Research & Reviews: Journal of Botany

ISSN: 2278-2222(online)
Volume 4, Issue 3
www.stmjournals.com

Phytochemical Evaluation of Vitex agnus-castus L Seeds

Collected from Different Geographical Regions
of the World
L Rajanna*, P Vijay Raghavan, GS Shailaja Sharma
Department of Botany, Bangalore University, Jnanabharathi Campus, Bangalore, Karnataka, India

Abstract
Vitex agnus-castus L (VAC) is an important medicinal plant. Seeds are used as a popular
treatment for the management of female reproductive system disorders. Other uses include the
treatment of hangovers, flatulence, fevers, benign prostatic hyperplasia, nervousness,
dementia, rheumatic conditions, cold, dyspepsia, spleen disorders, constipation and
promoting urination. VAC today is an important herb of commerce cultivated around the
world. VAC seeds were collected from different geographical location—USA, EU and India.
Phytochemical variations among these VAC seeds were studied using High Performance Thin
Layer Chromatography (HPTLC) and High Performance Liquid Chromatography
(HPLC)/fingerprinting techniques. HPTLC fingerprinting of VAC seeds were studied using
agnuside and casticin as marker compound. Casticin and agnuside were quantified in all the
six VAC seed samples by HPLC. Significant phytochemical variations were found among all
the VAC seed samples.

Keywords: Vitex agnus-castus L, phytochemical variations, high performance thin layer

chromatography, high performance liquid chromatography, casticin

*Authors for Correspondence: E-mail: lrajannabot@gmail.com

INTRODUCTION dry. The twigs of this shrub are very flexible

Vitex agnus-castus L (VAC) belongs to the
family Verbenaceae. It is an ornamental, large
deciduous shrub, native to Mediterranean
countries and central Asia, but widely grown
in North America. VAC is a shrub that has
been used for hundreds of years in Europe for
female reproductive system disorders [1] and
is well-tolerated and has established efficacy
in helping with some symptoms associated
with premenstrual syndrome [2, 3]. The major
active constituents of VAC are iridoid
glycosides, flavonoids, alkaloids, and essential
oils [4]. Its dominant pharmacological effect
on the body is inhibition of prolactin secretion
[4–6]. VAC plant parts are available in a
variety of dosage forms and its use is gaining
popularity in the United States.
VAC has long, finger-shaped leaves and
displays fragrant blue–violet flowers during
midsummer. Its fruit is a very dark-purple
berry that is yellowish inside, resembles a
peppercorn, and has an aromatic odor. Upon
ripening, the berry is picked and allowed to
and were used for furniture in ancient times.
References to VAC go back more than 2000
years, describing it as a healing herb. Ancient
Egyptians, Greeks, and Romans used it for a
variety of health problems. Use of VAC
continued into the middle ages, where folklore
persists that medieval monks chewed VAC
tree parts to maintain their celibacy, used the
dried berries in their food, or placed the berries
in the pockets of their robes in order to reduce
sexual desire; thus, the synonym of Monk’s
pepper [7].
Current promoted uses of VAC related to
treatment of disorders of the female
reproductive system such as short menstrual
cycles, premenstrual syndrome (PMS), and
breast swelling and pain
(mastodynia/mastalgia) [4, 6]. The
Commission E has approved the use of VAC
for irregularities of the menstrual cycle,
premenstrual complaints, and mastalgia.
Recent randomized, placebo-controlled studies
have been conducted and found VAC to be

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Phytochemical Evaluation of Vitex agnus-castus Seeds
effective and well-tolerated for the relief of
PMS symptoms, especially the physical
symptoms of breast tenderness/fullness,
edema, and headache [8].
The major constituents of VAC include the
following—Flavonoids: flavonol (kaempferol,
quercetagetin) derivatives, the major
constituent being casticin. Additional
flavonoids found include—penduletin,
orientin, chrysophanol D, and apigenin;
Water-soluble flavones—vitexin and
isovitexin; Alkaloids—viticin; Diterpenes—
rotundifuran (labdane-type), vitexilactone;
Iridoid glycosides—in the leaf—0.3%
aucubin, 0.6% agnuside (the p-
hydroxybenzoyl derivative of aucubin), and
0.07% unidentified glycosides. Essential oil of
leaves and flowers contain monoterpenes
(major chemicals found are limonene, cineole,
sabinene, α-terpineol, linalool, citronellol,
camphene, myrcene) and sesquiterpenes
(majority of them are β-caryophyllene, β-
gurjunene, cuparene, and globulol).
Depending on the maturity of the fruits used
and the distillation processes, the components
of the essential oil can vary greatly [9].
Since VAC is cultivated around the world for
use in different clinical indications, it is
important to establish phytoequivalence. VAC
is listed in US pharmacopeia as dietary
supplement. In the present investigation
phytochemical variation is studied by,
1. Comparison of High Performance Thin
Layer Chromatography (HPTLC)
fingerprint profile of VAC seeds using
casticin as marker compound;
2. Variation in casticin and agnuside content
in VAC seeds samples using High
Performance Liquid Chromatography
(HPLC).
VAC seeds were collected from different
geographical regions—California (n=1),
Florida (n=1), US; Vienna (n=1), EU; Turin
(n=1), EU; Krishnagiri (n=1) and Mettur
(n=1), India.
MATERIALS AND METHODS
Plant Material
The seeds of VAC were collected from
different geographical regions; such as US, EU
and India. It was identified and authenticated
RRJoB (2015) 21-28 © STM Journals 2015. All Rights Reserved
Rajanna et al.
by the National Institute for Science
Communication and Information Resources
(NISCAIR), New Delhi, India. The seeds of
VAC were powdered in a grinder to make a
fine powder and was sieved through mesh no.
120 and stored in air tight containers until used
for further analysis.
HPTLC Fingerprinting [10]
Equipment
A Cammag (Switzerland) HPTLC system
equipped with a sample applicator Linomat V,
Twin trough glass chamber (20x10 cm) with
SS lid, TLC scanner III, TLC visualizer and
Wincats—an integrated Software 4.02
(Switzerland) and Rotavapour.
Chemical and Reagents
Analytical grade methanol, toluene, ethyl
acetate, anisaldehyde, sulphuric acid were
obtained from Fisher Scientific Ltd.
(Bangalore, India). TLC Aluminium
precoated plate with Silica gel 60GF254 (20x10
cm2; 0.2 mm thick) used were obtained from
E. Merck Ltd. (Bangalore, India). Reference
standards—agnuside and casticin was
procured from Extrasynthese, France.
Sample and Standard Preparation
Sample preparation (T1–T6): 1 g of powdered
drug samples were extracted with 10 ml
methanol for 24 h by cold extraction method.
The extracts were filtered by Whatmann no. 1
filter paper and made up to 10 ml in a
volumetric flask. Filtrates were concentrated
to 5 ml on Rotavapour at 40 ºC and used for
HPTLC work.
Standard Preparation—Agnuside (S1)
5 mg of reference standard—agnuside was
dissolved in 3 ml of methanol and made up to
5 ml in standard volumetric flask.
Standard Preparation—Casticin (S2)
5 mg of reference standard—casticin was
dissolved in 3 ml of methanol and made up to
5 ml in standard volumetric flask.
Chromatography
Agnuside
TLC aluminium precoated plate with silica gel
60 GF254 (20 x 10 cm2; 0.2 mm thick) was used
with ethyl acetate: water: acetic acid (8: 1: 1)
as mobile phase. Methanol extract of samples
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Research & Reviews: Journal of Botany
Volume 4, Issue 3
ISSN: 2278-2222(online)

(T1–T2) and agnuside standard solution (S1)
were applied on the plate by using Linomat V
applicator. Camag twin trough glass chamber
(20x10 cm2) with SS lid was used for
development of TLC plate. The twin trough
glass chamber was saturated with mobile
phase for 30 min. TLC plate was developed to
8 cm distance above the position of the sample
application. The plate was removed from the
chamber and air dried at room temperature.
This plate was sprayed (derivitized) with
anisaldehyde–sulphuric acid reagent followed
by heating at 110 ºC for 10 min and HPTLC
fingerprint profile was snapped by Camag
TLC visualizer before derivitization in UV
254 nm and after derivatization
(Figures 1a and b).
Casticin
TLC aluminium precoated plate with silica gel
60 GF254 (20 x10 cm2; 0.2 mm thick) was used
with toluene: ethyl acetate (8: 2) V/V as
mobile phase. Methanol extract of samples and
casticin standard solution were applied on the
plate by using Linomat V applicator. Camag
twin trough glass chamber (20x10 cm2) with
SS lid was used for development of TLC plate.

Fig. 1(a): VAC Seed Samples—T1 = Krishnagiri, India, T2 = Mettur, India, T3 = California, US,
T4 = Florida, US, T5 = Vienna, EU and T6 = Turin, EU. S1 = Agnuside Reference Standard.
Visualized in UV 254 nm before Derivitization.

The twin trough glass chamber was saturated
with mobile phase for 30 min. TLC plate was
developed to 8 cm distance above the position
of the sample application. The plate was
removed from the chamber and air dried at
room temperature. This plate was sprayed
(derivitized) with anisaldehyde–sulphuric acid
reagent followed by heating at 110 ºC for 10
min and HPTLC fingerprint profile was

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Phytochemical Evaluation of Vitex agnus-castus Seeds Rajanna et al.

snapped by Camag TLC visualizer after

derivatization (Figure 2).

Fig. 1(b): VAC Seed Samples—T1 = Krishnagiri, India, T2 = Mettur, India, T3 = California, US,
T4 = Florida, US, T5 = Vienna, EU and T6 = Turin, EU. S1 = Agnuside Reference Standard.
Visualized after Derivitization.

Fig. 2: VAC Seed Samples—T1 = Krishangiri, India, T2 = Mettur, India, T3 = California, US,

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Research & Reviews: Journal of Botany
Volume 4, Issue 3
ISSN: 2278-2222(online)
T4 = Florida, US, T5 = Vienna, EU and T6 = Turin, EU. S1= Casticin Reference Standard.
Assay of Castisin by HPLC [11]
Test Solution: About 1000 mg of ground VAC
seeds were taken and placed in a 100 ml
volumetric flask, with stopper. It was extracted
twice with 40 ml of methanol using sonicator
for 15 min. Each supernatant was filtered and
transferred to a 250 ml round bottom flask.
The residues were rinsed with methanol, and
the resulting solution was filtered into the
flask. The combined extract was evaporated to
dryness. The residues was dissolved in
methanol, quantitatively transferred to a 20 ml
volumetric flask and diluted with methanol to
volume. It was filtered through a cellulose
membrane having 0.45 µ porosity.
Standard Solution: 10 mg of casticin
(Extrasynthese, France) was accurately
weighed in a 25 ml volumetric flask and
dissolved in about 10 ml of methanol and
diluted with methanol to the volume.
Fig. 3: HPLC Chromatogram Overlay of VAC Samples and Casticin Standard.
Assay of Agnuside by HPLC [11]
Test Solution
1000 mg of ground VAC seeds were taken and
placed in a 100 ml volumetric flask. It was
extracted twice with 40 ml of methanol, using
a hand homogenizer at 19,000 rpm for 2 min.
Centrifuged and each supernatant was
transferred to a 250 ml round-bottom flask.
The residue was rinsed with methanol and
filtered. The resulting solution was collected
RRJoB (2015) 21-28 © STM Journals 2015. All Rights Reserved
Chromatographic System: Shimadzu
prominence 20AD HPLC.
Detector: Photodiode array (PDA).
Chromatography Column and Stationary
Phase: Reverse phase C18 (250 mm x 4.6
mm), 5 µm.
Mobile Phase: Gradient mixture of methanol
and 5.88 g/l phosphoric acid in water.
Injection Volume: 10 µl.
Flow Rate: 1 ml per min.
Detection: UV, 348 nm
Procedure: 10 µl of the filtered standard
solution and 10 µl of the filtered test solution
were injected separately and the
chromatogram was recorded. The responses
were measured for the analyte peak. The
content of casticin in the VAC seeds was
calculated from the peak response of analytes.
The relative standard deviation for replicate
injections was not more than 0.08%.
into the flask. The combined extract was
evaporated to dryness, and the residue was
dissolved in 2 ml of solvent. The solution was
quantitatively transferred to a solid-phase
extraction cartridge (Supelco, SPE) to a
vacuum pressure not exceeding 300 mbar and
the eluate was collected. The round-bottom
flask was rinsed with 2 ml of solvent and then
the solution was passed through the cartridge,
vacuum was applied and the eluate was
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Phytochemical Evaluation of Vitex agnus-castus Seeds
collected. The cartridge was rinsed with 4 ml
of solvent, and the eluate was collected. The
eluates from the cartridge was combined,
transferred to a 10 ml volumetric flask, and
diluted with solvent to volume.
Standard Solution
About 5 mg of agnuside reference standard
was dissolved in 10 ml of methanol, with
sonication, diluted quantitatively, with
methanol to obtain a solution having a known
concentration of about 0.125 mg/ml and
filtered through a cellulose membrane having
0.45 µm porosity.
Fig. 4: HPLC Chromatogram Overlay of VAC Samples and Agnuside Standard.
RESULT AND DISCUSSIONS
HPTLC fingerprints of VAC sample (T1–T6)
shows variation. In Figures 1a and 1b, sample
T1 shows no band at Rf 0.75, band at Rf 0.5 of
sample T3 is fainter than other samples. Band
at Rf 0.4 corresponding to that of agnuside is
present in all the samples. In sample T4, band
at Rf 0.5 and Rf 0.4 are faint. Sample T5
shows no band at Rf 0.83. In Figure 2, samples
T1, T3, T4 and T5 showed no bands at Rf
0.33, Rf 0.41, Rf 0.75 and Rf 0.58,
respectively. Band at Rf 0.25 corresponding
to casticin is presents in all the samples.
RRJoB (2015) 21-28 © STM Journals 2015. All Rights Reserved
Rajanna et al.
Chromatographic System: Shimadzu
prominence 20AD HPLC.
Detector: PDA.
Chromatography Column and Stationary
Phase: Reverse phase C18 (250 mm x 4.6
mm), 5 µm.
Mobile Phase: Methanol and water (1:19).
Injection Volume: 10 µl.
Flow Rate: 1 ml per min.
Detection: UV, 258 nm.
Procedure: 10 µl of the filtered standard
solution and 10 µl of the filtered test solution
were injected separately and the
chromatogram was recorded. The analyte peak
responses were measured. The content of
agnuside in the substance being examined
from the peak response of analyte was
calculated.
Casticin and Agnuside Content
VAC samples were analysed using reverse
phase HPLC-PDA (Figures 3 and 4). The
retention time of casticin was at 10.9 min.
Peak corresponding to casticin was present in
all the samples. The casticin content in the
sample varied from 0.03% to 0.16% (Table 1).
The least was in sample T1 (0.03%) and
highest was in sample T4 (0.16%). Retention
time of agnuside was at 13.5 min. The peak
corresponding to agnuside content in the
sample varied from 0.009% to 0.03%. The
least was in sample T1 (0.009%) and highest
was in sample T4 (0.2%).
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Research & Reviews: Journal of Botany
Volume 4, Issue 3
ISSN: 2278-2222(online)
Table 1: Casticin and Agnuside Content in
VAC Samples.
VAC Casticin (%, Agnuside (%,
Samples w/w) w/w)
T1 0.03 0.009
T2 0.11 0.14
T3 0.09 0.07
T4 0.16 0.2
T5 0.13 0.12
T6 0.09 0.03
CONCLUSION
VAC is an important shrub of commerce
widely used in dietary supplement industry
and ayurveda. In ayurveda, VAC seed is
known as ‘Renuka’ (Sanskrit); however, Vitex
nigundo seeds are used as substitute here. It is
used in ayurvedic formulations like
Pramehamihira Taila, Vasachandanadi Taila,
Chandanadi Taila, Dashamularista,
Sarasvatarista, Mahayogaraja Guggula,
Anutaila, and Balasvagandha Lakshadi Taila
[12]. VAC is cultivated and used all over the
world as a medicinal plant. According to Hua-
Bin et al., HPLC fingerprinting technique is
capable of providing useful information
associated with the herbs’ quality, which can
be applied to be a platform for establishing the
qualities of herbal medicines [13]. Such
similar HPTLC fingerprinting evaluation of
medicinal plant collected from different
geographical locations have been carried out in
Asteracantha longifolia [14], Nicotiana
tabacum leaf [15] and root [16], Oroxylum
indicum [17] etc. In the present study, VAC
seeds collected from different geological
location viz., USA, EU and India showed
phytochemical variations. HPTLC profile of
VAC sample showed wide range of variation
(Table 1). Similarly, quantitative analysis of
casticin and agnuside showed wide range of
variation in content. Hence it is important to
establish phytoequivalence in VAC.
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Cite this Article
Rajanna L, Raghavan PV, Sharma GSS.
Phytochemical Evaluation of Vitex
agnus-castus L Seeds Collected from
Different Geographical Regions of the
World. Research and Reviews: Journal
of Botany. 2015; 5(3): 21–28p.
Page 28