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Bioactive ingredients of different extracts of Vitex agnus-castus L. Fruits from

Morocco and their antioxidant potential

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JJBS
Volume 14, Number 2, June 2021
ISSN 1995-6673
Pages 267 - 270
Jordan Journal of Biological Sciences

Bioactive ingredients of different extracts of Vitex agnus-castus

L. Fruits from Morocco and their antioxidant potential
Fatima El Kamari *, Driss Ousaaid, Amal Taroq, Yassine El Atki, Iman Aouam,
Badiaa Lyoussi, and Abdelfattah Abdellaoui.
Laboratory of Physiology Pharmacology and Environmental Health, Department of Biology, Faculty of Sciences Dhar Mehraz, Sidi
Mohamed Ben Abdellah University, B.P. 1796, Atlas, Fez, Morocco

Received: April 4, 2020; Revised: August 22, 2020; Accepted: September 11, 2020

Abstract

The current study aimed to examine the effect of different solvents on phenols and flavonoid contents and evaluate the
antioxidant activities of different extracts. At first, the Soxhlet extracts were performed with four solvents (ethanol,
methanol, ethyl acetate, and water) and were examined for their polyphenolic content, flavonoid content, and antioxidant
potentials using three assays (DPPH, FRAP, TAC). The dosage of phytochemical compounds (polyphenolic and flavonoid
contents) revealed that the highest values were established in ethanol extract (p<0.05). Additionally, the strongest
antioxidant activity measured by TAC and DPPH assays was established in ethanol extract with 400±00 mg AAE/g DW and
0.36 ± 0.07mg/mL, respectively, while the methanol extract showed the best antioxidant activity as measured by FRAP with
an IC 50 value of 2.98 ± 0.2 mg/mL; the lowest value was observed in ethyl acetate extract. The Vitex fruits possess
remarkable antioxidant potential, which may enhance their protective effects.

Keywords: Vitex agnus- castus, Antioxidant activity, Flavonoids, Polyphenols.

Arokiyaraj et al., 2009; Asdadi et al., 2015), while the

1. Introduction aromatic leaves of this plant are used as a spice.
V. agnus-castus L contain different bioactive
The failure of antioxidant defense systems is associated ingredients such as Iridoid glycosides, Flavonoids,
with oxidative stress, which induce the overproduction of Diterpenes and Essential fatty acids (Dugoua et al., 2008).
reactive oxygen species (ROS) (McCord, 2000). ROS V. agnus-castus is locally named “Angarf-lkrwaa” in the
reacts with different molecules such as membrane lipids, Imouzzer Ida Outanane region; the plant used as a
proteins, and DNA leading to the pathogenesis and sedative, antispasmodic, and an aphrodisiac (Abdelhai
progression of various diseases including cancer, Sijelmassi, 1991).
cardiovascular diseases, diabetes, tumors, rheumatoid The chemical contents and antioxidant capacities of
arthritis, and epilepsy (Halliwell and Guterridge, 2007; Ho different extracts of Vitex agnus-castus were determined
et al., 1992). Thus, there is a growing interest in finding and compared.
natural substances with antioxidant properties. Medicinal
herbs are considered as a good source of bioactive 2. Materials and Methods
compounds. They contain a large variety of functional
substances such as, polyphenols, flavonoids, vitamins, 2.1. Plant material
which provide protective effects (Hamli et al., 2017;
Rodríguez et al., 2013). Fruits of VAC were collected from Khenifra area
Vitex agnus-castus L. (VAC) is a medicinal plant that (Latitude: 32°56′05″ Nord; Longitude: 5°39′42″ Ouest;
grows in the Mediterranean region, Europe, and Asia; altitude: 827 m), (Middle Atlas, Morocco) in June-
(Ahangarpour et al., 2016). It belongs to the Verbenaceae October 2015 (flowering period). Professor A. Bari as a
family, commonly known as the chaste tree botanist identified our plant material (Department of
(Yushchyshena and Tsurkan, 2014). The uses of this Biology, FSDM, USMBA, Fez (Morocco).
medicinal herb are shown to have many biological 2.2. Extract preparation
activities. The fruits of this plant have been used to treat Before extraction, the Vitex fruits were washed and
various female conditions such as uterine cramps, then dried. Next, the dried fruits were powdered. The
menstrual disorders, lactation, and acne (Chhabra and extraction was performed with four solvents (ethanol,
Kulkarni, 2011). By-products of Vitex (essential oils and methanol, ethyl acetate, and water), and the solid to liquid
extracts) possess several biological activities including ratio was 1/20 using Soxhlet extractor for 8 h. The rotary
antimicrobial and antifungal activities (Ahmad et al., 2016; evaporator was used to concentrate all extracts then stored
in a refrigerator at 4°C.

*
Corresponding author e-mail: [email protected].
268 © 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 2

2.3. Dosage of total polyphenolic content (TPC) Table 1: Yield, total phenolic content and total flavonoids of
different extracts
The determination of TPC of extracts was assessed by
the method described by Jadouali et al., (2018) and Yield Phenols Flavonoids
detailed by Hamli et al., (2017) using Folin-Ciocalteu. Extract
(%) (mg GAE/g DW) (mg RE/g DW)
Results of TPC were calculated as mg GAE/g DW.
a
Ethanol 7.2 62.66 ± 2.5 58.16 ± 1.3 a
2.4. Determination of Total flavonoid content (TFC)
Methanol 6.3 46.66 ± 2.6b 31.7 ± 0.7b
To quantify TFC, we have chosen a modified Zhishen d
Ethyl acetate 2.04 21.50 ± 1.8 12.08 ± 1.1c
et al., (1999) method as detailed by Hamli et al., (2017).
Results were expressed as mg RE/g DW. Water 24 50.46 ± 1.2cb 16.07 ± 0.81d
2.5. Total antioxidant capacity test (TAC) a: comparison between the ethanol extract and all extracts, b:
comparison between the methanol extract and all extracts, c:
The test chosen to determine the TAC of extracts is comparison between the ethyl acetate extract and all extracts, d:
based on the method proposed by Prieto et al., (1999). comparison between the water extract and all extracts.
Briefly, each extract (25 µL) was appended to 1 mL of
Extraction process of active ingredients was assessed
phosphomolybdate solution. The color intensity was read
at wavelength 695 nm after incubating the mixture reaction by different solvents. As shown in (Table 1), the quantity
at 95 °C for 90 min used ascorbic acid as the standard of phenols of various extracts, measured by Folin–
Ciocalteu method, varied significantly from 21.50 ± 1.8 to
calibration. Results were expressed as mg AAE/g DW.
62.66 ± 2.5 mg GAE/g DW.
2.6. DPPH assay Results of flavonoids ranged from 12.08 ± 1.1 to 58.16
To examine the capacity to quench free radicals, we ± 1.3 mg RE/g DW. It is clear that the ethanol extract
have chosen DPPH assay as described by Wu et al., significantly contained the highest value of flavonoids
(2003). 0.1 mL of the extract/standard and 1.5 mL of (58.16 ± 1.3 mg RE/g DW), while ethyl acetate extract
DPPH solution (0.1 mmol) were mixed, then the mixture (12.08 ± 1.1) established the lowest value of TFC.
was intubated 30 min in the darkness. Decline in the 3.2. Antioxidant activities
intensity of coloration produced was read at 517 nm. The
DPPH scavenging activity was estimated by the following 3.2.1. Total antioxidant capacity (TAC)
equation: Results are documented in Figure 2; they revealed that
%Inhibition = [(A C –A S )/A C ] ×100 the best value of TAC was established in ethanol extract
Where Ac is the absorbance of the control, and As is the with a value equal to 408.33 ± 4.33(mg Eq AAE/g DW),
absorbance of the sample. BHT served as a positive control. while the ethyl acetate extract have the lowest antioxidant
capacity (147.4 ± 2.04 mg Eq AAE/g DW).
2.7. FRAP assay
Total antioxidant capacity(m g AAE/gDW)

The FRAP of extracts was examined using the 500

technique of Oyaizu (OYAIZU and M., 1986). Briefly, a Ethan.E
200 µL of the sample, 500 µL of phosphate buffer (0.2M, 400 Methan.E
pH 6.6), and 500 µL of potassium ferricyanide [K 3 Fe (CN) Ethyl.Ac.E
300 b
6]1% were mixed. Then, 500 µL of Trichloroacetic (TCA) d Water.E
10% was added and mixture was incubated during 20 min 200 c
at 50°C. The supernatant (2, 5 mL) after centrifugation,
500 µL of deionized water, and 100 µL of FeCl3 (10%) 100
were mixed. 700 nm was the wavelength used to read the
absorbance. The outcomes were deducted as the dose of 0
extract inhibiting 50% of FRAP.
.E
n.E

.E

.E

2.8. Statistical analysis

Ac
an

er
ha

at
eth

yl.
Et

W
h
M

Et

Statistical analysis was carried out by ANOVA one-

way followed by Tuckey-test, using the Graph Pad Prism 5 Figure 1. TAC of different extracts of V. fruits.
(Microsoft Software). Differences at P < 0.05 were
3.2.2. Antioxidant ability by DPPH
considered significant. The outcome was also subjected to
multivariate analysis (principal component analysis). Results of DPPH assay are presented in Table 2. The
IC 50 values of all tested samples through the DPPH
3. Results scavenging ability test ranged from 0.36 ± 0.07 mg/mL to
2.04 ± 0.21 mg/mL. All extracts inhibited the DPPH
3.1. Effects of solvent on extraction yield, polyphenol, radical as follows: ethanol > water > methanol > ethyl
and flavonoid contents acetate. The highest activity was obtained from ethanol
extract (IC 50 = 0.36 ± 0.07 mg/mL), while the lowest
Table 1 resumes the results of yields. The extraction ability was registered from ethyl acetate extract (IC 50 =
yield of water (24%) was significantly the highest 2.04 ± 0.21 mg/mL). However, the BHT exhibited the best
followed by ethanol (7.2%), methanol (6.3%), and ethyl antioxidant ability compared to all samples studied (0.10 ±
acetate (2.04%). 0.03 mg/mL).
 © 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 2 269
3.2.3. Ferric reducing power assay numerous biological parameters such as genotype, organ,
The highest and lowest reducing power values were ontogeny, and environmental conditions. Besides, the
obtained by methanol and aqueous extracts (2.98±0.2 - solvent used, polymerization, and interaction of active
3.54±0.22 mg/mL), respectively. Also, the best ability substances govern the solubility of phenolic compounds
tested by FRAP assay was established by BHT with value (Ksouri et al., 2008).
of 0.12 ± 0.01 mg/mL. In the same context, the ethanol was the highly
extractible solvent for flavonoids, while the lowest value
Table 1: Antioxidant activities of different extracts
was established in the ethyl acetate extract. The minimum
Extract DPPH FRAP yield of flavonoids extracted (7.12 ± 0.08 µg QE / mg of
Ethanol 0.36 ± 0.07 d
3.29 ± 0.41c extract) was also documented in the ethyl acetate extract of
bd Vitex agnus castus fruits from Manisa, Turkey (Sarikurkcu
Methanol 0.71 ± 0.33 2.98 ± 0.2dc
et al., 2009). Thus, the yield of flavonoids from fruits is
Ethyl Acetate 2.04 ± 0.21a 3.14 ± 0.21bcd controlled by the nature of the solvent (Gao and Liu,
Water 0.52 ± 0.02 cd
3.54 ± 0.22abc 2005). Hirobe et al., (1997) reported that the bark root of
BHT 0.10 ± 0.03ed 0.12 ± 0.01e plant (Vitex) also contained flavonoids.
In summary, according to the results obtained from all
a: comparison between the ethanol extract and all extracts, b: extracts, ethanol was the most proper organic solvent to
comparison between the methanol extract and all extracts, c:
extract molecules with antioxidant potential (Table 1).
comparison between the ethyl acetate extract and all extracts, d:
comparison between the water extract and all extracts, e:
An extract is qualified to have a strong antioxidant
comparison between the BHT and all extracts. effect if the IC 50 < 5 mg/mL (Abdillah et al., 2015). The
methanolic extract of VAC fruits collected from Antalya
3.3. Correlation of Antioxidant Activities with possesses strong antioxidant activity (Gökbulut et al.,
Flavonoids and Phenols Content 2010), which is in accordance of our data. The existence of
To find the influence bioactive compounds (phenols some anthraquinones could be responsible for this high
and flavonoids) on the antioxidant potential of Vitex agnus reducing power (Yen and Chuang, 2000). Therefore, the
castus extracts, we studied the relation between the methanolic extract could be rich in this class of secondary
antioxidant tests and the content of bioactive ingredients metabolites.
(polyphenols and flavonoids) using correlation test. Based The TPC and TFC correlate positively with antioxidant
on the data presented in Table 3, positive correlation was ability evaluated by TAC and negatively with DPPH,
recorded between phytochemicals and their ability to FRAP. Therefore, the activity of eliminating free radicals
scavenge free radicals (P < 0.01). The IC 50 DPPH from extracts could be attributed to the content of phenols
negatively correlated with TFC (r2 = -0.87) and phenols and flavonoids. Phenol compounds can contribute to
(r2 = -0.96). antioxidant activity, and they have been considered as anti-
inflammatory, anticancer, anticholinergic enzymes ,
Table 3: Pearson correlation coefficients between compounds and
antiviral, and antibacterial agents (Atki et al., 2019; Banji
antioxidant activities.
et al., n.d.; Jaiswal and Thakur, 2017; Ojo et al., 2017;
Antioxidant Flavonoids (TFC) Phenols (TPC) Rakass, 2018; Sagbo et al., 2017; Shawarb et al., 2017).
activities The correlation linked the FRAP and the bioactive
CAT 0.89 ** 0.95 ** substances was not significant, which indicates that the
FRAP assay in these extracts measures the activity of
DPPH -0.87 ** -0.96 ** certain phytochemical classes other than phenols and
FRAP -0.44 -0.34 flavonoids (Yen and Chuang, 2000).
* Correlation is significant at the P<0.05 level.
5. Conclusion
** Correlation is significant at the P<0.01 level.

Bioactive ingredients and the assessment of antioxidant

4. Discussion quality of Vitex agnus castus collected from Khenifra were
conducted in the present work. The outcome shows that
The present work was designed to evaluate, for the first chemical composition was affected by nature of solvent,
time, the TPC, TFC, and antioxidant capacities of Vitex and ethanol is evaluated as a proper solvent for extraction
agnus castus fruits extracts, which grow in wild habitats in of molecules, which exhibits the best antioxidant ability.
Khenifra area, Morocco. Therefore, from outcomes The outcomes revealed the possible application of
obtained from different extracts, water gives the highest evaluated Vitex agnus castus fruits extracts as the best
yield in comparison with other solvents. Our findings are source of bioactive molecules with health benefits.
similar to those obtained by Sağlam et al., (2007). In the
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