Vitex agnus-castus L.: Main Features and Nutraceutical Perspectives

Abstract

Medicinal plants are used worldwide due to their lower risk of side effects and eco-friendly, cost-effective production when compared to chemical drugs, encouraging researchers to further exploit the therapeutic potential of the former. One of the most popular medicinal plants is Vitex agnus-castus L., grown in tropical and sub-tropical regions, to which different health benefits have already been attributed. In this perspective article, the in vitro and in vivo therapeutic properties of V. agnus-castus L. have been analyzed and reviewed with a special focus on its health-promoting effects and potential nutraceutical applications.

Full text

Perspective
Vitex agnus-castus L.: Main Features and
Nutraceutical Perspectives
Eliana B. Souto 1, 2, *, Alessandra Durazzo 3, Amirhossein Nazhand 4, Massimo Lucarini 3,
Massimo Zaccardelli 5, Selma B. Souto 6, Amelia M. Silva 7,8 , Patricia Severino 9, 10, 11 ,
Ettore Novellino 12 and Antonello Santini 12, *
1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências
da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
2CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
3CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Roma, Italy;
alessandra.durazzo@crea.gov.it (A.D.); massimo.lucarini@crea.gov.it (M.L.)
4Department of Biotechnology, Sari Agricultural Science and Natural Resource University, 9th km of Farah
Abad Road, Sari 48181 68984, Mazandaran, Iran; nazhand.ah@gmail.com
5CREA-Research Centre for Vegetable and Ornamental Crops, Via Cavalleggeri 25,
84098 Pontecagnano (Salerno), Italy; massimo.zaccardelli@crea.gov.it
6Department of Endocrinology of Braga Hospital, Sete Fontes, 4710-243 São Victor, Braga, Portugal;
sbsouto.md@gmail.com
7Department of Biology and Environment, University of Trás-os-Montes e Alto Douro (UTAD),
Quinta de Prados, P-5001-801 Vila Real, Portugal; amsilva@utad.pt
8
Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of
Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal
9Industrial Biotechnology Program, University of Tiradentes (UNIT), Av. Murilo Dantas 300,
49032-490 Aracaju, Brazil; pattypharma@gmail.com
10 Tiradentes Institute, 150 Mt Vernon St., Dorchester, MA 02125, USA
11 Laboratory of Nanotechnology and Nanomedicine (LNMED), Institute of Technology and Research (ITP),
Av. Murilo Dantas, 300, 49010-390 Aracaju, Brazil
12 Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy;
ettore.novellino@unina.it
*Correspondence: ebsouto@ff.uc.pt (E.B.S.); asantini@unina.it (A.S.); Tel.: +351-239-488-400 (E.B.S.);
+39-81-253-9317 (A.S.)
Received: 14 May 2020; Accepted: 10 July 2020; Published: 16 July 2020


Abstract:
Medicinal plants are used worldwide due to their lower risk of side effects and eco-friendly,
cost-effective production when compared to chemical drugs, encouraging researchers to further
exploit the therapeutic potential of the former. One of the most popular medicinal plants is
Vitex agnus-castus L.
, grown in tropical and sub-tropical regions, to which different health benefits
have already been attributed. In this perspective article, the
in vitro
and
in vivo
therapeutic properties
of V. agnus-castus L. have been analyzed and reviewed with a special focus on its health-promoting
effects and potential nutraceutical applications.
Keywords:
chaste tree; Vitex agnus-castus L.; bioactive compounds;
in vitro
studies;
in vivo
studies;
nutraceuticals; health-promoting properties.
1. Introduction
Medicinal wild plants and herbs have been considered worldwide for centuries as valuable tools
in the management of different diseases, due to their ease of use and improved cost-effectiveness
when compared to chemical remedies obtained from synthesis [
1
]. Plants have recently been exploited
Forests 2020,11, 761; doi:10.3390/f11070761 www.mdpi.com/journal/forests

Forests 2020,11, 761 2 of 16
for nutraceutical purposes, as they play a key role in the development of food and plant-derived
phytocomplexes with medicinal properties, to be used in health conditions as preventive or curative
tools [
2
–
23
]. A popular medicinal plant with recognized beneficial effects on human health is
Vitex agnus-castus L.
, belonging to the Lamiaceae family (formerly included in the Verbenaceae family),
native to the Mediterranean area and diffused in Europe, Asia, and North Africa [
24
,
25
]. It has been
used by people in Italy, Iran, Greece, and Egypt for over 2500 years, mainly to treat gynecologic
disorders [
26
]. It is a globally famous plant known by different names, e.g. Fruit de gattilier (French),
Sauzgatillo (Spanish), Mönchspfefferfrüchte (German), Panj-angosht (Persian), Frutto di Agnocasto
(Italian), and Chaste tree (English) [27].
The term agnus-castus combines the Greek (
Forests 2020, 11, x FOR PEER REVIEW 2 of 17
cost-effectiveness when compared to chemical remedies obtained from synthesis [1]. Plants have
recently been exploited for nutraceutical purposes, as they play a key role in the development of
food and plant-derived phytocomplexes with medicinal properties, to be used in health conditions
as preventive or curative tools [2–23]. A popular medicinal plant with recognized beneficial effects
on human health is Vitex agnus-castus L., belonging to the Lamiaceae family (formerly included in
the Verbenaceae family), native to the Mediterranean area and diffused in Europe, Asia, and North
Africa [24,25]. It has been used by people in Italy, Iran, Greece, and Egypt for over 2500 years, mainly
to treat gynecologic disorders [26]. It is a globally famous plant known by different names, e.g. Fruit
de gattilier (French), Sauzgatillo (Spanish), Mönchspfefferfrüchte (German), Panj-angosht (Persian),
Frutto di Agnocasto (Italian), and Chaste tree (English) [27].
The term agnus-castus combines the Greek ἀ meaning pure, chaste) and the Latin (castus from
“castitas” meaning chastity), repeating the term chaste for “pure”, which refers to the anaphrodisiac
properties of this plant and its use by monks to maintain celibacy (thus the “monk’s pepper”
synonym). The word “castus” has been used for centuries to remark further the meaning or purity
associated with this plant. Homer, the semi-legendary author of the Iliad and the Odyssey epic
poems, defines agnus-castus as a “tendril for braiding”, explaining the origin of the genus “Vitex”.
Dioscorides, the Greek physician, used to suggest V. agnus-castus to decrease libido. Pliny the
Elder, the famous Roman author, naturalist, and natural philosopher, commented that this herb was
scattered on the beds of Athenian women when husbands went to war to ensure their loyalty.
Pietro Andrea Mattioli, an Italian physician and botanist of the XVI century, commented in his text
“Compendium de Plantis Omnibus una cum Earum Iconibus” (1571) with reference to the
properties of V. agnus-castus L. that: “… it forces the impulses of Venus when eaten either fried or
raw ... …..it is believed that not only eating or drinking it will make chaste men but even lying on it
...". V. agnus-castus is also known as “monk’s pepper” since the fruits of this plant have a bitter taste,
and the plant used to be cultivated by monks in their gardens as an anaphrodisiac, according to a
legend, to help them not betray their vow of chastity.
Vitex agnus-castus L. fruits have been traditionally consumed as food to enhance milk volume
and to treat flatulence and diarrhea as well as cyclic breast pain, menopause, acne, infertility,
premenstrual dysphoric disorder and other menstrual disorders (amenorrhea, dysmenorrhea) [28].
The European Medicines Agency and the German Health Commission have reported many
health benefits of this medicinal plant, including regulation of the menstrual cycle and treatment of
premenstrual syndrome, and mastalgia [29]. This paper is focused on a perspective analysis of the
health-promoting effects of V. agnus-castus L. and its nutraceutical potential. There are many
reported data on the different beneficial health-promoting potentials of this plant, including
antioxidant, immunomodulatory, cytotoxic, antimutagenic, antimicrobial, antifungal,
antinociceptive, opioidergic, antiepileptic, and anti-inflammatory properties, as well as benefits for
osteopenic syndromes, as shown in Figure 1 [30–34].
γν
o
Forests 2020, 11, x FOR PEER REVIEW 2 of 17
cost-effectiveness when compared to chemical remedies obtained from synthesis [1]. Plants have
recently been exploited for nutraceutical purposes, as they play a key role in the development of
food and plant-derived phytocomplexes with medicinal properties, to be used in health conditions
as preventive or curative tools [2–23]. A popular medicinal plant with recognized beneficial effects
on human health is Vitex agnus-castus L., belonging to the Lamiaceae family (formerly included in
the Verbenaceae family), native to the Mediterranean area and diffused in Europe, Asia, and North
Africa [24,25]. It has been used by people in Italy, Iran, Greece, and Egypt for over 2500 years, mainly
to treat gynecologic disorders [26]. It is a globally famous plant known by different names, e.g. Fruit
de gattilier (French), Sauzgatillo (Spanish), Mönchspfefferfrüchte (German), Panj-angosht (Persian),
Frutto di Agnocasto (Italian), and Chaste tree (English) [27].
The term agnus-castus combines the Greek ῦ meaning pure, chaste) and the Latin (castus from
“castitas” meaning chastity), repeating the term chaste for “pure”, which refers to the anaphrodisiac
properties of this plant and its use by monks to maintain celibacy (thus the “monk’s pepper”
synonym). The word “castus” has been used for centuries to remark further the meaning or purity
associated with this plant. Homer, the semi-legendary author of the Iliad and the Odyssey epic
poems, defines agnus-castus as a “tendril for braiding”, explaining the origin of the genus “Vitex”.
Dioscorides, the Greek physician, used to suggest V. agnus-castus to decrease libido. Pliny the
Elder, the famous Roman author, naturalist, and natural philosopher, commented that this herb was
scattered on the beds of Athenian women when husbands went to war to ensure their loyalty.
Pietro Andrea Mattioli, an Italian physician and botanist of the XVI century, commented in his text
“Compendium de Plantis Omnibus una cum Earum Iconibus” (1571) with reference to the
properties of V. agnus-castus L. that: “… it forces the impulses of Venus when eaten either fried or
raw ... …..it is believed that not only eating or drinking it will make chaste men but even lying on it
...". V. agnus-castus is also known as “monk’s pepper” since the fruits of this plant have a bitter taste,
and the plant used to be cultivated by monks in their gardens as an anaphrodisiac, according to a
legend, to help them not betray their vow of chastity.
Vitex agnus-castus L. fruits have been traditionally consumed as food to enhance milk volume
and to treat flatulence and diarrhea as well as cyclic breast pain, menopause, acne, infertility,
premenstrual dysphoric disorder and other menstrual disorders (amenorrhea, dysmenorrhea) [28].
The European Medicines Agency and the German Health Commission have reported many
health benefits of this medicinal plant, including regulation of the menstrual cycle and treatment of
premenstrual syndrome, and mastalgia [29]. This paper is focused on a perspective analysis of the
health-promoting effects of V. agnus-castus L. and its nutraceutical potential. There are many
reported data on the different beneficial health-promoting potentials of this plant, including
antioxidant, immunomodulatory, cytotoxic, antimutagenic, antimicrobial, antifungal,
antinociceptive, opioidergic, antiepileptic, and anti-inflammatory properties, as well as benefits for
osteopenic syndromes, as shown in Figure 1 [30–34].
ς
, meaning pure, chaste) and the Latin (castus
from “castitas” meaning chastity), repeating the term chaste for “pure”, which refers to the anaphrodisiac
properties of this plant and its use by monks to maintain celibacy (thus the “monk’s pepper” synonym).
The word “castus” has been used for centuries to remark further the meaning or purity associated
with this plant. Homer, the semi-legendary author of the Iliad and the Odyssey epic poems, defines
agnus-castus as a “tendril for braiding”, explaining the origin of the genus “Vitex”. Dioscorides,
the Greek physician, used to suggest V. agnus-castus to decrease libido. Pliny the Elder, the famous
Roman author, naturalist, and natural philosopher, commented that this herb was scattered on the
beds of Athenian women when husbands went to war to ensure their loyalty. Pietro Andrea Mattioli,
an Italian physician and botanist of the XVI century, commented in his text “Compendium de Plantis
Omnibus una cum Earum Iconibus” (1571) with reference to the properties of V. agnus-castus L. that:
“
. . .
it forces the impulses of Venus when eaten either fried or raw
. . . . . .
..it is believed that not only
eating or drinking it will make chaste men but even lying on it
. . .
". V. agnus-castus is also known as
“monk’s pepper” since the fruits of this plant have a bitter taste, and the plant used to be cultivated by
monks in their gardens as an anaphrodisiac, according to a legend, to help them not betray their vow
of chastity.
Vitex agnus-castus L. fruits have been traditionally consumed as food to enhance milk volume and
to treat flatulence and diarrhea as well as cyclic breast pain, menopause, acne, infertility, premenstrual
dysphoric disorder and other menstrual disorders (amenorrhea, dysmenorrhea) [28].
The European Medicines Agency and the German Health Commission have reported many
health benefits of this medicinal plant, including regulation of the menstrual cycle and treatment
of premenstrual syndrome, and mastalgia [
29
]. This paper is focused on a perspective analysis of
the health-promoting effects of V. agnus-castus L. and its nutraceutical potential. There are many
reported data on the different beneficial health-promoting potentials of this plant, including antioxidant,
immunomodulatory, cytotoxic, antimutagenic, antimicrobial, antifungal, antinociceptive, opioidergic,
antiepileptic, and anti-inflammatory properties, as well as benefits for osteopenic syndromes, as shown
in Figure 1[30–34].

Forests 2020,11, 761 3 of 16
Forests 2020, 11, x FOR PEER REVIEW 3 of 17
Figure 1. Scheme of the beneficial properties of Vitex agnus-castus L.
2. Main Substances of Nutraceutical Interest in Vitex agnus-castus
The chemical composition of Vitex agnus-castus L. includes many different chemical
compounds, among which are: vitexilactone, rolundifuran, ketosteroids, diterpenoids (vitexlactam,
vitexilactone, viteagnusin I, and rotundifuran), flavonoids (orientin, kaempferol, penduletin,
luteolin, artemetin, vitexin, and casticin), and iridoids (agnuside, agnusoside, agnucastosid A/B, and
aucubin) [35–37]. The identification and quantification of agnuside (Figure 2), together with
p-hydroxy benzoic acid, can be achieved by high-performance liquid chromatography HPLC) [38].
This method has been validated for extracts of the species Vitex negundo L. and Vitex trifolia L., with
limits of quantification and detection of 25 µg/mL and 10 µg/mL, respectively [38].
Figure 1. Scheme of the beneficial properties of Vitex agnus-castus L.
2. Main Substances of Nutraceutical Interest in Vitex agnus-castus
The chemical composition of Vitex agnus-castus L. includes many different chemical compounds,
among which are: vitexilactone, rolundifuran, ketosteroids, diterpenoids (vitexlactam, vitexilactone,
viteagnusin I, and rotundifuran), flavonoids (orientin, kaempferol, penduletin, luteolin, artemetin,
vitexin, and casticin), and iridoids (agnuside, agnusoside, agnucastosid A/B, and aucubin) [
35
–
37
].
The identification and quantification of agnuside (Figure 2), together with p-hydroxy benzoic acid,
can be achieved by high-performance liquid chromatography HPLC) [
38
]. This method has been
validated for extracts of the species Vitex negundo L. and Vitex trifolia L., with limits of quantification
and detection of 25 µg/mL and 10 µg/mL, respectively [38].

Forests 2020,11, 761 4 of 16
Forests 2020, 11, x FOR PEER REVIEW 4 of 17
(a) (b)
Figure 2. Chemical structure of vitexin (a) and of agnuside (b).
Gokbulut et al. measured the levels of vitexin (Figure 2), isolated from V. agnus-castus L. fruit
and leaf extracts, using the RP-HPLC-DAD (diode array detector) technique, and the results showed
that this flavonoid was present in considerable amounts (0.342 ± 0.0153% and 0.252 ± 0.0089%,
respectively) [39].
Agnuside together with casticin (a tetramethoxyflavone) have been used as fingerprint markers
to evaluate the quality of Japanese commercial products containing V. agnus-castus L. [40]. Another
marker validated for quality assessment was reported by Yahagi et al., using a liquid
chromatography–mass spectrometry (LC-MS)-based metabolomic technique and nuclear magnetic
resonance (NMR) spectroscopy to detect 3-O-trans-feruloyl tormentic acid, which was isolated from
the V. agnus-castus L. fruit extract [41]. In another study, using a rapid ultra-high performance liquid
chromatography diode array detector (UHPLC-DAD-QTOF-MS), seven markers of V. agnus-castus L.
fruit extract were claimed to be used as reference compounds for quality validation of medicinal
products containing this extract, in particular, vitetrifolin D (labdane diterpenoid),
5-hydroxykaempferol-3,6,7,4’-tetramethylether, casticin, isovitexin, and agnuside compounds [42].
A study conducted to compare the chemical markers occurring in food supplements and in V.
agnus-castus L. extracts, using the liquid chromatography electrospray triple quadrupole tandem
mass spectrometry (LC/ESI/(QqQ)MSMS) method in multiple reaction monitoring (MRM) mode as a
quantitative analysis, reported the presence of aucubin, orientin, luteolin-7-O-glucoside, agnuside,
isovitexin, homoorientin, and casticin compounds [43]. According to the findings from
RP-HPLC-DAD analysis, V. agnus-castus L. leaf and fruit extracts contained chlorogenic and caffeic
acid phenolic compounds with average concentrations of 0.27% and 0.32% (w/w), respectively [44].
Li et al. isolated eighteen compounds from V. agnus-castus L. fruit extract, using 1D/2D NMR and
mass spectrometry methods, and their chemopreventive potential was studied in Hepa 1c1c7 cells,
which showed NADP(H): quinone oxidoreductase type 1 (QR1) induction potential related, with
vitetrifolin D and vitexlactam C being the most promising [45]. In another study, the method of
supercritical carbon dioxide was used to detect the compounds dihydroselarene, α-terpinyl acetate,
trans-caryophyllene, sabinene, and 1,8-cineole [46]. Ono et al. applied HPLC coupled with NMR
analysis to determine the chemical constituents of V. agnus-castus L. fruits, reporting that
viteagnuside was the main compound present [47].
3. An Updated Snapshot of In Vitro and In Vivo Studies on Vitex agnus-castus
In-depth knowledge of the phytochemical composition of any potential medicinal plant is the
first step for the determination of its beneficial health properties. In the following, the main findings
Figure 2. Chemical structure of vitexin (a) and of agnuside (b).
Gokbulut et al. measured the levels of vitexin (Figure 2), isolated from V. agnus-castus L. fruit
and leaf extracts, using the RP-HPLC-DAD (diode array detector) technique, and the results showed
that this flavonoid was present in considerable amounts (0.342
±
0.0153% and 0.252
±
0.0089%,
respectively) [39].
Agnuside together with casticin (a tetramethoxyflavone) have been used as fingerprint markers to
evaluate the quality of Japanese commercial products containing V. agnus-castus L. [
40
]. Another marker
validated for quality assessment was reported by Yahagi et al., using a liquid chromatography–mass
spectrometry (LC-MS)-based metabolomic technique and nuclear magnetic resonance (NMR)
spectroscopy to detect 3-O-trans-feruloyl tormentic acid, which was isolated from the V. agnus-castus L.
fruit extract [
41
]. In another study, using a rapid ultra-high performance liquid chromatography diode
array detector (UHPLC-DAD-QTOF-MS), seven markers of V. agnus-castus L. fruit extract were claimed
to be used as reference compounds for quality validation of medicinal products containing this extract,
in particular, vitetrifolin D (labdane diterpenoid), 5-hydroxykaempferol-3,6,7,4’-tetramethylether,
casticin, isovitexin, and agnuside compounds [
42
]. A study conducted to compare the chemical markers
occurring in food supplements and in V. agnus-castus L. extracts, using the liquid chromatography
electrospray triple quadrupole tandem mass spectrometry (LC/ESI/(QqQ)MSMS) method in multiple
reaction monitoring (MRM) mode as a quantitative analysis, reported the presence of aucubin,
orientin, luteolin-7-O-glucoside, agnuside, isovitexin, homoorientin, and casticin compounds [
43
].
According to the findings from RP-HPLC-DAD analysis, V. agnus-castus L. leaf and fruit extracts
contained chlorogenic and caffeic acid phenolic compounds with average concentrations of 0.27%
and 0.32% (w/w), respectively [
44
]. Li et al. isolated eighteen compounds from V. agnus-castus L. fruit
extract, using 1D/2D NMR and mass spectrometry methods, and their chemopreventive potential was
studied in Hepa 1c1c7 cells, which showed NADP(H): quinone oxidoreductase type 1 (QR1) induction
potential related, with vitetrifolin D and vitexlactam C being the most promising [
45
]. In another
study, the method of supercritical carbon dioxide was used to detect the compounds dihydroselarene,
α
-terpinyl acetate, trans-caryophyllene, sabinene, and 1,8-cineole [
46
]. Ono et al. applied HPLC
coupled with NMR analysis to determine the chemical constituents of V. agnus-castus L. fruits, reporting
that viteagnuside was the main compound present [47].
3. An Updated Snapshot of In Vitro and In Vivo Studies on Vitex agnus-castus
In-depth knowledge of the phytochemical composition of any potential medicinal plant is the
first step for the determination of its beneficial health properties. In the following, the main findings

Forests 2020,11, 761 5 of 16
regarding beneficial health effects from
in vitro
and
in vivo
studies are reported. This information
gives an updated picture of the many activities of the compounds contained in this plant, outlining its
possible prospective application in the prevention and even in the treatment of pathological conditions.
3.1. Health-Promoting Activities of Vitex agnus-castus L. In Vitro
The
in vitro
health-promoting potential of V. agnus-castus L. has been reported in the past [
48
].
Table 1summarizes the main results of
in vitro
studies. V. agnus-castus fruit extract showed
in vitro
anticancer activity against HL-60 cells by arresting the cell cycle at the G2/M phase and by inducing
apoptosis [
49
]. Abdel-Lateef et al. [
50
] analyzed the chemical composition of V. agnus-castus L.
leaf extract and found it to be composed of phenolic acids, flavonoids, and iridoids, which could
significantly prevent HepG2 cell proliferation by inducing apoptosis through caspase-3 activation,
with the butanolic fraction being the most active (IC
50
=13.42
±
0.17 mg/mL). The cytotoxic effect of
agnuside was assayed in a colon cancer cell line (COLO 320 DM), showing an IC
50
value of 15.99
µ
g/mL,
and 76.1% cytotoxic activity at 200
µ
g/mL [
51
]. Cytotoxic activity of V. agnus-castus L. fruit extract
against other another human colon carcinoma cell line (COLO 201) was shown, by inducing activation
of caspase 3/9 leading to apoptosis [
52
]. Ribat et al. [
53
] described antiproliferative and anticancer
properties of
V. agnus-castus L.
fruit methanol extract, showing that normal rat embryonic fibroblast
cells were much less sensitive than AMN3 cells (mouse mammary gland adenocarcinoma cell line),
with IC
50
values of 1324
µ
g/mL and 129
µ
g/mL, respectively. A synergistic effect was observed with
co-administration of 5-fluorouracil and 10
µ
g/mL vitex (ethanolic extract of V. agnus-castus L. fruits),
using colon cancer cell lines [
54
]. Casticin extracted from V. agnus castus L. showed cytotoxic and
immunomodulatory properties by inhibiting phytohemagglutinin (PHA) induced T-cell proliferation,
phagocytosis, and chemotaxis [
55
]. Sarac et al. reported 56.18% and 72.25% antimutagenic activities for
the ethanolic extracts of V. agnus castus L. seed and leaf at the doses of 2.5 mg/plate and 0.125 mg/plate,
respectively [56].
Table 1. In vitro reported activities for V. agnus-castus L.
Condition Plant Part Extract Activity Effect Reference
In vitro Fruits Ethyl acetate Antioxidant
activity
Lipid peroxidation was inhibited by
casticin with an IC
50
value of 0.049 mM.
[57]
In vitro Leaves Supercritical
CO2
Antifungal
activity
The antifungal potential of essential oil
with an MIC value of 0.64 µL/mL. [58]
In vitro Leaves
Hydrodistillation
Antifungal
activity
The antimutagenic activity of V.
agnus-castus leaf extract against
Salmonella typhimurium.
[56]
In vitro Aerial parts
Methanolic
extract (Cr.
MeOH Ext.)
Antimicrobial
activity
The V. agnus-castus leaf essential oil
showed an antibacterial effect against
Staphylococcus aureus with an MIC value
of 0.31% v/v.
[59]
In vitro Seed n-hexane Antifungal
activity
Essential oil was effective against
Candida species with an MIC50 value of
1.75 mg/mL.
[60]
In vitro
Ripened fruits and
fruitless aerial
parts
Ethanol Anticancer
activity
Cytotoxic effect of V. agnus-castus fruit
extract against MCF-7 cancer cells with
IC50 =88 µg/mL.
[61]
In vitro Fruits
Chloroform–
methanol
(250:1)
Anticancer
activity
The cytotoxicity was due to
differentiation of the hematopoietic cell
line.
[62]
In vitro Fruits Ethanol Anticancer
activity
Enhanced apoptosis and decreased
intracellular ROS levels. [49]
In vitro Leaves Methanol Antioxidant
effect
The free radical scavenging effect of V.
agnus-castus methanol extract with an
IC50 value of 126.79 mg/mL.
[63]

Forests 2020,11, 761 6 of 16
The health of humans, animals, and the food chain is directly and indirectly influenced by
various pathogens. Thus, extensive studies have been conducted to control such microorganisms
using different approaches, among which the use of cost-effective and more effective natural plant
extracts or essential oil has attracted further attention; for example, V. agnus-castus L. has been
studied against several pathogenic species such as Staphylococcus aureus,Escheria coli,Bacillus subtilis,
and Pseudomonas aeruginosa. Habbab et al. [
64
] evaluated the antifungal activity of V. agnus-castus
flower and leaf essential oils against Aspergillus flavus and Penicillium escpansum, as well as the
antibacterial activity of V. agnus-castus L. seed and leaf essential oils against P. aeruginosa,E. coli,
and Klebsiella pneumonia.
Vitex agnus-castus L.
essential oil was shown to exhibit antibacterial activity
against Staphylococcus aureus [
65
].
Afarin et al.
[
66
] observed
in vitro
antimicrobial activity for
V. agnus-castus L.
essential oil at doses of 112.5 and 56.25
µ
g/mL against Candida albicans and S. aureus,
respectively. Katiraee et al. [
67
] found radical scavenging (IC
50
=27.16
µ
g/mL) and antifungal properties
for V. agnus-castus L. essential oil. Others reported that V. agnus-castus L. essential oil showed antifungal
potential against Sclerotinia sclerotiorum and Verticillium dahlia with LC
50
values of 3.322
µ
g/mL and
1.063
µ
g/mL, and 9.729
µ
g/mL and 7.313
µ
g/mL, respectively [
68
]. Stojkovi´c et al. [
69
] reported
antimicrobial potential for V. agnus-castus L. fruit and leaf essential oils attributed to the presence of
α
-pinene and 1,8-cineole. The administration of active ethyl acetate extract of V. agnus-castus L. leaf
exhibited antibacterial potential against methicillin-resistant S. aureus (MIC =0.312 mg/mL) owing to
steroids, terpenoids, and flavonoids [
70
]. Vitex agnus-castus L. leaf essential oil eliminated the cariogenic
bacteria Streptococcus mutans (MIC =15.6
µ
g/mL) [
71
]. In another study, alcoholic and aqueous extracts
had an antifungal effect on C. albicans isolated from clinical vaginal infections [
72
]. Vitex agnus-castus L.
seed essential oil showed antifungal activity against Candida species (
IC50 =1.072 mg/mL
) and also
antioxidant potential [73].
The anticancer activity of Vitex agnus-castus L. seed extracts against MCF-7 cells showed
DNA-damaging, cytotoxic, and apoptotic effects, and this extract also showed antioxidant
properties [
74
]. Rashed et al. [
75
] stated that the antioxidant effect of ethyl acetate extract of
V. agnus castus
could be attributed to flavonoids and tannins, as the main compounds. The antioxidant
effect of V. agnus-castus L. fruit and leaf extract was confirmed by the decolorization of the radical
monocation of 2,2
0
-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay [
76
].
Malta¸s et al.
described a H
2
O
2
scavenging effect for V. agnus-castus L. extract with antioxidant activity of
93.5 ±0.8%
[
77
]. In another study, using ethanolic extract of V. agnus-castus L. leaf, containing mainly
flavonoids and phenols as active ingredients, it was revealed that there was a direct correlation between
these compounds and antioxidant properties [
78
]. Ahmad et al. [
79
] reported that vitexcarpan, isolated
from the aerial parts of V. agnus-castus L. by extraction using ethyl acetate, showed anti-inflammatory
activity in activated human neutrophils. The presence of casticin in V. agnus-castus L. extract exhibited
anti-inflammatory potential with an efficacy of 69.51% and an IC50 value of 302.1 µg/mL [80].
Vitex agnus-castus L. fruit extract showed antiangiogenic activity due to the presence of
antiangiogenic compounds, confirmed by phytochemical analysis [81].
3.2. Health-Promoting Activities of Vitex agnus-castus L. in Animals
There are numerous therapeutic effects of V. agnus-castus L. described in animal models. Table 2
reports the results of the main animal model studies. Oral administration of V. agnus-castus L. extract
(200 mg/kg) in Sprague–Dawley rats with a mammary tumor for 15 days improved the oxidative status
of the mammary tumor tissue and treated tumor regression properties [82].

Forests 2020,11, 761 7 of 16
Table 2. Animal model reported activities for V. agnus-castus L.
Condition Plant Part Extract Activity Effect Reference
Animal model Leaves
Hydrodistillation
Antinociceptive
activity and
analgesic effect
The analgesic activity was
due to the activation of
muscarinic receptors of the
cholinergic system and
endogenous opioidergic
system.
[83]
Animal model Stems and
leaves Ethanol
Treatment of
polycystic ovary
syndrome
V. agnus-castus exhibited
hypoglycemic, antioxidant,
and antihyperlipidemic
activities in rats.
[84]
Animal model Fruits
Chloroform,
methanol, and
water
Antihyperlipidemic
activity
The V. agnus-castus extract
(500 mg/kg for 28 days)
decreased the levels of
VLDL, LDL, TG, and TC.
[85]
Animal model Fruits
Hexane, ethyl
ether, and
n-butanol
Protected against
nonalcoholic fat
liver disease
Prevented oxidative stress
and treated nonalcoholic fat
liver disease.
[86]
Animal model Berries Ethanol Anti-inflammatory
activity
Inhibited the production of
reactive oxygen species, the
release of cytokines, and the
formation of leukotriene.
[87]
Animal model Leaves Chloroform Antiangiogenic
activity
Prevented growth of
psoriasis, cataract, and
tumor.
[88]
Animal model Fruits Ethanol Antiaging effects
Improved
d-galactose-induced aging
symptoms, including
enhanced serum LH and
FSH levels, follicle
degeneration, and
endometrial atrophy.
[89]
Animal model Fruits Methanol Antiepileptic
activity
Reduced stage 5 duration
and after-discharge
duration.
[90]
Animal model Fruits Ethanol Osteoprotective
effects
Enhanced biomechanical
stability of bone via
connectivity density in the
orchidectomized rats and
improved the trabecular
microarchitecture.
[91]
Animal model Leaves Methanol Anti-inflammatory
effect
The V. agnus-castus methanol
extract (400 mg/kg) reduced
IL-6 and TNF-αlevels.
[92]
Animal model -
Methanol,
n-hexane, and
Ethyl acetate
Antioxidant and
antiapoptotic
effects
Vitexilactone extracted from
V. agnus-castus reduced
caspase-3 and apoptosis
marker expression in
Sprague–Dawley rats.
[93]
The administration of casticin extracted from V. agnus-castus L. fructus (1, 2, and 10 mg/Kg per day)
for two weeks protected inflammatory lung diseases in a mouse model due to an anti-inflammatory effect
by decreasing epithelium thickness, perivascular inflammatory cells, and peribronchial infiltration,
as well as reduced the numbers of total cells, lymphocytes, macrophages, and neutrophils [
94
].
The administration of V. agnus-castus L. fruit extract (165 mg/Kg/day) in rats controlled prostate
cancer by inducing apoptosis and showed an anti-inflammatory effect by inhibiting cyclooxygenase-2
activity [95]. Webster et al. reported that V. agnus-castus attenuates pre-menstrual syndrome [96].

Forests 2020,11, 761 8 of 16
Vitex agnus-castus administered for five days protected mice against lipopolysaccharide
(LPS)-induced acute lung damage, due to an antioxidant effect [
97
]. The methanol extract of
V. agnus-castus leaf showed an antiangiogenic effect in an ex vivo rat aorta, and also antioxidant
activity with an IC50 value of 126.79 µg/mL [88].
Moreover, administration of V. agnus castus ethanolic extract (8 and 80 mg/Kg/day) for three
months in ovariectomized rats improved memory and learning via decreasing uterine weight and
increasing the estrogen receptor ER
α
gene expression, respectively, suggesting a solution for memory
loss in postmenopausal women [
98
]. The hydroalcoholic extract of V. agnus-castus fruits (600 mg/Kg
twice a day) reduced age-related changes in a female mice model after 7 days [99].
V. agnus-castus extract was administered to a mouse model of permanent middle cerebral artery
occlusion for 30 days, and it was observed that the anti-inflammatory and estrogenic activities reduced
stroke injuries. Moreover, this study also reported neuroprotective activity by reducing matrix
metalloproteinase-9 (MMP-9), increasing interleukin 10, and improving adhesive removal and wire
hanging test performance [100].
3.3. Health-Promoting Activities of Vitex agnus-castus L. in Humans, with Particular Regard to Clinical Trials
Many clinical trials confirm the health-promoting effects of V. agnus-castus L. [
101
–
108
]. Table 3
reports the results of the main clinical trial studies. Naseri et al. [
109
] reported a reduction in menopausal
symptoms after taking V. agnus-castus L. extracts. The authors allocated the participants into two placebo
groups and a Vitex-treated group, and then assessed menopausal symptoms before and after an 8-week
intervention using the Greene Questionnaire [
110
]. After the intervention, the Vitex group showed
a reduction in vasomotor dysfunction, anxiety, and total menopausal disorder.
Yavarikia et al.
[
111
]
administered V. agnus-castus L. capsules to female participants three times a day for four months,
and obtained data with the Higham five-stage chart (for 1 month before the treatment and 4 months
during the treatment) [
112
] and a demographic questionnaire. They found a 47.6% decrease in bleeding
in the V. agnus-castus L. group. Oral administration of ethanolic extract of V. agnus-castus L. (4.0 mg),
dried as film-coated tablets, in Chinese women with premenstrual syndrome (PMS) reduced the
PMS score of the third cycle from 27.10 to 14.59 in the case group [
113
]. Daily administration of
V. agnus-castus L.
extract-containing tablets in Chinese women with premenstrual syndrome (PMS)
decreased the PMS scores of symptoms such as pain, food cravings, and water retention [
114
].
A self-assessment questionnaire was completed for the symptoms of PMS in women after taking
40 drops of V. agnus-castus L. extract for 6 days, the results of which showed mild to moderate relief in
PMS symptoms [
115
]. In a recent clinical trial, it was reported that the administration of
V. agnus-castus L.
in women with polycystic ovary syndrome reduced the level of dehy-droepiandrosterone sulfate
(DHEA-S) and normalized the menstrual cycle [
116
]. Healthy menopausal women co-administrated
V. agnus-castus L. and Nigella sativa with citalopram once a day for 8 weeks showed superior scores
in three of the Menopause-specific Quality of Life Questionnaire (MENQOL) domains: psychosocial
(p=0.001), physical (p=0.036), and vasomotor (p<0.001) [117].

Forests 2020,11, 761 9 of 16
Table 3. Clinical trials reporting V. agnus-castus L. activities.
Condition Activity Administration Effect Reference
Clinical trial
Treatment of
vasomotor
symptoms.
The administration of V.
agnus-castus (40 mg) once a
day for a month in women
with postmenopausal
symptoms.
Improvement of
sleep satisfaction. [118]
Clinical trial Treatment of
mastalgia.
The administration of V.
agnus-castus in patients with
mastalgia.
Reduction in
prolactin level after
three months.
[119]
Clinical trial
Treatment of
premenstrual
syndrome.
Administration of V.
agnus-castus extract (20 mg)
once a day for three
menstrual cycles in Japanese
women.
The symptoms of
premenstrual
syndrome were
improved.
[120]
Clinical trial
Treatment of
premenstrual
syndrome.
The administration of V.
agnus-castus (40 mg) once a
day for three months in
migrainous women with
premenstrual syndrome.
The symptoms of
premenstrual
syndrome were
reduced in 66
women.
[121]
Clinical trial
Treatment of
premenstrual
syndrome.
The administration of V.
agnus-castus extract Ze 440
(20 mg) once a day.
The symptoms of
premenstrual
syndrome were
relieved in women.
[122]
Clinical trial
Treatment of
menopausal
syndrome.
The administration of V.
agnus-castus extract (40
drops) once a day for 8
weeks in women.
Hot flashes were
positively
influenced by Vitex
in women.
[123]
Clinical trial
Treatment of
premenstrual
syndrome.
The co-administration of V.
agnus-castus with Hypericum
perforatum twice a day for 16
weeks in women with
premenstrual syndrome.
Symptoms such as
hydration clusters
and anxiety were
alleviated.
[124]
Clinical trial
Treatment of
premenstrual
syndrome.
The administration of V.
agnus-castus extract (40
drops) for 4 months.
The pregnancy rate,
endometrial
thickness,
ovulation, and
fertility were
increased in
women.
[125]
Clinical trial
Prolactin-inhibiting
activity.
The daily administration of
Agnus-castus extract
(BP1O95E1) at a
concentration of 480 mg for
two weeks in healthy male
subjects.
Decreased
prolactin profile
levels.
[126]
Vitex agnus-castus L. extracts have reportedly exhibited many health-promoting effects, but some
limitations have been shown for human health such as agitation, headache, nausea, tachycardia,
fatigue, dry mouth, urticaria, and gastrointestinal problems [
78
]. In a study by Owolabi et al. [
127
],
it was also reported that increasing the dose and long-term administration of the Vitex genus induced
toxicity in rats.

Forests 2020,11, 761 10 of 16
4. Conclusions
Considering the naturally occurring bioactive compounds with therapeutic potential that it
contains, V. agnus-castus L. is one of the best-selling and most widely used medicinal herbs worldwide.
Nevertheless, there is a need for further
in vivo
and
in vitro
research and more clinical trials to assess
the beneficial health compounds content of this plant and the related mechanisms of action in the
treatment of various diseases, as it is so far been largely limited to animal model trials and
in vitro
studies. The clinical trial studies in humans focus on a limited number of health conditions, suggesting
the need to exploit what is observed in animal trials and possible in prospective assess also possible
applications in humans. The beneficial properties of V. agnus-castus L. trigger interest in the possibility
of developing novel nutraceutical formulations, which can help to support health conditions before the
need of a pharmacological therapy, in particular for individuals who do not qualify for a conventional
drug-based treatment. It should be noted also that there is a need for more comprehensive clinical
trials over a long timeframe in order to draw definitive conclusions about the findings related to the
various effects and consequences of a long-term consumption of V. agnus-castus L.
Author Contributions:
E.B.S., A.D., A.N., M.L., E.N., and A.S. conceived and designed the work. E.B.S., A.D.,
A.N., M.L., M.Z., A.M.S., P.S., E.N., and A.S. wrote the work. A.D., A.N., M.L., M.Z., S.B.S., A.M.S., and P.S.
validated and elaborated data information and figures. All authors have read and agreed to the published version
of the manuscript.
Funding:
The authors acknowledge the support of the research project: Nutraceutica come supporto
nutrizionale nel paziente oncologico, CUP: B83D18000140007. E.B.S. acknowledges the sponsorship of
the projects M-ERA-NET-0004/2015-PAIRED and UIDB/04469/2020 (strategic fund); A.M.S. acknowledges
UIDB/04033/2020 (CITAB), receiving support from the Portuguese Science and Technology Foundation, Ministry
of Science and Education (FCT/MEC) through national funds, and co-financed by FEDER under the Partnership
Agreement PT2020.
Conflicts of Interest: The authors declare no conflict of interest.
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