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High performance thin layer chromatography (HPTLC) and high performance

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 Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

High performance thin layer chromatography (HPTLC) and high

performance liquid chromatography (HPLC) for the qualitative and
quantitative analysis of Calendula officinalis—Advantages and
limitations
Christine M. Loescher a, David W. Morton a, Slavica Razic b, Snezana Agatonovic-Kustrin a,∗
a
School of Pharmacy and Applied Science, La Trobe Institute of Molecular Sciences, La Trobe University, Edwards Rd, Bendigo 3550, VIC, Australia
b
Department of Analytical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Chromatography techniques such as HPTLC and HPLC are commonly used to produce a chemical finger-
Received 5 February 2014 print of a plant to allow identification and quantify the main constituents within the plant. The aims
Received in revised form 28 March 2014 of this study were to compare HPTLC and HPLC, for qualitative and quantitative analysis of the major
Accepted 26 April 2014
constituents of Calendula officinalis and to investigate the effect of different extraction techniques on the
Available online 2 May 2014
C. officinalis extract composition from different parts of the plant. The results found HPTLC to be effective
for qualitative analysis, however, HPLC was found to be more accurate for quantitative analysis. A com-
Keywords:
bination of the two methods may be useful in a quality control setting as it would allow rapid qualitative
Calendula
Fingerprint profiling analysis of herbal material while maintaining accurate quantification of extract composition.
Quality control © 2014 Elsevier B.V. All rights reserved.
HPLC
HPTLC

1. Introduction chlorogenic acid are commonly associated with anti-oxidant activ-

Calendula officinalis, commonly known as Marigold, has been
traditionally used for its anti-inflammatory effects. Extracts of
C. officinalis have also been found to have anti-oxidant, anti-
fungal, anti-oedema, anti-diabetic and wound healing properties
[1,2]. The major constituents of C. officinalis include steroids, ter-
penoids, triterpenoids, flavonoids, phenolic acids and carotenes
[3,4]. Faradiol, rutin, caffeic acid and chlorogenic acid have all
been isolated from C. officinalis and have shown biological activ-
ity in the body [4–8]. The most potent anti-inflammatory effects
of C. officinalis have been attributed to the faradiol monoesters
[9]. Faradiol belongs to the triterpenoid family and has shown
anti-inflammatory effects similar to indomethacin, a non-steroidal
anti-inflammatory (NSAID), at an equimolar dose [5]. Rutin, one
of the major flavonoids found in many vegetable materials, has
also been associated with anti-inflammatory effects as well as anti-
bacterial, and hepatoprotective activity [6,8]. Synthetic derivatives
of rutin such as troxerutin are also medically used to strengthen
blood vessels [10]. Caffeic acid, a phenolic compound, and its ester
∗ Corresponding author. Tel.: +61 3 5444 7360; fax: +61 3 5444 7878.
E-mail address: s.kustrin@latrobe.edu.au (S. Agatonovic-Kustrin).
ities [7].
The aims of this study were to compare HPTLC and HPLC, for
qualitative and quantitative analysis of the major constituents of C.
officinalis and to investigate the effect of different extraction tech-
niques on the C. officinalis extract composition from different parts
of the plant. The fingerprinting profile was used to look at the differ-
ences in composition depending on extraction technique, different
parts of the plant, such as petal, flower heads, and leaves, as well
as determine if a significant difference in composition between the
African and English Marigold could be identified.
Due to the complex nature of herbal medicines, chromato-
graphic methods are commonly used to develop a fingerprint;
a profile of the different constituents within the herbal product
[11]. Fingerprinting has the potential to identify the plant, quantify
active ingredients, and to detect impurities or contaminants such as
herbicides. High performance liquid chromatography (HPLC) and
gas chromatography (GC) are commonly used for fingerprinting
as they can successfully separate out the different constituents
of the extract and can provide both qualitative and quantitative
data [11,12]. However, HPLC is expensive to run, uses large quan-
tities of solvent, and columns may be sensitive to very high or
low pH mobile phases. GC is limited to the use of volatile com-
pounds, such as essential oils in herbal preparations [11]. Thin layer

http://dx.doi.org/10.1016/j.jpba.2014.04.023
0731-7085/© 2014 Elsevier B.V. All rights reserved.
 C.M. Loescher et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59
chromatography (TLC) has been used for the qualitative analysis of
herbal medicines and is still commonly used to characterise and
track components visually or as an initial separation technique
[11,13,14]. With advances in technology in recent years, TLC has
become more sophisticated and with the development of high per-
formance thin layer chromatography (HPTLC), has the potential to
provide both qualitative and quantitative data [15].
Despite much work on the effect of C. officinalis extracts on cell
cultures being reported [2,16,17] there is little data available on the
concentration of active ingredients present in different parts of the
C. officinalis plant [18]. Moreover, it can also be difficult to differ-
entiate between the English Marigold (C. officinalis) and the African
Marigold (Tagetes erecta L.) which, although different species, can
often be confused with each other [19].
2. Experimental
2.1. Reagents and solutions
All samples and mobile phases were prepared using analytical
grade reagents. Dried C. officinalis flowers were obtained from an
All Rare Herbs (Mapleton, Australia) and standardised CO2 C. offici-
nalis extract was sourced from The Herbarie (Stoney Hill Farm, Inc,
Prosperity, SC, USA).
Wild calendula flowers and leaves were collected from a local
garden in Bendigo (Victoria, Australia). African Marigold seeds (Mr.
Fothergill’s Seeds & Bulbs Pty Ltd, Sydney, Australia) were bought
from the local nursery and grown in a growth cabinet for 6 months
at a temperature of 20 ◦ C with flowers harvested when required.
Standard solutions of rutin 97% (Alfa Aesar, Heysham, UK),
chlorogenic acid 95% (Sigma-Aldrich, Saint Louis, Missouri, USA),
caffeic acid 98% (Sigma-Aldrich) and faradiol >99% (PhytoLab,
Dutendorfer, Germany) were prepared using absolute ethanol
(Merck, Darmstadt, Germany). A mixed standard containing rutin,
chlorogenic acid and caffeic acid was also made.
2.2. Sample collection and preparation
All fresh flower samples were dried in a 50 ◦ C oven overnight
before use. The dried flower samples were manually separated into
petals and flower heads for analysis. All samples were ground into
a fine powder for analysis using a coffee grinder (Sunbeam, Botany,
Australia).
2.3. Extraction
Between 1 and 4 g of sample was placed into an 18 × 55 mm
cellulose extraction thimble (Whatman, Little Chalfont, UK) and
then refluxed in a Soxhlet apparatus for 8 h using 50 mL of absolute
ethanol as solvent. They were then concentrated to about 1:10 w/v
using a rotor evaporator. All samples were filtered using 1.0 ␮m
syringe filters (Millipore, Molsheim, France) before use. Samples
were stored in the fridge to prevent sample degradation.
2.4. High performance thin layer chromatography (HPTLC)
NC Silica (F254 10 × 20 cm) HPTLC plates (Merck) and were acti-
vated in an oven at 50 ◦ C for 30 min before use. Samples were
applied using a Linomat 5 (Camag, Muttenz, Switzerland) semi-
automated applicator with a 100 ␮L syringe (Hamilton, Bonaduz,
Switzerland) which was programmed using Planar chromatogra-
phy manager-winCATS (Camag). Sixteen lanes were applied per
plate with a 6 mm band width, 11.3 mm between bands and 8 mm
from the bottom of the plate. Volumes applied varied between 0.2
and 5.0 ␮L with the syringe rinsed with absolute ethanol between
samples.
53
Table 1
The gradient mobile phase used for HPTLC plate development. The percentage of
each solvent required, migration distance of the solvent front and drying time
between mobile phases are all displayed.
Hexane (%) Ethyl acetate (%) Water (%) Migration (mm) Drying time (min)
0 80 20 60 5
66 34 0 75 2
100 0 0 90 2
A mixture of hexane (BDH Chemicals Ltd. Poole, England), ethyl
acetate (Merck), and acetic acid (Merck) was previously used as a
mobile phase in HPTLC separations. The 3-step gradient elution was
set up with hexane, ethyl acetate containing 2% glacial acetic acid
and Milli-Q water (Bedford, Massachusetts, USA) using the WinCats
(Camag) software (Table 1). An AMD2 developer (Camag) was used
for the development of the plates.
After plate development a photo was taken with a TLC visu-
aliser containing a 12 bit camera (Camag) using UV wavelengths of
366 nm, 243 nm, or white light before derivatisation. Then, either
10% H2 SO4 (BDH Chemicals Ltd.) in Milli-Q water or 2-aminoethyl
diphenylborinate (Natural product reagent A) (Sigma-Aldrich) was
sprayed over the plate in a thin layer using quick-fit airbrushing
glassware. Plates were then placed in a 50 ◦ C oven for 30 min to
complete derivatisation before another photo was taken. Quan-
titative HPTLC analysis was performed using VideoScan software
(Camag) with a minimum peak width of 7 pixel, peak heights of
400 pixel, and a minimum area of 1000 pixel, using a filter width of
7 nm.
2.5. High performance liquid chromatography (HPLC)
A pentafluorophenyl with TMS end capping (PFP) column,
100 × 4.60 mm (Phenomenex, Sydney, Australia) was used with
a Shimadzu HPLC system (Tokyo, Japan) which included a SCL-
10Avp system controller, LC-10ATvp pump, DGU-14 A degasser,
SIL-10ADvp auto injector and a SPD-10Avp UV–vis detector. Mobile
phase was prepared using and acetonitrile (Merck) and a 2% aque-
ous acetic acid solution in a ratio of 15:85. The mobile phase was
degassed using a vacuum filter and 47 mm Magna Nylon Mem-
brane Filters (Thomas Scientific, Swedesboro, NJ, USA). The detector
wavelength was 254 nm, the flow rate was 0.5 mL/min, and the
injection volume was 0.5 ␮L. Samples were run for a total of
15 min.
3. Results and discussion
For HPTLC, chromatographic conditions were based on iso-
cratic separation with different mobile phase combinations of
hexane, ethyl acetate and acetic acid [5,20]. The mobile phase
was successful at separating caffeic acid and faradiol. Polar sol-
vents, such as water, were successful at migrating the polar
standards, chlorogenic acid and rutin, but produced a spiky sol-
vent front. A gradient elution of 15:85 acetonitrile and water
with 2% acetic acid was found to produce baseline separation of
rutin, caffeic acid and chlorogenic acid with a clean solvent front
(Table 1).
Table 1 Both HPLC and HPTLC use UV light for the detection
of analyte compounds. On the HPTLC plate chlorogenic acid and
caffeic acid produced blue bands at Rf values of 0.16 and 0.61,
respectively, when observed under 366 nm UV light (Fig. 1A), while
Rutin, and faradiol could not be detected and were made detectable
through derivatisation with 2-aminoethyl diphenylborinate, as a
specific, and sulphuric acid, as universal reagent. 2-aminoethyl
diphenylborinate allowed rutin to be visualised as a green band
(Rf 0.12) under UV 366 nm and chlorogenic and caffeic acid became
54 C.M. Loescher et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59

Fig. 1. A developed HPTLC plate seen before derivatisation (A), and after derivatisation with H2 SO4 (B). Lanes 1 and 16 contain the standardised extract. Lanes 2—rutin,
faradiol and lane 15—caffeic and chlorogenic acid. All other lanes contain a mixture of the four standards at increasing concentrations from left to right. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)

a more vibrant blue but it had little effect on faradiol. Sulphuric acid
allowed all standards to be seen with faradiol appearing as a brown
band at Rf 0.72, however, it decreased the visibility of chlorogenic
acid (Fig. 1B).
Detection and base line separation was achieved for HPLC for
chlorogenic acid, caffeic acid and rutin with retention factors of
3.30, 4.45 and 7.55 min, respectively. Faradiol could not be detected
using HPLC (Fig. 2).
Semi-automated applicators have allowed greater control of
substance application onto HPTLC plates. In order to assess the
extent of variation, 4 identical plates were set up and run under the
same conditions. Using a two way ANOVA with repetition (n = 4),
it was found that the results were statistically different (P = 0.02).
Thus, it cannot be assumed that replicate plates are the same and
further analysis is required if data from different plates is to be com-
pared. The accuracy of the image analysis (i.e. variation between
replicate images), was assessed by comparing multiple images of
the same plate. Although no significant variation was found (P val-
ues 0.7–0.9), at times the software would recognise a single peak
as a double peak (Fig. 3).
The HPLC method shows linearity for all standards over the
entire working range (0.05–2 mg/mL) (Fig. 4a). However, the linear
range of the HPTLC method is 0.05–0.1 mg/␮L for chlorogenic acid
and rutin, and 0.05–0.5 mg/␮L for caffeic acid (Fig. 4b). Calibration
for the HPLC and HPTLC methods were compared by analysing a
solution of mixed standards at known concentrations (Fig. 6), hav-
ing 0.565 mg/mL of chlorogenic acid, 0.735 mg/mL of caffeic acid
and 0.830 mg/mL of rutin. It was found that HPLC gave results
closest to the true values (0.548, 0.662 and 0.789 mg/mL)
while HPTLC results showed greater variation from the true
values.
Different extraction methods were tested to see how they affect
the chemical fingerprint and concentration of the four marker com-
pounds. A standardised C. officinalis supercritical CO2 extract, dried
C. officinalis extracts of the flower, petals, flower head and leaves,
a locally grown C. officinalis flower, and African Marigold extracts
were compared using HPTLC and HPLC separation for both qualita-
tive and quantitative analysis.
HPTLC was able to identify and separate the selected standards
and allowed comparisons between samples to be made (Fig. 5). All
 C.M. Loescher et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59 55

Fig. 2. HPLC chromatogram of 1 mg/mL standards of chlorogenic acid (3.30 min), caffeic acid (4.45 min) and rutin (7.55 min).

samples except the standardised extract and the African Marigold
had a green band at Rf 0.12 indicative of rutin. The caffeic acid band
(Rf 0.61) was also seen in all samples except in the leaf and African
Marigold extracts. The chlorogenic acid band (Rf 0.16) was seen in
all samples before derivatisation (not shown) but was not as clear
on the derivatised plate. All samples contained a brown band at
Rf 0.74 which was similar in appearance to the faradiol band at Rf
0.72. Other significant bands were seen at Rf 0.64 and 0.70 which
appeared red in colour. An additional blue band at Rf 0.48 was seen
in the standardised extract and a blue band at Rf 0.82 was only seen
in the African Marigold.
As base line separation could not be achieved with the HPLC
analysis of the extracts, only semi-quantitative data for rutin,
chlorogenic acid and caffeic acid was obtained (Fig. 6). Despite
a large amount of variation seen within the extracts, all sam-
ples contained similar ratios of all three standards. Chlorogenic
acid was unable to be quantified in the standardised extract. The
average amount of chlorogenic acid, caffeic acid and rutin were
0.31–1.03 mg/mL, 0.07–0.75 mg/mL and 0.72–3.19 mg/mL, respec-
tively, across all extracts.
HPTLC offers significant advantages in that it uses less sol-
vent, and has the ability to simultaneously run multiple samples
thus saving time and cost [21,22]. HPLC methods, on the other
hand, are generally considered more robust, and are capable of
higher degrees of precision on replication and quantitation. With
advances in HPTLC technology other aspects of fingerprinting such
as the use of gradient mobile phases and detection also need to
be considered to determine the effectiveness of HPTLC as a qual-
ity control technique and potential applications in a quality control
setting.
Both techniques found isocratic mobile phases insufficient at
separating all of the standards. For HPTLC, the polar standards rutin
and chlorogenic acid failed to migrate from the origin, while fara-
diol remained on the column when using HPLC.

Fig. 3. Two images of the same plate. Image on the left shows the software picking up single peaks for faradiol in lanes 14 and 15, but double peaks in the image on the right,
and a double peak for caffeic acid in lane 15, but only a single peak in the right image.
56 C.M. Loescher et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59
Fig. 4. HPTLC (a) and HPLC (b) calibration curves of chlorogenic acid ( ), caffeic acid
( ), and rutin ( ) using peak area. Error bars show the 95% confidence intervals
(n = 6). (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
To overcome this problem, gradient elutions were trialled for
both methods, with only HPTLC being successful in providing good
separation for all standards used. A polar solvent like water allows
the migration of the polar standards in HPTLC but might cause
spikey solvent fronts and co-elution which will make identifica-
tion of substances difficult [23]. A gradient elution was found to be
useful at restricting the migration of the polar solvent, in this case
water, and ensuring adequate drying time which allowed a straight
solvent front to be produced. The use of gradient elution was also
explored using HPLC. However, a rising baseline was seen as the
mobile phase composition changed. This is a common limitation of
in the use of gradient elution in HPLC where UV–vis as a baseline
reading is established from the initial mobile phase used [24]. This
made the detection of faradiol difficult as it got lost in the baseline.
It was therefore decided an isocratic mobile phase would be used
for the separation of the standards with faradiol excluded from the
HPLC analysis.
While both methods use UV detection, HPTLC used specific set
(available) wavelengths of 336 nm, 254 nm, and/or white light to
illuminate bands on the HPTLC plate. If samples were unable to
be detected under these wavelength conditions, compounds could
be chemically altered (i.e. be derivatised) to allow detection [23].
One of the major advantages of HPTLC was that samples could be
run simultaneously in lanes side by side and results were able to
be seen directly on the plate in the form of coloured bands. This
allowed for easy identification of standards present and compar-
isons of samples (Fig. 2). The plate was then photographed and
software used to produce chromatograms which could then be used
for further analysis. HPTLC however, often requires derivatisation
to allow samples to be seen under the specific wavelengths of light.
In this study, natural product reagent A was useful for detecting
most of the standards, except faradiol. This was to be expected as
natural product reagent A is specific for phenolic compounds [25].
Sulphuric acid, a universal agent, allowed all compounds to be seen,
however, this decreased the visibility of chlorogenic acid due to its
degrading nature (Fig. 2B).
Another advantage of HPTLC is that images of plates can be taken
before and after derivatisation which gives a large pool of data for
analysis. Derivatisation however, must be used with caution as it
is difficult to get an even coating of the derivative compound on
the plate which may then lead to the problems if quantification of
results is required [22].
While HPTLC derivatisation can be time consuming, HPLC is also
very time consuming as it requires samples to be run individually.
HPLC UV–vis also requires a wavelength to be set for detection. This
caused additional problems for faradiol as it was ideally detected at
210 nm while the other standards were detected at 254 nm. Other
difficulties seen with the UV detector included determining if a
sample was being detected, remaining on the column, coming out
on the solvent front, or getting lost in a noisy base line. There are
however, many other detection methods available for HPLC such as
a photo diode array (PDA), mass spectroscopy (MS) and evaporative
light scatter (ELS) which can provide more adequate information
to allow the identification of substances in a complex mixture [24].
The UV–vis detector is the most widely used detector for HPLC
and although there may be initial difficulties in determining the
optimum wavelength to be used for a set of standards, optimi-
sation is fairly straightforward [24]. Both HPTLC and HPLC face
similar limitations when developing a method for fingerprint pro-
filing, however, HPTLC has the advantage of having both coloured
bands and retention factors to identify individual substances. It also
allows samples to be run simultaneously making it a quick simple
process of comparing chemical profiles.
The introduction of automated applicators for HPTLC has greatly
increased the repeatability of sample application, allowing sam-
ple volumes to be applied more evenly and accurately [22]. Renger
et al. [23] recommended that the relative standard deviation (RSD)
of an applicator should be no higher than 1–2%. The main difference
between traditional TLC and HPTLC is the smaller more consistent
particle sizes of the silica on the HPTLC plates which allows cleaner
and quicker separation of substances [26]. However, small incon-
sistencies in the powder coating on the plates can occur and further
contribute to variations seen between plates [23]. A plate compar-
ison was done using a two way ANOVA and significant variation
was seen between plates (P < 0.5). This suggests the need to per-
form calibration with each plate if quantification is required. An
internal or external standard may be beneficial as it would allow a
response factor to be determined and would allow for the natural
variation of the stationary phase between plates [22]. There was
no difference found between the images taken of the same plate (P
values 0.7–0.9), indicating consistency in the imaging of the cam-
era. However, the software may also have difficulty determining if
peaks are overlapping or only a single peak is present (Fig. 6). The
need for manual alterations of the data is of concern as it opens
the method up to more uncertainties and the potential to influence
results.
Even though quantitative calibration data in TLC is usually best
fitted with a polynomial regression (Fig. 4a), there is often a linear
range within working range that can be established with sat-
isfactory linear regression. The linear range of the method was
established as 0.05–0.1 mg/mL for chlorogenic acid and rutin and
 C.M. Loescher et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59 57

Fig. 5. Derivatised plate of C. officinalis extracts. Lanes 2–3 petal extracts, lanes 4–5 flower heads extracts, lanes 6–7 leaf extracts, lanes 8–9 whole flower extracts, lanes
10–11 wild flower extracts, lanes 12–13 African Marigold extracts, lanes 14–15 standardised extract. Lanes 1 and 16 have been removed for clarity. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

0.05–0.5 mg/mL for caffeic acid. Even though the calibration of caf-
feic acid was found to be linear over a greater concentration range,
this may not have been due to a higher saturation point or greater
sensitivity of the compound. As seen in Fig. 3, caffeic acid appears
to create two bands and significant tailing on the HPTLC plate.
This may have been due to the degradation of the substance on
the plate and therefore quantification is uncertain. The degrada-
tion of caffeic acid can be inhibited by the addition of antioxidants
to the extraction solvents. Studies indicated that polyphenols are
responsible for oxidative degradation of caffeic acid and its deriva-
tives [27]. Only a single peak was seen for caffeic acid in the HPLC
analysis and therefore HPLC quantification is likely to be more reli-
able (Fig. 4).
HPLC was found to have a linear calibration over the entire
working range (0.05–2 mg/mL) which would help explain why it
is seen as the preferred technique in the literature (Fig. 4b). The
use of a scanning densitometer for HPTLC detection would provide
more data on a given band allowing for greater accuracy of the
quantification [26]. While HPTLC and high resolution plate imag-
ing has the potential to quantify, current techniques still require
improvement to enable them to become competitive with tradi-
tional methods. A combination of the two techniques was used

Fig. 6. HPLC chromatogram of dried C. officinalis extract. Peak areas used for quantification and retention times for chlorogenic acid (3.30 min), caffeic acid (4.48 min) and
rutin (7.63 min).
58 C.M. Loescher et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 52–59
to determine the chemical composition of C. officinalis and how
differences in the natural variation of the plant effected the com-
position and levels of the chosen standards. Growth conditions of
the plant, location of growth, part of the plant used and the age
of the plant can all cause variation in the final chemical compo-
sition [28,29]. Other factors such as the extraction method used
can also greatly affect the composition of the final pharmaceutical
product [7,30].
The extraction method and solvent used can have a great effect
on what substances are extracted from a plant. This was illustrated
by the difference seen in the lab extraction samples and the stan-
dardised extract fingerprints. An ethanol solvent was used for the
lab extractions and it was found to contain a range of polar and non-
polar substances. The standardised extract, however, was produced
using supercritical CO2 extraction, then dissolved in a coconut oil
base and contained mostly nonpolar substances as seen with the
limited number of bands at lower Rf values (Fig. 5). Rutin was found
to be present in the HPLC analysis of the standardised extract but
not on the HPTLC plate. This may have been caused by the lower
sensitivity of HPTLC or by interference from the noisy baseline, seen
in all the HPLC extract chromatograms (Fig. 6), causing a false read-
ing and therefore it was not a true reflection of the amount of rutin
present in the standardised extract. The difference in composition
of the standardised extract compared to the other extracts is likely
because different extraction methods are better at extracting dif-
ferent compounds. Supercritical CO2 is better for the extraction of
volatile and low molecular weight compounds and prevents the
loss or degradation of compounds through heat [31]. The standard-
ised extract was also standardised for faradiol, which could change
the levels of other compounds in the sample. An understanding of
how an extraction can influence the yield of a particular chemical is
important in ensuring the most active combination of ingredients
in a particular herbal plant ends up in the final therapeutic product.
It also highlights the importance of understanding the extraction
procedure used to ensure valid comparisons are made between
different products and plant materials.
Where the plant was grown, and which part of the plant used
can also have an influence on the levels of particular constituent of
the plant. In this study, dried C. officinalis of known species from
Queensland were compared to locally grown wild C. officinalis. The
flower heads, petals and leaves of C. officinalis were also compared.
The qualitative analysis using HPTLC found both C. officinalis species
to have very similar fingerprint profiles as did the petal and flower
head profiles. The leaves however, were found to have a differ-
ent fingerprint with a red band at Rf 0.7 to be of greater intensity
and other bands to be less distinct compared to the other extracts
(Fig. 5). This is not surprising as it is the flowers that are usually
used. A study by Ercetin et al. [32] also found that the C. offici-
nalis flower extracts contained higher anti-oxidant activity than
the leaf extracts. The red band seen in the leaves may be due to
the presence of flavonoids as they have previously been reported
to produce an orange–yellow band under 366 nm UV light although
further testing would be required for conformation [33]. Faradiol
was potentially seen in all samples, however, it was observed to be
present at slightly higher Rf values in the extracts than the standard
alone (Rf 0.74 and 0.72, respectively). Faradiol is usually present in
plants as faradiol esters such as faradiol laurate, faradiol myristate
and faradiol palmitate [14]. This may have caused the band shift,
however, further investigation would be required to determine if
this is the case. The similarities in fingerprints allow comparisons
to be made without concern for which part of the flower is used.
This will also allow the part of the flower which contains more of
a particular constituent to be used without causing problems with
overall species identification. Caution may need to be taken when
looking at leaf extracts as there is the possibility that it may cause
confusion with adulteration.
As small differences in chemical levels do not appear to affect
the overall fingerprint of C. officinalis, there is the potential that
adulteration from different species can also be detected using this
technique. The C. officinalis chemical profiles were compared to
African Marigolds, T. erecta, which has previously been confused
with C. officinalis [19]. The African Marigold was found to contain a
blue band at Rf 0.82 which was not seen in the C. officinalis extracts
and also did not contain any of the quantified standards (Fig. 5). The
blue band is likely to be another phenolic compound as described
by Maleš and Medić-Šarić [33]. The blue band may allow African
Marigold to be identified in a mixture of the two species, however,
further studies would be required to determine the sensitivity of
the analysis to contamination of African Marigold in C. officinalis
products.
The method of extraction was found to cause differences in the
chemical profile of the extracts, however, where the plant was
grown or what part of the plant was used had little effect on the
final profile. Quantitatively large variation was seen between the
extractions which was most likely due to the extraction technique.
Differences could be seen between African Marigold and C. offici-
nalis indicating that this technique has the potential to be used for
detecting adulteration of Calendula herbal products.
The use of HPTLC for qualitative analysis allowed quick determi-
nation on the effects of the extraction method used, plant species,
and part of the plant used, on chemical composition. Quantitative
data from HPLC showed that there was a great amount of variation
seen in the Soxhlet extraction method and there was no real differ-
ence observed in the type of plant used. Although there is still much
work required, a combination of both HPTLC and HPLC may provide
much useful information for the quality control of herbal medicines
to ensure safe and affective products are available in the market-
place. The combination of HPTLC and HPLC has the potential to be
used in a quality control setting to provide a cheap and rapid anal-
ysis of herbal compounds but also provide accurate quantification
when required.
4. Conclusion
Recent advances in technology have allowed the improvement
of thin layer chromatography so that HPTLC analysis is able to
provide useful qualitative data for the quick determination of key
standards in samples, with easy comparisons to be made between
different extracts. Quantitatively, however, HPTLC data resulted in
greater variation in the reported concentrations which may be due
in part to limitations in the sensitivity of the software. The results
of this study clearly show that HPLC remains a more robust method
and is preferred for quantitative analysis.
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