Latin American Journal of Pharmacy

(formerly Acta Farmacutica Bonaerense)

Regular Article
Received: March 1, 2013
Revised version: July 13, 2013
Accepted: July 14, 2013

Lat. Am. J. Pharm. 32 (7): 1082-7 (2013)

New HPLC Method for Quality Control of -Escin

in Aesculus hippocastanum L. Hydroalcoholic Extract
Priscila A. DE ALMEIDA 1, Michele C. ALVES 1, Hudson C. POLONINI 1, Lidiane S. DUTRA 1,
Magda N. LEITE 1, Ndia R.B. RAPOSO 1, Anderson de O. FERREIRA 2 & Marcos A.F. BRANDO 1,2*
1

Ncleo de Pesquisa e Inovao em Cincias da Sade (NUPICS), Faculdade de Farmcia,

Universidade Federal de Juiz de Fora, Rua Jos Loureno Kelmer, s/n,
36036-330 Juiz de Fora MG, Brasil
2 Ortofarma Laboratrio de Controle de Qualidade, BR 040, Empresarial Park Sul, 39,
36120-000 Matias Barbosa MG, Brasil

SUMMARY. The major phytoconstituent present in Aesculus hippocastanum L. is a complex mixture of

triterpene saponin glycosides known as escin. The objective of this study was to develop and validate a
simple and fast HPLC method for quantification of -escin in A. hippocastanum L. hydroalcoholic extract.
It was proposed, developed and validated a method by liquid chromatography with UV detection (HPLCUV). The method was validated for specificity, linearity, limits of detection and quantification, precision,
accuracy and robustness and was proven adequate for quality control of this natural product.

INTRODUCTION
Medicinal plants have been for a long time a
major source of therapeutic agents for the pharmaceutical and cosmetic industries 1. One of
these plants that have been gaining relevance is
the Aesculus hippocastanum L. (Hippocastanaceae), also known as horse chestnut. It is
widely cultivated around the world as an ornamental plant, and it also has several ethnopharmacological uses 2.
The major phytoconstituent present in A.
hippocastanum is a complex mixture of triterpene saponin glycosides known as escin 3 ,
among other compounds such as flavonoids,
tannins and oligosaccharides 2. To the present,
horse chestnut have been proved to be effective
in the treatment of conditions such as chronic
venous insufficiency 4, hemorrhoids 5, inflammation 6 and cerebral ischemic damage 7,8, being
these biological activities mainly due to escin 8,9.
Escin (Fig. 1) is a natural mixture of triterpene saponins, which mainly consist of escin Ia,
escin Ib (-escin), isoescin Ia and isoescin Ib (escin) 10, being that -escin is the major active
component 7, when compared to -escin. Only
a few reports deal with the pharmacological ac-

tivity of -escin. Although it has less bioactivity

than -escin, commercial preparations available
in different countries contain both - and -escin 11.

Figure 1. Chemical structures of -escin and -escin

10.

KEY WORDS: Aesculus hippocastanum, High pressure liquid chromatography, Quality control.
*

Author to whom correspondence should be addressed. E-mail: marcosbrand2012@gmail.com

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ISSN 0326-2383

Latin American Journal of Pharmacy - 32 (7) - 2013

In the analytical context, the determination

of marker coumpounds in herbal medicines is
not only important, but also a necessary issue.
Analysis of saponins can be quite challenging
because of their complexity, amphiphilic nature
and tendency to form mixed aggregates in solution, which can turn separation and purification
into difficult processes 12.
Numerous analytical methods have been reported for the determination of escin including
spectrophotometry 13, thin-layer chromatography (TLC)densitometry 14, gas chromatography
(GC) 15, hyphenated techniques such as liquid
chromatography coupled do mass spectrometry
(LCMS) 10,11 or nuclear magnetic resonance
(LCNMR) 15 and capillary electrophoresis (CE)
16. Many of these methods have limitations such
as the necessity of well-trained person and unavailability of equipment in routine laboratories,
due to the high cost 17.
A most achievable technique, the high performance liquid chromatography with ultraviolet
detection (HPLC-UV), for its turn, has not been
cited in literature with the objective to determine -escin, probably due to the fact that the
molecule lack a strong UV chromophore. In this
light, the objective of this study was to develop
and validate a simple and fast HPLC method to
the quality control of this raw material, using
UV detection.
MATERIAL AND METHODS
Reagents, reference standard and materials
The methanol used in the preparation of the
mobile phase was HPLC grade (Panreac, Spain).
The analytical grade reagents used were: anhydrous acetic acid (Neon, Brazil), chlorophorm
(Sigma, USA), propanol (Sigma, USA) and hydrochloric acid (HCl) (Sigma, USA). Ultrapure
water obtained in an AquaMax-Ultra 370 Series
(Young Lin, Korea) was used throughout analysis. As a work standard, -escin (Sigma, USA)
was used, and it was previously standardized (n
= 6) against a reference standard (United States
Pharmacopeia, nominal content 99.09%) and the
relative potency found was 98.70%. As raw material, hydoalcoholic extract of A. hippocastanum (Catedral, Brazil) was used. All the mobile phases were filtered in a 0.45-m filter
membrane (Sartorius, Germany) and degassed
by an ultrasonic apparatus (Cristfoli, Brazil) for
30 min before use. All volumetric glassware
used was previously calibrated.
Chromatographic conditions
The HPLC analyses were performed in a

Young Lin (Korea) chromatography system

composed by: quaternary pump (YL 9110), photodiode array detector (YL 9160), automatic injector (YL 9150), column compartment (YL
9130) and software controller (Clarity). Chromatographic separation was achieved using a
CN column, 250 x 4.6 mm, 5 m particle size
(Kromasil, France), connected with a pre-column (CN, 4.0 x 3.0 mm, 5 m, Phenomenex,
USA). The chromatographic conditions maintained constants were: injection loop of 20 L;
mobile phase consisted solely of methanol at
1.0 mL/min (isocratic elution) with pH adjusted
to 6.0 with 0.1 M acetic acid; column compartment at 20 C; and UV detection at 210 nm.
Standard and Sample Solutions
Standard
An accurately weighed (analytical digital balance AY220, Shimadzu, Japan) amount of the escin standard (0.010g) was dissolved and diluted in methanol to obtain a work solution of 200
g/mL.
Sample
Sample solution was prepared using adequate volumes (variable accordingly to validation test) of hydroalcoholic extract of A. hippocastanum, which were transferred to a beaker
in which 20.0 mL 0.1 M HCl were added. The
mixture was subjected to agitation for 15 min
using a magnetic stirrer. The content of the
beaker was poured into a separatory funnel of
250 mL. This was followed by the extraction of
-escin by adding 70.0 mL of a mixture of
propanol:chloroform (2:5, v/v). After stirring vigorously for 2 min and then a 5-min rest period,
the organic phase was separated from the aqueous phase. To the remaining aqueous phase,
another portion of the extractor liquid was
added and the procedure was repeated twice,
making up three extractions. The organic phases
were combined in a round bottom flask, and
then they were rotaevaporated to dryness under
reduced pressure. The residue in the flask was
then resuspended with methanol, and subsequent washings were performed, transferring
the washings to a 50 mL volumetric flask up to
its volume.
Validation
After the method development, the validation tests were performed according to the International Conference on Harmonization (ICH)
guidelines 18 and Instituto Nacional de Metrologia, Normalizao e Qualidade Industrial (INMETRO) 19, comprising the following parame1083

DE ALMEIDA P.A., ALVES M.C., POLONINI H.C., DUTRA L.S., LEITE M.N., RAPOSO N.R.B., FERREIRA A. de O. & BRANDO M.A.F.

ters: specificity, linearity, limit of detection

(LOD), limit of quantification (LOQ), precision,
accuracy, and robustness.
Specificity
The specificity was determined by comparison among the chromatograms of the sample
(89.47 g/mL), the standard (100 g/mL) and a
hydroalcoholic solution at 60% (ethanol:water,
60:40, v/v) that mimetizes the ethanol content of
the sample and that was prepared following the
same procedure conducted for the sample.
Linearity
The test was conducted from the plotting of
three standard curves constructed each one
from five concentrations, in order to assess the
linear relationship between the concentration of
the analyte and the obtained areas. From the
Standard Solution, aliquots of 4.0, 4.5, 5.0, 5.5,
and 6.0 mL were withdrew and diluted to 10.0
mL in volumetric flasks, resulting in the following concentrations: 80.0, 90.0, 100.0, 110.0, and
120.00 g/mL (n = 3). For the sample, aliquots
of 4.0, 4.5, 5.0, 5.5, and 6.0 mL were withdrawn
from the sample solution and diluted to 10.0 mL
in volumetric flasks, resulting in the following
concentrations: 76.6, 86.1, 95.7, 105.3 and 114.9
g/mL (n = 3). The data for each concentration
level were statistically evaluated by analysis of
variance (ANOVA) 20,21 and subjected to the
least squares method to determine the correlation coefficient of the calibration curve.
Limits of Detection and Quantification
The limit of detection (LOD) and the limit of
quantification (LOQ) were determined from
three standard calibration curves and were calculated as shown in Eq. [1] and Eq. [2], respectively:
[1]
[2]
where a is the slope of the calibration curve
and S is the standard deviation of the y-intercept.
Precision
The test was designed to assess the degree
of dispersion among the series of measurements
obtained by the same analyst (intra-assay precision, repeatability) and between two analysts
and two days (within-lab variations, intermediate precision), for a solution at 100% of the
work concentration. Repeatability was determined by analyzing six replicates consecutively
by a single analyst in a single day. The intermediate precision was also performed in six repli-

1084

Levels
Factors

X1: wavelength of detection (nm)

X2: mobile phase flow rate

-1

+1

208
0.8

210
1.0

212
1.2

Table 1. Factor and levels for the experimental design

performed to evaluate the robustness of the method.

cates, but in two days, by different analysts. An

injection precision of less than 5% coefficient of
variation (CV) was considered appropriate.
Accuracy
The determination of accuracy was performed by the method of standard additions.
The sample solely and the sample spiked with
80, 100, and 120% of the nominal standard work
solutions concentrations (n = 3 for each concentration level) were injected into the chromatograph. The result was expressed as percentage
of recovery, compared with the analytical curve
obtained.
Robustness
The test was performed to measure the ability of the method to withstand small changes of
the analytical parameters selected. For this purpose, a 22 experimental design with triplicate in
the central point was conducted, as one can see
in Table 1.
RESULTS AND DISCUSSION
To the best of the authors knowledge, there
is none analytical methodology in official compendia or in the scientific literature for quantification of -escin in the hydroalcoholic extract of
A. hippocastanum. Although saponins can be
determined in natural products by several analytical techniques, the HPLC is the most diffused
one, despite the fact they are usually difficult to
detect by UV since most of them lack a strong
UV chromophore. Traditionally, their detection
has been performed at lower UV wavelengths
ranging from 200 to 210 nm, limiting the selection of solvents and the gradients that can be
used 22.
Acetonitrile-water gradients have been the
mobile phase of choice when using C-18
columns, although methods using methanol as
the organic modifier have been used too, with
or without the addition of an acid or buffer.
However, the problematic detection of saponins
by UV absorvance has sustained the development of HPLC-MS methods 23. The MS detection,
however, is expensive and time consuming, and
so we developed a method for -escin determi-

Latin American Journal of Pharmacy - 32 (7) - 2013

nation using UV detection, the most widespread

detection worldwide.
Initially, we have set a C18 column and a
methanolic mobile phase as initial chromatographic conditions, obtaining good responses
for the analyte determination. The scanning of
the best wavelength of detection showed that
210 nm was appropriate for purposes. We also
evaluated the influence of the pH of the mobile
phase and the temperature of the column compartment, and we observed that the best responses were obtained setting the pH at 6.0 and
the column temperature at 20 C. Using the conditions chosen, the validation studies were performed. For specificity, the results are shown in
Fig. 2. As one can observe in Fig. 2, the solvent
peak does not interfere with the interest compounds analysis. There is no overlap of peaks

Figure 2. Chromatograms obtained for the specificity

test. Injections of: A, hydroalcoholic solution; B, escin
standard; C, Aesculus hippocastanum L. extract; D,
standard (above), sample (below) and placebo overlapped. Chromatographic conditions: CN column (250
x 4,6 mm, 5 m particle size), mobile phase composed by methanol 100% (pH=6.0) at flow of 1.0 mL
min1 (isocratic elution), column compartment at 20
C, and UV detection at 210 nm.

of standard or sample compared to other from

solvents used. In Fig. 2D one can see the overlap of co-eluting peaks from the samples and
standards, confirming that there is indeed specificity in the method.
The results of the linearity are also shown in
Table 2. The study of the linearity of the
method was subjected to the least squares
method and obtained a correlation coefficient
greater than 0.99 (R2 = 0.9913). The linearity
was also evaluated through analysis of variance
21. The test of significance of regression returned
the values of Fcalculated (MSmodel / MSresidual) =
1481.65, larger than Fcritical 0.05, v1=1, v2=13 = 4.67,
which confirms the existence of a significant linear relationship between the two variables, with
95% confidence. The test of lack of fit value returned Fcalculated (MSlack of fit / MSpure error) = 2.91,
smaller than Fcritical 0.05, v1=3, v2=10 = 3.71, with 95%
confidence, which confirms that there is no lack
of fit in the model. Thus, the generated results
when one uses the equation of the model to
foresee the -escin concentration in the A. hippocastanum fluid extract will be very close to
the real values in the samples.
The LOD and LOQ for the samples were calculated from calibration plots and the results
were expressed in Table 3. For precision and
accuracy tests (Table 3), all coefficients of variation found were within specification limits recommended ( 5%). For accuracy, the averages
recoveries lie within acceptance criteria of 98102% for this parameter.
In order to evaluate the method robustness,
a 22 experimental design with triplicate in the
central point was conducted. The coefficient of
variation from the assays was low (1.55%). It
shows that the active recovery in method is not
affected by neither factors tested and the
method can be considered robust (Table 4).
Considering all the validation and taking all
these data into account, one can say that from a
practical standpoint the method is considered
valid for the intended goals.
CONCLUSION
Under the conditions described, the method
for quantitative determination of -escin in A.
hippocastanum L. hydroalcoholic extract is in
accordance to the validation parameters required by the ICH and INMETRO guidelines.
The method was simple, selective, precise, accurate and fast, providing the reliability required
for an analytical method to be used in the quality control of this natural product.

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DE ALMEIDA P.A., ALVES M.C., POLONINI H.C., DUTRA L.S., LEITE M.N., RAPOSO N.R.B., FERREIRA A. de O. & BRANDO M.A.F.

Parameter

Calibration
plots

Results
Slope

Intercept

Linear coefficient

Range (g/mL)

7357.19

+134965.13

0.9913

80-120

Source

Sum of Square (SS)

Degrees of freedom

Mean of Square (MS)

1011

1.62 1011

Residual

1.42 1009

13

1.09 1008

Lack of fit

6.64 1008

2.21 1008

Pure error

7.61 1008

10

7.60 1007

Total

1.64 1011

14

1.17 1010

Model
Analysis of
variance

1.62

Table 2. Results from the test of linearity (n = 3).

Parameter

LOD (g/mL)
LOQ (g/mL)

Value

n=3
n=3

0.20
1.08

Especification

Precision

CV (%) Intra-day, First day

CV (%) Intra-day, Second day
CV (%) Inter-day

n = 12
n = 12
n = 24

3.08
1.52
2.90

CV 5%

Accuracy

Average recovery (%)

n = 15

99.18

98.0-102.0%

Table 3. Results for LOD, LOQ, precision and accuracy tests. CV: coefficient of variation.

Matrix X
Experiment

1
2
3
4
5
6
7

X1

X2

X12

-1
1
-1
1
0
0
0

-1
-1
1
1
0
0
0

1
-1
-1
1
0
0
0

Assay (%)

98.93
100.73
103.23
103.09
102.36
102.87
101.18

4039
3684
3456
3493
3397
3514
3504

1.604
1.725
1.829
1.879
1.800
1.780
1.882

Table 4. Factors, levels and contrast coefficients of the matrix for the experimental design conducted in the robustness study. X1: wavelength of detection (nm); X2: mobile phase flow rate; N: number of theoretical plates;
S: symmetry of the chromatographic peak. All experiments provided good symmetry of the analytical peak (>
1.0) and column efficiency (number of theoretical plates/meter > 2000).

Acknowledgements. The authors would like to

thank CAPES, FAPEMIG and UFJF for the financial
support.

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