Varaprasad Bobbarala et al.

/ Drug Invention Today 2009, 1(1),39-45

Available online through
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Research Article

Vitex agnus castus Molecular Marker Compounds Extraction and Optimization Using HPLC
with ELS Detector
1Somasekhar Penumajji, 2*Varaprasad Bobbarala, and 3K. Chandrasekhar Naidu
1Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Hyderabad
2For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam-17

3Department of Botany, Andhra University, Visakhapatnam-3

Received on: 20-08-2009; Revised on: 21-09- 2009; Accepted on:15-10-2009

ABSTRACT
Vitex agnus castus extracts in different solvent ratios were analyzed using High Performance Liquid Chromatography (HPLC) with Evapora-
tive Light Scattering Detector (ELSD). The markers of our interest in Vitexagnus castus are Agnuside, Casticin and Vitexyl acetone. To
correlate the retention time of the molecular marker compounds with Vitex agnus castus extract were compared with standard molecular
markers of Agnuside, Casticin and Vitexyl acetone using same conditions of that of extract and were analyzed statistically. Apart from the
results ELS Detector is regarded as a valuable alternative to UV detection system for liquid chromatographic analysis of substances that does
not contain a strong chromophore. As the markers do not contain strong chromophore, it is difficult to identify it UV detection system in High
performance liquid chromatography. So an alternative is the Evaporative Light scattering Detector which is an effective tool for the identifi-
cation of the compounds which do not contain chromophores. Present study concludes that ELS Detection system is more efficient than the
UV/Vis detection system.

Keywords: Vitexagnus castus, Evaporative light scattering detector, High Performance Liquid chromatography.

INTRODUCTION
Vitexagnus castus commonly called Vitex, and is widely cul-
tivated in warm temperate and subtropical regions for its aromatic
foliage and flowers. The leaves, flowers, and/or berries may be con-
sumed as a decoction, traditional tincture, cider vinegar tincture, syrup,
elixir, or simply eaten straight off the plant as a medicinal food. The
berries are considered a tonic herb for both the male and female repro-
ductive systems. The leaves are believed to have the same effect but
to a lesser degree1, 2. This plant is commonly called monk’s pepper
because it was originally used as anti-libido medicine by monks to aid
their attempts to remain celibate. It is believed to be an aphrodisiac,
hence the name chaste tree. Clinical studies have shown its beneficial
effects in the management of premenstrual stress syndrome (PMS) 3,
4, 5 and infertility. The use of extracts of the plant is recommended in
Germany 6.
The markers of our interest are Agnuside, Casticin and Vitexyl
acetone do not contain strong chromophore, it is difficult to identify
it by UV detection system in High performance liquid chromatogra-
phy. So an alternative is the Evaporative Light Scattering Detector
(ELSD) which is an effective tool for the identification of the com-
pounds which do not contain chromophores. Evaporative light scat-
tering detection is a process by which an HPLC solvent stream is
nebulized and the resultant droplets entrained in a gas stream. Mo-
bile phase is then evaporated from the droplets. When an analyte is
*Corresponding author.
Dr. Varaprasad Bobbarala
Scientist In-Charge,
For U Biosciences/IMMA Labs,
A/4A, Park lane Residency, East point colony,
Visakhapatnam, A.P-530017, India.
Tel.: + 91-9949129539
E-mail:varaprasadphd@rediffmail.com
Drug Invention Today Vol.1.Issue 1.November 2009
less volatile than the mobile phase, it remains in the gas stream as a
“dry” solute particle and flows to the detection region of the ELS
detector where a light beam intersects the stream. The particles scat-
ter the light beam, and the detector measures the effects of the scat-
tering. ELSD is compatible with virtually all modes of chromatogra-
phy, including flow injection analysis. It responds to all compounds
that are, relative to the mobile phase, sufficiently nonvolatile at the
conditions of analysis. Applications for ELSD include combinatorial
libraries of small molecules, natural product extracts and libraries,
phospholipids, food products, and related materials. In particular,
ELSD is used to detect pharmaceuticals lacking chromophores, car-
bohydrates, polysaccharides, lipids, vegetable oils, fatty acids, oli-
gomers, or hydrocarbons. For molecules without chromophores and
those that do not fluoresce, ELSD provides effective detection. An
ELS detector augments the absorbance detector’s function by de-
tecting compounds that do not have a UV/Vis chromophore. You can
also use ELSD as a qualitative tool to demonstrate the purity or com-
plexity of a sample, rather than to quantitate an individual analyte.
This detection system is well suited for the plant derived molecules 7,
8, 9, 10, 11, 12, 13, 14.
MATERIALS AND METHODS
Vitexagnus castus whole plant is ground to a free flowing
powder in a pulverizer. 1.5g of the powdered whole plant material was
weighed into 50ml flasks. 20ml of the solvent was added into the
powdered whole plant material in each flask dichloromethane/metha-
nol (50:50), 100% methanol, 80% ethyl acetate/methanol, 60% ethyl
acetate/methanol, 50% ethyl acetate/methanol, 40% ethyl acetate/
methanol, 20% ethyl acetate/methanol individually. Allow it to soak at
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 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),39-45
room temperature for 12 hours or overnight. Stir the mixture for 12
hours at room temperature. Filter through a Wattman filter paper and
collect the filtrate. Return the residue to the reaction flask. Add 20ml
more of the solvent. Stir the mixture for 4 hours at room temperature.
Filter and collect the filtrate. Combine both the filtrates. Concentrate
the filtrate using a rotary evaporator under vacuum keeping the tem-
perature of the water bath below 50°C, to near dryness. Dry the sample
completely under high vacuum using a vacuum freeze drier. The re-
sulting off-white to pale yellow material is collected and stored in air
tight container.

The extracts obtained from different solvent extractions were

analyzed using high performance liquid chromatography (HPLC) with
Evaporative Light Scattering Detector (ELSD).

Molecular marker compounds in Vitexagnus castus: Chromatogram 02

The markers of our interest inVitexagnus castus are Agnuside,
Casticin and Vitexyl acetone. To correlate the retention time of the
molecular marker compounds with Vitexagnus castus extract, stan-
dard molecular Agnuside, Casticin and Vitexyl acetone are injected in
HPLC using same conditions of that of extract. The quantitation of
Agnuside, Casticin and Vitexyl acetone standards are done by inject-
ing different volumes. All chromatograms are overlaid and the area Zoning of the chromatogram:
under the peaks is noted and a calibration curve is drawn and the
value of relative standard deviation R2 must be 0.99 otherwise the
method need to fine tuned.
RESULTS
Chromatogram 01 shows the chromatographic pattern of the
extract. Chromatogram 02 shows the Solvent B composition which is
indicated by red line. The Composition of the solvent B is initially 5%
which becomes 100% in 20 minutes and then it stays 100% till the end
of run.
Based on the intensity of the peaks the chromatogram is
divided into 2 zones, Zone 1 is from 2 to 6 minutes, Zone 2 is from 7 to
11 minutes and Zone 3 is from 15 to 26 minutes (Chromatogram03).

Standard chromatogram of Vitexagnus castus:

During the process of HPLC method development the ex-

tract of Vitexagnus castus obtained with 100% methanol extraction
was used for HPLC analysis.

Chromatogram 03

Chromatogram 01
Chromatogram 04
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 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),39-45
Chromatogram04 showing the chromatographic patterns in
ELSD, UV254nm and UV310nm.
Retention time correlation with Molecular marker compounds:

The markers of our interest inVitexagnus castus are Agnuside,

Casticin and Vitexylacetone. To correlate the retention time of the
molecular marker compounds with Vitexagnus castus extract, stan-
dard molecular markers Agnuside, Castacin and Vitexylacetone are
injected in HPLC using same conditions of extract. From chromato-
grams 05 and 06 it is evident that the peak at 8.8 min corresponds to
Agnuside and the peak at 16.1 min corresponds to Casticin and the
peak at 18.7 correspond to Vitexylacetone.

Chromatogram 07

Graph 01: Calibration curve for Agnuside

Chromatogram 05
Chromatogram07: Overlay of chromatograms of Agnuside
at different concentrations.From the calibration curve the R2value is
found to be 0.99 (graph 01)

Quantitation of Casticin:
The quantitation of Casticin is done by injecting different
volumes 1µl, 2µl, 3µl, 4µl, 6µl, 8µl, and 10µl (chromatogram08). All
chromatograms are overlaid and the area under the peaks is noted
and a calibration curve is drawn and the value of R2 was found out to
be 0.99 (graph02).

Chromatogram 06

Chromatogram05 showing the retention time of Agnuside,

Castacin and Vitexylacetone reference standard. Chromatogram06
showing the overlay of Agnuside, Castacin and Vitexylacetone refer-
ence standard with the extract.
Quantitation of Agnuside:
The quantitation of Agnuside is done by injecting different
volumes 1µl, 2µl, 3µl, 4µl, 8µl, 12µl, and 20µl (chromatogram07). All
chromatograms are overlaid and the area under the peaks is noted
and a calibration curve is drawn and the value of R2 was found out to
be 0.99 (graph01).
Drug Invention Today Vol.1.Issue 1.November 2009 Chromatogram 08 39-45
 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),39-45

Graph 02: Calibration curve for Casticin Graph 03: Calibration curve for Vitexylacetone

Chromatogram08: Overlay of chromatograms of Casticin at From the calibration curve the R2value is found to be 0.99(graph03).
different concentrations. From the calibration curve the R2value is Chromatogram09: Overlay of chromatograms of Casticin at different
found to be 0.99(graph02) concentrations.

Quantitation of Vitexylacetone: Molecular Markers Optimization:

The quantitation of Vitexylacetone is done by injecting dif- Following are the HPLC Chromatograms of the Vitexagnus
ferent volumes 1µl, 2µl, 3µl, 4µl, 6µl, 8µl, and 12µl (chromatogram09). castus extracts obtained in different solvents.
All chromatograms are overlaid and the area under the peaks is noted
and a calibration curve is drawn and the value of R2 was found out to
be 0.99 (graph03).

Chromatogram 09
Chromatogram 10

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 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),39-45
Chromatogram12: Extraction with Ethyl acetate: methanol
(80:20). Chromatogram13: Extraction with Ethyl acetate: methanol
(60:40).

Chromatogram 11
Chromatogram 14
Chromatogram10: Extraction with dichloromethane: metha-
nol (50:50). Chromatogram11: Extraction with 100% methanol.

Chromatogram 15
Chromatogram14: Extraction with Ethyl acetate: methanol
(50:50). Chromatogram15: Extraction with Ethyl acetate: methanol
Chromatogram 12 (40:60).

Chromatogram 13 Chromatogram 16

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 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),39-45

Chromatogram16: Extraction with Ethyl acetate: methanol

(20:80). Chromatogram17: Overlay of fingerprints of Vitexagnus castus
extracted with varying compositions of ethyl acetate / methanol mix-
tures in Zone-1.

Graph04

Chromatogram 18

Graph05

Graph 04 showing the Agnuside optimization in different

solvent extractions. Statistical analysis reveals that the recovery of
Agnuside is maximum in 40%ethylacetate extraction. Graph05 show-
ing the Casticin optimization in different solvent extractions. Statisti-
cal analysis reveals that the recovery of Casticin is maximum in
60%ethylacetate extraction.

Chromatogram 19
Chromatogram18: Overlay of fingerprints of Vitexagnus castus ex-
tracted with varying compositions of ethyl acetate/methanol mixtures
in Zone-2. Chromatogram19: Overlay of fingerprints of Vitexagnus
castus extracted with varying compositions of ethyl acetate/metha-
nol mixtures in Zone-3.

Graph06
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 Varaprasad Bobbarala et al. / Drug Invention Today 2009, 1(1),39-45
Graph07
Graph06 showing the Vitexyl acetone optimization in differ-
ent solvent extractions. Statistical analysis reveals that the recovery
of Vitexyl acetone is maximum in 40%ethylacetate extraction. Graph 07
is multiparameter extraction optimization.
Thus it is evident from the statistical analysis that 46.6 %
ethyl acetate in methanol is the optimum solvent composition with
which we can get the maximum recovery of Agnuside, Castacin and
Vitexylacetone markers and minimum recovery of the polysaccharides.
DISCUSSION
ELS Detector is regarded as a valuable alternative to UV
detection system for liquid chromatographic analysis of substances
that does not contain a chromophore. The markers of our interest of
in the extraction optimization of Vitexagnus castus are Agnuside,
Casticin and Vitexyl acetone. As the markers do not contain chro-
mophore, it is difficult to identify it UV detection system in High
performance liquid chromatography. So an alternative is the Evapora-
tive Light scattering Detector which is an effective tool for the identi-
fication of the compounds which do not contain chromophores.
Vitexagnus castus whole plant is ground to a free flowing powder and
different solvents were added into the powdered whole plant material
Source of support: Nil, Conflict of interest: None Declared
Drug Invention Today Vol.1.Issue 1.November 2009
in each flask and allowed to soak at room temperature, Filter and
collect the filtrate. Concentrate the filtrate using a rotary evaporator
under vacuum Dry the sample completely under high vacuum using a
vacuum freeze drier. Each extract is injected into the HPLC with UV
and ELSD; the resulting chromatograms are evaluated for optimiza-
tion.
The results of the present study conclude that ELS Detec-
tion system is more efficient than the UV/Vis detection system, It is
evident that 46.6% ethyl acetate in methanol is the optimum solvent
composition with which we can get the maximum recovery of Agnuside,
Casticin and Vitexyl acetone markers. The chromatograms clearly re-
vealed that the marker peaks are not observed in UV spectrum; whereas
perfect separations as well as identification of peaks were observed
in ELSD chromatogram.
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