2023.08.03.31
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Evaluation of the phytochemical content and
antioxidant properties of different extracts of Achillea odorata L.

Hind Amira1
, Hassiba
Benabdallah*1
, Walid Mamache1
, Fatima Benchikh1
,
Roumaissa Ounis1
, Bensouissi Chawki2
and Smain Amira1







1- Laboratory of Phytotherapy Applied to Chronic
Diseases, Department of Biology and Animal Physiology, Faculty of Nature and
Life Sciences, University of Setif 1, 19000, Algeria. [email protected] [email protected]; [email protected] ;
[email protected] ;
[email protected] ; [email protected]
2-Biotechnology Research Center (CRBt), UV 03 BP E73, Nouvelle
Ville Ali Mendjli, Constantine, Algeria. [email protected] ()
*Corresponding author: [email protected]
Available from:
http://dx.doi.org/10.21931/RB/2023.08.03.31ABSTRACT
The present study reports the chemical
profile and antioxidant activity of Achillea odorata L. aerial parts of
different extracts obtained using five solvents of varying polarities (decocted,
hydro-ethanol, hydro-methanol, hydro-ethyl acetate and hydro-chloroform
extracts). The chemical profile and the antioxidant potential were evaluated by
measuring the total phenolic content, total flavonoid content, total tannin
content, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS)
radical-scavenging, reducing the power of FeCl3 and phenanthroline assays.
The highest phenolic content and antioxidant potential were generally shown by
the methanol extract (AME). AME exhibited the highest values for total
polyphenols content (424.94 ± 0.58 mg gallic acid equivalent /g of dry extract),
ABTS (13.46 ± 1.153 µg/mL), reducing the power of FeCl3 (9.918 ± 0.019
µg/mL) and phenanthroline (6.22 ±0.04 µg/mL). These results suggest that A.
odorata L. possesses potential antioxidant compounds that could be
tested as drug candidates against oxidative-related pathological processes.
Keywords: Achillea odorata L., Antioxidant
activity, Free radical scavenging, Polyphenols, Reducing power.
INTRODUCTION
Reactive
oxygen species (ROS), byproducts of aerobic metabolism, are critical players in
several crucial signaling pathways 1. Robust scientific data,
however, demonstrates that high ROS concentrations are harmful and are to blame
for DNA mutations, lipid oxidation, and protein peroxidation. According to
research, oxidative stress may play a significant role in developing or
progressing some diseases, including cancer, diabetes mellitus, inflammatory
diseases, and neurodegenerative disorders 2. Humans have discovered
natural medicines, primarily plants, for various ailments. As a result, the
development of new drugs has long been based on natural compounds 3.
Although the pharmaceutical sector has extensively used
organic chemistry, natural products are more in demand these days to replace
synthetic ones with unfavorable side effects. This has led scientists to revert
to nature as a critical source for developing new, safer, and more efficient
molecules 4. Because of their antioxidant activity, antioxidants
such as polyphenols, vitamins, terpenoids, and nitrogen compounds can
significantly impact the body when consumed as fruits, vegetables, aromatic and
medicinal plants 5. These large quantities of organic substances,
which plants produce, are crucial parts of our food. These substances are
valued by nutritionists, consumers, and researchers because they are suitable
for human health 6.
One of the
Asteraceae family’s more recent evolutionary genera is Achillea odorata
L. This plant is a significant medicinal plant since it has been used for
various medical conditions, including as a spasmolytic, choleretic, wound
therapy, and anti-inflammatory agent 7. More than 100 species of the
Achillea genus, which is widespread worldwide, have been identified 8.
Most of its distribution is in North America, various regions of Europe, the
Middle East, the Mediterranean, Eastern and Western Asia, Australia and New
Zealand 9,10. This genus has been used as an anti-inflammatory 11,
anti-diabetic 12, anti-rum, stimulating tonic, and for
ethnoveterinary treatments (cattle, poultry and dogs) 13,14.
Moreover,
previous papers reported the antioxidant properties 14,15 of the Achillea
species’ plant extracts and essential oils. Due to the various antioxidant
potentials of chemicals with varying polarities, both extraction yield and
antioxidant activity of plant-origin extracts primarily rely on the solvent 16.
Comparative research is necessary to choose the best solvent for each substrate
to maximize antioxidant activity. While using different polarity substances,
one can learn more in-depth details about the characteristics of the extracts.
The present study was undertaken to realize for the first time a
comparative analysis of the phytochemical content and antioxidant properties of
different extracts from A. odorata L. Five solvents were chosen:
methanol 50%, ethanol 50%, ethyl acetate 50%, chloroform 50%, and decocted water
as extract solvents which permit comparison of the antioxidant properties among
the polarity based solvent extracts of medicinal plants. We should note that the
ABTS and the reducing power activities of A. odorata L. have
never been documented in the literature.
MATERIALS AND METHODS
Plant material
A. odorata L. plant was
collected in June 2020 from Jijel (Algeria). The identification and taxonomic
classification of the plant were developed by Professor Smain Amira, Department
of Animal Biology and Physiology, University Setif 1, Algeria. The voucher
number 302 AO 16/06/20 Jij/SA was deposed at the Phytotherapy Applied to
Chronic Diseases laboratory. After being thoroughly cleaned, the plant’s aerial
part was dried naturally. The air-dried plant was crushed into an excellent
powder using an electric grinder.
Chemicals
All chemicals were of analytical grade and purchased
from Sigma (Paris, France). All chemicals were analytical-grade and were bought
from Sigma and Aldrich.
Plant extraction
The current investigation used decoction
and four different solvents to extract chemicals with various polarities. Green
chemistry’s typical empirical techniques were used to obtain the decocted
extract. To remove polar chemicals, methanol and ethanol were used. Semi-polar
chemicals were extracted using ethyl acetate, and nonpolar compounds were
extracted using chloroform.
Preparation of decocted extract (ADE)
50 g of the plant powder was boiled for 10
minutes in 500 mL of water and then cooled. The homogenate was obtained and
filtered through muslin after the decoction period. The resulting filtrate
underwent further filtration using filter paper before being put onto plates
and baked in an oven at 38°C 17. The resulting extract (ADE) was
then kept at 4°C in a covered bottle to prevent any molecular breakdown from
light.
Preparation of hydro-ethanol (AEE), hydro-methanol
(AME), hydro-ethyl acetate (AEAE) and hydro-chloroform (AChE) extracts
(50%)
The extracts of A. odorata L.
were prepared using different solvents with different polarities (methanol,
ethanol, ethyl acetate, chloroform) according to Markham 18
However, 50 g of ground material of the
plant was macerated in 50% solvent (ethanol, methanol, ethyl acetate,
chloroform) at room temperature and protected from light for 72 hours, with
maximum agitation. Then, the mixture is filtered and evaporated using a rotary
evaporator (BÜCHI) at a temperature of 40-50°C. This mixture was then dried in
an oven at 38°C. The extracts obtained (AEE, AME, AEAE, AChE, respectively) were
stored at 4°C in a protected bottle.
Phenolics and pigments content determination
Calculation of the plant extraction yield
The yield of the plant extraction is the
ratio between the weight of the extract and the weight of the plant to be
treated. After extracting the active ingredients from the A. odorata L.
plant, the yield is calculated using the following formula (1):

Where: Y= yield of the extract in
percentage. WE = weight of the extract in grams. Wp = weight of the plant in
grams.
Determination of total phenolic content
Total phenolic content (TPC) was assessed
by Folin Ciocalteu reagent 19. A volume of 100 µL of each extract
was mixed with 500 µL of Folin Ciocalteu reagent (diluted 10 times). After 4
min, 400 µL of 7.5% Na2CO3 solution was added. The
completed mixture was agitated and allowed to react at room temperature in the
dark for one hour. The reaction mixture’s absorbance was then measured at 760
nm. A gallic acid standard curve determined different extracts’ total amounts
of polyphenols. The results were represented as mg of gallic acid equivalent
(GAE) for each gram of dried plant extract.
Determination of total flavonoid content
Total
flavonoid content (TFC) was determined using aluminum chloride assay 20.
Briefly, 1 mL of AlCl3 (2%) was combined with 1 mL of each examined extract or
standard (quercetin). After 10 min of incubation, the absorbance against a
prepared blank was measured at 430 nm. Using a calibration curve for quercetin,
the results were reported as mg of quercetin equivalent per gram of dry plant
extract weight (mg QE/g DE).
Determination of total tannin content
Total tannin
content (TTC) was estimated using Folin Ciocalteu reagent (FCR), taking tannic
acid as standard 21. The reaction mixture was made by combining 0.5
mL of extract, 2.5 mL of 10% Folin-Ciocalteu reagent mixed with water and 2.5 mL
of 7.5% Na2CO3. The same procedure was prepared blank
without a sample. The samples were incubated in a thermostat at 45°C for 45 minutes. The absorbance was
determined by using a spectrophotometer at λmax = 765 nm. The samples were produced in triplicate for each analysis,
and the mean absorbance value was calculated. The calibration curve was created by repeating the
procedure using the tannic acid standard solution. The concentration of the TTC
was determined (mg/mL) from the calibration line based on the observed
absorbance. The total tannin content is expressed in mg tannic acid equivalent
(TAE) per gram of dry extract.
Evaluation of in vitro antioxidant
activities
ABTS radical scavenging activity
The
antiradical activity of the extracts using the ABTS radical was evaluated
according to the method of Re et al. 22.
Briefly, ABTS cation was generated by reacting 2 mM of ABTS with 2.45 mM of K2S2O8.
The mixture was then kept at ambient temperature for 16 hours in complete
darkness. Distilled water was used to dilute the ABTS+ solution to an
absorbance of 0.700 at 734 nm before use. After mixing 40 µL of each sample at
various concentrations with 160 µL of the ABTS+ solution for 10 minutes, the
absorbance at 734 nm was measured. The percentage of inhibition was calculated
using the following formula (2). Results were compared to the standards BHA and
α-tocopherol.

Reducing the power of FeCl3
To estimate
the possible reducing power capacity of A. odorata L., the
protocol of Oyaizu 23 was used. A total of 40 µL of phosphate buffer
(pH = 6.6; 0.2 M) was added to 10 µL of sample solution (extract or standard)
in methanol, with various concentrations, followed by the addition of 50 µL of
potassium ferricyanide (1%). At 50°C, the mixture was incubated for 20 min. The
absorbance was then measured at 700 nm after adding 50 µL of trichloroacetic
acid solution (10%), 40 µL of distilled water, and 10 µL of ferric chloride
solution (0.1%). The obtained results were expressed as A0.50 values
and compared with α-tocopherol.
Phenanthroline assay
To evaluate
the phenanthroline antioxidant assay, we used the protocol described by
Szydlowska-Czerniaka et al. 24. A total of 50 µL of FeCl3
(0.2%) was mixed with 10 µL of various dilutions of sample solutions (extract
or standard) in methanol, followed by the addition of 30 µL of O-phenanthroline
(0.5%) and adjusted with 110 µL of methanol. The absorbance at 510 nm was
measured following 20 min of incubation at 30°C. The obtained results were
expressed as A0.50 values and compared with the used antioxidant
positive control (BHA).
Statistical
analysis
The results are represented
as the mean ± SD (standard deviation). The different IC50 and A0.5
values are calculated using the Graph pad Prism 7 software according to
the log (concentration) vs. normalization (absorbance) method. The analysis of
variance (one-way ANOVA) was performed to compare the different values, followed
by the Tukey test (multiple comparisons). The difference is considered
statistically significant at the risk of 5% (P<0.05).
RESULTS
Phytochemical study of the extracts
Extraction yield
Extraction yields obtained for the various
extracts are shown in Table 1. AME extract demonstrated the most significant
yield of 16.6%, then AQE (16.1%) and AEE (14.9%). While AEEA and AChE showed a
lower extraction yield. When combined, the yield using polar solvents (AME, ADE
and AEE) was 45.6%. In comparison, the yield from nonpolar solvents (AEAE and
AChE) was only 12.2%, suggesting that A. odorata L. extracts had
more polar components.
Total polyphenols, flavonoids and tannins
content
The present
study quantified the contents of total phenolic, flavonoids, and tannins in
extracts of A. odorata L. The obtained results are
summarized in Figure 1. The methanol extract recorded the highest total
phenolic content with a value of 424.94 ± 0.58 mg GAE/g DE, followed by the ADE
and AEE with a value of 372.88 ± 1.91 and 349 ± 0.03 mg GAE/g DE, respectively.
At the same time, the ethyl acetate and chloroform extracts gave the lowest
value (113.78 ± 0.33 and 97.01 ± 0.14 mg GAE/g DE), respectively.

Figure 1. Yield extraction,
total phenolics, flavonoids and tannins contents of A. odorata’s
extract. ADE:
decocted extract, AME: hydro-methanolic extract, AEE: hydro-ethanolic extract, AEAE:
hydro-ethyl acetate extract, AChE: hydro-chloroformic extract, GAE: gallic acid
equivalent, QE: quercetin equivalent, TAE: tannic acid equivalent, DW: dry
weight. results are expressed as means ± sd (n=3). ****; p≤0.0001 compared to AME
or AEE.
Furthermore, the AEE showed the highest
levels of flavonoids and tannins (154.71 ± 0.35 mg QE/ g DE and 72.62 ± 0.30 mg
TAE/ g DE, respectively) followed by the AME (98.33 ± 0.85 mg QE/ g DE and
47.15 ± 0.32 mg TAE/ g DE, respectively), then ADE with value close to the
latter extract (86.01 ± 1.02 mg QE/ g DE and 34.87 ± 0.93 mg TAE/ g DE,
respectively). Moreover, AEAE and AChE showed a small amount of flavonoids and
tannins, as shown in Table 1.
Antioxidant activity
ABTS
radical cation decolorization assay
According to
the present work, the ability of A. odorata L. extracts to
scavenge the radical ABTS is shown in Figure 2. Compared to standards, all
extracts exhibited high antioxidant activity and in the following order: AME
(IC50 = 13.46 µg/mL) > AEE (IC50 = 15.34 µg/mL) >
ADE (IC50 = 16.38 µg/mL) > AEAE (IC50 = 18.84 µg/mL)
> AChE (IC50 = 20.25 µg/mL). The AME and AEE showed a significant
activity similar to standards (BHA and α-tocopherol).

Figure 2. Free radical scavenging (ABTS) activity of A.
odorata L. extracts. ADE: decocted extract, AME:
hydro-methanolic extract, AEE: hydro-ethanolic extract, AEAE: hydro-ethyl
acetate extract, AChE: hydro-chloroformic extract. Data were presented as IC50
means±SD (n=3) (ns: not significant, *; P≤0.05, **; P≤0.01, ***; P≤0.001,
****; P≤0.0001) vs BHA as standard.
Reducing power (reducing power of FeCl3
and phenanthroline)
The reducing
power of FeCl3 and phenanthroline activity of the extracts was
determined and compared to standard (BHT or α-tocopherol). The results were in the following order of activity for
reducing power of FeCl3: AME (IC50 = 9.91 µg/mL) > ADE (IC50
= 19.50 µg/mL > AEE (IC50
= 20.02 µg/mL) > AEAE (IC50
= 24.38 µg/mL) > AChE (IC50
= 39.61 µg/mL) (Figure 3), and
in the following order for phenanthroline assay: AME (IC50 = 6.22 µg/mL) > ADE (IC50
= 8.50 µg/mL > AEE (IC50
= 12.16 µg/mL) > AEAE (IC50 = 24.05 µg/mL) > AChE (IC50
= 24.75 µg/mL) (Figure 4). The
highest reducing power was shown by AME, which is the best and relatively
closer compared with standards.

Figure 3. Reducing power of FeCl3 of A.
odorata L. extracts. ADE: decocted extract, AME: hydro-methanolic
extract, AEE: hydro-ethanolic extract, AEAE: hydro-ethyl acetate extract, AChE:
hydro-chloroformic extract. Data were presented as IC50 means±SD
(n=3) (ns: not significant, ****; P≤0.0001) vs α-tocopherol
as standard.

Figure 4. Phenanthroline activity of A. odorata L. extracts. ADE: decocted
extract, AME: hydro-methanolic extract, AEE: hydro-ethanolic extract, AEAE:
hydro-ethyl acetate extract, AChE: hydro-chloroformic extract. Data were
presented as IC50 means±SD (n=3) (ns: not significant, *; P≤0.05,
**; P≤0.01, ****; P≤0.0001) vs BHA as
standard.
DISCUSSION
Achillea genus is used traditionally for
treating many disorders and is viewed as a popular plant in many countries in
the world and may be considered a functional food; this feature has been
attributed to its high bioactive content and diversity of terpenes, flavonoids,
coumarins, phenolic acids7. The distinctive structures and
functional groups of plant phytochemicals are known to affect their polarity
and solubility in the different extraction solvents used. Thus, the yields
would be dependent on such characteristics. Methanol is frequently reported to
generate the highest yields when used as an extracting solvent 25.
Moreover, the extraction yield for most extract plants was directly
proportional to the polarity of solvents. For instance, methanol and chloroform
gave the highest and lowest extraction yields, respectively. This finding
agrees with Do et al. 26, who demonstrated the superiority of polar
solvents with hydroxyl groups such as methanol and water in extracting solid
mass from plant samples. In this study, the overall crude extraction yield,
which ranged from 4.0% to 16.6% (Table 1), was lower than those reported by
Boutennoun et al. 11 (18.2%) and higher than the yield
extraction found by Benali et al. 14 (0.37-1.04%) for the
essential oil of the same plant. This could be explained by the different
concentrations and types of solvents and extraction methods used in those
studies.
A. odorata L. extracts were examined for their total
polyphenols, flavonoids and tannins; the results revealed that they have high
amounts of polyphenols. The highest polyphenol level has been identified in
AME. These findings were lower than the previous study realized by Boutennoun et
al. 11. These results are in agreement with those of Spingo et
al. 33, who suggested that polar solvents are the
best-extracting media for polyphenols and flavonoids, which may be due to an
increase in polarity of flavonoids upon conjugation through glycosides with
hydroxyl groups that enhances their solubility in polar solvents. The chemical
makeup of the extraction media used significantly impacts the extraction yield
of phenolics and their antioxidant efficiency 28. These factors
include plant part/type, genetic makeup of the species, agroclimatic
conditions, harvest timing, and post-harvest processing. The type of extraction
solvent chosen significantly impacts the accuracy of botanical chemicals
separated from plant material 29. For the extraction of phenolic substances
in a prior study, polar and nonpolar solvents like methanol, ethanol, acetone,
propanol, ethyl acetate, and water were frequently employed 30. The
solubility of these phenolic compounds in the solvent used for the extraction
procedure affects the recovery of phenolics from plant sources.
Polyphenolic compounds are the primary
constituents present in the plant and mainly possess a potent antioxidant
activity 34. Hence, the antioxidant activities of the plant sample
were evaluated via three complementary techniques: ABTS radical cation
decolorization, reducing power and phenanthroline assays to give better
conclusive results of the antioxidant properties of the extracts.
The IC50 of ABTS scavenging
assay and A0.5 of phenanthroline and reducing power assays of each
extract were compared to the IC50 and A0.5 of α-tocopherol or BHA as standards. The parameters IC50 and A0.5
are used to express the antioxidant activity of a molecule. The lower value of
IC50 or A0.5 means that the molecule has the highest
antioxidant activity. The antioxidants can be classified according to the
following scale: The extract that has an IC50 or A0.5 of
less than 50 μg/mL is a potent antioxidant, the one that
has an IC50 or A0.5 between 50- 100 μg/mL is a powerful antioxidant, the sample that has an IC50
or A0.5 between 101-150 μg/mL is a medium antioxidant, while the
extract with an IC50 or A0.5 greater than 150 μg/mL is a weak antioxidant 35. The figures show that all
extracts tested in the present study are potent antioxidant agents since their
IC50 and A0.5 are less than 50 μg/mL.
The
oxidation of ABTS by potassium persulfate results in the stable radical known
as the ABTS reagent 36. At 734 nm, the ABTS blue/green chromophore
decolorizes due to being scavenged by antioxidant molecules. The drop in ABTS
absorbance in the presence of the tested extract shows the proportion of ABTS
inhibition. The capacity of antioxidant substances to generate stable radicals
is frequently linked to their scavenging abilities 37. BHA and α -tocopherol are potent antioxidants, especially with significant
antiradical activity, according to several authors 38,39. The AME
and AEE showed considerable activity similar to standards (BHA and α-tocopherol). Typically, extracts produced using solvents with high
polarity exhibit more excellent radical scavenging activity. The polar phase of
the extract contributes to the inhibition of ABTS radical through simple
electron transfer 40. These high radical scavenging potential
results could be attributed to the oxygenated monoterpenes dominated in
Achillea species 14. The oxygenated monoterpenes possess a solid
radical scavenging potential 41.
A reducing
power test is frequently used to evaluate an antioxidant’s capacity to give an
electron. Numerous studies have demonstrated a clear link between some plant
extracts’ reducing power and antioxidant properties. In the reducing power of
FeCl3, the presence of antioxidant reductants in the extracts causes
a reduction of the Fe3+/ferricyanide complex to its ferrous form. It is
monitored by measuring the formation of Perl’s Prussian blue of ferrous form at
a wavelength of 700 nm 35. In this study, AME showed the highest
reducing power, which is the best and relatively closer than standards. It can
be observed from these results that the change in solvent polarity alters its
ability to dissolve antioxidant compounds, and it is evident from the above
finding that the polar extracts have more activity than the nonpolar ones.
However, this is in agreement with the report of Hertog et al. 43
that methanol is a widely used and effective solvent for the extraction of
antioxidants and phenolic compounds. Comparing our data with published
literature is tricky because studies on the antioxidant activity of A. odorata
L. are minor and almost unobtainable.
The composition of phenolic
compounds in the samples with many hydroxyl groups may cause a difference in
antioxidant activity. These groups were in charge of the significant
antioxidant action. In addition, these activities were influenced by the
extraction method and circumstances (temperature, polarity, solvent
concentration, and time) 44.
CONCLUSION
A. odorata L. collected from the Jijel region (Algeria) showed a considerable quantity of total phenolic
compounds and flavonoids. A significant capacity of A. odorata L. extract to scavenge the ABTS free radical was observed. Moreover, a strong
reducing power was exhibited. The most potent antioxidant activity was obtained
for AME. Our work also reveals that different solvents have different extraction
efficiencies, which suggests that when addressing the antioxidant potential of
any sample, the solvent effect should be considered. It is generally known that the antioxidant capacity of plants is
primarily due to phenolic chemicals found in the plant kingdom. A. odorata L.
extracts are good sources of antioxidants; it is, therefore, possible to
valorize this plant in the pharmaceutical and food industries.
ACKNOWLEDGMENTS
The Algerian Ministry of Higher Education and
Scientific Research (MESRS) has supported this research.
CONFLICT OF INTEREST The authors declare that they have no conflicts of interest. | AUTHOR CONTRIBUTION Hind Amira: Conceptualization; Data curation; Formal
analysis; Investigation; Methodology; Software; Writing-original draft;
Writing-review & editing. Hassiba Benabdallah: Supervision; Validation;
Visualization; Writing-review & editing. Walid Mamache: Co-Supervision;
Validation; Visualization; Formal analysis; Writing-review & editing.
Fatima Benchikh: Conceptualization; Visualization; Writing-original draft;
Writing-review & editing. Chawki Bensouici: Data curation; Methodology;
Resources; Validation. Romaissa Ounis: Data curation; Formal analysis;
Investigation. Smain Amira: Project administration; Validation; Visualization;
Writing-review & editing
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Received: 28 May 2023/ Accepted: 15 July 2023 / Published:15
September 2023
Citation: Amira H, Benabdallah H, Mamache W,
Benchikh F, Ounis R, Chawki B,
Amira S. Evaluation
of the phytochemical content and antioxidant properties of different extracts
of Achillea odorata L. Revis Bionatura
2023;8 (3) 31. http://dx.doi.org/10.21931/RB/2023.08.03.31