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plants
Communication
Variability of Secondary Metabolites of the Species
Cichorium intybus L. from Different Habitats
Nenad M. Zlati´c * and Milan S. Stankovi´c ID
Department of Biology and Ecology, Faculty of Science, University of Kragujevac, 34000 Kragujevac, Serbia;
mstankovic@kg.ac.rs
*Correspondence: nzlatic@gmx.com; Tel.: +381-34-336223
Received: 26 July 2017; Accepted: 8 September 2017; Published: 11 September 2017
Abstract:
The principal aim of this paper is to show the influence of soil characteristics on
the quantitative variability of secondary metabolites. Analysis of phenolic content, flavonoid
concentrations, and the antioxidant activity was performed using the ethanol and ethyl acetate
plant extracts of the species Cichorium intybus L. (Asteraceae). The samples were collected from one
saline habitat and two non-saline habitats. The values of phenolic content from the samples taken
from the saline habitat ranged from 119.83 to 120.83 mg GA/g and from non-saline habitats from
92.44 to 115.10 mg GA/g. The amount of flavonoids in the samples from the saline locality varied
between 144.36 and 317.62 mg Ru/g and from non-saline localities between 86.03 and 273.07 mg Ru/g.
The IC
50
values of antioxidant activity in the samples from the saline habitat ranged from 87.64 to
117.73
µ
g/mL and from 101.44 to 125.76
µ
g/mL in the samples from non-saline habitats. The results
confirmed that soil types represent a significant influence on the quantitative content of secondary
metabolites. The greatest concentrations of phenols and flavonoids and the highest level of antioxidant
activity were found in the samples from saline soil. This further corroborates the importance of
saline soil as an ecological factor, as it is proven to give rise to increased biosynthesis of secondary
metabolites and related antioxidant activity.
Keywords: saline habitats; secondary metabolites; adaptation; different solvents
1. Introduction
The abiotic factor with the most harmful effect upon the productivity and growth of plants is soil
salinity. Increased concentrations of salt in soil exert a negative influence on plants due to the following
reasons: water absorption is more difficult, the growth of the plant may be hindered, and plant
metabolism as well as both physiological and chemical processes may be disturbed to a significant
extent [
1
]. In order to successfully adapt to saline soil, plants develop a series of specific mechanisms
which enable them to respond appropriately to salinity-induced stress [
2
]. The mechanisms develop at
molecular, cellular, metabolic, and physiological levels [3].
Salinity-induced stress causes the production of reactive oxygen species (ROS), such as hydrogen
peroxide, hydroxyl radicals, and superoxide anions in a variety of cells resulting in oxidative stress [
4
].
Increased production of ROS leads to oxidative damage of cellular membranes, proteins, carbohydrates,
and DNA [
5
]. Plants activate enzymatic and nonenzymatic antioxidant systems for the purpose of
protection from the negative influence of free radicals. The activation of enzymatic systems includes
superoxide dismutase, catalase, and glutathione reductase, whereas carotenoids, flavonoids, and other
phenolic compounds belong to nonenzymatic systems of protection [
4
]. Different ecological factors
that directly influence the plant primary metabolism have different effects. Correspondingly, the
differentiation of secondary metabolism occurs. The primary function of secondary metabolites is
their participation in the process of plant adaptation to the effects of ecological factors [
6
]. From an
Plants 2017,6, 38; doi:10.3390/plants6030038 www.mdpi.com/journal/plants
Plants 2017,6, 38 2 of 9
evolutionary viewpoint, biosynthesis of secondary metabolites in plants was considered as an
ecophysiological response of a plant to different influences of abiotic factors in a certain habitat,
including salinity [
7
]. Secondary metabolites participate in the process of plant adaptation to the
ecological conditions of environment and their quantity in plant organs varies depending on both the
abiotic and biotic factors that a plant is exposed to [8].
A great number of investigated medicinal plants that are used in the fields of pharmacy and
medicine belong to the family Asteraceae. Within this plant family there are several important genera,
which abound in medicinal plant species, and such genera are: Artemisia,Achillea,Inula, and Matricaria.
These genera show the intense biological activity applied in the treatment of respiratory, digestive,
cardiovascular, and some other types of diseases [
9
]. Apart from therapeutical effects, the active
metabolites of the species that belong to this family manifest antimicrobe, antiviral, antiproliferative,
and antioxidant activity in both in vitro and in vivo conditions [10,11].
The genus Cichorium encompasses approximately nine species. Among the best known
medicinal species that belong to the genus Cichorium are: C. intibus,C. spinosum, and C. dubium.
Chicory (C. intybus L., Asteraceae) is a perennial herbaceous plant up to 120 cm high. Its stem is
erect and branched. The lower leaves are curled, whereas the upper ones are bare and lanceolate.
The flowers are blue in colour and grow either individually or in groups. The fruit is achene, smallish
and ovoid in shape. C. intybus populates meadows, saline habitats, areas by the road, the edges of
forests, and the territories of Europe, West Asia, and North Africa. Due to the limited distribution
and wider application, the successful growth of this species is enabled on territories outside its initial
area [12].
Cichorium intybus is well known in the folk and modern medicine for its anticancer [
13
],
antioxidant [
14
], antidiabetic [
15
], anti-inflammatory [
16
], antimicrobial [
17
], anthelmintic [
18
],
analgesic [
19
], cardiovascular [
20
], gastroprotective [
21
], hepatoprotective [
22
], immunological [
23
],
reproductive effects [
24
], wound healing abilities [
25
], and many other pharmacological
applications [
17
,
26
]. It is also important to mention the numerous active substances of this species:
alkaloids, coumarins, caffeic acid derivatives, sesquiterpene lactones, flavonoids, terpenoids, and
volatile compounds [26].
Secondary metabolites, apart from other adaptive mechanisms, have significance in relation to the
reaction of plants to the chemical stress induced by increased quantity of salt in soil in the process of
neutralisation of consequences of toxicity for the purpose of ecophysiological adaptation. Taking the
previously stated fact into account, the species C. intybus was selected as a plant suitable for analysis,
with its suitability stemming from the fact that it grows in habitats with both normal mineral regime
and with saline soils. Accordingly, by means of sampling the species from one habitat with saline soil
and two habitats with normal mineral regime as well as by analysing both the quantity of secondary
metabolites from the group of phenolic compounds and their antioxidant activity, a comparison was
performed in order to determine their significance in terms of adaptation to toxic effects of salt.
2. Results and Discussion
Total phenolic content, flavonoid concentration, and antioxidant activity
in vitro
were determined
using ethanol and ethyl acetate extracts of whole C. intybus plant from saline and non-saline habitats.
In order to extract the active substances of different polarity, the extraction solvents of different polarity
were used.
2.1. The Total Quantity of Phenolic Compounds
The results of the analysis of the total quantity of phenolic compounds, flavonoid concentration,
and antioxidant activity in ethanol and ethyl acetate extracts of the aboveground plant parts of the
species C. intybus are shown in Table 1.
Plants 2017,6, 38 3 of 9
Table 1.
Total phenolic content, flavonoid concentration, and antioxidant activity of the analysed
species in ethanol and ethyl acetate extracts.
Locality
Type of Analysis
Total Phenolic Content
(mg of GA/g of Extract)
Flavonoid Content
(mg of Ru/g of Extract)
Antioxidant Activity
IC50 (µg/mL)
Type of Extract
Ethanol Ethyl Acetate Ethanol Ethyl Acetate Ethanol Ethyl Acetate
Oblaˇcinska slatina
120.83
±
1.02
119.83 ±1.34
144.36
±
0.83
317.62 ±2.04
117.73
±
1.71
87.64 ±1.90
Ivanjica 95.53 ±0.97 115.10 ±1.50
129.00
±
1.18
273.07 ±1.56
120.53
±
2.21
101.44 ±1.53
Kragujevac 92.44 ±1.12 96.55 ±1.45 86.03 ±0.59 176.09 ±1.39
121.05
±
1.66
125.76 ±2.33
Each value is the average of three analyses ±standard deviation.
The total quantity of phenolic compounds in ethanol extracts ranged from 92.44 to
120.83 mg GA/g of extract, whereas the values in the ethyl acetate extracts varied from 96.55 to
119.83 mg GA/g of extract. The results showed that the greatest quantity of phenolic compounds in the
ethanol extracts was found in the sample from the locality Oblaˇcinska slatina (120.83 mg GA/g), while
the smallest quantity was observed in the sample collected in Kragujevac (92.44 mg GA/g). Similarly,
the highest concentration of phenolic compounds in the ethyl acetate extracts was measured in the
samples from the locality Oblaˇcinska slatina (119.83 mg GA/g), whereas the lowest concentration
was found in the samples collected in Kragujevac (96.55 mg GA/g). In the extracts obtained using
moderate and lower solvents (ethanol and ethyl acetate) the order of concentrations for phenolic
compounds is: Oblaˇcinska slatina > Ivanjica > Kragujevac.
The analyses showed that the extract obtained using solvents of lower polarity contained the
highest concentration of active compounds. The total quantity of phenolic compounds was greater in
the ethanol extracts made from the solvent of moderate polarity and the samples collected from the
locality of Oblaˇcinska slatina (120.83 mg GA/g of extract). Consequently, it was shown that a certain
group of phenolic compounds is important for the adaptation to saline habitats [27].
The total quantity of phenolic compounds in the aboveground plant parts of the species C. intybus
varied depending on the type of locality from which the plant material was taken. The substrate of
Oblaˇcinska slatina contains high concentrations of salt. Contrary to this, the substrates of Ivanjica
and Kragujevac contain low salt concentrations. Due to this difference in salt concentrations, greater
quantities of phenolic compounds (120.83 mg GA/g) were measured in the plant extract of the
sample from Oblaˇcinska slatina. The obtained results for the quantity of phenolic compounds
of the species C. intibus are in accordance with the previously recorded values [
28
]. It has been
confirmed that the quantity of polyphenols in plants belonging to the species Mentha pulegium
increases due to the stress caused by greater concentrations of salt in ground substrate [
29
]. Similarly,
the examination of the species Nigella sativa demonstrated the increase in the total quantity of
phenols due to saline treatment [
30
]. Salinity-induced increases in the concentration of the group
of phenolic acids (protocatechuic, chlorogenic, and caffeic acids) has been observed in the species
Matricaria chamomilla [31].
The plants sampled in Ivanjica contained higher concentrations of phenolic compounds in
comparison with the samples from Kragujevac. These localities differ in altitude, as Ivanjica is
located at an altitude of 997 m and Kragujevac at an altitude of 194 m. Previously performed studies
showed that the plant species in habitats with increased intensity of light contain phenolic compounds,
which have considerable influence on the adaptational abilities of plants populating these habitats.
The increased quantity of phenolic compounds in plants plays a protective role from ultraviolet-B
radiation, which is more intense at higher altitudes [32,33].
Plants 2017,6, 38 4 of 9
2.2. The Total Quantity of Flavonoids
The results of the analysis of the total quantity of flavonoids in ethanol and ethyl acetate extracts
of aboveground plant parts of the species C. intybus are shown in Table 1. The total quantity of
flavonoids in the ethanol extracts ranged from 86.03 to 144.36 mg Ru/g of extract, whereas the quantity
in the ethyl acetate extracts varied from 176.09 to 317.62 mg Ru/g of extract. The results showed
that the greatest quantity of flavonoids in the ethanol extracts was found in the sample from the
locality Oblaˇcinska slatina (144.36 mg Ru/g), while the smallest quantity was observed in the sample
collected in Kragujevac (86.03 mg Ru/g). Similarly, the highest concentration of flavonoids in the ethyl
acetate extracts was measured in the sample from the locality Oblaˇcinska slatina (317.62 mg Ru/g),
whereas the lowest concentration was found in the sample collected in Kragujevac (176.09 mg Ru/g).
In the extracts obtained using moderate and lower solvents (ethanol and ethyl acetate) the order of
concentrations for phenolic compounds is: Oblaˇcinska slatina > Ivanjica > Kragujevac.
The concentration of flavonoids in plant extracts depends on the polarity of solvents used in
the extract preparation. Based on the obtained values for concentration of flavonoids, the highest
concentration of these compounds is observed in the extracts obtained using solvents of low polarity.
High concentrations of flavonoids in ethyl acetate extracts may be attributed to their high solubility in
this solvent [
34
]. Accordingly, the analysis of whole plant extracts indicated that ethyl acetate was the
most effective solvent for the extraction of flavonoids from the species C. intybus and that therefore
solvents with low and moderate polarity should be used to this end.
Apart from solvent polarity, what proved to be relevant in terms of the quantity of flavonoids is the
type of locality from which samples were taken. The greater quantity of flavonoids was observed in the
extract made from the samples collected from the locality of Oblaˇcinska slatina (317.62 mg Ru/g) the
substrate of which contains high concentrations of salt. Previous studies confirmed that certain plants
from saline habitats synthetize greater concentrations of flavonoids as a response of the secondary
metabolism to the adaptation of species to the increased level of salt in the substrate [35,36].
The concentration of flavonoids differs in the localities of Ivanjica and Kragujevac, with Ivanjica
having higher concentrations of flavonoids in both types of extracts. This study revealed a difference in
the quantity of flavonoids in the samples collected at different altitudes. Increases in altitude may cause
increases in the plant production of flavonoids. UV-B radiation increases the production of flavonoids
in barley [
37
]. The greater intensity of light in a habitat is accompanied by a larger accumulation of
secondary metabolites in certain plant organs [32].
2.3. Antioxidant Activity
The obtained values for antioxidant activity of the ethanol and ethyl acetate extracts made from
the aboveground plant parts of the species C. intybus are shown in Table 1. Antioxidant activity of the
ethanol extracts from different localities ranged from 117.73 to 121.05
µ
g/mL, while the antioxidant
activity of the ethyl acetate extracts varied from 87.64 to 125.76
µ
g/mL. The results showed that the
highest level of antioxidant activity of both the ethanol and ethyl acetate extracts was observed in the
samples from the locality Oblaˇcinska slatina, with values of 87.64
µ
g/mL and 117.73
µ
g/mL for each
type of extract, respectively. The lowest values of both the ethanol and ethyl acetate extracts were
obtained from the samples collected in Kragujevac, with values of 121.05
µ
g/mL and 125.76
µ
g/mL for
each type of extract, respectively. In the extracts obtained using moderate and lower solvents (ethanol
and ethyl acetate) the order of antioxidant activity is: Oblaˇcinska slatina > Ivanjica > Kragujevac.
The extraction of antioxidant substances of different chemical structure was achieved using
solvents of different polarity. The largest capacity to neutralize DPPH (2,2-dyphenyl-1-picrylhydrazyl)
radicals was measured in the ethyl acetate extract from the saline habitat Oblaˇcinska slatina, which
neutralized 50% of free radicals at concentrations of 87.64
µ
g/mL. It is well known that the phenolic
content of plants may contribute directly to their antioxidant activity [
34
] due to its role in scavenging
free radicals.
Plants 2017,6, 38 5 of 9
The plants sampled from the locality Oblaˇcinska slatina had the highest concentrations of phenols
and flavonoids. This indicates that secondary metabolites of the phenolics group in C. intybus are the
key active substances for the expression of antioxidant activity. Earlier studies on major secondary
metabolites in upper parts of C. intybus demonstrated the presence of sesquiterpene lactones as well as
caffeic acids derivates (chicoric acid, chlorogenic acid, isochlorogenic acid, dicaffeoyl tartaric acid).
Significant biological activity of these phenol acids and flavonoids has already been confirmed for
both in vitro and in vivo model systems. The antioxidant activity has been reported as well [26].
Numerous studies have shown increased activity of antioxidant enzymes in plants located in
habitats with saline substrates. Neutralisation of free radicals in the species Phragmites karka increases
when the plant is exposed to higher concentrations of salt [
38
]. Similar results have been obtained for
the species Hordeum vulgare [39].
In comparison with the locality of Kragujevac, Ivanjica stands out in terms of its greater ability to
neutralise free radicals. This is attributable to the differences in the type of habitat and altitude.
These results are in accordance with the findings that the extracts of the samples collected at
higher altitudes have greater antioxidant capacity than the extracts of the samples taken at lower
altitudes [40,41].
2.4. Correlation between Phenolic Compounds, Flavonoids, and Antioxidant Activity
The obtained results for the total quantity of phenolic compounds and flavonoids, as well as
for the level of antioxidant activity of the ethanol and ethyl acetate extracts of the species C. intybus
were statistically analysed for the purpose of determining the degree of correlation. The values of
correlation point to the significant link between the analysed parameters.
The correlation between the total quantity of phenolic compounds, flavonoids, and the values of
antioxidant activity measured in both ethanol and ethyl acetate extracts is established in C. intybus
(Table 2). It can be noticed that an increase in the phenol content of extract decreases the IC
50
value,
i.e., increases their scavenging DPPH free-radical activity (negative correlation). This is important
in the light of the fact that the lower IC
50
values imply higher levels of antioxidant activity. Similar
conclusions have been reached in other studies [42,43].
Table 2.
The coefficient of correlation (p< 0.05) between the total phenolic compounds (TPC), total
flavonoids (TF) and the values for antioxidant activity (AA).
(r) AA Ethanol AA Ethyl Acetate
TPC Ethanol −0.999 * −0.835
TPC Ethyl acetate −0.760 −0.985
TF Ethanol −0.800 −0.994
TF Ethyl acetate −0.832 −0.999 *
* Correlation is significant at the 0.05 level.
3. Materials and Methods
3.1. Plant Material
Plants were sampled from natural populations found in different localities with saline and
non-saline substrate. The plant material was sampled from three different localities: one saline
(Oblaˇcinska slatina, Serbia) and two non-saline ones (Ivanjica and Kragujevac, Serbia). The sampling
was performed in the phase of flowering in August 2014 (Table 3). The collected samples were identified
in the Department of Biology and Ecology of the Faculty of Science in Kragujevac. Aboveground plant
parts were dried in a dark room at room temperature. Dry samples were ground in a blender and thus
kept in dark vials until analysis.
Plants 2017,6, 38 6 of 9
Table 3. The basic characteristics of the localities in which the species C. intybus was sampled.
Locality Type of Habitat Altitude Latitude and Longitude
Oblaˇcinska slatina Meadow, Hygrophilous habitat 285 m 43◦18017.76” N
21◦4100.340” E
Ivanjica Meadow, Mesophilous habitat 997 m 43◦28032.55” N
20◦10029.11” E
Kragujevac Meadow, Mesophilous habitat 194 m 44◦01031.18” N
20◦54050.62” E
3.2. Preparation of Plant Extracts
Dried plant material (10 g) was put into Erlenmeyer flasks filled with 200 mL of ethanol or ethyl
acetate and thus left at room temperature. The extract was filtrated after 48 h using Whatman No. 1
filter paper. The plant extracts were condensed to dry on a rotary vacuum at 40
◦
C. The obtained
extracts were placed in sterile containers and kept in the fridge at 4
◦
C. The total quantity of phenolic
compounds, flavonoids, as well as the level of antioxidant activity were determined in the extract
concentration of 1 mg/mL using ethanol or ethyl acetate as solvents.
3.3. Chemicals
Organic solvents and sodium hydrogen carbonate were purchased from Zorka pharma Sabac,
Serbia. 2,2-dyphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma Chemicals Co., St. Louis, MO,
USA. Folin-Ciocalteu phenol reagent, 3-tert-butyl-4-hydroxyanisole (BHA), and aluminium chloride
hexahydrate (AlCl
3·
6H
2
O) were purchased from Fluka Chemie AG, Buchs, Switzerland. All other
solvents and chemicals were of analytical grade.
3.4. Determination of Total Phenolic Contents
The total phenolic content was determined using the spectrophotometric method [
44
]. First,
0.5 mL of methanol solution (1 mg/mL) of extract, 2.5 mL of 10% Folin-Ciocalteu’s reagent dissolved
in water, and 2.5 mL 7.5% NaHCO
3
were used to obtain the reaction mixture. Then, the samples were
incubated at 45
◦
C for 15 min. The absorbance was measured at
λmax
= 765 nm. The samples were
prepared in triplicate and the mean value of absorbance was obtained. The blank was prepared with
methanol solution. The same procedure was repeated for the gallic acid and the calibration curve was
constructed. The total phenolic content was expressed as gallic acid equivalent (mg of GA/g).
3.5. Determination of Flavonoid Concentrations
The concentration of flavonoids was determined using the spectrophotometric method [
45
].
The sample contained 1 mL of methanol solution of the extract in the concentration of 1 mg/mL and
1 mL of 2% AlCl
3
solution dissolved in methanol. The samples were incubated at ambient temperature
for an hour. The absorbance was measured at
λmax
= 415 nm. The samples were prepared in triplicate
and the mean value of absorbance was obtained. The same procedure was repeated for the rutin and
the calibration line was constructed. Concentration of flavonoids in extracts was expressed in terms of
rutin equivalent (mg of Ru/g).
3.6. Evaluation of DPPH Scavenging Activity
The efficiency of the plant extract to neutralise DPPH (1,1-diphenyl-2-picrylhydrazyl radical) free
radicals was determined using the spectrophotometric method previously described [
46
] with adequate
modifications [
47
]. The plant extract was dissolved in methanol to obtain the concentration 1 mg/mL.
Dilutions were made to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.99, and
0.97 mg/mL. Diluted solutions (1 mL each) were mixed with 1 mL of DPPH methanolic solution
Plants 2017,6, 38 7 of 9
(80 mg/mL). The absorbance was recorded at 517 nm. The control samples contained methanol and
DPPH reagents. The percentage inhibition was calculated using the equation: % inhibition = 100
×
(A of
control
−
A of sample)/A of control)), whilst IC
50
values were estimated based on the sigmoidal curve
presenting the dependence of the percent of DPPH scavenging on sample concentration. Antioxidant
activity was expressed as the half-maximal inhibitory concentration (IC
50
values in mg/mL). In the
presented results, antioxidant efficiency of the extract increased with decreasing IC
50
values. The data
were presented as mean values ±standard deviation (n= 3).
3.7. Statistical Analysis
All experimental measurements were carried out in triplicate and are expressed as the average of
three analyses
±
standard deviation. Results were analysed statistically using IBM, SPSS, Statistics,
ver. 19, Armonk, NY: IBM Corp. Pearson
'
s correlation coefficient (r) was used to evaluate relationships
between contents and antioxidant properties of chicory extracts.
4. Conclusions
The results of this research show that there is a significant difference in the quantity of secondary
metabolites and their activity in the species C. intybus, which populates both saline and non-saline
habitats. The total quantity of phenolic compounds and flavonoids increases due to the presence of salt
in the substrate. The level of antioxidant activity was higher in the samples taken from saline habitats,
and the result implies that this is a mechanism of plant adaptation to the increased concentrations of
salt in the substrate. Plants may adapt to the stressful conditions in the habitat by means of synthesis
regulation and accumulation of secondary metabolites. The plants tolerant to salt stress are sources of
secondary metabolites and may be highly applicable in pharmaceutical and food industries.
Acknowledgments:
This investigation was supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia III41010. The authors acknowledge Ana Vuˇci´cevi´c for manuscript lecturing.
Author Contributions:
Nenad M. Zlati´c partly conducted field work, organised and performed experiments,
planned the effective presentation of data, and wrote the paper; Milan S. Stankovi´c proposed the theme, partly
conducted the field work, and provided guidance and supervision to organise the experiments and analyse the
data; both authors revised the paper in accordance with the instructions.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Munns, R. Comparative physiology of salt and water stress. Plant Cell Environ.
2002
,25, 239–250. [CrossRef]
[PubMed]
2. Bohnert, J.H.; Shen, B. Transformation and compatible solutes. Sci. Hortic. 1999,78, 237–260. [CrossRef]
3.
Gupta, B.; Huang, B. Mechanism of salinity tolerance in plants: Physiological, biochemical, and molecular
characterization. Int. J. Genom. 2014. [CrossRef] [PubMed]
4.
Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev.
Plant Biol. 2004,55, 373–399. [CrossRef] [PubMed]
5.
Van Breusegem, F.; Dat, F.J. Reactive oxygen species in plant cell death. Plant Physiol.
2006
,141, 384–390.
[CrossRef] [PubMed]
6.
Kliebenstein, D.J.; Osbourn, A. Making new molecules—Evolution of pathways for novel metabolites in
plants. Curr. Opin. Plant Biol. 2012,15, 415–423. [CrossRef] [PubMed]
7.
Navarro, J.M.; Flores, P.; Garrido, C.; Martinez, V. Changes in the contents of antioxidant compounds in
pepper fruits at different ripening stages, as affected by salinity. Food Chem. 2006,96, 66–73. [CrossRef]
8.
Ramakrishna, A.; Ravishankar, G.A. Influence of abiotic stress signaling on secondary metabolites in plants.
Plant Signal. Behav. 2011,6, 1720–1731. [PubMed]
9.
Mukherjee, S.K. Medicinal plants of Asteraceae in India and their uses. In Proceeding of National Seminar;
Gupta, S.K., Mitra, B.R., Eds.; Ramakrishna Mission Ashrama: Kolkata, India, 2006; pp. 43–49.
Plants 2017,6, 38 8 of 9
10.
Jayaraman, S.; Manoharam, S.M.; Illanchezian, S. In-vitro antimicrobial and antitumor activities of
Stevia rebaudiana (Asteraceae) leaf extracts. Trop. J. Pharm. Res. 2008,7, 1143–1149. [CrossRef]
11.
Kasim, L.S.; Ferro, V.A.; Odukoya, O.A.; Drummond, A.; Ukpo, G.E.; Seidel, V.; Gray, A.I.; Waigh, R.
Antimicrobial agents from the leaf of Struchium sparganophora (Linn) Ktze, Asteraceae. J. Microbiol. Antimicrob.
2011,3, 13–17.
12.
Gaji´c, M. Genus Cichorium L. In Flora of Serbia; Josifovi´c, M., Ed.; Serbian Academy of Sciences and Arts:
Belgrade, Serbia, 1975; Volume 7, pp. 266–268.
13.
Lee, K.T.; Kim, J.I.; Park, H.J.; Yoo, K.O.; Han, Y.N.; Miyamoto, K.I. Differentiation-inducing effect of
magnolialide, a 1
β
-hydroxyeudesmanolide isolated from Cichorium intybus, on human leukemia cells.
Biol. Pharm. Bull. 2000,23, 1005–1007. [CrossRef] [PubMed]
14.
Mehmood, N.; Zubair, M.; Rizwan, K.; Rasool, N.; Shahid, M.; Ahmad, V.U. Antioxidant, antimicrobial and
phytochemical analysis of Cichorium intybus seeds extract and various organic fractions. Iran J. Pharm. Res.
2012,11, 1145–1151. [PubMed]
15.
Pushparaj, P.N.; Low, H.K.; Manikandan, J.; Tan, B.K.; Tan, C.H. Antidiabetc effects of Cichorium intybus in
streptozotocin-induced diabetic rats. J. Ethnopharmacol. 2007,111, 430–434. [CrossRef] [PubMed]
16.
Ripoll, C.; Schmidt, B.; Ilic, N.; Poulev, A.; Dey, M.; Kurmukov, A.G. Antinflammatory effects of a
sesquiterpene lactone extract from chicory (Cichorium intybus L.) roots. Nat. Prod. Commun.
2007
,2,
717–722.
17.
Das, S.; Vasudeva, N.; Sharma, S. Cichorium intybus: A concise report on its ethnomedicinal, botanical, and
phytopharmacological aspects. Drug Dev. Ther. 2016,7, 1–12.
18.
Miller, M.C.; Duckett, S.K.; Andrae, J.G. The effect of forage species on performance and gastrointestinal
nematode infection in lambs. Small Rumin. Res. 2011,95, 188–192. [CrossRef]
19.
Wesołowska, A.; Nikiforuk, A.; Michalska, K.; Kisiel, W.; Chojnacka-Wójcik, E. Analgesic and sedative
activities of lactucin and some lactucin-like guaianolides in mice. J. Ethnopharmacol.
2006
,107, 254–258.
[CrossRef] [PubMed]
20.
Nayeemunnisa, A. Alloxan diabetes-induced oxidative stress and impairment of oxidative defense system
in rat brain: Neuroprotective effects of Cichorium intybus.Int. J. Diabetes Metab. 2009,17, 105–109.
21.
Gürbüz, I.; Üstün, O.; Ye¸silada, E.; Sezik, E.; Akyürek, N.
In vivo
gastroprotective effects of five Turkish folk
remedies against ethanol-induced lesions. J. Ethnopharmacol. 2002,83, 241–244. [CrossRef]
22.
Gilani, A.H.; Janbaz, K.H. Evaluation of the liver protective potential of Cichorium intybus seed extract on
acetaminophen and CCl4-induced damage. Phytomedicine 1994,1, 193–197. [CrossRef]
23.
Kim, J.H.; Mun, Y.J.; Woo, W.H.; Jeon, K.S.; An, N.H.; Park, J.S. Effects of the ethanol extract of
Cichorium intybus on the immunotoxicity by ethanol in mice. Int. Immunopharmacol.
2002
,2, 733–744.
[CrossRef]
24.
Behnam-Rassouli, M.; Aliakbarpour, A.; Hosseinzadeh, H.; Behnam-Rassouli, F.; Chamsaz, M. Investigating
the effect of aqueous extract of Cichorium intybus L. leaves on offspring sex ratio in rat. Phytother. Res.
2010
,
24, 1417–1421. [CrossRef] [PubMed]
25.
Süntar, I.; Küpeli-Akkol, E.; Keles, H.; Yesilada, E.; Sarker, S.D.; Baykal, T. Comparative evaluation of
traditional prescriptions from Cichorium intybus L. for wound healing: Stepwise isolation of an active
component by
in vivo
bioassay and its mode of activity. J. Ethnopharmacol.
2012
,143, 299–309. [CrossRef]
[PubMed]
26. Al-Snafi, A.E. Medicinal importance of Cichorium intybus—A review. IOSR J. Phram. 2016,6, 41–56.
27.
Ksouri, R.; Megdiche, W.; Debez, A.; Falleh, H.; Grignon, C.; Abdelly, C. Salinity effects on polyphenol
content and antioxidant activities in leaves of the halophyte Cakile maritima.Plant Physiol. Biochem.
2007
,45,
244–249. [CrossRef] [PubMed]
28.
Montefusco, A.; Semitaio, G.; Marrese, P.P.; Iurlaro, A.; de Caroli, M.; Piro, G.; Dalassandro, G.; Lenucci, M.S.
Antioxidants in varieties of chicory (Cichorium intybus L.) and wild poppy (Papaver rhoeas L.) of southern
Italy. J. Chem. 2015. [CrossRef]
29.
Queslati, S.; Karray-Bouraoui, N.; Attia, H.; Rabhi, M.; Ksouri, R.; Lachaal, M. Physiological and antioxidant
responses of Mentha pulegium (Pennyroyal) to salt stress. Acta Physiol. Plant 2010,32, 289–296. [CrossRef]
30.
Bourgou, S.; Kchouk, M.E.; Bellila, A.; Marzouk, B. Effect of salinity on phenolic composition and biological
activity of Nigella sativa.Acta Hortic. 2010,853, 57–60. [CrossRef]
Plants 2017,6, 38 9 of 9
31.
Cik, J.K.; Klejdus, B.; Hedbavny, J.; Baˇckor, M. Salicylic acid alleviates NaCl-induced changes in the
metabolism of Matricaria chamomilla plants. Ecotoxicology 2009,18, 544–554.
32.
Alonso-Amelot, M.E.; Oliveros-Bastidas, A.; Calcagno-Pisarelli, M.P. Phenolics and condensed tannins in
relation to altitude in neotropical Pteridium spp. A field study in the Venezuelan Andes. Biochem. Syst. Ecol.
2004,32, 969–981. [CrossRef]
33.
Li, Y.; Gao, J.; Zhang, L.; Su, Z. Responses to UV-B exposure by saplings of the relict species Davidia involucrata
Bill are modified by soil nitrogen availability. Pol. J. Ecol. 2014,62, 101–110. [CrossRef]
34.
Stankovi´c, M.; Topuzovi´c, M.; Soluji´c, S.; Mihajlovi´c, V. Antioxidant activity and concentration of phenols
and flavonoids in the whole plant and plant parts of Teucrium chamaedrys L. var. glanduliferum Haussk.
J. Med. Plants Res. 2010,4, 2092–2098.
35.
Ksouri, R.; Megdiche, W.; Falleh, H.; Abdelly, C. Influence of biological, environmental and technical
factors on phenolic content and antioxidant activities of Tunisian halophytes. C. R. Biol.
2008
,331, 865–873.
[CrossRef] [PubMed]
36.
Stankovi´c, M.S.; Petrovi´c, M.; Godjevac, D.; Daji´c-Stevanovi´c, Z. Screening inland halophytes from the central
Balkan for their antioxidant activity in relation to total phenolic compounds and flavonoids: Are there any
prospective medicinal plants? J. Arid. Environ. 2015,120, 26–32. [CrossRef]
37.
Liu, L.; Gitz, C.D.; McClure, W.J. Effects of UV-B on flavonoids, ferulic acid, growth and photosynthesis in
barley primary leaves. Physiol. Plant 1995,93, 725–733. [CrossRef]
38.
Abideen, Z.; Qasim, M.; Rasheed, A.; Adnan, M.Y.; Gul, B.; Khan, M.A. Antioxidant activity and polyphenolic
content of Phragmites karka under saline conditions. Pak. J. Bot. 2015,47, 813–818.
39.
Unal, B.T.; Aktas, L.Y.; Guven, A. Effects of salinity on antioxidant enzymes and proline in leaves of barley
seedlings in different growth stages. Bulg. J. Agric. Sci. 2014,20, 883–887.
40.
Alonso-Amelot, M.E.; Oliveros-Bastidas, A.; Calcagno-Pisarelli, M.P. Phenolics and condensed tannins
of high altitude Pteridium arachnoideum in relation to sunlight exposure, elevation, and rain regime.
Biochem. Syst. Ecol. 2007,35, 1–10. [CrossRef]
41.
Ganzera, M.; Guggenberger, M.; Stuppner, H.; Zidorn, C. Altitudinal variation of secondary metabolite
profiles in flowering heads of Matricaria chamomilla cv. BONA. Planta Med.
2008
,74, 453–457. [CrossRef]
[PubMed]
42.
Stankovi´c, M.S. Total phenolic content, flavonoid concentration and antioxidant activity of
Marrubium peregrinum L. extracts. Kragujevac J. Sci. 2011,33, 63–72.
43.
Zlati´c, N.M.; Stankovi´c, M.S.; Simi´c, Z.S. Secondary metabolites and metal content dynamics in
Teucrium montanum L. and Teucrium chamaedrys L. from habitats with serpentine and calcareous substrate.
Environ. Monit. Assess. 2017,189, 110. [CrossRef] [PubMed]
44.
Singleton, V.L.; Orthofer, R.; Lamuela, R.R.M. Analysis of total phenols and other oxidation substrates and
antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 1999,299, 152–178.
45.
Quettier, D.C.; Gressier, B.; Vasseur, J.; Dine, T.; Brunet, C.; Luyckx, M.C.; Cayin, J.C.; Bailleul, F.; Trotin, F.
Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and
flour. J. Ethnopharmacol. 2000,72, 35–42. [CrossRef]
46.
Takao, T.; Watanabe, N.; Yagi, I.; Sakata, K. A simple screening method for antioxidant and isolation of
several antioxidants produced by marine bacteria from fish and shellfish. Biosci. Biotechnol. Biochem.
1994
,
58, 1780–1783. [CrossRef]
47.
Kumarasamy, Y.; Byres, M.; Cox, P.J.; Jaspars, M.; Nahar, L.; Sarker, S.D. Screening seeds of some Scottish
plants for free radical scavenging activity. Phytother. Res. 2007,21, 615–621. [CrossRef] [PubMed]
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