/z 255 [M+H-CH3]+corresponds to CH3 loss.Ten, the fragment at m/z 180 [M+H-
CH3-C6H6]+ (loss of a benzene group), followed by fragments at m/z 163 [M+H-CH3-C6H6-H2O]+and 149 [M+H-
CH3-C6H6-2H2O]+. Finally, the fragment at m/z 119 [M+H-CH3-C6H6-2H2O-CO]+ indicates the loss of carbon
monoxide. Peak 13 features a molecular ion at m/z 342 [M+H]+. Its UV profile (absorption bands at 215 and 287)

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suggests a flavanone as reported by Portet et al. (2008). Fragments at m/z 325 [M+H-CH3] and m/z 311 [M+H-
2CH3] reveal the subsequent loss of 2 CH3-, indicating a methoxylated compound. Fragmentation of the m/z 311 ion
yielded a fragment ion at m/z 281, originating from the loss of a CO molecule. The absence of significant H 2O loss
indicates a flavanone skeleton (Portet et al., 2008). Thus, peak 13 would be a dimethoxyflavanone.Among the
compounds identified in the ethereal extract of V. amygdalina, several phenolic acid derivatives such as cinnamic
acids (peaks 5 and 8) and a benzoic acid (peak 11) were detected. In addition to phenolic acids, fragésine (a lignan)
is highlighted by peak 6. Indeed, fragmentation of fragesin showed a molecular ion at m/z 371 [M+H] + and a
daughter ion at m/z 353 [M+H-H2O] (Gao et al., 2015).

Antioxidant activity of ethereal leaf extracts

A compound's antioxidant activity is explained by its ability to resist oxidation. In the DPPH TLC test, compounds
with antioxidant activity show up as active yellow-pale fingerprints on a violet background in the chromatograms. In
the presence of free-radical scavengers (or reducers), the violet DPPH radical is reduced by H-transfer to its reduced
form, yellow 1,1-diphenyl-2-picrylhydrazine (DPPH-H) (Takao et al., 1994).It should also be remembered that
DDPH also acts as a developer of free-radical scavenging phytocompounds. Ethereal extracts of v. amygdalina
leaves contain coumarins capable of reducing (or scavenging) DPPH. The anti-free radical properties of these
extracts were therefore revealed by TLC. The frontal ratios (Rf) of the compounds corresponding to the said activity
zones have been recorded in Table 3.
Table 3:- Rf of different antiradical phytocompounds in ethereal leaf extracts against DPPH.
Retention factor (Rf)
VA 0,08 ; 0,13 ; 0,25 ; 0,42 ; 0,52 ; 0,92
Comparing the chromatographic profiles of the phytochemical screening and those of the DPPH TLC test, we found
that these zones of anti-free radical activity were mainly those of coumarins. Indeed, coumarins are known for their
remarkable antioxidant powers (Lin et al., 2008).

Spectrophotometric antioxidant profile of ethereal leaf extracts

Figure 2 shows the results of the antioxidant activity evaluation test, which compares the DPPH-reducing
concentrations of the various samples studied with those of the reference antioxidant, Vitamin C. Leaf extracts from
the fractions tested showed DPPH reduction rates of over 50% from 0.03 mg/mL.

Analysis of variance (ANOVA) applied to the mean of the percentages of reduction at 0.25 mg/mL gives a value of
P < 0.05. The differences between the percentage reduction values are therefore significant. In addition, VA ethereal
extracts showed good anti-radical (or reducing) activity close to that of vitamin C (96.558 ± 0.002%) at the same
concentration. Comparing our results with those reported by N'guessan (2013) in a study of ethyl acetate and n-
butanol extracts of V. amygdalina, we find that the reduction percentages are relatively close.
120

100
% DPPH Reductions

0,0156 mg/mL
80
0,03125 mg/mL
60 0,0625 mg/mL
0,125 mg/mL
40
0,25 mg/mL
20 0,5 mg/mL
1 mg/mL
0
VA VIT C
Extracts

Figure 2:-Synopsis of the quantified antioxidant activity of Vernonia amygdalina ethereal leaf extracts.

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VA: ethereal extract of V.amygdalinaleaves; VIT C: vitamin C

The concentrations of ethereal leaf extracts required to reduce 50% of the DPPH concentration were determined
graphically. The lower the CR50, the higher the antioxidant activity. It should be noted that vitamin C was used as
the reference antioxidant. The CR50 values determined are shown in Table 4.

Table 4:- CR50 values (mg/mL) for ethereal leaf extracts.

Extrait
VA VIT C
CR50 (mg/mL) 0,01995 ±0,2x10-5% 0,016150 ±0,3x10-6%
In view of the CR50 values presented in Table 4, the ethereal extract of V. anygdalina leaves shows remarkable
DPPH radical reduction capacities, compared with that of vitamin C. Extracts with CR 50 values close to those of VA
vitamin C (0.01995 ± 0.2x10-5 % mg/mL).

Conclusion:-
The present study focused on the phytochemical and antioxidant analysis of an ethereal extract from the leaves of
Vernoniaamygdalina from Côte d'Ivoire. Phytochemical screening by TLC revealed the presence of coumarins and
flavonoids in the selective extract of V. amygdalina leaves. In addition, LC-MS/MS analysis enabled structural
characterization of phytoconstituents such as auraptene, imperatorine, coumarin, aleuritine, apigenin, scopoline and
others. From the qualitative TLC test to the quantitative DPPH radical scavenging spectrophotometry test, it
emerged that the ethereal extract of V. amygdalina showed a good antioxidant profile. Moreover, the antioxidant
capacities revealed are close to those of vitamin C. The phytochemical approach thus provides a rational explanation
for the use of V. amygdalina leaves in endogenous and popular therapeutic practices in Côte d'Ivoire.

Competing interests
Authors have declared no competing interests exist.

References:-
1. Akissi Z. L. E. (2021). Etude chimique de plantes médicinales de Côte d’Ivoire, cas : de Asparagus africanus
Lam., de IcacinamanniiOliv. Et de Olyralatifolia L. Thesis 323 p.
2. Kostova I. (2006). Synthetic and Natural Coumarins as Antioxidants. Mini-Reviews in Medicinal Chemistry; 6:
365-74.
3. Lü J-M, Lin PH, Yao Q, Chen C. (2010). Chemical and molecular mechanisms of antioxidants: experimental
approaches and model systems. Journal of Cellular and Molecular Medicine: 14: 840-60.
4. Adou D. A, N’guessan A. H. O, Mamyrbékova-Békro J. A, Békro Y-A. (2022). Evaluation of the antioxidant
potential and aglyconecoumarins contents of ethereal extracts of 4 medicinal plants from Côte d’Ivoire. Journal of
Pharmacognosy and Phytochemistry 2022; 11(6): 46-49
5. Newman, D.J., Cragg, G.M., 2020. Natural Products as Sources of New Drugs over the Nearly Four Decades
from 01/1981 to 09/2019. J. Nat. Prod. 83, 770-803.
6. Grinkevitch N. I., Safronitch L. N., (1993). Analyse chimique des plantes medicinales. Vischayachkola,
MoscouRussie: 175 p.
7. Dekker M. (2003). Semiconductor photochemistry and photophysics. Molecular and Supramolecular
Photochemistry, New York: 361 p.
8. Newman D., Cragg G. et Snader K. (2003). Natural products as sources of new drugs over the period 1981-2002.
Journal of Natural Products.66, 1022-1037.
9. Békro Y. A., Mamyrbekova-Békro J. A., Boua B. B., Tra Bi F. H., Ehilé E. E. (2007). Etude ethnobotanique et
screening phytochimique de Caesalpiniabenthamiana(Baill.) Herend. Et Zarucchi (Caesalpiniaceae). Sciences et
Nature :4 (2): 217-225.
10. Cazes J. (2010). Coumarins: TLC Analysis. Encyclopedia of Chromatography, Third Edition. CRC Press,
Telor& Francis Group, Lndon-New York: 511-513.
11. N’Gama K. C. C. (2013). Etude phytochimique et effet d'extraits de GmelinaarboreaRoxb. (Verbenaceae) de
Côte d’Ivoire sur la stabilité osmotique d’érythrocytes. Thèse de Doctorat, Université NanguiAbrogoua, Cote
d’Ivoire. 152p
12. Takao T., Kitatami F., Watanabe N., Yagi A., Sakata K. (1994). A simple screening method for antioxydants and
isolation of several antioxydants produced by marine bacteria from fish and shell fish. Bioscience, Biotechnology
and Biochemistry: 58, 1780-1783.

1427
ISSN: 2320-5407 Int. J. Adv. Res. 12(11), 1421-1428

13. Blois M. (1958). Antioxydant determinations by the use of a stable free radical. Nature 181: 1199 -1200.
14. Kabran G., R., M. (2014). Etude chimique et cytotoxique de dix plantes de Côte d'Ivoire, utilisées dans le
traitement traditionnel du cancer du sein. Thèse de doctorat, Université NanguiAbrogoua, Côte d'Ivoire, 265 p.
15. Etekpo D. S., N’Gaman-Kouassi C. C., Mamyrbekova-Békro J. A., Békro Y-A. (2018). Antioxidant profiles of
alcoholic tinctures from Heterotisrotundifolia (sm.) Jacq.-Fél. (Melastomacaceae) by DPPH radical
trapping.European Journal of Biomedical and Pharmaceutical Sciences: 5 (10), 39-45.
16. Tanoh K. S., N’gaman-Kouassi C. C., Boa D., Mamyrbekova-Bekro J. A., Bekro Y-A. (2019). Activité
antioxydante des extraits bruts hydroéthanoliques et hydroacétoniques des organes de quatre plantes médicinales de
Côte d’Ivoire. Nature et Technologie: 11 (2), 28-34.
17. Wagner H et Bladt S. (1996). Plant drug analysis in thin layer chromatography, Springer Verlag Berlin Edatlas
2nd edition: 384 p.
18. Em Canh Pham, Vien Van Doan, Tuong Vi Le Thi, Cuong Van Ngo, Lenh Vo Van (2024) In vivo and in silico
antihypertensive, anti-inflammatory, and analgesic activities of Vernoniaamygdalina Del. leaf extracts. Heliyon, 10
(19), e38634
19. Oluwaseun Ruth Alara, Nour Hamid Abdurahman, SitiKholijah Abdul Mudalip, Olusegun AbayomiOlalere
(2017), phytochemical and pharmacological properties of Vernoniaamygdalina: A REVIEW. Journal of Chemical
Engineeringand Industrial Biotechnology, 2 , 80-96.
20. PortetBénédicte, Nicolas Fabre, Raoul Rozenberg, Jean-Louis Habib-Jiwan, Claude Moulis (2008). Analysis of
minor flavonoids in Piper hostmannianum var. berbicense using liquid chromatography coupled with atmospheric
pressure chemical ionization mass spectrometry. Journal of Chromatography A; 1210 (1), 45-54.
21. Gao Wei, Clemens Kirschbaum, Juliane Grass, Tobias Stalder (2016). LC–MS based analysis of endogenous
steroid hormones in human hair. The Journal of Steroid Biochemistry and Molecular Biology; 162, 92-99.
22. Lin H. C., Tsai S. H., Chen C. S., Chang Y. C., Lee C. M., Lai Z. Y., Lin C. M. (2008). Structure–activity
relationship of coumarin derivatives on xanthine oxidase-inhibiting and free radical-scavenging activities.
Biochemicalpharmacology : 75, 1416–1425.
23. N’Guessan H. A. (2013). Composition qualitative, quantitative et activités anti oxydante et anti proliférative de
10 plantes médicinales de Côte d’Ivoire. Thèse de doctorat, Université NanguiAbrogoua, Côte d'Ivoire : 212 p.

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