IN VITRO ANTIOXIDANT ACTIVITY OF DIFFERENT EXTRACTS OF ALGERIAN ALLIUM PLANT ...

12 Pages·2017·0.13 MB·English

Manacer et al. 80- 91 Revue des BioRessources Vol 7 N° 1 Juin 2017

IN VITRO ANTIOXIDANT ACTIVITY OF DIFFERENT EXTRACTS OF
ALGERIAN ALLIUM PLANT (Allium triquetrum L.)
MENACER A1,*, BOUKHATEM M N 2, BENHELAL A3 and SAÏDI F 1
Laboratoire Biotechnologies, Environnement et Santé, Département de Biologie et Physiologie Cellulaire,
Faculté des Sciences de la Nature et de la Vie, Université Saad Dahleb- Blida 1, Blida, Algeria.
2 Département de Biologie et Physiologie Cellulaire, Faculté des Sciences de la Nature et de la Vie, Université
Saad Dahleb- Blida 1, Blida, Algeria.
3
Abstract To enrich the knowledge of new bioactive molecules and their natural resources, the present study
aims to value an endemic plant growing in Algeria, named Allium triquetrum L., by evaluating the antioxidant
activity of aqueous and methanolic extracts from the leaves (AEL, MEL) and the bulbs (AEB, MEB), by using
DPPH scavenging and FRAP methods, with a search of main chemical groups and determination of total
phenolic compounds. Phytochemical study revealed mainly, the presence of flavonoids, saponins and glycosides.
Furthermore, AEL has presented the greatest levels of polyphenols (14.31 ± 0.36 mg GAE/g dw) and flavonoids
(22.12 ± 0.33 mg QE/g dw). The aqueous extracts have a high antioxidant capacity comparing to the methanolic
extracts (EC50: AEL = 0.97 mg/ml, AEB = 1.36 mg/ml). Our results indicate clearly that Allium triquetrum
extracts show important antioxidant activity and could be used as a new source of natural antioxidants molecules
Keywords Antioxidant activity; Allium triquetrum L.; extracts; polyphenols; flavonoids.
ALGÉRIENNE (Allium triquetrum L.) IN VITRO
Résumé: ssances sur les nouvelles molécules bioactives et leurs ressources naturelles,
cette présente étude a pour objectif de valoriser une plante endémique qui pousse en Algérie nommée Allium
triquetrum aqueux et méthanoliques des feuilles (AEL,
MEL) et des bulbes (AEB, MEB) en utilisant la méthode du piégeage du radical libre DPPH et la méthode de
FRAP, avec la recherche de principaux groupes chimiques et la détermination du taux de composés phénoliques.
AEL a présenté la plus grande teneur en polyphénols (14.31 ± 0.36 mg GAE/g dw) et flavonoïdes (22.12 ± 0.33
mg QE/g dw). Les extraits aqueux présentent une activité antioxydante élevée en comparaison avec les extraits
méthanoliques (EC50: AEL = 0.97 mg/ml, AEB = 1.36 mg/ml). Nos résultats montrent clairement que les
extraits d'Allium triquetrum présentent une activité antioxydante importante et pourrait être utilisé comme une
nouvelle source de d'agents anti-oxydants naturels.
Mots clés Activité antioxydante, Allium triquetrum L.; extraits; polyphénols; flavonoïdes

Introduction needs. These exogenous antioxidants are
An antioxidant can be defined as any commonly called dietary antioxidants [4].
substance that when present at low Dietary antioxidants compounds include
concentrations compared to that of an flavonoids, phenolic acids, carotenoids and
oxidizable substrate, significantly delays or tocopherols that can inhibit Fe3+/AA
inhibits the oxidation of that substrate [3]. induced oxidation, scavenge free radicals,
The body makes some of the antioxidants, and act as reductants [5]. Medicinal and
it uses them to neutralize free radicals. aromatic plants are still a most important
These antioxidants are called endogenous part of complementary and alternative
antioxidants. However, the body relies on medicine in the developing countries. The
external (exogenous) sources, primarily the use of medicinal herbs as antioxidant and
diet, to obtain the rest of the antioxidants it anti-inflammatory is common in our
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Auteur Correspondant, A. Menacer : [email protected]
Manacer et al. 80- 91 Revue des BioRessources Vol 7 N° 1 Juin 2017

country. For xample, garlic (Allium number of diseases: coronary heart disease,
sativum) and shallots (Allium ascalonicum) cancer, obesity, diabetes, disturbances of
have antioxidant and free radical- the gastrointestinal tract, hyper-
scavenging characteristics and identifiable cholesterolemia and inflammatory diseases
odors at low concentrations. They contain [6].
two main classes of antioxidant Many researchers have studied the
compounds: flavonoids (flavones and chemical composition, antioxidant activity
quercetins) and sulfur-containing and other biological properties of several
compounds (allyl-cysteine, diallyl sulfide species of Allium genus, especially
and allyl trisulfide) [5]. cultured as garlic and onion, but few
Allium is the most representative genus of studies have been conducted on
the Liliaceae family, which includes 700 spontaneous species as Allium triquetrum
species of widely distributed bulbous L. This is a very early blooming species
perennials and biennials. At present Allium which grows vigorously in cultivations. It
spp. plants are considered the most is characterized by green striped, white,
important vegetables consumed fresh or in pendulous flowers looking like small lilies.
different cooked dishes in the world. These It possesses several vernacular names (e.g.,
plants are used as common foods, and for triangle onion, triangular-stalked garlic,
the treatment of many diseases, due to their three-cornered leek), which refer to
content of phytonutrients. In fact, the different taxa. The plant is used as a main
edible parts of Allium spp. plants are used ingredient in salads and soups, because of
for the treatment and prevention of a its mild taste similar to onion [7].
In order to contribute to the search for new
bioactive molecules and new naturals
sources, this work aims to study a
spontaneous endemic plant named Allium
triquetrum L. (Algeria), by determining of
its phytochemical components, and
evaluating of in vitro antioxidant
proprieties of aqueous and methanolic
extracts of its two parts (leaves and bulbs)
with their reducing capacity. Polyphenolics
and flavonoids contents of different
extracts have also been estimated
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1- Material and methods evaporator (Laborota 4001- efficient
This work has been undertaken at the Heidolph 2, Schwabach, Germany) to
laboratory of physicochemical analyzes of obtain dry concretes and kept at 4 °C until
Saidal group (Medea, West of Algeria). further use [11].
Plant material
Methanolic extracts (MEL and MEB)
The harvest of A. triquetrum was carried
These extracts were obtained by
out in the region of Mitidja (Blida,
maceration of the plant powder (20 g) in
Algeria) during the flowering period
500 ml of methanol under magnetic
(March-May, 2013). Its identification was
stirring for 24 h (initially, the plant powder
performed at the laboratory of Plant
was defatted with petroleum ether to
Biology, Department of Biology and
remove the lipids). Methanol enriched by
cellular physiology at the University of
polar substances was evaporated by a
Blida 1 and the Department of Botany of
vacuum rotary evaporator at 40° C to
the National College of Agronomy (ENSA
obtain dry concrete. These dry concretes
El-Harrach, Algiers) (Fig. 1). The parts of
were conserved at 4° C, within 1 month,
the plant were separated, washed, cut into
until further use [11].
small pieces and then dried and protected

from light at room temperature (25-28 °C).
Evaluation of antioxidant activity of the
The plant material is finally reduced to
extracts
powder.
The antioxidant activity of the extracts was
Phytochemical tests evaluated by two methods: DPPH (2,2-
The chemical groups of the two parts of diphenyl-1-picrylhydrazyl) free radical
the plant (leaves and bulbs), were scavenging and ferric reducing antioxidant
identified by precipitation and staining power assay (FRAP) [12-13-15].
reactions according to the methods
described in the literature [8,9,10].
DPPH radical scavenging capacity
Preparation of extracts The DPPH assay has many advantages
In order to determine the antioxidant comparing to other methods, such as good
efficacy of different parts of the plant, the stability, credible sensitivity, simplicity
aqueous and methanolic extracts were and feasibility [12]. The protocol of Atoui
prepared from two parts (leaves and bulbs), and al. (2005) [13] with slightly
using the protocol of Juvekar and Halade modification was used to evaluate this
(2006) with slightly modification [11]. capacity. A quantity of 2 ml of methanolic
solution of DPPH (4%) was added to 1 ml
Aqueous extracts (AEL and AEB)
of each extract at different concentrations.
20 g of the powder of each part of the plant
After, the mixtures were incubated at room
were placed in 500 ml of boiling distilled
temperature in the dark for 30 minutes and
water (100 °C) under magnetic stirring, for
the decrease in the absorbance was
30 minutes. The aqueous extracts are then
determined at 515 nm using a UV-Visible
cooled, filtered by Whatman filter paper 4,
spectrophotometer Lambda 25
evaporated using a vacuum rotary
(PerkinElmer, Waltham, Massachusetts,
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USA). Methanol presents a blank and Juntachote and al. [16]. To 0.5 ml of each
DPPH solution a negative control. A sample, 5 ml of distilled water and 0.5 ml
positive control was presented by gallic of Folin-Ciocalteu's were added. After 3
acid (GA). The percentage of DPPH min, 0.5 ml of sodium carbonate at 20%
scavenging activity was determined as were added and incubated for one hour at
follows: room temperature in the dark. The OD was
    read at 760 nm using a UV-Visible
spectrophotometer (Perkin-Elmer Lambda
 
 
25). Standard solutions of GA with

A is the absorbance of negative control different concentrations were used to
0
and A is the sample absorbance. obtain a standard curve and the results
1
The efficient concentration EC50 were expressed as milligram gallic acid
(otherwise called the inhibitory equivalents per gram of dry weight of
concentration IC50) value was determined extract (mg GAE/g dw) [16].
for each sample. This is defined as the
Estimation of total flavonoids
concentration of substrate that causes 50%
The method of aluminum trichloride AlCl
loss of the DPPH activity (color) [14]. 3
was adopted to quantify total flavonoids in
Ferric Reducing Antioxidant Power different extracts. To 1 ml of each sample,
FRAP assay 1 ml of 2% w/v AlCl in methanol was
3
Reducing power was determined by the added and incubated at room temperature
method prescribed by Yen and Chen [15]. for 30 minutes. Absorbance was read at
The 1 ml of each sample at various 430 nm. Quercetin (Q) was used as a
concentrations was mixed with a phosphate standard and the results were expressed in
buffer (2.5 ml, pH 6.6) and potassium milligram of quercetin equivalents per
ferricyanide K [Fe(CN) ] (2.5 ml, 1%), gram of dry weight of extract (mg QE/g
3 6
and the mixture was incubated at 50° C for dw) [17].
30 minutes. Next, 2.5 ml of
Statistical analysis
trichloroaceticacid (10%) were added to
The Microsoft Office Excel 2007 program
the reaction mixture, which was then
was used to realize: linear regressions,
centrifuged at 3000 RPM (rotation per
histogram and curves of antioxidant
minute), for 10 min. The upper layer of the
activity, calibration curves, and means ±
solution (2.5 ml) was mixed with distilled
standard deviations.
water (2.5 ml) and ferricchloride (0.5 ml,
Comparison between groups was
1%), and the optical density (OD) was
conducted by one-way analysis of variance
measured at 700 nm (UV-Visible
spectrophotometer, Perkin-Elmer Lambda
multiple comparison test. Differences
25). A high absorbance will indicate an
between groups with P<0.05 were
increase in reducing power. GA was used
considered statistically significant [18].
as a reference antioxidant substance [15].
Statistical analysis was performed by
Estimation of total polyphenols probit analysis method using XLStats 2013
The determination of total polyphenols is software (Pros statistical software,
accomplished by the method described by Addinsoft, Paris, France).
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2- Results and discussion The results of the phytochemical study are
Phytochemical screening shown in Table 1.

Table 1 Results of phytochemical screening of Allium triquetrum L.
Chemical compound Leaves Bulbs
Alkaloids - -
Anthocyanins - -
Anthracenosids (Combined forms) - +
Anthracenosids (Oxidative forms) - -
Anthraquinones - +
Cardiotonic heterosides ++ ++
Coumarins - -
Flavonoids +++ +++
Glycosides ++ +++
Polyphenols ++ +
Iridoids ++ +
Tannins - -
Mucilage + ++
Phenolic derivatives ++ +
Quinones - -
Saponosids ++ +++
Terpenes + +
-: absent / +: slightly present / ++: moderately present / +++: highly present

The phytochemical screening of leaves and and leek (A. porrum). However, the species
bulbs of A. triquetrum showed the presence of this genus contains big amounts of other
of polyphenols, flavonoids, phenolic molecules such as coumarins, amine acids,
derivatives, saponosides, cardiotonic tannins, sterols, etc [19,20,21]. The study
heterosides, mucilage, glycosides and of Najjaa et al. (2011) has shown the
iridoids, trace amount of terpens and presence of a high amount of iridoids and
absence of alkaloids, tannins, quinones, coumarins with the absence of
anthraquinones, anthracenosids, coumarins anthracenosids and anthraquinons in the
and anthocyanins. Some combined forms leaves of A. roseum and A. ampeloprasum
of anthracenosids were present in the [8]. Another interesting study of Egyptian
bulbs. leek (A. ampeloprasum var. kurrat)
This work confirmed that A. triquetrum showed the presence of alkaloids, tannins
contains various chemical compounds as steroids, flavonoids, terpenoids and
well as compared to the different species of saponins [22].
Allium genus. Polyphenols, flavonoids, The richness and diversity of secondary
saponins and glycosides are the most metabolites in the plants of the genus
important secondary metabolites in Allium Allium explains their use for the treatment
spp., as garlic (A. sativum), onion (A. cepa)
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and prevention of many diseases especially The linear regressions for antioxidant
cancer diabetes and hypercholesterolemia. power of different extracts and control
were used to calculate the EC50 values.
Antioxidant activity
The results of radical scavenging assays
DPPH radical scavenging capacity
and EC50 values are given in Fig. 2 and
Table 2, respectively.

120 GA
y
vit100 AEL
cti AEB
a
ng 80 MEL
gi
n MEB
e
v 60
a
c
s
cal 40
di
a
r
f 20
o
%
0
0 0,5 1 1,5 2 2,5 3 3,5
concentration (mg/ml)
Fig. 2 Curves of DPPH radical scavenging activity (%) of control, aqueous and methanolic
extracts of Allium triquetrum L. according to different concentrations

Table 2. EC50 values of control, aqueous and methanolic extracts of Allium triquetrum L.
Control/Extract EC50 Values (mg/ml)
GA 0.62
AEL 0.97
AEB 1.36
MEL 1.98
MEB 2.45

The outcomes of this study showed that A. 1.36 mg/ml, respectively. While, the
triquetrum is endowed with good lowest activities are given by the
antioxidant activity in comparing with the methanolic extracts of bulbs (MEB 57.3%)
positive control GA which showed an and leaves (MEL 68.3 %) which EC50 are
inhibitory rate equal to 99.56% at 3 mg/ml equal to 2.45 and 1.98 mg/ml, respectively.
with EC50 equal to 0.62 mg/ml. AEL has This study confirms the results obtained in
the highest radical scavenging activity previous researches [23,24]. Chang and al.
(90%) followed by AEB (83.3%), their (2013) showed that the DPPH free radical
EC50 values are equal to 0.97 mg/ml and scavenging activity of Allium spp. ranged
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from 67.34% to 90.03% for A. fistulosum, trisulphide appeared to be the main
while it ranged from 63.63% to 88.33% for antioxidative compounds. In addition, the
A. sativum. Furthermore, the leaf extracts antioxidant activity is also related to other
had higher DPPH free radical scavenging bioactive compounds: dietary fibers,
activity than that of the stem, bulb and microelements (especially Se) and
root. In contrast, Tugbobo and al. (2015) polyphenols [23,25,26]. Phenolic
found that DPPH scavenging ability of the compounds are large groups of secondary
extracts was higher in aqueous extract than metabolites that are able to neutralize or
ethanolic extract as 41.08% was obtained quench the free radicals. Flavonoids and
for aqueous extract while 39.46% was their derivatives are the largest group of
obtained for ethanolic extract at same polyphenols found in plants that possess
concentration. strong antioxidant activities due to the
The antioxidant activity of Allium spp. has scavenging reactive oxygen species and
been attributed mainly to a variety of inhibition of oxidative stress [27].
sulphur containing compounds and their
Ferric Reducing Power FRAP assay
precursors. Scientific evidence shows that
The results are plotted in Fig. 3.
allicin, diallyl disulphide and diallyl

2,5
GA
m 2 AEL
n
0
0 AEB
71,5
at MEL
y
sit 1 MEB
n
e
d
al
c0,5
pti
O
0
0 0,25 0,5 0,75 1 1,25 1,5 1,75
Concentration (mg/ml)

Fig. 3 Ferric reducing power of control, methanolic and aqueous extracts
of Allium triquetrum L.
Similarly to DPPH radical scavenging extracts have the lower reductive capacity
capacity, FRAP assay also showed that the (OD: MEL = 0.72, MEB = 0.4).
different extracts of A. triquetrum possess Also, the experiment of Tugbobo and al.
an antioxidant activity but which is less (2015) has confirmed that aqueous extract
important compared to the control GA (OD of Allium sativum has higher reducing
at 3 mg/ml equal to 2.5). The higher potential than ethanolic extract [24]. On
reducing power is shown by the AEL with the other hand, Nencini and al. (2007) has
an OD equal to 1.22 followed by AEB (OD proved that the flowers of species growing
equal to 1.02), while the methanolic wild showed the higher reducing power.
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Interesting results were shown even by the the type, quality, part (leaves, stalk, flower,
leaves, while the antioxidant capacity of seeds) of the plant, location of habitat,
the bulbs was lower [28]. climatic conditions, soil characteristics,
Reducing power is a novel antioxidation etc. Extraction method and solvent agent
defence mechanism; the two mechanisms (water, alcohol, etc.) are also important
available to affect this property are by factors, considerably affecting plant
electron transfer and hydrogen atom antioxidants capacity.
transfer. This is because the ferric-to-
Total polyphenols and flavonoids
ferrous ion reduction occurs rapidly with
content
all reductants with half reaction reduction
potentials above that of Fe3+/Fe2+, the The equations of calibration curves of
quercetin (y = 0.004 x + 0.013; R2 = 0.996)
values in the Ferric reducing antioxidant
and gallic acid (y = 0.049 x + 0.099; R2 =
property (FRAP) assay will express the
0.992) were used for the estimation of total
corresponding concentration of electron-
polyphenols and flavonoids respectively.
donating antioxidants [29].
The results are presented in Fig. 4 and Fig.
According to the literature [30], the
5.
antioxidant activity of plants depends on

) 16
w D
d
g 14
E/ C
A 12
B
G
g 10 A
m
n ( 8
o
ati 6
r
nt
e 4
c
n
o 2
C
0
AEL MEL AEB MEB
Extract
Fig. 4 Total polyphenols of various extracts of Allium triquetrum L expressed as means ±
standard deviations (Means with different letter are significantly different (PB0.05) according

This study showed the presence of dw for AEB. The methanol extracts
important levels of phenolic compounds in showed somehow lower concentrations,
the different extracts with a significant MEL presents 10.46 ± 0.04 mg GAE/g dw
differences between their means (P<0.05). of polyphenols and 14.27 ± 0.20 mg QE/g
The largest concentration is presented by dw of flavonoids, however their amounts
the AEL, the total of polyphenols and in MEB are equal to 8.59 ± 0.18 mg
flavonoids which equal to 14.31 ± 0.36 GAE/g dw and 12.79 ± 0.10 mg QE/g dw,
mg.GAE/ g dw and 22.12 ± 0.33 mg QE/g respectively. On the other hand, we noticed
dw, respectively, compared to 12.09 ± 0.13 that leaves contain higher levels comparing
mg GAE/g dw and 16.05 ± 0.13 mg QE/g to the bulbs.
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25
D
)
w
d
g 20
E/ C
Q
g 15 B
m A
(
n
o
ati 10
r
nt
e
c 5
n
o
C
0
AEL MEL AEB MEB
Extract
Fig. 5 Total flavonoids of various extracts of Allium triquetrum L expressed as means ±
standard deviations. (Means with different letter are significantly different (PB0.05) according

The content of phenolic acids and
Conclusion
flavonoid varied in different solvents.
This work showed that the Algerian plant
Turkmen and al. (2006) reported that
A. triquetrum L. like the most Allium
solvents with different polarity (ethanol
species contains various chemical
and water) have significant effect on
compounds such us flavonoids, saponins
polyphenols content [31]. In addition,
and glycosides, and is endowed with good
antioxidant activity is higher in more polar
to moderate antioxidant activity. The
extraction solvents. Settharaksa and al.
aqueous extracts have a highest antioxidant
(2014) showed that water was the best
capacity and showed the greatest levels of
solvent for phenol and flavonoid extraction
polyphenols and flavonoids comparing to
[32].
the methanolic extracts. The difference
These results justify the difference between
between antioxidant capacities of its
potential of antioxidant activity for
different extracts is related to phenolic
different extracts of A. triquetrum.
content which is correlated to the plant part
Phenolics and flavonoids were the major
or portion and to the solvent and extraction
contributors to antioxidant activity and
method. A. triquetrum can be used as a
their higher content was associated with
new source of natural antioxidants
higher antioxidant activity [33]. Since the
molecules. However, these results require
antioxidant activity of phenolics is mainly
further research work, in order to identify
due to their redox properties, this allows
and characterize the bioactive substances
them to act as reducing agents, hydrogen
involved, in particular phenolic
donors and singlet oxygen quenchers In
compounds, and to know their mechanisms
addition, to their physiological roles in
of action.
plants as an important contributor to the
survival of plant species they have benefits
as antioxidants for human health [29].
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