Algeria
Chlef, Algeria
Algeria
Chlef, Algeria
Chlef, Algeria
Introduction. Myrtus communis, Aristolochia longa, and Calycotome spinosa are medicinal plants frequently used in Algeria. Some plants can cause a fragility of the erythrocyte membrane and lead to hemolysis. Therefore, we aimed to study the cytotoxicity of aqueous extracts from the aerial part of these species against red blood cells. Study objects and methods. The hemolytic effect was determined spectrophotometrically by incubating an erythrocyte solution with different concentrations of the aqueous extracts (25, 50, 100, and 200 mg/mL) at 37°C during one hour. In addition, we performed phytochemical screening and measured the contents of polyphenols and flavonoids. Results and discussion. After one hour of incubation of human red blood cells with the aqueous extracts at different concentrations, the hemolysis percentage showed a significant leak of hemoglobin with A. longa (68.75 ± 6.11%; 200 mg/mL), the most toxic extract followed by C. spinosa (34.86 ± 5.06%; 200 mg/mL). In contrast, M. communis showed very low cytotoxicity (20.13 ± 3.11%; 200 mg/mL). Conclusion. These plants are sources of a wide range of bioactive compounds but their use in traditional medicine must be adapted to avoid any toxic effect.
Myrtus communis, Aristolochia longa, Calycotome spinosa, folk medicine, phenolic compounds, alkaloids, hemoglobin, cell toxicity, hemolytic activity
INTRODUCTION
Medicinal plants are an important pool of molecules
with therapeutic potential for drug innovation [1].
According to Estella et al., vulgarization of traditional
herbal remedies is confronted with many predicaments
due to the lack of information on their therapeutic and
toxicological properties to guarantee their rational
use [2]. According to Calixto [3], plants contain
hundreds of phytotherapeutic agents with adverse effects
and some of them are very toxic if inappropriately used.
In fact, Kharchoufa et al. have identified more than
89 toxic medicinal plants used as treatment in the North-
Eastern region of Morocco [4]. These plants contain
toxic compounds: alkaloids followed by glucosides,
terpenoids, proteins, and phenolics. Their toxicity can
lead to serious adverse reactions or interactions with
other plants. On the other hand, a misidentification
of plants can lead to a toxicity that may also result
from an uncontrolled or excessive use [5]. Therefore,
before formulating and marketing a herbal medicine,
appropriate scientific studies are essential, including
those into pharmacological properties, toxicity, and side
effects [6].
Algeria has more than 3000 species belonging
to several botanical families distributed all along
the Mediterranean, Saharan, and tropical regions.
Calycotome spinosa (L.) belongs to the Fabaceae family,
Aristolochia longa belongs to the Aristolochiaceae
family, and Myrtus communis belongs to the Myrtaceae
family [7]. Algeria is the only country that hosts
both species, M. communis (L.) in the North and
Myrtus novelli in the South [8]. For several centuries,
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M. communis has been used in folk medicine as
treatment for many diseases due to its broad spectrum
of pharmacological and therapeutic effects [9]. Species
of Aristolochia are known for their toxicity and pose
potential health risk associated with their content of
aristolochic acids [10].
Since these species are widely used in the littoral
zone of Algeria in traditional and folk medicine,
there is a need for research into their toxicity. In this
context, we aimed to evaluate the hemolytic effect on
human erythrocyte cells induced by aqueous extracts
of M. communis (Rayhan), A. longa (Bereztem), and
C. spinosa (Guendoul). The last two species have been
rarely studied.
STUDY OBJECTS AND METHODS
Medicinal plants Myrtus communis (L.), Calycotome
spinose (L.), and Aristolochia longa (L.) were collected
in many areas in the littoral of Algeria (March 2018),
namely Damous (Tipaza), Benni Haoua (Chlef), and
Bissa (Chlef) (Fig. 1). These species were identified
by Dr. Belhacini, a teacher and researcher at Hassiba
Benbouali University of Chlef (Algeria).
Preparation of aqueous extracts. For each species
we used dried and powdered plant aerial parts according
to traditional use in these areas, namely leaves for
M. communis and A. longa and leaves and flowers for
C. spinosa. Aqueous extracts were prepared by a
decoction of the plant material. In particular, 10 g of the
plant material was boiled with 100 mL of distilled water
for 15 min and then the solution was filtered and dried
at 39°C.
Phytochemical screening. Phytochemical tests were
performed on 5% infusion to detect certain secondary
metabolites according to Takaidza et al. and Behbahani
et al. [11, 12].
Determination of total phenol contents. A mixture
of 250 μL of Folin Ciocalteu phenol reagent, 50 μL of
the sample, and 500 μl of 20% Na2CO3 was prepared.
The volume was adjusted to 5 mL with distilled water
while shaking vigorously. After 30 min incubation,
absorbance was read at 765 nm. A calibration curve of
gallic acid (0–1 mg/mL) was done in parallel. The results
were expressed in mg of gallic acid equivalent/g of dry
matter (mg EAG/g DM) [13].
Determination of total flavonoid contents. The
flavonoid assay was performed according to the method
of Hmid et al. [14]. 1 mL of each extract was mixed with
1 mL of 2% AlCl3. After 10 min incubation, absorbance
was read at 430 nm. The flavonoid concentrations
were calculated using a calibration curve established
with quercetin (0–40 μg/mL) and expressed in mg of
quercetin equivalent/g of dry matter (mg EQ/g DM).
Hemolytic activity determination. A phosphate
buffered saline (PBS) solution with pH = 7.4 was
prepared by mixing the following compounds in
appropriate concentrations: Na2HPO4 (10 Mm), K H2PO4
(1.8 Mm), KCl (2.7 Mm), and NaCl (137 Mm) [15].
A concentration range for each extract (M. communis,
C. spinose, and A. longa) was prepared by diluting
in PBS: 25, 50, 100, and 200 mg/mL. An erythrocyte
suspension was prepared from the blood of a healthy
donor in a heparin tube. After centrifugation at
2400 rpm for 10 min, the plasma was removed and
the pellet was washed twice with PBS and then filled
up with the same volume of plasma removed. The
erythrocyte suspension was diluted 20 times with PBS.
Erythrocyte hemolysis assay. The hemolytic effect
test of the species studied was carried out according
to the method described by Haddouchi et al. and Guo-
Xiang and Zai-Qun [16, 17]. We mixed 2950 μL of the
erythrocyte suspension with 50 μL of aqueous extract
for each species in a hemolysis tube. The operation
was repeated three times for each concentration. The
tubes were incubated at 37°C for one hour. During this
period, 500 μL of each test was taken every 15 min
(in 15, 30, 45, and 60 min) and added to 1.5 mL of PBS
and then centrifuged again at 2400 rpm for 10 min. The
absorbance of the hemoglobin leak in the supernatant
was read at 548 nm against a blank containing PBS.
A negative control tube was prepared under the same
experimental conditions, 2950 μL of the erythrocyte
suspension and 50 μL of the PBS buffer solution. On
the other hand, a total hemolysis tube was prepared
Figure 1 1 – Myrtus communis; 2 – Calycotome spinose; 3 – Aristolochia longa
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containing 250 μL of the erythrocyte suspension and
4750 μL of distilled water. Each test was repeated
three times. The hemolysis rate of various extracts was
calculated as a percentage (%) of total hemolysis after
15, 30, 45, and 60 min of incubation, according to the
following formula:
% Hemolysis=A(extract at 60 min)-A(negative control
at 60 min)/A(Total hemolysis at 60 min) (1)
Statistical analysis. Statistical analysis was done
by One Way ANOVA. The data obtained were analyzed
using the student’s t-test. A P value less than 0.01 was
considered statistically significant.
RESULTS AND DISCUSSION
Phytochemical screening. The phytochemical
screening allowed us to highlight the presence of some
secondary metabolites (saponosides, tannins, alkaloids,
flavonoids, and anthocyanins). The phytochemical
tests carried out on the infused flowers and leaves of
the selected plants are shown in Table 1. The results
obtained after shaking the infusion for 15 min showed
that Myrtus communis and Aristolochia longa were
rich in saponosides because the foam was greater than
1 cm. In the Calycotome spinosa leave and flower
infusion, the foam was unstable in the order of a few
mm. The appearance of the orange and pink color after
the addition of isoamyl alcohol indicated the presence
of flavones in the C. spinosa leave and flower infusion.
The purplish pink color indicated the presence of
flavonones in the leaves of M. communis. In the A. longa
infusion, the result was negative. The precipitate in the
C. spinosa and M. communis infusions, which were
previously acidified with sulfuric acid, after adding
some drops of the Mayer reagent indicated the presence
of alkaloids. However, the test was negative for A. longa.
The appearance of a pink and red coloration after adding
ammonia to the HCl-infused A. longa and C. spinosa
indicated the presence of anthocyanins. However, this
secondary metabolite was absent in the M. communis
leave infusion.
Contents of total polyphenols and flavonoids.
The amount of polyphenols in the dry matter was
expressed in mg gallic acid equivalent (mg EAG/g
MS) and determined by the equation: y = 0.940x + b;
R2 = 0.981. T he a mount of flavonoids i n t he d ry m atter
was expressed in mg of quercetin equivalent (mg EQ/g
MS) and determined by the equation: y = 0.055x + b;
R2 = 0.996 (Table 2). The total polyphenol content in
the dry matter was 234.89 ± 0.80, 283.68 ± 0.60, and
346.27 ± 2.00 mg GEA/g for C. spinosa, A. longa,
and M. communis, respectively. The content of total
flavonoids in the dry matter was 10.50 ± 0.03, 34.86 ±
0.06, and 31.02 ± 0.19 mg EQ/g for A. longa, C. spinose,
and M. communis, respectively (Table 2).
Hemolytic activity. In the negative control tube
(tube containing only PBS and erythrocyte suspension),
the hemolysis rate was constant and did not exceed
2.77 ± 0.35% after one hour of incubation. On the
Table 1 Results of phytochemical screening tests for Myrtus communis, Calycotome spinose, and Aristolochia longa
Samples Saponoside Test Tanin Test Flavonoid Test Anthocyanin Test Alkaloid test
Myrtus
communis (leaves)
Foam > 1cm Catechin tannins Flavonones – +
Calycotome spinose
(leaves and flowers)
Foam 0.2 cm – Flavones – +++
Aristolochia longa
(leaves)
Foam > 1,8 cm Catechin tannins +/– + –
“+” Present, “++” Moderate presence, “+++” High presence, “+/–” Presence not evident, “–” Absent
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other hand, a total hemolysis of red blood cells was
clearly observed in the total hemolysis tube. Indeed, we
recorded a hemolysis rate that reached 99.86 ± 10.32% at
60 min.
For the aqueous extract of the M. communis leaves,
we observed a significantly low hemolysis rate during
the first 15 minutes (P < 0.01). The hemolysis rates
were 5.07 ± 0.21, 7.85 ± 1.20, and 12.57 ± 2.89% for
the concentration of 25 mg/mL; 6.04 ± 1.90 (P < 0.01),
6.46 ± 0.77, and 20.42% for 50 mg/mL; 8.05 ± 1.41,
11.32 ± 5.72, and 19.51 ± 6.71 for 100 mg/mL; and
12.01 ± 0.21, 12.22 ± 0.26, and 20.14 ± 3.11% (P < 0.01)
for 200 mg/mL, respectively, compared to total
hemolysis (Fig. 2).
For C. spinosa, we found a significant increase
in hemolysis rates over time (15, 30, 45, 60 min).
Also, the rates were considerably higher with higher
concentrations of the extract. For the concentrations
of 25 and 50 mg/mL, hemolysis rates ranged between
6.52 ± 3.78 and 17.12 ± 1.50%, as well as 7.50 ± 2.95
and 22.36 ± 2.12%, respectively. However, a significant
hemolytic effect was recorded in 100 (45 min) and
200 mg/mL (15, 45, and 60 min) of the C. spinosa
extract. This rate increased from 8.14 ± 1.23% at 15 min
to 23.61 ± 8.94% at 60 min in the presence of a
100 mg/mL concentration and from 24.44 ± 3.95% at
15 min to 34.86 ± 5.05% at 60 min in the presence of a
200 mg/mL concentration (Fig. 3).
For the extract of the A. longa leaves (Fig. 4), we
found an increase in hemolysis rates over time (15,
30, 45, and 60 min). As the concentration increased,
the percentage of hemolysis increased as well. At
concentrations of 25 and 50 mg/mL, a hemolysis
percentage ranged from 5 (15 min) to 6.71% (60 min)
and from 7.22 (15 min) to 18.47% (60 min), respectively.
The hemolysis rate was significant at 15 and 45 min
(P < 0.01).
On the other hand, we observed an important
hemolytic effect of the A. longa aqueous extract at
concentrations of 100 and 200 mg/mL. This rate went
Table 2 Polyphenol and flavonoid content in Myrtus
communis, Calycotome spinose, and Aristolochia longa
Species Polyphenols
mg GEA/g DM
Flavonoids
mg EQ/g DM
Myrtus communis
Calycotome spinosa
Aristolochia longa
346.27 ± 2.00
234.89 ± 0.80
283.68 ± 0.60
31.02 ± 0.19
34.86 ± 0.06
10.50 ± 0.03
TH: Total Hemolysis. NC: Negative Control.
The means of 3 replicates. P < 0.01. ** significant
Figure 2 Hemolytic effect of four concentrations
of Myrtus communis extract at 15, 30, 45, and 60 min
Time, min
Hemolysis, %
TH: Total Hemolysis. NC: Negative Control.
The means of 3 replicates. P < 0.01. ** significant
Figure 3 Hemolytic effect of four concentrations
of Calycotome spinosa extract at 15, 30, 45, and 60 min
Time, min
Hemolysis, %
TH: Total Hemolysis. NC: Negative Control.
The means of 3 replicates. P < 0.01. ** significant
Figure 4 Hemolytic effect of four concentrations
of Aristolochia longa extract at 15, 30, 45, and 60 min
Time, min
Hemolysis, %
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from 22.12 ± 1.95 (15 min) to 40.23 ± 9.13% (60 min)
and from 41.71 ± 0.75 (15 min) to 68.75 ± 6.11% (60 min),
respectively. This increase in hemolytic effect remained
inferior to total hemolysis.
Hemolytic effect of the plants studied at 60 min.
Fig. 5 shows the evolution of the hemolytic effect or the
leakage of Hb after 60 min for the A. longa, C. spinose,
and M. communis extracts at four concentrations (25, 50,
100, and 200 mg/mL) in a PBS buffer medium (pH 7.4)
containing an erythrocyte suspension incubated at 37°C,
compared to a negative control tube (PBS + suspension)
and a total hemolysis tube (distilled water + suspension).
TH: Total Hemolysis. NC: Negative Control. The
means of 3 replicates. P < 0.01. ** significant
The M. communis species showed a significantly low
hemoglobin leakage rate compared to the other species,
as well as a 20.14 ± 3.11% total hemolysis. This species
had a lesser effect on the cell membrane of erythrocytes
(P < 0.01). However, C. spinosa caused a significant
intermediate leakage of hemoglobin, compared to
M. communis and A. longa, at 200 mg/mL (60 min),
namely in the range of 34.86 ± 5.06% (P < 0.01).
Nevertheless, the most important cytotoxic effect on
red cells was produced by the aqueous extract of the
A. longa leaves, where the leakage rate was 68.75 ±
6.11% at 200 mg/mL (60 min) and close to total
hemolysis (P > 0.01), indicative of the species’ high
toxicity. These results are phenotypically observable in
the supernatant.
For millennia, humans have been searching for drugs
in barks, seeds, fruit organs and other parts of plants
to heal themselves and alleviate pain [18]. Nowadays,
several studies have been conducted on plants to create
new drugs and, to some extent, to evaluate their toxicity
and identify their components. Polyphenolic compounds
of M. communis L. extracts are grouped in three major
chemical classes: phenolic acids, tannins, and flavonoids
[19]. Our results of the phytochemical screening of
C. spinosa are consistent with those reported by Cherfia
et al., who identified polyphenols, flavonoids, alkaloids,
tannins, and saponosides [20]. The Aristolochia species
are a source of various active compounds such as
aristolochic acid, alkaloids (aporphines, protoberberines,
protopines), quinolines, amides, chlorophylls, terpenoids,
lignans, flavonoids, tetralones, and steroids [21].
The polyphenol and flavonoid contents that we
found in the M. communis leaves were higher than
those obtained by Bouaziz et al., who reported
157.70 ± 2.65 mg EAG/g MS and 2.64 ± 0.22 mg EQ/g
of dry matter [22]. In another study, the hydromethanolic
extract of C. spinosa leaves had a polyphenol content of
228.42 ± 8.86 and a flavonoid content of 4.87 ± 0.12 [23].
According to Djeridane et al., the methanolic extract of
A. longa contained 1.47 ± 0.20 mg/g EAG polyphenols
and 0.81 ± 0.02 mg/g EQ flavonoids [24]. Our results
were in agreement with Merouani et al., who found
396.88 ± 8.86 mg/g EAG polyphenols and 9.92 ±
0.23 mg/g EQ flavonoids [25].
Plants contain toxic compounds in high doses,
which makes the evaluation of their hemolytic power
indispensable for their correct use in traditional therapy,
as well as for choosing the right mode of administration
and preserving the integrity of membranes. According
to Haddouchi et al., the hemolysis test should be
performed even if a plant has a powerful antioxidant
power, since its use in traditional medicine and in
pharmacological preparations will be impossible in the
presence of their hemolytic effect, which is an indicator
of cytotoxicity [16]. “Free radicals induce several
effects on erythrocytes, such as hemolysis, fluidity
of the membrane, changes in morphometry and lipid
peroxidation, among others. Erythrocytes potentially
promoting the oxidative process are extremely sensitive
to oxidative damage because of the polyunsaturated
fatty acid content in their cell membranes and their high
content of oxygen and hemoglobin” [26].
Many secondary metabolites were revealed in
our extracts that may be a cause of cytotoxicity. We
found a major lysis of red blood cells treated with
C. spinosa, which was more prominent when treated
with Aristolchia, testifying to severe toxicity. We found
Figure 5 Hemolytic effect of four concentrations of Myrtus communis, Calycotome spinose,
and Aristolochia longa extracts at 60 min
60 min
Hemolysis, %
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very few studies on A. longa and C. spinosa, trying to
examine a relationship between the extracts’ chemical
composition and toxicity.
According to Bissinger et al., saponins – a secondary
metabolite identified in aqueous extracts of the plants
under our study – may lead to the stimulation of
hemolysis as well as to suicidal erythrocyte death [27].
Alkaloids are present in many plants which may be toxic
and affect human health [28]. Mahdeb et al. reported
that alkaloids are capable of disrupting the permeability
of the membranes of erythrocytes [29].
As stated by Galati and O’Brien, many adverse
effects were associated with dietary polyphenol
consumption or exposures such as hemolytic
anemia [30]. The authors added that before using these
polyphenols for therapy, they need to be assessed for
safety.
According to Grollman et al., the toxicity of
Aristolochia longa is due to a toxin that is a major
component of all Aristolochia species, namely
the aristolochic acid responsible for nephropathic
syndromes, although the therapeutic use of Aristolochia
has rarely taken into account its intrinsic toxicity
before [31]. These findings corroborate the study of
Touiti et al., which showed that Aristolochia longa was
incriminated in nephrotoxicity [32].
CONCLUSION
Some herbs used in traditional therapy in high
doses can reveal toxic properties and harm human
health. It appears essential to determine their hemolytic
capacity as a marker of toxicity for rational adaptation
to traditional therapy. We found that Aristolochia longa
and Calycotome spinosa caused significant lyses of
red blood cells and a potent leakage of hemoglobin.
Therefore, these species cannot be used without
control as a therapeutic or pharmacological tool to
treat diseases. Furthermore, it is important to perform
antitumoral tests on cancer cells with these plant
extracts or their chemical compounds to develop anticancer
drugs.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
CONTRIBUTION
L. Gadouche conceived and designed the analysis,
performed the biological experiments, and wrote
the paper. A. Zidane and K. Zerrouki contributed to
data analysis and revised the paper. K. Azouni and
S. Bouinoune performed the biological experiments. All
the authors revised the manuscript for publication.
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