South African Journal of Botany 151 (2022) 288 294

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Artemisia campestris dried leaf extracts: Effects of different extraction

methods and solvents on phenolic composition and biological activities
Rafika Metouia,b, Hedi Mighria, Jalloul Bouajilab, Mansour Znatib,c, Hajer El-Jania,
Ahmed Akrouta,*
a
Laboratory of Separation, Analyses and Valorisation of Natural bioactive Compounds, Arid Regions Institute, 4119 Medenine, Gabes University, Tunisia
b
Faculte de Pharmacie de Toulouse, Laboratoire des IMRCP UMR CNRS 5623, Universite Paul-Sabatier, Toulouse F-31062, France
c
Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity (LR11ES39), Team: Medicinal Chemistry and Natural Products and Reactivity, Faculty of
Science of Monastir, 5019, Monastir University, Tunisia

A R T I C L E I N F O A B S T R A C T

Article History: In the present study, the effects of three techniques of extraction (maceration, fractionation and reflux) using
Received 7 June 2022 successively four solvents (dichlormethane (DM), ethyl acetate (EtAc), butanol (But) and water (W)) on phy-
Revised 12 September 2022 tochemical contents (total polyphenols and total flavonoids) and biological activities (antioxidant, anti-
Accepted 1 October 2022
inflammatory, anti-cholinesterase, anti-xanthine oxidase and cytotoxic) of Artemisia campestris dried leaves
Available online 13 October 2022
extracts were investigated. The results showed that the highest yield of crude extract was obtained by the
Edited by: L Sebastiani reflux method. The maceration method was shown to be the best one for the extraction of total polyphenols
(345.8 mg GAE/g dry extract) and total flavonoids (296.3 mg QE/g dry extract) when butanol and ethyl ace-
Keyword:
tate were used as solvents, respectively. LC-MS analysis revealed the presence of 29 phenolic compounds.
Artemisia campestris
The ethyl acetate extract obtained with maceration method was the most rich one in these compounds (20
Extraction method
compounds) amoung them six have the highest amounts (ranged from 1228.9 to 19,898.2 mg/g DE) and only
Antioxidant
Cytotoxicity luteolin was detected in this extract at 9874.9 mg/g DE. Likewase this same compound displayed the stron-
Anti-xanthine oxidase gest anti-xanthine oxidase, antioxidant (ABTS assay) and cytotoxic activities (against MCF-7 and OVCAR cell
Anti-cholinesterase lines) with IC50 of 5.0 and 4.4 mg/L, and inhibition percentage of 84.4 and 93.0% at 50 mg/L, respectively. The
ethyl acetate extract obtained by the fractionation method showed the highest antioxidant (DPPH method)
and anti-cholinesterase activities with IC50 of 11.0 mg/L and inhibition percentage of 55.9% at 50 mg/L,
respectively.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction of the natural sources of bioactive substances, as well as the structure,

Plants may confer a variety of health benefits. Many of the Tuni-
sian plants have medicinal properties and there is an increasing
demand for herbal remedies because of undesirable effects of syn-
thetic drugs. Herbal preparations for medicinal usages contain vari-
ous types of bioactive compounds (terpenoids, polyphenols,
saponins, anthraquinones, alkaloids, etc.) which could be extracted
and isolated by different extraction methods. The amount and the
nature of these extractable active compounds depend on several
parameters: nature of the solvent and its concentration, pH, solvent-
to-solid ratio, pressure, technique of separation, number of steps,
time and temperature of extraction, nature of plant material and its
origin, moisture content, and particle size and shape of the plant
matrix (Tiwari et al., 2011; Shahrajabian et al., 2021;
Mahomoodally et al., 2018). Considering the diversity in composition
* Corresponding author.
E-mail address: akirite@yahoo.fr (A. Akrout).
type and physicochemical properties of these compounds, a universal
extraction protocol is not conceivable, and specific processes must be
designed and optimized for each source of bioactive substances.
Artemisia campestris L. is a perennial scarcely aromatic herb
belonging to Asteraceae family, widespread in the south of Tunisia,
commonly known as "T’gouft". The leaves of A. campestris are widely
used in traditional medicine as decoction for their antivenin, anti-
inflammatory, antirheumatic, antimicrobial and antifungal properties
and to treat gastric disturbances, diarrhea, abdominal cramps, hyper-
tension and rheumatism (Al-Snafi, 2015). The phytochemical screen-
ing of this species revealed the presence of tannins, polyphenols,
flavonoids, saponosides, essential oil and minerals (Akrout et al.,
2011; Belhattab et al., 2011; Boulanouar et al., 2013; ksouri et al.,
2014). Various biological activities have been reported for different
extracts of A. campestris such as antitumor, antioxidant, antibacterial,
antifungal, inhibition of the scorpion venom and protective effect of
against aspirin-induced gastric lesions (Akrout et al., 2010; Ben Nasr
et al., 2014; Djidel and khennouf, 2014). Several reports have shown

https://doi.org/10.1016/j.sajb.2022.10.002
0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.
R. Metoui, H. Mighri, J. Bouajila et al.
that polyphenols especially flavonoids exhibit important physiologi-
cal functions such as antioxidant (Chandrasekara and Shahidi., 2011),
anti-cancer (Tsai et al., 2010) and anti-inflammatory (Kao et al.,
2007).
The objective of the present study was to investigate the effects of
extraction solvents (dichloromethane, ethyl acetate, butanol and
water) and methods (maceration, fractionation and reflux) on pheno-
lic contents (total polyphenols and total flavonoids) and biological
activities (antioxidant, anti-inflammatory, anti-cholinesterase, anti-
xanthine oxidase (XOD), anti- superoxide dismutase (SOD) and cyto-
toxicity) in dried leaves of A. campestris. To the best of our knowledge,
no previous study has been reported concerning these aspects.
2. Materials and methods
2.1. Chemicals
All chemicals used were of analytical reagent grade. All reagents
were purchased from Sigma, Aldrich, Fluka (Saint-Quentin France).
2.2. Collection of plant material
The aerial parts of approximately 20 individual plants of A. cam-
pestris collected during the full vegetative stage just before the flow-
ering stage (August) from natural population in Beni- Khedache
(mountainous region in South-East of Tunisia) and voucher speci-
mens (AC0813) collected were identified, processed and deposited in
the range ecology laboratory of Arid Regions Institute (IRA), Mede-
nine, Tunisia. After the harvest, the plant material was cleaned from
dust, air-dried under shade (20 26 °C) on the laboratory bench for
two weeks. Then, the leaves were separated from the other parts and
used for the extraction.
2.3. Preparation of extracts
Three extraction methods (maceration, fractionation and reflux)
were used to prepare crude extracts from dried leaves of A. campest-
ris. Each extraction method was performed in triplicate.
2.3.1. Maceration method
Samples (200 g of the dried leaves of A. campestris) were mixed
with 1 L DM in a stoppered container and kept in contact away from
light during 72 h at room temperature (20 26 °C) with occasionally
manual agitation. The mixture was then filtered and the filtrate was
kept away from light at room temperature. The same sample was
treated with the same protocol twice. Filtrates were collected
together and the solvent was totally evaporated under reduced pres-
sure at 40 °C by rotary evaporator to obtain the the dry extract of the
DM maceration (MDM) which was weighed and stored in amber vial
at 4 °C for further analysis. Then, the same sample was successively
treated with EtAc, But and W under the same conditions as DM to
obtain the extracts of different solvents (MEtAc, MBut and MW,
respectively). All extracts were kept in amber vials and stored at 4 °C
for further analysis.
2.3.2. Fractionation method
Samples (200 g) of the dried leaves of A. campestris were mixed
with 1 L (70% aqueous methanol) in a stoppered container and kept
in contact away from light during 72 h at room temperature (20 26 °
C) with occasionally manual agitation. The mixture was then filtered
and the filtrate was kept in the dark at room temperature and the
same sample was treated with the same protocol twice. Filtrates
were collected together and concentrated under reduced pressure at
40 °C using rotary evaporator. The aqueous residue was then succes-
sively fractioned with DM, EtAc and But to obtain FDM, FEtAC and
FBut extracts, respectively. The residual aqueous solution was also
289
South African Journal of Botany 151 (2022) 288 294
evaporated to dryness to obtain FW extract. All extracts were kept in
amber vials and stored at 4 °C for further analysis.
2.3.3. Reflux method
200 g of dried leaves of A. campestris were immersed with 1 L of
DM in a round-bottomed flask which is connected to a condenser.
The mixture was heated during 1 h after boiling point. Then, the mix-
ture was filtered and the filtrate was concentrated by evaporating
the solvent under reduced pressure at 40 °C by rotary evaporator to
obtain the dry extract of the reflux DM (RDM). The same sample was
treated successively under the same conditions with the following
solvents of croissant polarity: EtAc, But and W to obtain REtAc, RBut
and RW extracts, respectively. All extracts were kept in amber vials
and stored at 4 °C for further analysis.
2.3.4. Determination of total polyphenol and total flavonoid contents
and biological activities
The total polyphenol and the total flavonoid contents (Folin-Cio-
calteu method and aluminum trichloride method, respectively), the
anti-oxidant activity (DPPH and ABTS assays), the anti- Alzheimer
activity (anti-acetylcholinesterase assay), the anti-inflammatory
(anti-5-lipoxygenase assay) and thy cytotoxic activitie against MCF-7
and OVCAR cell lines (MTT assay), were determined according to the
protocols used by Bekir et al. (2013). The anti-xanthine oxidase activ-
ity and the anti-superoxide dismutase activity were evaluated
according to the methods used by Kammoun et al. (2015).
2.3.5. LC-MS analysis
LC-MS analysis was carried-out by a Shimadzu ultra-fast liquid
chromatography system equipped with an online DGU-20AS degas-
ser, a CTO-20AC column oven, a LC-20ADXR binary pump, a SIL-
20ACXR auto-sampler and a SPD-M20A diode array detector with
13 mm flow cell. Separation was made using a Discovery BioC18 col-
umn (250 mm £ 4.6 mm, 5 mm) (Supelco). The mobile phase was
composed by solvent A: formic acid (0.1% in water) and solvent B:
formic acid (0.1% in methanol) with the following conditions:
0 45 min with 10% B, 45 55 min with 100% B, the initial conditions
were held for 10 min as a re-equilibration step. The flow rate of the
mobile phase was 0.5 mL/min, the column temperature was main-
tained at 40 °C and the injection volume was 5 mL.
The HPLC system was equipped with a Shimadzu mass spectrom-
eter coupled to an electro spray ionization source (ESI). The mass
spectrometer was used in negative ion mode with a capillary voltage
of 3.5 V, a dry gas flow rate of 12 L/min, a nebulizing gas flow of
1.5 L/min, a block source temperature of 400 °C, a dissolving line (DL)
temperature of 250 °C, the full scan spectra from 50 to 2000 Da and a
voltage detector of 1.35 V.
LC-MS analysis of phenolic compounds in A. campestris seeds
extracts was determined by a validated method analysis using thirty
three standards (Bennour et al., 2020; Mighri et al., 2019).
2.3.6. Statistical analysis
All data were expressed as means § standard deviations of tripli-
cate measurements. Statistical comparisons were performed by the
ANOVA test with a statistical significance level set at p < 0.05.The
Pearson correlations measure the relation between variables. All sta-
tistical analyses were carried out using SPSS (IBM SPSS Statistics
20.0).
3. Results and discussion
3.1. Extraction yield
The extraction yield percentages of A. campestris dried leaves are
presented in Table 1. The yields varied according to the used solvent
and the extraction method. In fact, the reflux method gave the
R. Metoui, H. Mighri, J. Bouajila et al.
Table 1
Extraction yield, total polyphenol and total flavonoid contents in A. campestris dried
Extraction method Solvent Extract code
M Dichloromethane MDM
F FDM
R RDM
M Ethyl acetate MEtAc
F FEtAc
R REtAc
M Butanol MBut
F FBut
R RBut
M Water MW
F FW
R RW
M: Maceration; F: Fractionation; R: Reflux.
Within each column, values with different upper letters means significantly different at level p < 0.05.
highest yields (4.2 § 0.1 to 15.0 § 0.0%) compared to other methods
independently to solvent types. Among the used solvents, water dis-
played the highest yield (15.0 § 0.0%) with the reflux method
whereas EtAc provided the lowest yield (0.3 § 0.0%) with fraction-
ation method. Except water, the extract amounts obtained by frac-
tionation were found to be lower than those of other methods
whatever the used solvent. The results showed that all solvents
exhibited almost, the same yield of crude extract (3.7 4.9%) when
the maceration method was used. The significant differences of
extraction yield percentage among different solvents may be attrib-
uted to the difference of the polarity of the used solvents as well as
the availability of different extractable phytoconstituents present in
A. campestris dried leaves.
For the effectiveness of extracting technique, the results showed
that yields of the reflux extraction were better than other extraction
methods due probably to the temperature effect. In fact, temperature
affects many physical properties including viscosity, diffusivity, solu-
bility and surface tension whose contribute to obtain high amount of
crude extract (Jayanthi and Latitha, 2013), although at the same time,
the increases in temperature might cause the degradation and the
decomposition of phytochemicals compounds. On the other hand, it
should be remember that the relatively low yields obtained by the
fractionation method compared to other methods could be partially
explained by the fact that the extraction of these phytochemicals
from the 70% methanolic extract by liquid-liquid partition was not be
completely achieved.
3.2. Total polyphenol content
The total polyphenol contents (TPC) in the A. campestris dried
leaves extracts were analyzed using the Folin-Ciocalteu method. The
amounts of TPC, determined from the regression equation of the cali-
bration curve and expressed in mg gallic acid equivalent per g of
dried extract (mg GAE/g DE), are presented in Table 1. These amounts
varied from 79.3 § 0.6 mg GAE/g DE in the RW extract to
345.8 § 3.0 mg GAE/g DE in MBut extract. MBut and MEtAC extracts
displayed the highest amounts of total polyphenols (345.8 § 3.0 and
322.1 § 2.9 mg GAE/g DE, respectively). RDM, MDC, RBut, FDM and
FEtAC extracts also showed high amounts on TPC ranged from
238.3 § 3.1 to 285.2 § 5.9 mg GAE/g DE. Among water extracts, with
reflux and fractionation methods, the RW and MW extracts displayed
almost the same amount on TPC (178§5.9 and 181.5 § 4.7 mg GAE/g
DE, respectively). Whatever the method of extraction used, DM, EtAc
and But seems to be the most appropriate solvents for the extraction
of high amounts on total polyphenols from dried leaves of A. campest-
ris. The TPC are significantly different between the extraction meth-
ods with the exception of MDM and FDM extracts (238.3 § 3.1 and
243.3 § 5.2 GAE/g DE) and MW and FW (178.0 § 5.9 and
South African Journal of Botany 151 (2022) 288 294
Leaves extracts according to used solvent and method of extraction.
Extraction yield (%) Total polyphenol (mg GAE/g DE) Total flavonoid (mg GAE/g DE)
4.9 § 0.0e 243.3 § 5.2d 144.5 § 0.5f
0.9 § 0.0h 238.3 § 3.1d 193.2 § 2.8b
6.2 § 0.0c 277.9 § 4.8c 99.5 § 1.7 g
3.7 § 0.2f 322.1 § 2.9b 296.3 § 0.3a
0.3 § 0.0i 145.8 § 4.6 g 183.7 § 1.7c
9.2 § 0.0b 285.2 § 5.9c 114.7 § 5.5 g
3.9 § 0.0f 345.8 § 3.0a 177.4 § 3.4d
1.5 § 0.0 g 219.7 § 3.7e 154.1 § 3.2e
4.2 § 0.1e 272.4 § 4.5c 194.5 § 1.7b
4.1 § 0.7ef 181.5 § 4.7f 62.1 § 2.1i
5.4 § 0.0d 178.0 § 5.9f 59.2 § 0.5i
15.0 § 0.0a 79.3 § 0.6h 86.5 § 0.0h
181.5 § 4.7 GAE/g DE) obtained by reflux and maceration methods
which displayed almost the same amounts on total polyphenols.
However, it should be noted that with reflux method, the TPC
amounts in DM, EtAc and But extracts are not significantly different
(values ranged between 272.4 § 4.5 and 285.2 § 5.9 mg GAE/g DE)
showing no effect of the increasing solvent polarity. Those can be
attributed to the thermal effect in decomposition of some polyphe-
nols at high temperature which accelerate their oxidation and other
degenerative reactions (Sultana et al., 2009). Only with maceration
method, the amounts on TPC increases significantly with solvent
polarity except water explained by its high polarity which favor the
extraction of more other polar biomolecules such as polysaccharides
and proteins which can interfere with polyphenols during analysis.
Djidel and khennouf (2014) evaluated the polyphenols contents of
the aqueous extract of the aerial parts of A. campestris (65.5 § 0.0 mg
GAE/g of dry plant). This value is comparable to that found in our
study with the fractionation method. Also, our results are in agree-
ment with those found by Saidan et al. (2015) who showed that the
TPC found in Orthosiphon stamineus leaves extracts obtained by the
maceration method were higher than those of the reflux method.
3.3. Total flavonoid content
The total flavonoid contents (TFC) in the A. campestris dried leaves
extracts were analyzed. The amounts of TFC, determined from regres-
sion equation of calibration curve and expressed in mg quercetin
equivalent per g dried extract (mg QE/g DE), are presented in Table 1.
These amounts varied according to the solvent and the method of
extraction from 59.2 § 0.5 mg QE/g DE in FW extract to
296.3 § 0.3 mg GAE/g DE in REtAC extract. Whatever the solvent
used, except EtAc, the obtained extracts by reflux presented higher
amounts of TFC than other extraction methods, whereas those pro-
vided by fractionation method displayed the lowest TFC. The highest
amount of TFC was displayed by the MEtAc extract. FDM and RBut
extracts also showed high amounts of TFC (193.2 § 2.8 and
194.5 § 1.7 mg QE/g DE, respectively). Whatever the extraction
method used, water extracts displayed the lowest amounts of TFC
(59.2 § 0.5 - 86.5 § 0.0 mg QE/g DE), whereas extracts obtained by
the reflux method with DCM and EtAc, presented the lowest amounts
on TFC (99.5 § 1.7 and 114.7 § 5.5 mg QE/g DE, respectively) but, in
return those obtained by water and But were the highest (86.5 § 0.0
and 194.5 § 1.7 mg QE/g DE, respectively). It seems that the heating
and polar solvent promote the extraction of TFC from the dried leaves
of A. campestris. The difference of TFC between extracts could be
explained by the nature of flavonoids in dried leaves of A. campestris
and the ability of the solvent and the method used to extract them.
According to our results, and since the highest amounts on flavonoid
were extracted by DCM, EtAc and But which are solvents with a
290
R. Metoui, H. Mighri, J. Bouajila et al. South African Journal of Botany 151 (2022) 288 294

moderate polarity, it seems that the majority of flavonoids extracted
from A. campestris dried leaves could be aglycones or mono and di-
glycosyl flavonoids (Dib et al., 2016). The relatively high levels of TFC
in RBut and RW extracts may be attributed to the ability of these two
solvents to extract more polar flavonoids such as tri-gylcosyl flavo-
noids when they are used at their boiling temperature which are
equal or more than 100 °C. In a previous study on A. campestris,
Ksouri et al. (2014) determined the amount of total flavonoids in
water and EtAc extracts from the A. campestris aerial parts obtained
by maceration and reported the values of 67.4 and 63.8 mg QE/g
DE, respectively which were lower than those obtained in this
study. These differences may be due to environmental factors or
the part of plant used. Indeed, total flavonoids contents are
known to vary from the different parts of the same plant
(Romani et al., 2003).
3.4. Contents on individual phenolic compounds determined by LC-MS
analysis
The phenolic compounds identified in A. campestris leaves
extracted with maceration, fractionation and reflux methods by using
various solvents are presented in Table 2. Among thirty three tested
standards, twenty nine compounds were detected: fifteen phenolic
acids (quinic acid, gallic acid, protocatchuic acid, chlorogenic acid, 4-
O-caffeoylquinic acid, caffeic acid, syringic acid, p-coumaric acid,
trans-frulic acid, o-coumaric acid, 3,4-di-Ocaffeolyquinic acid, ros-
marinic acid, 4,5-di-O-caffeolyquinic acid, trans-cinnamic acid and
1,3-di-O-cafeylquinic acid), five flavones (cirsiliol, luteolin, apigenin,
cirsilineol and acacetin), two flavonols (kaempferol and quercetin),
one flavanol ((-)-epicatechin), two flavonol glycosides (quercitrin
and rutin), two flavone glycosides (luteolin-7-O-glucoside and apige-
trin), one flavanone (naringenin) and one flavanone glycoside
(naringin). Only nine compounds were shared between all extracts at
chic acid, cirsiliol,
varying amounts: quinic acid, caffeic acid, protocate
cirsilineol, kaempferol, naringenine and apigenin. In fact those com-
pounds reached particularly higher amounts in AcEt extract obtained
with maceration method, except quinic acid. In return, two chemical
compounds were detected only in AcEtM extract such us naringine
(9.78 mg/g DE) and luteolin, strangely in appreciable amount
of 9874.9 mg/g DE. This last compound has been proven to be
health benefits due especially to its antioxidant properties and
apoptotic cell death induction (Manzoor et al., 2017;
Swaminathan et al., 2019).
Remarkable higher amounts are also given in aqueous extract
obtained with reflux method for caffeic acid (2435.21 mg/g DE), 3,4-
di-O-cafeylquinic acid (20,958.00 mg/g DE) and 4,5-di-O-cafeylquinic
acid (25,795.10 mg/g DE). The DCM extracts obtained with macera-
tion and reflux methods showed lower number (12 13 compounds)
of phenolic compounds present generally, in lower amounts when
compared to other extracts.
With maceration method, whatever the solvent used, the
amounts of quinic acid have always been the highest (346.6 to
1432.50 mg/g DE). this compound was considerate as a precursor of
shikimic acid and other secondary compounds synthesis (4-O-caf-
feoylquinic acid, 1,3-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic
acid and 4,5-di-O-caffeoylquinic acid) and a versatile chiral starting
material for the synthesis of new pharmaceuticals (Marsh et al.,
2009).
Rutin and 3,4-di-O-caffeylquinic acid have been previously identi-
fied in aqueous and methanoic extracts of the aerial part of A. cam-
pestris by fractionation method. In addition, cirsilineol and cirsiliol
have been identified in the ethyl acetate and methanolic extracts
obtained from the fractionation method of the aerial part of A. cam-
pestris (ksouri et al., 2014).

Table 2
Amounts of phenolic compounds (mg/g DE) determined by LC/MS in different extracts of A. campestris dried leaves.

Extraction method and used solvent

Compounds Dichloromethane Ethyl acetate Butanol Water

M F R M F R M F R M F R

Quinic acid 346.6 333.57 4.19 1336.30 219.55 359.28 1432.50 56.76 489.29 1270.91 553.41 1212.64
Gallic acid 3.718 2.09 3.95 5.19
Protocatechic acid 2.12 36.43 3.32 263.12 107.72 174.00 42.85 126.89 93.51 145.04 32.30 85.61
Chlorogenic acid 273.95 475.13 189.64 201.54 1202.94 167.97 1887.32 698.52 1100.76
4-O-cafeylquinic acid 154.49 221.40 112.78 104.59 1539.67 88.66 3979.97 454.63 2435.21
Caffeic acid 4.76 10.93 1.78 128.87 93.97 60.92 50.00 70.10 13.50 70.30 17.15 47.18
Syringic acid 0.12 4.94 3.22 5.94 3.36 2.29 0.31 9.96 2.01
1,3-di-O-cafeylquinic acid 6.46 4.68
(-)-Epicatechine 12.16 1.37
p-coumaric acid 0.003 40.99 19.87 6.50 1.65 1.68 1.78 1.80 3.24 1.34
trans-ferulic acid 0.57 94.53 69.73 15.57 13.65 7.25 1.71 63.54 11.56
Rutin 203.43 87.00 337.30 1444.51 1394.67 2957.23 183.57 675.03 421.22 3564.43
o-coumaric acid 0.23 78.11 16.84 0.72 2.11 3.54 0.86 6.072 0.79
Luteoline-7-O-glucoside 17.45 0.29 4.39 6.82 5.42 2.63 6.23 12.89 9.26
3,4-di-O-cafeylquinic acid 937.31 10,922.14 3072.56 4659.36 8712.49 617.72 13,341.87 763.45 20,958.00
Quercitrine 3.81 15.75 48.68 26.81 19.79 18.29 15.69
Rosmarinic acid 29.66 35.34 4.41 23.18 18.38
Naringin 9.77
Apigetrin 3.95
4,5-di-O-cafeylquinic acid 3.02 1665.06 86.45 9354.38 3816.18 10,468.45 3182.12 133.05 10,881.03 926.52 25,795.1
trans-cinnamic acid 3.56 14.71 8.83 5.51 0.23
Quercetin 3.54 64.45 161.28 27.91 44.31 16.49 7.87 3.25 12.01
Kaempferol 6.02 10.37 6.28 3176.40 891.44 329.62 13.04 23.08 473.42 17.41 25.77 5.91
Naringenin 51.39 60.05 127.58 1041.46 157.07 75.02 3.37 8.90 185.90 0.65 24.75 1.19
Apigenin 3.89 7.71 5.52 1345.01 60.68 40.40 3.31 3.65 83.43 4.00 590.80 2.07
Luteolin 9874.90
Cirsiliol 6137.02 8730.42 8372.23 19,898.18 6708.40 5314.08 738.21 457.80 6984.24 833.37 8142.33 162.37
Cirsilineol 830.47 833.72 987.99 2409.70 38.34 212.47 44.87 6.59 360.96 40.86 952.17 17.20
Acacetin 2.08 2.78 4.94 13.71 0.71 3.368 2.66
M: Maceration; F: Fractionation; R: Reflux.

291
R. Metoui, H. Mighri, J. Bouajila et al. South African Journal of Botany 151 (2022) 288 294

3.5. Biological activities
Several compounds have been isolated and identified from differ-
ent extracts of this species, such as flavonoids (10). The shoots of A.
campestris were the subject of a previous phytochemical investiga-
tion leading to the identification by LC/MS of 39 molecules including
coumarins, flavones, flavonols, phenolic acids and sesquiterpenes.
The major phenolic compounds are principally luteolin-7-O-rutino-
side, rhamnetin, isorhamnetin, hydroxycoumarin, kaempferolrutino-
side and three di-O-caffeoylquinic acid isomers (11).
extracts (except REtAc and RW), exhibited potent antioxidant activity
with an IC50 ranged from 8.9 to 15.8 mg/L two or three times less
than positive control. The variation on antiradical activity of DCM
extracts was not significantly different between the extraction meth-
ods, whereas slightly significant different was shown between
extracts obtained by fractionation method except butanol.
Our results were in agreement with those reported by Konate 
et al. (2010) who reported that the EtAc extract obtained by fraction-
ation method of the Sida acuta Burn.f. plant showed the highest anti-
oxidant activity than other extracts.

3.5.1. Antioxidant activity 3.5.2. XOD inhibitory activity

Dried leaves extracts of A. campestris were assessed for antioxi-
dant activity using two different spectrophotometric tests DPPH and
ABTS assays and were compared to the positive control, ascorbic
acid. The results are presented in Table 3. The FEtAc extract exhibited
the highest antiradical activity, when DPPH test was used, with an
IC50 value of 10.9 § 2.0 mg/L, which represents 2.5 times less than
the activity of ascorbic acid (4.7 § 0.0 mg/L). MDCM, FDCM, MBut,
RBut and FBut extracts also showed potent antiradical activity with
an IC50 ranged from 14.4 § 1.1 to 15.8 § 0.0 mg/L. Some extracts
showed moderate activity such as MW and MEtAc with an IC50 of
18.6 § 0.1 and 19.0 § 1.1 mg/L, respectively, whereas the extracts
obtained by reflux method such as RDCM, REtAc and RW extracts dis-
played weak activity with an IC50 ranged from 25.1 § 1.1 mg/L to
31.6 § 1.1 mg/L. Only the FW extract showed a very weak antioxidant
activity with an IC50>50 mg/L. It seems that both maceration and
fractionation are the best methods to be used for the extraction of
antioxidant compounds using EtAc, DCM and But as solvents. Our
results are in concordance with those found by Djidel and khen-
nouf (2014) who announced that the evaluation of DPPH scavenging
activity of the ethyl acetate extract of A. campestris aerial parts
obtained by maceration method is the most active extract with IC50
of 5.8 mg/L. In fact, especially in the EtAc extract, activity may be
related to the compositional richness in various phenolic compounds
and mainly the presence of the luteolin. As found with DPPH assay,
some of A. campestris extracts showed a potent activity using ABTS
test. Indeed, the MEtAc extract showed the strongest activity
(IC50=4.4 § 0.1 mg/L) which was similar to that found by the positive
control ascorbic acid (IC50 = 4.3 § 0.1 mg/L). The rest of tested
The XOD inhibition by dried leaves extracts of A. campestris is
reported for the first time in this study. All extracts exhibited a weak
XOD inhibitory activity (IC50>50 mg/L) (Table 2) except MEtAc, MBut
and MDCM extracts obtained by the maceration method which
showed a relatively potent activity with an IC50 of 5.0 § 2.7; 8.9 § 0.1
and 14.5 § 0.7 mg/L, respectively. These values are relatively lower
than that of the reference allopurinol (1.1 § 0.6 mg/L). Our results are
in agreement with those of Konate  et al. (2010) who showed that the
ethyl acetate extract obtained by fractionation extraction of the plant
Cienfuegosia digitata Cav gave the highest anti-XOD activity than
other extracts. We noticed also that some of our extracts exhibited
higher anti-XOD activity than that of other plant extracts such as
Amyema scandens extracts with an IC50=13.0 § 0.4 mg /L
(Bosisio et al., 2000).
3.5.3. Anti-5-lipoxygenase activity
According to our knowledge, no studies on the anti-5-lipoxyge-
nase activity of A. campestris dried leaves extracts have been earlier
reported. The results expressed as inhibition percentage at 10 mg/L
are ranged from 2.3 § 0.1 for REtAc extract to 24.6 § 2.0% for FEtAc
extract (Table 2). Compared to the NDGA standard (54.0%), the MEtAc
extract and all those obtained by the fractionation method showed
higher activity ranged from 19.1 § 0.8 to 24.6 § 2.1%. The other
extracts could be considered as lowly inflammatory activity with an
inhibition percentage varying from 2.3 to 9.8% given particularly by
extracts of reflux method. We note that our results are in agreement
with the study of Konate  et al. (2010) who showed that the EtAc

Table 3
Antioxidant, anti-XOD, anti-SOD, anti-5-LOX, anti-AchE and cytotoxic activities of controls and A. campestris dried leaves extracts according to used solvent and method of
extraction.

Extraction Solvent/Control Antioxidant activity Cytotoxic activity

method
DPPH ABTS Anti-XOD activity Anti-5-LOX activity Anti-AChE activity OVCAR MCF-7
IC50 (mg/L); IP (%)1 IC50 (mg/L); IP (%)1 IC50 (mg/L); IP (%)1 IP (%)2 IP (%)1 IP (%)1 IP (%)1

M Dichloromethane 15.8 § 0.0c 9.5 § 0.1b 14.5 § 0.7d 9.3 § 0.6d 22.2 § 0.5e 75.3 § 7.1c 76.7 § 2.7d
F 14.4 § 1.1bc 9.3 § 0 .5b >50.0; 18.6 § 1.5 22.1 § 1.0b 43.0 § 2.2c 76.0 § 1.8c 71.6 § 1.7e
R 25.1 § 1.1e 8.9 § 1.2b >50.0; 26.6 § 0.2 3.20 § 0.0f 12.8 § 1.2 g 75.7 § 2.0c 90.5 § 1.6b
M Ethyl acetate 19.0 § 1.1d 4.4 § 0.1a 5.0 § 2.7b 19.1 § 0.8c 33.7 § 2.8d 84.4 § 1.5b 93.0 § 0.5a
F 10.9 § 2.0b 9.7 § 1.2b >50.0; 36.8 § 4.2 24.6 § 2.1b 55.9 § 3.4b 74.7 § 1.2c 84.7 § 1.1c
R 31.6 § 0.1f >50.0; 50§0.0 >50.0; 23.7 § 0.1 2.3 § 0.5h 14.3 § 0.5 g 29.3 § 3.4e 44.6 § 2.4f
M Butanol 15.1 § 0.0c 15.8 § 1.8d 8.9 § 0.1c 9.4 § 0.8d 32.4 § 1.0d 24.9 § 0.4f 24.2 § 1.8 g
F 15.4 § 1.1c 10.9 § 0.0c >50.0; 34.2 § 3.3 23.0 § 2.0b 50.9 § 2.8b 27.4 § 1.6e 13.5 § 5.5h
R 15.4 § 1.1c 11.0 § 0.1c >50.0; 35.1 § 1.2 9.8 § 0.1d 14.0 § 0.7 g 76.7 § 5.6c 87.4 § 1.1c
M Water 18.6 § 0.1d 13.1 § 0.0d >50.0; 52.7 § 1.1 8.4 § 0.5d 20.1 § 1.9f 37.8 § 4.8d 44.1 § 2.1f
F >50.0; 5.0 § 0.95 9.1 § 0.0b >50.0; 5.45 § 0.9 19.6 § 0.0c 48.2 § 2.7b 6.6 § 2.0 g 5.1 § 0.3i
R 25.1 § 0.8e >50.0; 19.1 § 5.5 >50.0; 6.7 § 0.3 5.5 § 0.4e 0.0h 0.0h 25.4 § 4.6 g
Ascorbic acid 4.7 § 0.0a 4.3 § 0.1a
Allopurinol 1.12 § 0.6a
NDGA 54.0 § 4.2a
Galantamine 85.5 § 0.0a
Tamoxifen 98.8 § 4.9a 98.5 § 5.6a
M: Maceration; F: Fractionation; R: Reflux; IP: Inhibition percentage.
Within each column, values with different upper letters means significantly different at level p < 0.05.
1
IP at 50 mg/L.
2
IP at 10 mg/L.

292
R. Metoui, H. Mighri, J. Bouajila et al.
Table 4
Pearson coefficients between quality indices (Yield, TPC, TFC, DPPH, ABTS, AntiXOD, Anti-5-LOX, Anti-AchE OVCAR and MCF-7).
Yield TPC TFC
TPC 0.32
TFC 0.47 0.53
DPPH 0.56 0.13 0.56
ABTS 0.70 0.47 0.44
Anti-XOD 0.04 0.56 0.55
Anti-5-LOX 0.71 0.16 0.44
Anti-AChE 0.79 0.01 0.30
OVCAR 0.55 0.41 0.64
MCF-7 0.29 0.31 0.56*
extract of the plant Digitata cav resulting from the fractionation
method is the most active one on the anti-5-lipoxygenase activity.
3.5.4. Anti-cholinesterase activity
To the best of our knowledge, this is the first report on the anti-
cholinesterase activity of A. campestris dried leaves extracts using
acetylcholinesterase enzyme. At 50 mg/L, only the extracts obtained
by the fractionation method showed a highest anti-cholinesterase
activity with an inhibition percentage ranged from 43.0 § 2.2 to
55.9 § 3.7% when compared to the galantamine standard (85.5%).
The other extracts exhibited a weak activity with an IP <33.7%. What-
ever the solvent used, the extracts obtained by the reflux method
showed the lowest anti-cholinesterase activity (0< IP <14.3%). Our
results are in harmony from those found by Kchaou et al. (2016), who
showed that the But, EtAc, DCM and hexane extracts obtained by
fractionation method of Zygophyllum album plant exhibited the high-
est anti-cholinesterase activity.
3.5.6. Cytotoxicity/anti-cancer assay
Cytotoxicity was evaluated on two cell-lines: MCF-7 breast cancer
and OCVAR ovarian cancer at a concentration of 50 mg/L of A. cam-
pestris dried extract using MTT test. To the best of our knowledge,
this is the first report on the cytotoxicity activity of A. campestris
dried leaves extracts against these two cell lines. By comparing the
IP% of the various products tested at the used concentration towards
both cellular lines, it was shown, that sensitivity of OVCAR and MCF-
7 cell lines varied in the same order with the various tested extracts
(Table 2). The MEtAc extract exhibited the highest cytotoxic activity
against OVCAR and MCF-7 cell lines with inhibition percentages of
84.4 § 1.5 and 93.0 § 0.5%, respectively. The last value was almost
close to the Tamoxifen standard with IP of 98.8 § 5.6%. All DCM
extracts (MDCM, FDCM and RDCM), as well as FEtAc and Rbut
extracts displayed also a potent cytotoxic activity with IP ranged
from 71.6 § 1.7 to 90.5 § 1.6%. The rest of A. campestris extracts
(REtAc, Fbut, MW, FW and RW) showed a PI< 50%.
We notice that for cold extraction methods (maceration and frac-
tionation), the most active extracts against both MCF7 and OVCAR
cells were EtAc extracts (74.7 93.0%) whereas But extracts displayed
lower activity (13.5 24.9%). In contrast, for reflux method, DCM and
But extracts showed the highest activity ranged from 75.3 to 90.3%
while the EtAc extract exhibited lower activity (29.3 and 44.6%,
respectively).
3.6. Pearson correlations
According to Pearson correlation test applied to all extracts
(Table 4), no correlations (R2< 0.50) were established between yield
extracts of A. campestris dried leaves and antioxidant, Anti-XOD,
Anti-5-LOX, Anti-AChE and MCF-7 activities and their analyzed phy-
tochemicals (TPC and TFC). Those can be explained by the presence
in these tested extracts of other substances. Although numerous
South African Journal of Botany 151 (2022) 288 294
DPPH ABTS Anti-XOD Anti-5-LOX Anti-AChE OVCAR
0.40
0.58 0.49
0.33 0.55 0.03
0.21 0.67 0.09 0.92
0.56 0.59 0.38 0.19 0.11
0.43 0.36 0.29 0.02 0.14 0.95
studies have effectively, confirmed the biological activities of A. cam-
pestris such as antioxidant, antimicrobial, antitumor, antidiabetic,
anthelmintic, hepatoprotective, nephroprotective, insecticidal, anti-
venomous, antiulcer and allelopathic (Akrout et al., 2004; Al-
Snafi, 2015; Megdiche-Ksouri et al., 2015; Akkari et al., 2016;
Sefi et al., 2010), the phytochemical investigations of A. campestris
shoots are leading to the identification of other various molecules
including coumarins, sesquiterpenes (Ksouri et al., 2014), alkaloids,
saponins (Pereira et al., 2018; Metoui et al., 2017) and fatty acids
(Carvalho et al., 2011). These different activities can be due to the
synergistic action of these different molecules. In return, the strong
biological activities of some extracts which prompt us to try to isolate
and identify the molecules responsible for these activities, TPC, TFC
and DPPH shown a positive correlation (0.55<R2<0.58) with Anti-
XOD activity. Those are in agreement with studies which concluded
that flavonoids are reported to be antioxidants inhibitors of xanthine
oxidase (Gonzalez et al., 1995; Nile and Khobragade (2011)). In the
same way, the OVCAR inhibitory activity revealed a positive correla-
tion with TFC and the potential antioxidant activity disclosed by
DPPH and ABTS tests (0.56<R2<0.64) while the MCF-7 was correlated
only to the TFC. We can suggest that this anti-cancer activity may be
related to the high amount of TFC content.
4. Conclusion
The chemical composition (Total polyphenols and total flavo-
noids) and the biological activities (antioxidant, anti-inflammatory,
anti-cholinesterase, anti-XOD and cytotoxic activities) of the A. cam-
pestris dried leaves extracts were shown to be strongly affected by
the method of extraction and the solvent used. The fractionation and
the maceration methods were shown to be the best method for the
extraction of the high amounts of these active substances that exhib-
ited strong antioxidant, anti-XOD and cytotoxic activities when
dichloromethane, ethyl acetate and butanol were used successively
as solvents for the extraction.
These findings lead us to conclude that the A. campestris, dried
leaves could be considered as a natural source forthe extraction of
natural antioxidant, natural anti-XOD and natural cytotoxic com-
pounds. Therefore, further work should be performed to isolate and
identify the biomolecules responsible for these activities from
dichloromethane, ethyl acetate and butanol extracts.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not -for-profit sectors.
293
R. Metoui, H. Mighri, J. Bouajila et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors would like to think the Institution of Research and
higher Agricultural Education (Ministry of Agriculture), Tunisia for
the financial support.
References
Akrout, A., El-Jani, H., Amouri, S., Neffati, M., 2010. Screening of antiradical and antibac-
terial activities of essential oils of Artemisia campestris L., Artemisia helba Alba Asso,
and Thumus capitatus Hoff. Et Link. growing wild in the southern of Tunisia. Recent
Res. Sci Technol. 1, 29–39.
Akrout, A., Gonzalez, L.A., El-Jani, H., Madrid, P.C., 2011. Antioxidant and antitumor
activities of Artemisia campestris and Thymelaea hirsuta from southern of Tunisia.
Food Chem. Toxicol. 49, 342–347.
Al-Snafi, A.L., 2015. The pharmacological importance of Artemisia campestrris: a review.
Asian J. Pharm. Res. 5, 88–92.
Akkari, H., B’chir, F., Hajaji, S., Rekik, M., Sebai, E., Hamza, H., Darghouth, M.A., Gharbi, M.,
2016. Potential anthelmintic effect of Capparis spinosa (Capparidaceae) as related to
its polyphenolic content and antioxidant activity. Vet. Med. 6, 308–316.
Bekir, J., Mars, M., Souchard, J.P., Bouajila, J., 2013. Assessment of anti-oxidant, anti-
inflammatory, anti-cholinesterase and cytotoxic activities of pomegranate (Punica
granatum) leaves. Food Chem. Toxicol. 55, 470–475.
Belhattab, R., Boudjouref, M., Barroso, J.G., Pedro, L.P., Figueiredo, A.C., 2011. Essential oil
composition from Artemisia campestris grown in Algeria. J. Environ. Biol. 5, 429–432.
Ben Nasr, H., Hammami, T.S., Mahmoudi, L., Zeghal, K., 2014. Aqueous leaves extract of
Artemisia campestris inhibition of the scorpion venom induced hypertension. J.
Med. Plants Res. 8, 538–542.
Bennour, N., Mighri, H., Eljani, H., Zammouri, T., Akrout, A., 2020. Effect of solvent evap-
oration method on phenolic compounds and the antioxidant activity of Moringa
oleifera cultivated in Southern Tunisia. S. Afr. J. Bot. 129, 181–190.
Boulanouar, B., Abdelaziza, G., Aazza, S., Gagob, C., Miguel, G.M., 2013. Antioxidant
activities of eight Algerian plant extracts and two essential oils. Indust. Crops Prod.
46, 385–389.
Bosisio, E., Mascetti, D., Cabalion, P., 2000. Screening of plants from New Caledonia and
Vanuatu for inhibitory activity of xanthine oxidase and elastase. Pharm. Biol. 38,
18–24.
Carvalho, I.S., Teixeira, M.C., Brodelius, M., 2011. Fatty acids profile of selected Artemi-
sia spp. plants: health promotion. LWT-Food Sci. Technol. 44, 293–298.
Chandrasekara, A., Shahidi, F., 2011. Determination of antioxidant activity in free and
hydrolyzed fractions of millet grains and characterization of their phenolic profiles
by HPLC- DAD-ESI-MSn. J. Funct. Foods 3, 144–158.
Djidel, S., khennouf, S., 2014. Radical scavenging, reducing power, lipid peroxidation
inhibition and chelating properties of extracts from Artemisia campestris L. Aerial
parts. Annu. Res. Rev. Biol. 10, 1691–1702.
Dib, I., Angenot, L., Mihamou, A., Ziyyat, A., Tits, M., 2016. Artemisia campestris L.: eth-
nomedicinal, phytochemical and pharmacological review. J. Herbal Med. 7, 1–10.
Gonzalez, A.G., Bazzocchi, I.L., Moujir, L., Ravelo, A.G., Correa, M.D., Gupta, M.P., 1995.
Xanthine oxidase inhibitory activity of some Panamian plants from Celastraceae
and Lamiaceae. J. Ethnopharmacol. 46, 25–29.
Jayanthi, P., Latitha, P., 2013. Comparison of conventional and sound assisted methods
for extraction of eichhornia crassipes (Mart. Solms). Asian J. Pharm. Clin. Res. 6,
143–146.
El Euch, S.K., Bouajila, J., Bouzouita, N., 2015. Chemical composition, biological and
cytotoxic activities of Cistus salviifolius flower buds and leaves extracts. Indust.
Crops Prod. 76, 1100–1105.
294
South African Journal of Botany 151 (2022) 288 294
Kao, T.H., Wu, W.M., Hong, C.H., Wu, W.B., Chen, B.H., 2007. Anti-inflammatory effect of
isoflavone powder produced from soy bean cake. J. Agric. Food Chem. 55, 11068–
11079.
Ksouri, W.M., Trabelsi, N., Mkadmini, K., Bourgou, S., Noumi, A., Snoussi, M., Barbria, R.,
Tebourbi, O., Ksouri, R., 2014. Artemisia campestris phenolic compounds have anti-
oxidant and antimicrobial activity. Indust. Crops Prod. 63, 104–113.
Konate , K., Souza, A., Coulibaly, A.Y., Meda, N.T., Kiendrebeogo, M., Lamien-Meda, A.,
Millogo-Rasolodimby, J., Lamidi, M., Nacoulma, O.G., 2010. In vitro antioxidant, lip-
oxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosi
adigitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae). Asian J. Biol. Sci. 13
(22), 1092–1098.
Kchaou, M., Salah, H., Mhiri, R., Allouche, N., 2016. Anti-oxidant and anti- acetylcholin-
esterase activities of Zygophyllum album. Bangladesh J. Pharmacol. 11, 54–62.
Mahomoodally, M.F., Mollica, A., Stefanucci, A., Aumeeruddy, M.Z., Poorneeka, R.,
Zengin, G., 2018. Volatile components, pharmacological profile, and computational
studies of essential oil from Aegle marmelos (Bael) leaves: a functional approach.
Ind. Crops. Prod. 126, 13–21.
Manzoor, M.F., Ahmad, N., Manzoor, A., Kalsoom, A., 2017. Food based phytochemical
luteolin their derivatives, sources and medicinal benefits. Inter. J. Agric. Life Sci. 3
(11), 1.
Marsh, K.B., Boldingh, H.L., Shilton, R.S., Laing, W.A., 2009. Changes in quinic acid
metabolism during fruit development in three kiwifruit species. Funct. Plant Biol.
36, 463–470.
Megdiche-Ksouri, W., Trabelsi, N., Mkadmini, K., Bourgou, S., Noumi, A., Snoussi, M.,
2015. Artemisia campestris phenolic compounds have antioxidant and antimicro-
bial activity. Ind. Crops Prod. 63, 104–113.
Metoui, R., Bouajila, J., Znati, M., Cazaux, S., Neffati, M., Akrout, A., 2017. Bioactive fla-
vones isolated from Tunisian Artemisia campestris L. Leaves. Cell. Mol. Biol. 63, 86–
91.
Mighri, H., Akrout, A., Bennour, N., Eljeni, H., Zammouri, T., Neffati, M., 2019. LC/MS
method development for the determination of the phenolic compounds of Tuni-
sian Ephedra alata hydro-methanolic extract and its fractions and evaluation of
their antioxidant activities. S. Afr. J. Bot. 124, 102–110.
Nile, S.H., Khobragade, C.N., 2011. Phytochemical analysis, antioxidant and xanthine
oxidase inhibitory activity of Tephrosia purpurea Linn. root extract. Indian J. Nat.
Prod. Res. 2, 52–58.
Pereira, C.G., Barreira, L., Bijttebier, S., Pieters, L., Marques, C., Santos, T.F., et al., 2018.
Health promoting potential of herbal teas and tinctures from Artemisia campestris
subsp. maritime: from Traditional Remedies to Prospective Products. Sci. Rep. 8,
468. https://doi.org/10.1038/s41598-018-23038-6.
Romani, A., Vignolini, P., Galardi, C., Aroldi, C., Vazzana, C., Heumler, D., 2003. Polyphe-
nolic content in different plant parts of soy cultivars grown under natural condi-
tions. J. Agric. Food Chem. 51, 5301–5306.
Saidan, N.H., Hamil, M.S.R., Memon, A.H., Abdelbari, M.M., Hamdan, M.R., Mohd, K.S.,
Abdul Majid, A.M.S., Ismail, Z., 2015. Selected metabolites profiling of Orthosiphon
stamineus Benth leaves extracts combined with chemometrics analysis and corre-
lation with biological activities. BMC Complement. Altern. Med. 15, 350. https://
doi.org/10.1186/s12906-015-0884-0.
Sefi, M., Fetoui, H., Makni, M., Zeghal, N., 2010. Mitigating effects of antioxidant proper-
ties of Artemisia campestris leaf extract on hyperlipidemia, advanced glycation end
products and oxidative stress in alloxan-induced diabetic rats. Food Chem. Toxicol.
48, 1986–1993.
Shahrajabian, M.H., Sun, W., Cheng, Q., 2021. Plant of the millennium, caper
(Capparis spinosa L.), chemical composition and medicinal uses. Bull. Natl. Res.
Cent. 45 (1) NA, available https://link.gale.com/apps/doc/A669406454/AONE?
u=anon~b22ef8b6&sid=googleScholar&xid=337c69b3.
Sultana, B., Anwar, F., Ashraf, M., 2009. Effect of extraction solvent/technique on the
antioxidant activity of selected medicinal plant extracts. Molecules 14, 2167–2180.
Swaminathan, A., Basu, M., Bekri, A., Drapeau, P., Kundu, T.K., 2019. The dietary flavo-
noid, luteolin, negatively affects neuronal differen- tiation. Front. Mol. Neurosci.
12, 1–7.
Tiwari, P., Kumar, B., Kaur, M., Kaur, G., Kaur, H., 2011. Phytochemical screening and
extraction: a review. Inter. Pharm. Sci. 1 (1), 98–106.
Tsai, Y.C., Lin, C.L., Chen, B.H., 2010. Preparative chromatography of flavonoids and sap-
onins in Gynostemma pentaphyllum and their antiproliferation effect on hepatoma
cell. Phytomedicine 18 (1), 2–10.