The Influence of Altitude on the Polyphenols Content and Antioxidant
Capacity of Northern Moroccan 'Dellahia' Prickly Pear Juice
KOALAGA YEWAGA DRAMANE1,2,*, MESSAOUDI ZERHOUNE1, IBRIZ MOHAMMED2,
AIT HADDOU LHOUSSAIN1
1Laboratory of Pomology, Department of Arboriculture-Viticulture,
National School of Agriculture,
B.P. S/40, Meknes, 50001,
MOROCCO
2Laboratory of Vegetable Production and Agro-Industry, Department of Biology, Faculty of Sciences,
Ibn Toufail University,
University Campus, BP 133, Kenitra,
MOROCCO
*Corresponding Author
Abstract: - Moroccan cactus exhibits high genetic variability with several cultivars. The 'Dellahia' prickly pear
variety, prevalent in northern Morocco and noted for its green pulp, is among the least valued cactus varieties,
primarily consumed fresh. This study aimed to assess the impact of altitude on total phenolic acids and flavonoid
content (TPC and TFC) and the antioxidant activity of 'Dellahia' prickly pear juice from northern Morocco.
Significant differences in TPC ranged from 91.29 to 130.45 mg GAE/Kg of juice from the Mestassa and Wahran
sites (at 119 m and 482 m altitude, respectively). TFC also varied slightly, from 18.8 to 19.1 mg RE/Kg of juice.
Variations in antioxidant activity were evident in both DPPH• and ABTS+ assays, with DPPH• inhibition
percentages ranging from 8.85% to 19.14% and ABTS+ inhibition from 41.07% to 54.35%. However, the influence
of altitude on these parameters was inconclusive, as samples from higher altitudes did not consistently yield lower
or higher values. Other factors such as soil composition, sunlight, and farming practices may influence these
results.
Key-Words: - Opuntia ficus-indica, Dellahia, altitude, polyphenols, flavonoid, antioxidants, DPPH, ABTS.
Received: April 9, 2024. Revised: September 2, 2024. Accepted: September 23, 2024. Published: October 30, 2024.
1 Introduction
There are over 1,500 species and 130 genera in the
Cactaceaes family, 300 of which are in the Opuntia
genus, [1]. The prickly pear cactus, Opuntia ficus
indica, is found in the Mediterranean region, Central
and South America, Central and South Africa, the
Middle East, and India, [2], [3], [4]. Cactus has been
used fresh or processed for human consumption, as
well as a functional constituent for food and
pharmaceutical products due to the important content
of bioactive compounds like polyphenols, [5], [6],
[7], [8]. The edible pulp and pericarp of the fruit
might be soft green, greenish-white, canary yellow,
lemon yellow, red, cherry red, or purple, [9]. The
cactus pear crop has several ecological and economic
benefits. Unfortunately, Morocco's output suffers a
significant loss due to a lack of monetization
opportunities. Cactus pear is also gaining popularity
in numerous countries because of its ecological,
environmental, and socioeconomic benefits,
including erosion and desertification control, and
fruit-producing feed, [10]. Its ecological benefits are
due to its crassulacean acid metabolism, which
facilitates CO2 absorption at night, reducing water
loss during photosynthesis, [1], [4], [11]. Cactus pear
is suited for growth in marginal dry and semi-arid
environments because of its low water requirements
and high-water use efficiency ratio, [12], [13]. The
Food and Agriculture Organization recommends
cactus pear as a potential crop in light of global
climate change, [12], [14]. According to [15], cactus
pear is a low-input crop that can be grown
sustainably and yield fruits and cladodes that are
edible to both people and animals. There are many
different species of Moroccan cacti, and they exhibit
a high degree of genetic variation, [10]. They are
categorized according to the following: when they
flower (early or late), what color the blossoms are
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(yellow, orange, or pink), what color the fruit and
pulp are (green, yellow, orange, red, or purple), what
shape the fruit is (oval, round, or rectangular), what
their organoleptic characteristics are [16], and how
much antioxidant they contain, [17], [18]. The
‘Dellahia’ prickly pear, which is common in northern
Morocco, is distinguished by its green pulp. Because
of the poor oil content of its seeds, it is one of the
least valuable cactus kinds. As a result, its fruits are
mostly consumed raw. The purpose of this study is to
discuss the effect of altitude on total polyphenols and
flavonoid content, and antioxidant activity in the fruit
juice of this variety in northern Morocco to
reevaluate the many options for reducing the loss of
excess production.
2 Materials and Methods
2.1 Vegetal Materials
Prickly pear fruits were collected in August 2016
from four sites at different altitudes in northern
Morocco as shown in Table 1 (Appendix). The global
view of the study region and the sampling sites are
shown respectively in Figure 1 and Figure 2.
Fig. 1: View of the study region
Fig. 2: Sampling sites
2.2 Chemicals and Reagents
Folin-Ciocalteu reagent (2N), DPPH• (2,2-Diphenyl-
1-picrylhydrazyl), ABTS+ (2,2' Azinobis -3
Ethylbenzothiazoline 6 Sulfonic Acid), rutin, gallic
acid and methanol were obtained from Sigma Aldrich
Corp. (Merck KGaA, Darmstadt, Germany). Sodium
carbonate (Na2CO3), sodium nitrite (NaNO2), sodium
hydroxide (NaOH), potassium persulphate (K2S2O8),
aluminum chloride (AlCl3) and Trolox (6-hydroxy-
2aluminumtramethylchroman-2-carboxylic acid,
purity ≥ 97%) were purchased from Fisher Scientific
SA. (Boulevard Sebastien Brant - F67403 Illkirch
Cedex – France). All of the other reagents were of
analytical grade unless otherwise stated.
2.3 Determination of Total Polyphenols and
Flavonoid Content
2.3.1 Determination of Total Polyphenols
Content (TPC)
The total polyphenols content of samples (TPC) was
determined spectrophotometrically using the Folin-
Ciocalteu reagent method depicted by [19] with
minor modifications. Indeed, this reagent is reduced
to tungsten and molybdenum oxide in an alkaline
medium giving a blue color in the presence of
polyphenols. Briefly, the assay was carried out by
adding 1 mL of Folin-Ciocalteu reagent (50%) to 0.2
mL of sample extract into a 10 mL test tube. After
shaking well, the mixture, 0.8 mL of 7.5% sodium
carbonate solution (Na2CO3) was added to the
mixture. The final mixture was incubated in the dark
at room temperature for 30 minutes. Then, we
measured the absorbance at 765 nm with an S-22
UV/VIS spectrophotometer. A calibration curve was
established using gallic acid. The gallic assay is
prepared using various concentrations of gallic acid
solutions (from 0 to 1 µg/mL). The gallic range
undergoes the same treatment as the sample to be
analyzed. Total polyphenol content is determined
graphically using the gallic range calibration curve,
which is a straight line with the following equation :
Y = aX +b
Where :
- Y : measured absorbance
- X : total polyphenol concentration required.
- a and b : constants
Then, we expressed the total polyphenols content
(TPC) in mg gallic acid equivalent (mg GAE / Kg of
juice).
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2.3.2 Determination of Total Flavonoid Content
(TFC)
The total flavonoid content (TFC) of samples was
determined spectrophotometrically following the
method depicted by [19] with some minor
adjustments. This method is characterized by the
formation of a flavonoid-aluminum complex, whose
maximum absorbance is 430 nm. The assay was
carried out by adding 1 mL of sample extract to 4 mL
of double distilled water into a 10 mL test tube. Then,
we added 0.3 mL of 5% NaNO2 solution. After 5
minutes, we added 0.3 mL of 10% methanolic AlCl3
solution. At the 6th minute, we added 2 mL of 1M
NaOH solution. The mixture was made up to 10 mL
with double distilled water, and the final mixture was
shaken very well. Then, we immediately measured
the absorbance at 510 nm with an S-22 UV/VIS
spectrophotometer. The calibration curve was
established using rutin. A calibration curve was
established using rutin. The rutin assay is prepared
using various concentrations of rutin solutions (from
0 to 1 µg/mL). The rutin range undergoes the same
treatment as the sample to be analyzed. Total
flavonoid content is determined graphically using the
rutin range calibration curve, which is a straight line
with the following equation :
Y = aX +b
Where :
- Y : measured absorbance
- X : total flavonoid concentration required.
- a and b : constants
Then, we expressed the total flavonoid content (TFC)
as mg rutin equivalent (mg RE/Kg of juice).
2.4 Determination of Antioxidant Activity
2.4.1 Determination of Antioxidant Activity by
the DPPH Method
Free radical scavenging activity of the methanolic
extracts was determined spectrophotometrically
following the DPPH• stable radical method depicted
by [20] with some minor adjustments. When reacting
with an antioxidant compound, the DPPH• radical is
reduced by donating hydrogen. This reduction is
featured by color changes, from deep violet to dark
yellow. Briefly, we added 3.9 mL of a freshly
prepared methanolic DPPH• solution (0.1M) to 0.1
mL of samples or positive control of Trolox. We used
an equal amount of methanol (4 mL) as a negative
control. We performed all measurements in triplicate.
After incubating at room temperature in the dark for
30 min, we measured the absorbance at 515 nm with
an S-22 UV-Vis spectrophotometer. Then, we
calculated the DPPH• inhibition percentage as the
absorbance decrease of the antioxidant samples
relative to the control. We determined the Trolox
equivalent antioxidant capacity (TEAC) by using the
equation established from linear regression after
plotting known solutions of Trolox (20–800 μM). At
last, we expressed the antioxidant activity as the
DPPH• free radical inhibition percentage according
to the following formula:
% Inhibition (DPPH•) = [(ODcont - ODsamp)/ODcont]×100
With:
- ODcont: Optical Density (absorbance) of the control
(pure methanol)
- ODsamp: Optical Density (absorbance) of the sample
The effective concentration required to inhibit
50% of the free radical (IC50) is calculated using the
equation of the curve obtained by plotting the
different inhibition percentages of free radical DPPH•
as a function of different sample concentrations of
total polyphenols contents. Generally, the equation of
this curve is linear. We expressed the IC50 in mg/mL
of juice.
2.4.2 Determination of Antioxidant Capacity by
the ABTS Method
The antioxidant capacity was also determined using a
second method based on the ABTS+ scavenging
potential as depicted by [21] with some minor
adjustments. First, we generated the ABTS+ radical
by reacting 7 mM ABTS and 2.45 mM potassium
persulphate (K2S2O8). After incubation in the dark at
room temperature for 16 hours, we diluted the
solution with 80% methanol to obtain an absorbance
of 0.70 ± 0.02 at 734 nm. Then, we added 3.9 mL of
this ABTS+ solution to 0.1 mL of the sample. The
mixture was carefully stirred, and incubated at room
temperature for 6 min. Then, we immediately
measured the absorbance of the reactive mixture at
734 nm with an S-22 UV-Vis spectrophotometer and
compared it to the antioxidant power of Trolox used
as a reference. We performed all determinations in
triplicate. We determined the inhibition percentage
of ABTS+• free radical as the decrease in absorbance
of the samples over the control. We also determined
the Trolox equivalent antioxidant capacity (TEAC)
by using the equation established from linear
regression after plotting known solutions of Trolox
(20–800 μM). At last, we expressed the antioxidant
activity as the ABTS+• free radical inhibition
percentage according to the following formula:
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%Inhibition (ABTS+) = [(ODcont- ODsamp)/ODcont]×100
With:
- ODcont: Optical Density (absorbance) of the control
(pure methanol)
- ODsamp: Optical Density absorbance of the sample
The effective concentration required to inhibit
50% of the free radical (IC50) for the ABTS+ assay
was determined by the same graphical method
described previously for the DPPH• assay.
2.5 Statistical Analysis
We used SPSS 20 software and Microsoft Excel 2019
to perform descriptive statistical analysis (based on
the calculation of the mean and standard deviation for
each parameter studied), analysis of variance (one-
way ANOVA test of variation: "sites"), and
comparison of means. We used the Student-Newman-
Keuls test to determine significant differences
between means with a 95% confidence interval (P =
.05).
3 Results and Discussions
3.1 Total Polyphenols (TPC) and Total
Flavonoid Contents (TFC)
Total polyphenols and flavonoid contents (TPC and
TFC) exhibited by the different samples studied are
displayed in Table 2 (Appendix). The calibration
curves of gallic acid and rutin are displayed in Figure
3 and Figure 4, respectively.
Phenolic compounds, also known as polyphenols,
are metabolic products widely distributed in plant
foods; they possess many biological and
pharmacological properties that may offer protection
against chronic diseases. These compounds have an
antioxidant effect superior to that of vitamins; they
can neutralize the effects of oxidative free radicals
[22]. For instance, studies have provided evidence
that oral administration of citric acid during insulin-
induced hypoglycemia can attenuate increases in
oxidative stress biomarkers in brain and liver tissue,
reduce increases in serum aminotransferases, and
provide histological protection against liver damage
[23]. In our study, as displayed in Table 2
(Appendix), we noticed no significant difference
between the samples regarding the TPC, with values
ranging from 91.29 ± 6.01 to 130.45 ± 12.31 mg
GAE/Kg of juice for Mestassa (119 m asl) and
Wahran (482 m asl) samples, respectively. This
means that the effect of altitude on the total
polyphenols content of 'Dellahia' prickly pear juice
from the study area is not significant. The values
found in our study are higher than those reported by
[24] for the Moroccan prickly pear cultivar Moussa
characterized by its yellow pulp (7.76 mg GAE/Kg of
juice) and Moroccan cultivar El Asri with red pulp
(15.34 mg GAE/Kg of juice). In recent studies, [25]
and [26] reported higher values of total phenolic
content in prickly pear juice ranging from 310.0 to
511 ± 2.9 mg GAE/Kg of juice and 130 to 180 mg
GAE/L of juice respectively.
Fig. 3: Gallic acid calibration curve
Fig. 4: Rutin calibration curve
3.1.1 Total Polyphenols Content (TPC)
Investigations on red and yellow cultivars from the
Kingdom of Saudi Arabia showed that the pulps
juices of red cactus contain high total phenolic
contents equivalent to 1065.15 mg GAE/100 ml of
juice, whereas the juices from the pulps of yellow
cactus have lower total phenolic contents (667.82 mg
GAE/100 ml of juice), [27]. These values remain
higher than those found in our study. In general, our
findings are in agreement with the literature
considering that previous studies reported a content
of phenolic compounds, in Opuntia spp. juice,
ranging from a minimum of about 22 mg GAE/Kg to
a maximum of about 660 mg GAE/Kg, [4], [26],
[28], [29], [30], [31]. Fruits such as grapes, apples,
pears, cherries, and berries contain up to 200-300 mg
of polyphenols per 100 g of fresh weight. Products
made from these fruits also contain significant
amounts of polyphenols, [32], [33]. The TPC of
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‘Dellalia’ prickly pear juice in the present study
was less than that of the other juices such
as turnip juice (772 mg/L), red grape juices
(1728 mg/L), red wine (1869 mg/L) [34], and
pomegranate (1284 to 9476 mg GAE/L of juice). In
light of these values, prickly pear and its by-products
remain interesting sources of TPC.
3.1.2 Total Flavonoid Content (TFC)
Flavonoids constitute the largest part of polyphenols
(2/3), and phenolic acids such as gallic acid and
caffeic acid represent the remaining part (1/3), [35].
Flavonoids are classified into several groups,
anthocyanins, flavonols, flavones, and flavanones
being the most important, [36]. Multiple types of
flavonoids have been reported in the Opuntia cactus,
their kinds and contents differ depending on the
variety and degree of ripening, [37]. Today, the
properties of flavonoids are widely studied in the
medical field for their anti-viral, anti-tumor, anti-
inflammatory, anti-allergic, and anti-cancer activities,
[38].
In our study, flavonoids constitute less than 1/5
of total polyphenols. As displayed in Table 2
(Appendix), we noticed a significant difference
between the samples regarding their total flavonoid
content. The samples from Tizakhte (highest altitude,
713 m asl), show the highest total flavonoid content
(19.1 ± 0.1 mg RE/Kg of juice) followed by the
samples from Mestassa with 18.9 ± 0.2 mg RE/Kg of
juice (lowest altitude, 119 m asl). The samples of
Wahran and Boujibar situated at respectively 482 and
572 m asl have the same flavonoid content (18.8 ±
0.01 mg RE/Kg of juice). Based on this study, we can
establish the effect of altitude on flavonoid content;
however, the variability found between samples from
different altitudes could also be due to other factors
not investigated in our study such as growing
conditions for example.
TFC ranging from 50.24 to 52.58 mg RE/Kg
respectively for Achefri and Amouslem cultivars at
the Arbaa Sahel site in southern Morocco (327 m asl)
and 67.02 to 69.95 mg RE/Kg for the same cultivars
respectively from Asgherkis site also in south
Morocco (709 m asl) have been reported by [10].
These values are higher than those found in our study
but confirm that the effect of altitude on the flavonoid
content of prickly pear fruits is significant.
Investigations on red and yellow cultivars from the
Kingdom of Saudi Arabia showed that the pulps
juices of red cactus contain high total flavonoid
contents equivalent to 159.49 mg RE/100 ml of juice,
whereas the juices from the pulps of yellow cactus
have lower total phenolic contents (80.35 mg RE/100
ml of juice), [27]. These values remain higher than
those found in our study. In recent studies, higher
values of TFC in different cultivars of prickly pear
juice have been reported ranging from 47 to 87 mg
QE/Kg of juice, [25], [31]
In general, the TFC reported in this study was
lower than previously reported values in cactus pear
fruits [25], [29], [39], [40], probably because we
processed only the pulp without the skin, which
should show a higher phenolic content. This
explanation is in agreement with data reported in the
literature on the polyphenol composition of the skin
and pulp of several fruits [17], considering that
polyphenols might tend to accumulate in the dermal
tissues of the plant body due to their potential role in
protection against UV radiation, acting as attractants
in fruit dispersal, and as chemical defense against
pathogens and predators, [41]. The TFC of ‘Dellalia’
prickly pear juice in this study was less than that
of apple juice (92 mg RE/L of juice) [42], but pretty
similar to pomegranate juice TFC (14,45 to 56.99 mg
RE/L of juice), [43]. In light of these values, prickly
pear and its by-products remain interesting sources of
TFC.
3.2 Antioxidant Activity
Antioxidants are compounds that protect cells from
the oxidative effects of reactive oxygen species, and
an imbalance between these reactive oxygen species
and antioxidants results in oxidative stress. Oxidative
stress can cause cellular damage associated with a
variety of diseases, including diabetes, cancer,
cardiovascular disease, neurodegenerative disorders,
and aging. Oxidative stress can also damage many
biological molecules, with proteins and DNA
molecules being important targets of cellular damage.
Antioxidants protect cells from damage caused by
these free radicals by interfering with the radical-
generating systems and enhancing the function of
endogenous antioxidants, [44]. Phenolic compounds,
and polyphenols in particular, are natural molecules
with numerous physiological properties, given their
ability to protect cells against damage caused by free
radicals, [45], [46]. From a dietary point of view,
their intake has a beneficial effect on human health,
improving intestinal inflammation, [47], [48] and
indirectly interfering with specific signaling proteins,
which mediate gene regulation in response to
oxidative stress and inflammation, [49], [50].
In general, in this work, the antioxidant activity
with ABTS+• was higher than that obtained with
DPPH• radical (Table 3, Appendix) with a percentage
of inhibition of ABTS+• radical ranging from 41.07 ±
4.47 and 54.35 ± 0.37 respectively for Tizakhte (713
m asl) and Mestassa (119 m asl) samples while the
percentage of inhibition of the DPPH- radical ranged
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from 8.85 ± 1.13 to 19.14 ± 2.73 respectively for
Boujibar (572 m asl) and Tizakhte (713 m asl)
samples. These tests showed that the richest sample
in total phenolic content was not the one that showed
the highest value in free radical scavenging activity
against both free radicals ABTS+• and DPPH•. The
different behavior against both free radicals, which
was also observed in other fruits’ extracts, could be
explained by the fact that many antioxidants react
rapidly while other radicals may react slowly, or even
be inert, to DPPH• due to their steric inaccessibility,
[51]. Overall, the results obtained are of the same
order as those previously reported [17], [51], [52],
but do not confirm that fruits with the highest level of
total polyphenols have the greatest free radical
scavenging capacity. Indeed, this apparent
relationship between total polyphenols and
antioxidant capacity was not found in some Mexican
Opuntia fruits, [28].
In addition, a pairwise correlation between TPC,
TFC, antioxidant activity, and altitude was performed
to obtain an overall perspective of the free radical
scavenging capacity and respective chemical
components on the one hand, and the effect of
altitude on the different parameters on the other hand
(Table 4, Appendix). The Pearson correlation test
was used for this purpose. This correlation test
showed that the chemical parameter with a significant
correlation with the ABTS+• test was total flavonoid
(R=0.885). In contrast, the ABTS+• and DPPH• test
values were not significantly correlated with TPC
(correlation coefficients of 0.411 and 0.400,
respectively). Our results are in contrast to the data
reported in the literature by [53] who showed that the
correlations between ABTS+• and DPPH• tests with
TPC and TFC were also high, demonstrating that
both tests can be considered to measure the free
radical scavenging capacity of these fruits. The same
author reported that the chemical parameter with the
highest correlation with the ABTS+• test was TPC
(0.9818), while the DPPH• test had the highest
correlation with TFC (0.9735).
The effective concentration to inhibit 50% of the
free radical (IC50) was calculated using the equation
of the curve obtained by plotting the different
percentages of free radical inhibition as a function of
TPC sample concentrations. No significant
differences were noticed regarding the IC50 values for
the ABTS+• test. The values found ranged from 90 ±
13 to 120 ± 16 mg GAE/g of juice for Mestassa (119
m asl) and Tizakhte (713 m asl) samples,
respectively. As for the IC50 of the DPPH• test, a
significant difference was noticed between the
samples, with Tizakhte fruits being more effective
(0.222 ± 0.013 mg/mL juice) while Boujibar fruits
being the least effective (0.765 ± 0.034 mg/mL
juice). Overall, the IC50 values obtained from the
ABTS+• assay ranged from 0.090 ± 0.013 to 0.120 ±
0.016 mg/mL of juice for the Mestassa (119 m asl)
and Tizakhte (713 m asl) samples, respectively.
These values are lower than those obtained by the
DPPH• assay, demonstrating that all samples are
more efficient in free radical scavenging capacity by
the ABTS+• assay than by the DPPH• assay.
Overall, the correlation between the studied
parameters and altitude was not significant according
to the Pearson correlation test at 95% and 99%
confidence intervals. As shown in Table 4
(Appendix), the correlation coefficients between
altitude and the studied parameters ranged from
0.038 and 0.557 for the inhibition percentage of
ABTS+• and DPPH•, respectively. In light of these
results, the effect of altitude on TPC and TFC, and
antioxidant activity could not be determined.
4 Conclusions
In summary, our study confirmed that prickly pear
fruit juice is an important source of polyphenols and
flavonoids. Values found are relatively higher or
lower than those reported in the literature. A
significant difference in total polyphenols, and
flavonoid content was observed. Nevertheless, the
effect of altitude on these phytochemicals’ content
could not be established because samples from the
highest altitude did not present the lowest or highest
content of phytochemicals. For instance, Tizakhte
samples located at the highest altitude (713 m)
contain 110.96± 9.76 mg GAE/Kg of juice while
Boujibar, Wharan, and Mestassa samples respectively
located at 572m, 482m, and 119 m above sea level
contain respectively 109.66±6.79, 130.45±12.31, and
91.29±6.02 mg GAE/Kg of juice. As for free radical
scavenging activity, the same observation was made
using both tests, ABTS+ and DPPH•: a significant
difference was noticed between samples but this
difference is not relevant to the altitude. Indeed, the
Pearson correlation test shows that the parameters
studied in our assay are not correlated to the altitude.
Several factors could probably explain this lack of
correlation as growth conditions like the influence of
dew on crops from high altitude, that make them
grow at similar conditions as if they were at sea level.
Other factors as soil composition or sunlight might
probably influence these parameters. Further
investigations should be carried out with a disposal
able to master the influence of external factors.
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Acknowledgments:
The authors extend their appreciation to the National
School of Agriculture of Meknes and the Ibn Toufail
University of Kenitra.
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Contribution of Individual Authors to the
Creation of a Scientific Article (Ghostwriting
Policy)
Conceptualization : D.Y.K., Z.M., M.I. and L.A.H;
Data curation : D.Y.K., L.A.H, and Z.M. ; Formal
analysis : D.Y.K., Z.M., and M.I. ; Investigation :
K.Y.D., Z.M., and L.A.H. ; Project administration :
Z.M., and M.I. ; Methodology : D.Y.K., Z.M., and
L.A.H. ; Resources : Z.M., M., and D.Y.K. ;
Software : Z.M., and D.Y.K. ; Supervision : Z.M.,
and M.I. ; Validation : Z.M., and M.I. ; Writing—
original draft : D.Y.K. All authors
have read and agreed to the published version of the
manuscript.
Sources of Funding for Research Presented in a
Scientific Article or Scientific Article Itself
No funding was received for conducting this study.
Conflict of Interest
The authors declare that they have no conflict of
interest.
Creative Commons Attribution License 4.0
(Attribution 4.0 International, CC BY 4.0)
This article is published under the terms of the
Creative Commons Attribution License 4.0
https://creativecommons.org/licenses/by/4.0/deed.en_
US
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APPENDIX
Table 1. Geographic coordinates and climatic characteristics of the study sites
Altitude (m)
Latitude
Mean Temperature (°C)
Annual Rainfall (mm)
Tizakhte
713
35.00597
10 – 30
700 – 800
Boujibar
572
35.01616
10 – 30
600 – 700
Wahran
482
35.03564
10 – 30
500 – 600
Mestassa
119
35.10460
10 – 30
400 – 500
Table 2. Total Polyphenols and Flavonoid contents (TPC and TFC) (Mean ± SD; n = 3)
Values in the same line followed by the same letter are not significantly different with a 95% confidence interval.
Table 3. Inhibition percentage and IC50 for DPPH• and ABTS+ assays (Mean ± SD; n = 3)
Mestassa
Wahran
Boujibar
Tizakhte
% Inhibition DPPH•
10.29 ± 1.37a
16.4 ± 3.52b
8.85 ± 1.13a
19.14 ± 2.73b
% Inhibition ABTS+
54.35 ± 0.37a
54.17 ± 2.42a
45.95 ± 5.70a,b
41.07 ± 4.47b
DPPH• IC50 (mg/mL)
0,326 ± 0,017a
0,634 ± 0,019b
0,765 ± 0,034c
0,222 ± 0,025d
ABTS+ IC50 (mg/mL)
0,090 ± 0,013a
0,094 ± 0,090a
0,090 ± 0,011a
0,120 ± 0,016a
Values on the same row followed by the same letter are not significantly different with a 95% confidence interval
Table 4. Pearson Correlation matrix for TPC, TFC, free radical scavenging capacity, and altitude
% Inhibition
DPPH•
TPC
% Inhibition
ABTS+
TFC
Altitude
IC50_ABTS+
IC50_DPPH•
% Inhibition
DPPH•
1
TPC
0.400
1
% Inhibition
ABTS+
0.179
0.411
1
TFC
0.074
0.316
0.885**
1
Altitude
0.557
0.510
0.038
0.091
1
IC50_ABTS+
0.693*
0.101
0.335
0.405
0.509
1
IC50_DPPH•
0.343
0.406
0.860**
0.776**
0.122
0.509
1
* P-value = .05; statistically significant correlation at the 95% confidence level
** P-value = .01; statistically significant correlation at the 99% confidence level
Mestassa
Wahran
Boujibar
Tizakhte
Total Phenol Content (TPC)
91.29 ± 6.02 a
130.45 ± 12.31 b
109.66 ± 6.79 c
110.96 ± 9.76 c
Total Flavonoid Content (TFC)
18.97 ± 0.2 a,b
18.81 ± 0.1 a
18.80 ± 0.1 a
19.11 ± 0.1 b
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