JPC – Journal of Planar Chromatography – Modern TLC

https://doi.org/10.1007/s00764-019-00004-0

ORIGINAL RESEARCH PAPER

A new thin-layer chromatography–direct bioautography assay

for the qualitative and quantitative determination of peroxidase
inhibitors in plant extracts
Reham S. Darwish 1 & Eman Shawky 1 & Hala M. Hammoda 1 & Fathallah M. Harraz 1

# Akadémiai Kiadó, Budapest, Hungary 2020

Abstract
Thin-layer chromatography (TLC) hyphenated to bioassays is a modern tool used for discovery of drugs with diverse biological
activities from plant extracts which justifies the continuous need for developing such methods. A new TLC–bioautography assay
for detecting peroxidase enzyme inhibitors was developed and validated in this study. Peroxidases are related to pathogenesis of
many diseases such as neurodegenerative disorders and rheumatoid arthritis and are considered as inflammatory markers. The
developed TLC–bioautography method was based on reaction of peroxidase with hydrogen peroxide and the subsequent
formation of blue color by the reaction between the released oxygen and benzidine. Compounds with peroxidase inhibition
activity are observed as yellowish spots against a blue background. The developed method was validated according to the ICH
guidelines. Quantitative estimation of peroxidase inhibitors was achieved by applying image analyses techniques, showing good
linearity (R2 = 0.998), accuracy (% recovery 96.6–99.7%), intra-day precisions (RSD% = 0.95–2.35) and inter-day precision
(RSD% = 1.80–3.20) as well as low LOD (0.28 μg/spot) and LOQ (0.85 μg/spot). The developed method was applied to
different Juniperus species fractions as a demonstrative illustration. Overall, the method proved to be efficient, simple and rapid
for identification of peroxidase inhibitors in complex matrices and can be applied in high-throughput screening of plant extracts.

Keywords TLC–bioautography . Validation, antiperoxidase . High-throughput screening . Juniperus

Introduction
Natural products (NPs) are now considered an appreciated
source of diverse chemical structures to be exploited in the
discovery of new drugs. The identification of bioactive com-
pounds considering the complexity of plant extracts remains
to be a challenging task. Effect directed analysis (EDA) is now
widely used in the screening of natural products [1], where the
advances in thin-layer chromatography (TLC) hyphenations
allowed for bioprofiling, and characterization of active com-
ponents within the complex matrices of plant extracts [2].
TLC–bioautography has reached a broad scope of enzy-
matic inhibitory activities such as: acetylcholinesterase [3,
4], xanthine oxidase [5], α- and β-glucosidases [6], lipase
* Eman Shawky
shawkyeman@yahoo.com; eman.m.shawky@alexu.edu.eg
1
Department of Pharmacognosy, Faculty of Pharmacy, Alkhartoom
square, Alexandria University, Alexandria 21521, Egypt
[7] and tyrosinase [8]. The current wide application of TLC
direct bioautography (DB) emphasizes the continuous need
for the development of novel bioautographic methods for the
detection of versatile bioactivities.
Meanwhile, peroxidases represent a family of isoenzymes
(heme-containing enzymes) present in almost all living organ-
isms that catalyze the oxidation of a number of substrates
(hydrogen donors) by hydrogen peroxide [9]:
Peroxidase
H2 O2 þ AH2 → 2H2 O þ A
Some peroxidases are related to pathogenesis of some dis-
eases such as: atherosclerosis, neurodegenerative disorders,
cancer, diabetes, renal diseases, rheumatoid arthritis, chronic
sinusitis, gastric ulcers, colitis, pancreatitis and chronic peri-
odontitis [10, 11]. Some peroxidase enzymes are considered as
inflammatory markers such as the myeloperoxidase which uti-
lizes H2O2 to oxidize chloride, resulting in the formation of the
pro-inflammatory oxidant, hypochlorous acid (HOCl), a
microbiocidal which oxidizes many biological substrates
resulting in damage of tissues [10, 11]. Recently a variety of

tissue specific peroxidases inhibitors are used to treat various
types of diseases [10–13]. Peroxidase and antiperoxidase ac-
tivities are generally evaluated in-vitro using spectrophotomet-
ric methods that depend on the presence of hydrogen donors
(chromogens), such as: pyrocatechol-aniline and guaiacol
which during the peroxidase biocatalytic process, are convert-
ed to a pink-colored compound with λmax of 510 nm for the
former and a brown-colored compound with a λmax of 470 nm
for the later [14, 15]. The lack of any previous reports for a
bioautographic method for detection of peroxidase inhibitors
prompted the attempt to develop and validate such a procedure.
Fractions of different Juniperus species were selected as the
focus of this study. Plants belonging to Juniperus, Family
Cupressaceae, are used traditionally to cure digestive disor-
ders, chronic arthritis, gout, diabetes and they were proven to
have several pharmacological activities including anti-inflam-
matory, hypoglycemic, diuretic, antiseptic, and antirheumatic
features [16, 17]. Juniperus well-known bioactive components
include flavonoids, biflavonoids, sesquiterpenes, diterpenes
and lignans which exhibit various biological activities [18].
The presented work aimed at development and validation
of a novel bioautographic method for qualitative and quanti-
tative evaluation of the antiperoxidase activity of plant ex-
tracts or fractions and investigation of antiperoxidase activity
of different fractions of J. communis, J. horizontalis and J.
chinensis.
Materials and methods
Chemicals
Horseradish peroxidase enzyme type II, essentially salt free
(150 u/mg), and quercetin standard were purchased from
Sigma-Aldrich (Darmstadt, Germany). Acetic acid, ethyl ace-
tate, dichloromethane, n-butanol, chloroform and methanol
were purchased from Merck (Darmstadt, Germany), n-hexane
from BDH (Poole, England).
Normal phase silica gel 60 TLC plates (20 cm × 20 cm) and
reversed phase-C18 modified silica gel TLC plates (20 cm ×
20 cm) were purchased from Merck (Darmstadt, Germany).
Plant collection and extraction
Non-flowering aerial parts of Juniperus communis, J.
horizontalis and J. chinensis were collected on March 2017
from the International Garden at Cairo, Egypt. Plants’ identi-
fication was confirmed by Dr. Therese Labib, specialist of
plant identification in El Orman Garden, Cairo, Egypt. Three
voucher specimens (No. JC-45 J. communis, JH-46 J.
horizontalis and JCH-47 J. Chinensis) were kept in the her-
barium of Pharmacognosy Department, Faculty of Pharmacy,
Alexandria University.
JPC-J Planar Chromat
100 g of air dried grinded aerial parts of J. communis, J.
horizontalis and J. chinensis were separately macerated in
1.3 L 95% ethanol, filtered and concentrated under reduced
pressure. After that, each ethanol extract was re-dissolved in a
mixture of water and methanol (1:1) to get a hydroalcoholic
extract, and then liquid–liquid extraction was done using n-
hexane, methylene chloride, ethyl acetate and n-butanol, sub-
sequently. Finally, concentration of each fraction to dryness
under reduced pressure was carried out. Methylene chloride
fractions were re-dissolved in methylene chloride, while ethyl
acetate and butanol fractions were re-dissolved in methanol to
prepare suitable concentration of each fraction as illustrated in
(Table 1) each fraction was filtered using syringe filters.
High–performance thin–layer chromatography
TLC chromatography: The samples were applied bandwise by
means of a CAMAG (Switzerland) Linomat V automated
spray-on band applicator equipped with a 100 μL syringe.
Samples and standard solutions were applied to pre-coated
TLC plates (10 cm × 20 cm) which were obtained from TLC
plates (20 cm × 20 cm) after cutting using a ruler and a cutter.
Application was done from the left to the right of the plate at a
distance of 1.5 cm from the bottom and the margins of the
plate. Standard plate is the plate of quercetin (the model ana-
lyte in this study) which was used to construct the calibration
curve and to calculate some of validation parameters of the
studied method. On the other hand, sample plates are plates of
different fractions which were used to evaluate the
antiperoxidase activity of the studied plants. For standard plate
bandwidth was set to 6 mm and inter-band spaces to 4 mm to
get 18 tracks for six different concentrations each was applied
in triplicate manner. While for sample plates, bandwidth was
set to 8 mm and inter-band spaces to 5 mm to get 16 tracks; 14
tracks for samples and the last two tracks were for application
of standard with concentration of 4 and 6 μg/spot for working
calibration method.
The mobile phase that provided the best separation results
was chloroform:methanol:acetic acid, 8.5:1.5:0.15 (v/v)
(system I) for methylene chloride fractions and ethyl
acetate:methanol:water:acetic acid, 45:4:2:0.35 (v/v) (system
II) for ethyl acetate and butanol fractions. The inclusion of an
acid (acetic acid) in the chromatographic system was important
to suppress the ionization of the acidic compounds being
chromatographed. Development of plates was performed by
ascending chromatography over a distance of 90 mm in a
CAMAG twin trough chamber (10 × 20 cm) at room
temperature. Saturation of the development chamber with
mobile phase was done 15 min before the run. After
development, hot air (40 °C) was used for plates drying. All
mobile phases were freshly prepared with analytical grade
chemicals in volumes sufficient to supply the tank for each run.
JPC-J Planar Chromat

Table 1 Preparation of samples

and their final concentrations for Fraction Plant Weight Volume of solvent Concentration
peroxidase inhibition assay
Methylene chloride
Methylene chloride fractions J. communis 5 mg 0.5 mL 10 mg/mL
J. horizontalis 27 mg 0.5 mL 54 mg/mL
J. chinensis 76 mg 0.5 mL 152 mg/mL
Methanol
Ethyl acetate fractions J. communis 38 mg 4 mL 9.5 mg/mL
J. horizontalis 24 mg 4 mL 6 mg/mL
J. chinensis 28 mg 4 mL 7 mg/mL
Butanol fractions J. communis 27 mg 1 mL 27 mg/mL
J. horizontalis 22 mg 1 mL 22 mg/mL
J. chinensis 6 mg 1 mL 12 mg/mL

Revealing reagents preparation
Peroxidase enzyme solution preparation: 10 mg/mL solution of
peroxidase in cold phosphate buffer (pH 7) was prepared. Then
50 mL of this solution was diluted by its mixing with 50 mL of
phosphate buffer (pH 7). Benzidine was prepared by dissolving
4 g in 700 mL MeOH, H2O2 solution was prepared by dilution
of 10 mL of 20 V H2O2 with 250 mL of distilled water.
solution for 3 s then the enzyme was activated by putting the
plate in the oven at 35 οC for 2 min [19]. After that the plate was
dipped in a container containing H2O2 solution then it was re-
moved and finally dipped in another container containing ben-
zidine solution.
Validation

Validation of the developed and optimized methods was done

The image processing methodology
Image acquisition and image analysis software
Images of all studied plates were taken by scanning TLC
plates using a flatbed scanner Canon-MF 4750. ImageJ
(http://rsb.info.nih.gov/ij/download.htmlImageJ) was used as
image analysis software package. The image analysis method
was developed and validated for the quantitation of the
peroxidase inhibition activity of different fractions of J.
communis, J. horizontalis and J. chinensis.
Effect-directed analysis by TLC
Development of a novel method for assessment of peroxidase
inhibition activity
Calibration function was built as following: successive spots
from the stock solution of quercetin (1 mg/mL) were applied
in the following order 3, 4, 5, 6, 7, and 8 μl. Each of these
calibration levels was applied in triplicate. The standard plate
was developed in a mobile phase composed of ethyl
acetate:methanol:water:acetic acid, 45:4:2:0.35 (v/v) (system
II). Peak area was plotted against standards concentration in
μg/spot in order to construct the calibration curve, after that
subjection of the calibration data least-squares regression analy-
sis was done. Thereafter, peroxidase inhibition activity of the
samples was detected by immersion of plates in the enzyme
according to the ICH guidelines [20] individually. Validation
parameters were linearity, sensitivity (LOD, LOQ), precision,
specificity, and accuracy. Quercetin standard plate (normal phase
silica plate, system II) was used for the validation of the method.
Sensitivity of methods was determined in terms of the limit of
detection (LOD) and limit of quantification (LOQ) values that
based on the residual standard deviation of a regression line and
slope. Intra-day precision was determined by independent repeat-
ed analysis of five different volumes of standard solution
(quercetin) each repeated triplicate under the same conditions by
using the same apparatus and the same analytical method in the
same laboratory and on the same plate. Inter-day precision includ-
ed the analysis of the standard three times a day over 3 days by a
different analyst. The results of repeatability and intermediate
precision are expressed as relative standard deviation (RSD) (%).
Accuracy of peroxidase inhibition activity was carried out by
standard addition technique, as two spots representing two con-
centration levels 4 μg and 6 μg of quercetin (model analyte)
were applied over two tracks containing ethyl acetate fraction of
J. communis (pre-analyzed sample), respectively. After that the
plate was developed in system II. Addition experiments were
performed in triplicate and the accuracy was calculated as the %
of analyte recovered. Three analyses per concentration were
performed and mean + SD was determined.
The specificity of the methods was determined to ensure
that assays have the capability of determination analytes with-
out any interference from matrix elements that may have ef-
fect on the assay.

Fig. 1 Mechanism of oxidation of
benzidine by peroxidase/H2O2
into a blue colored complex
Quantitative determination of peroxidase inhibition activity
Samples; methylene chloride, ethyl acetate and n-butanol frac-
tions were prepared as mentioned. Each sample was spotted in
two tracks of volume of 4 and 6 μL. The development of
sample plates was done in a CAMAG twin trough chamber
using the suitable mobile phase system as we mentioned.
After immersion as discussed, plates were dried followed by
its scanning using a flatbed scanner Canon-MF 4750.
Fig. 2 TLC chromatograms of methylene chloride fractions of Juniperus
communis (tracks 1, 2), Juniperus horizontalis (tracks 3, 4) and Juniperus
chinensis (tracks 5, 6) were developed in system I and TLC
chromatograms of ethyl acetate and butanol fractions Juniperus
communis (tracks 7, 8 and 13, 14), Juniperus horizontalis (tracks 9, 10
JPC-J Planar Chromat
Results and discussion
Development and validation of a TLC–bioautography
method for evaluation of peroxidase inhibitory
activity of plant extracts
The comprehensive chemical and biological profiling of dif-
ferent Juniperus fractions was the target of this study. In this
regard, a novel bioautographic method coupled to a validated
and 15, 16) and Juniperus chinensis (tracks 11, 12 and 17, 18) were
developed in system II, under UV lamp 254 nm (a) and (c), after
dipping in peroxidase enzyme followed by dipping in H2O2 solution
and benzidine solution, separately and respectively (b) and (d)
JPC-J Planar Chromat

Fig. 3 TLC bioautograms of

quercetin applied on normal
phase (mobile phase, ethyl
acetate:methanol:water:acetic
acid, 45:4:2:0.35 (v/v) (system II))
and reversed phase-C18 modified
silica plates (mobile phase, water:
methanol, 7:3 (v/v))

TLC method was developed for evaluation of peroxidase in- where peroxidase inhibitors were observed as yellow spots
hibition activity of plant extracts/fractions. The new method against a blue background (Fig. 3).
depends on oxidation of benzidine by peroxidase enzyme in The developed method was then validated where quercetin
presence of hydrogen peroxide into a blue colored compound. was used as a chemical standard for the quantification of per-
Benzidine is oxidized by the peroxidase/H2O2 system, oxidase inhibitory activity. Quercetin was chosen after testing
resulting in formation of benzidine cation free radical. This and trying several phenolic compounds for their peroxidase
radical is in rapid equilibrium with the blue-colored charge- inhibitory activity. The linear regression data showed good
transfer complex of benzidine with benzidine diimine as illus- linear relationship over the concentration range of 0.85–9 μg
trated in (Fig. 1) [21–23]. /spot for peroxidase inhibition method where the correlation
Bleaching the blue color of the oxidized benzidine by con- coefficient of the software data indicated a very good linearity
stituents with peroxidase inhibition activity resulted in the for the method (R2 > 0.99) (Table 2). The method showed
appearance of inhibitors as yellowish spots against a blue high precision with low %RSD values (Table 2) and low
background. More intensive area around the bleached spot values of LOD and LOQ indicated that the methods was sen-
on the TLC plate indicates a stronger peroxidase inhibition sitive (Table 2) and % recovery values indicated high accuracy
capacity (Fig. 2b and d). of the studied method (Table 3).
The developed bioautographic assay was tested on the two
most commonly used TLC layers; silica and C18 modified
Quantitative evaluation of different Juniperus
silica plates. Quercetin (1 mg/mL) was used as standard to
fractions for their peroxidase inhibition activities
evaluate the bioautographic assay on TLC layers. Positive
results could be observed with the two TLC layers types
The quantitative evaluation of the antiperoxidase activity for
the different studied fractions was performed using image J
software package. The proposed quantitative methodology is
Table 2 Regression statistic parameters, limit of detection, limit of
quantification, intra-day and inter-day precision of the TLC–DB method based on measurements of TEA (total extract area); i.e., the
total areas under a video-scan of an extract/fraction
Regression equation Y = 2133X–2444
Correlation coefficient R2 = 0.998 Table 3 Accuracy of the TLC–DB method
LOD 0.280
Model Matrix Added Expected Found % %
LOQ (μg /spot) 0.85 analyte amount amount amount amount Recovery RSD
Linearity range (μg/spot) 0.85–9 (μg/ (μg/ (μg/spot) (μg/
Precision spot) spot) spot)
(intra-day precision) RSD% (inter-day precision) RSD%
Quercetin 3.85 4 7.85 7.83 99.7 2.21
1.67 2.30 7.77 98.9
2.18 3.20 7.66 97.5
1.41 2.01 3.85 6 9.85 9.73 98.7 1.84
0.95 1.80 9.66 98.0
2.35 3.10 9.52 96.6
 JPC-J Planar Chromat

Table 4 Quantitative evaluation

of different extracts and fractions Plant Peroxidase inhibition (SAC)
of the three plants for peroxidase
inhibition Applied sample volume Average SAC
4 μL 6 μL

Methylene chloride fractions J. communis 0.140 0.141 0. 141

J. horizontalis 0.031 0.032 0.032
J. chinensis 0.039 0.041 0.040
Ethyl acetate fractions J. communis 0.217 0.219 0.218
J. horizontalis 0.136 0.139 0.138
J. chinensis 0.193 0.195 0.194
n-Butanol fractions J. communis 0.042 0.045 0.044
J. horizontalis 0.059 0.062 0.061
J. chinensis 0.115 0.118 0.117

(approximation of the sum of all the peaks) which can be
considered as the sum of antiperoxidase activity of all extract
ingredients. The TEA value is compared to areas obtained for
standards, and then calculated using the experimentally deter-
mined Eqs. (1) and (2). The only requirement is working inside
the linear range of the peak area response, both for the standard
and extract/fraction measured [3, 24]. Having the linear cali-
bration curve for a reference compound, one can compute the
equivalent amount of the standard, giving the same
antiperoxidase activity (TEA) as the extract/fraction. This can
be called the standard activity coefficient (SAC). For example
if the SAC equals 2, this mean that the dry extract/fraction is
twice as active as the pure reference compound.
SAC ¼ MS =ME ð1Þ
Where MS and ME are the quantities of standard and ex-
tract/fraction, respectively, in μg/spot; MS and ME are in the
linear range, and both have the same antiperoxidase activity
(TEA). The standards and crude extracts/fractions cannot be
applied at levels resulting in the same TEA, because of differ-
ent linearity ranges. Additionally, at lower concentrations,
some compounds can be applied below the detection limit.
Furthermore, both samples are spotted in the middle of their
linearity range and the SAC is calculated from the formula
containing the TEA of an extract/fraction and calibration
curve coefficients of a standard:

Fig. 4 TLC and Image J program

chromatograms of (a) methylene
chloride fraction and (b) ethyl
acetate fraction of Juniperus
communis, after dipping in
peroxidase enzyme, H2O2 and
benzidine, separately and
respectively
JPC-J Planar Chromat

quantitative estimation of xanthine oxidase inhibitors and superox-
SAC ¼ ðTEA–IÞ= S* ME ð2Þ
Where TEA is total extract or fraction area; I is intercept
from the standard’s curve equation;
S-slope from the standard’s curve equation; and ME is ap-
plied microgram of extract or fraction. Due to introduction of
parameters from the standard’s curve equation, the SAC be-
comes independent of the applied amount of extract or fraction.
Studying SAC values of the different fractions (Table 4,
Fig. 2a–d) revealed that fractions of the highest peroxidase
inhibition activity are methylene chloride and ethyl acetate
fractions of J. communis (Fig. 4). Ethyl acetate fraction of J.
communis showed the highest peroxidase inhibition activity
with SAC value of 0.218 (Table 4).
Conclusion
This study presented the development and validation of a novel
bioautographic peroxidase assay which provides many advan-
tages over other spectrophotometric assays, as it speeds up the
whole screening procedure, detects the active zones which can
facilitate their isolation and characterization. On the other
hand, other assay methods are only able to determine whether
the whole plant extract/fraction has activity or not. The devel-
oped method was successfully applied to discover the potential
peroxidase inhibitors in different Juniperus species. The meth-
od was found to be rapid, simple, sensitive, accurate and pre-
cise and suitable for high-throughput analysis of plant extracts.
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