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 [7] and tyrosinase [8]. The current wide application of TLC
direct bioautography (DB) emphasizes the continuous need
Natural products (NPs) are now considered an appreciated for the development of novel bioautographic methods for the
source of diverse chemical structures to be exploited in the detection of versatile bioactivities.
discovery of new drugs. The identification of bioactive com- Meanwhile, peroxidases represent a family of isoenzymes
pounds considering the complexity of plant extracts remains (heme-containing enzymes) present in almost all living organ-
to be a challenging task. Effect directed analysis (EDA) is now isms that catalyze the oxidation of a number of substrates
widely used in the screening of natural products [1], where the (hydrogen donors) by hydrogen peroxide [9]:
advances in thin-layer chromatography (TLC) hyphenations
Peroxidase
allowed for bioprofiling, and characterization of active com- H2 O2 þ AH2 → 2H2 O þ A
ponents within the complex matrices of plant extracts [2].
TLC–bioautography has reached a broad scope of enzy- Some peroxidases are related to pathogenesis of some dis-
matic inhibitory activities such as: acetylcholinesterase [3, eases such as: atherosclerosis, neurodegenerative disorders,
4], xanthine oxidase [5], α- and β-glucosidases [6], lipase cancer, diabetes, renal diseases, rheumatoid arthritis, chronic
sinusitis, gastric ulcers, colitis, pancreatitis and chronic peri-
odontitis [10, 11]. Some peroxidase enzymes are considered as
* Eman Shawky inflammatory markers such as the myeloperoxidase which uti-
[email protected]; [email protected] lizes H2O2 to oxidize chloride, resulting in the formation of the
pro-inflammatory oxidant, hypochlorous acid (HOCl), a
1
Department of Pharmacognosy, Faculty of Pharmacy, Alkhartoom microbiocidal which oxidizes many biological substrates
square, Alexandria University, Alexandria 21521, Egypt resulting in damage of tissues [10, 11]. Recently a variety of
 JPC-J Planar Chromat

tissue specific peroxidases inhibitors are used to treat various 100 g of air dried grinded aerial parts of J. communis, J.
types of diseases [10–13]. Peroxidase and antiperoxidase ac- horizontalis and J. chinensis were separately macerated in
tivities are generally evaluated in-vitro using spectrophotomet- 1.3 L 95% ethanol, filtered and concentrated under reduced
ric methods that depend on the presence of hydrogen donors pressure. After that, each ethanol extract was re-dissolved in a
(chromogens), such as: pyrocatechol-aniline and guaiacol mixture of water and methanol (1:1) to get a hydroalcoholic
which during the peroxidase biocatalytic process, are convert- extract, and then liquid–liquid extraction was done using n-
ed to a pink-colored compound with λmax of 510 nm for the hexane, methylene chloride, ethyl acetate and n-butanol, sub-
former and a brown-colored compound with a λmax of 470 nm sequently. Finally, concentration of each fraction to dryness
for the later [14, 15]. The lack of any previous reports for a under reduced pressure was carried out. Methylene chloride
bioautographic method for detection of peroxidase inhibitors fractions were re-dissolved in methylene chloride, while ethyl
prompted the attempt to develop and validate such a procedure. acetate and butanol fractions were re-dissolved in methanol to
Fractions of different Juniperus species were selected as the prepare suitable concentration of each fraction as illustrated in
focus of this study. Plants belonging to Juniperus, Family (Table 1) each fraction was filtered using syringe filters.
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- High–performance thin–layer chromatography
matory, hypoglycemic, diuretic, antiseptic, and antirheumatic
features [16, 17]. Juniperus well-known bioactive components TLC chromatography: The samples were applied bandwise by
include flavonoids, biflavonoids, sesquiterpenes, diterpenes means of a CAMAG (Switzerland) Linomat V automated
and lignans which exhibit various biological activities [18]. spray-on band applicator equipped with a 100 μL syringe.
The presented work aimed at development and validation Samples and standard solutions were applied to pre-coated
of a novel bioautographic method for qualitative and quanti- TLC plates (10 cm × 20 cm) which were obtained from TLC
tative evaluation of the antiperoxidase activity of plant ex- plates (20 cm × 20 cm) after cutting using a ruler and a cutter.
tracts or fractions and investigation of antiperoxidase activity Application was done from the left to the right of the plate at a
of different fractions of J. communis, J. horizontalis and J. distance of 1.5 cm from the bottom and the margins of the
chinensis. 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
Materials and methods studied method. On the other hand, sample plates are plates of
different fractions which were used to evaluate the
Chemicals antiperoxidase activity of the studied plants. For standard plate
bandwidth was set to 6 mm and inter-band spaces to 4 mm to
Horseradish peroxidase enzyme type II, essentially salt free get 18 tracks for six different concentrations each was applied
(150 u/mg), and quercetin standard were purchased from in triplicate manner. While for sample plates, bandwidth was
Sigma-Aldrich (Darmstadt, Germany). Acetic acid, ethyl ace- set to 8 mm and inter-band spaces to 5 mm to get 16 tracks; 14
tate, dichloromethane, n-butanol, chloroform and methanol tracks for samples and the last two tracks were for application
were purchased from Merck (Darmstadt, Germany), n-hexane of standard with concentration of 4 and 6 μg/spot for working
from BDH (Poole, England). calibration method.
Normal phase silica gel 60 TLC plates (20 cm × 20 cm) and The mobile phase that provided the best separation results
reversed phase-C18 modified silica gel TLC plates (20 cm × was chloroform:methanol:acetic acid, 8.5:1.5:0.15 (v/v)
20 cm) were purchased from Merck (Darmstadt, Germany). (system I) for methylene chloride fractions and ethyl
acetate:methanol:water:acetic acid, 45:4:2:0.35 (v/v) (system
Plant collection and extraction II) for ethyl acetate and butanol fractions. The inclusion of an
acid (acetic acid) in the chromatographic system was important
Non-flowering aerial parts of Juniperus communis, J. to suppress the ionization of the acidic compounds being
horizontalis and J. chinensis were collected on March 2017 chromatographed. Development of plates was performed by
from the International Garden at Cairo, Egypt. Plants’ identi- ascending chromatography over a distance of 90 mm in a
fication was confirmed by Dr. Therese Labib, specialist of CAMAG twin trough chamber (10 × 20 cm) at room
plant identification in El Orman Garden, Cairo, Egypt. Three temperature. Saturation of the development chamber with
voucher specimens (No. JC-45 J. communis, JH-46 J. mobile phase was done 15 min before the run. After
horizontalis and JCH-47 J. Chinensis) were kept in the her- development, hot air (40 °C) was used for plates drying. All
barium of Pharmacognosy Department, Faculty of Pharmacy, mobile phases were freshly prepared with analytical grade
Alexandria University. 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 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
Peroxidase enzyme solution preparation: 10 mg/mL solution of dipped in a container containing H2O2 solution then it was re-
peroxidase in cold phosphate buffer (pH 7) was prepared. Then moved and finally dipped in another container containing ben-
50 mL of this solution was diluted by its mixing with 50 mL of zidine solution.
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. Validation

Validation of the developed and optimized methods was done

The image processing methodology
according to the ICH guidelines [20] individually. Validation
parameters were linearity, sensitivity (LOD, LOQ), precision,
Image acquisition and image analysis software
specificity, and accuracy. Quercetin standard plate (normal phase
silica plate, system II) was used for the validation of the method.
Images of all studied plates were taken by scanning TLC
Sensitivity of methods was determined in terms of the limit of
plates using a flatbed scanner Canon-MF 4750. ImageJ
detection (LOD) and limit of quantification (LOQ) values that
(http://rsb.info.nih.gov/ij/download.htmlImageJ) was used as
based on the residual standard deviation of a regression line and
image analysis software package. The image analysis method
slope. Intra-day precision was determined by independent repeat-
was developed and validated for the quantitation of the
ed analysis of five different volumes of standard solution
peroxidase inhibition activity of different fractions of J.
(quercetin) each repeated triplicate under the same conditions by
communis, J. horizontalis and J. chinensis.
using the same apparatus and the same analytical method in the
same laboratory and on the same plate. Inter-day precision includ-
Effect-directed analysis by TLC ed the analysis of the standard three times a day over 3 days by a
different analyst. The results of repeatability and intermediate
Development of a novel method for assessment of peroxidase precision are expressed as relative standard deviation (RSD) (%).
inhibition activity Accuracy of peroxidase inhibition activity was carried out by
standard addition technique, as two spots representing two con-
Calibration function was built as following: successive spots centration levels 4 μg and 6 μg of quercetin (model analyte)
from the stock solution of quercetin (1 mg/mL) were applied were applied over two tracks containing ethyl acetate fraction of
in the following order 3, 4, 5, 6, 7, and 8 μl. Each of these J. communis (pre-analyzed sample), respectively. After that the
calibration levels was applied in triplicate. The standard plate plate was developed in system II. Addition experiments were
was developed in a mobile phase composed of ethyl performed in triplicate and the accuracy was calculated as the %
acetate:methanol:water:acetic acid, 45:4:2:0.35 (v/v) (system of analyte recovered. Three analyses per concentration were
II). Peak area was plotted against standards concentration in performed and mean + SD was determined.
μg/spot in order to construct the calibration curve, after that The specificity of the methods was determined to ensure
subjection of the calibration data least-squares regression analy- that assays have the capability of determination analytes with-
sis was done. Thereafter, peroxidase inhibition activity of the out any interference from matrix elements that may have ef-
samples was detected by immersion of plates in the enzyme fect on the assay.
 JPC-J Planar Chromat

Fig. 1 Mechanism of oxidation of

benzidine by peroxidase/H2O2
into a blue colored complex

Quantitative determination of peroxidase inhibition activity Results and discussion

Samples; methylene chloride, ethyl acetate and n-butanol frac- Development and validation of a TLC–bioautography
tions were prepared as mentioned. Each sample was spotted in method for evaluation of peroxidase inhibitory
two tracks of volume of 4 and 6 μL. The development of activity of plant extracts
sample plates was done in a CAMAG twin trough chamber
using the suitable mobile phase system as we mentioned. The comprehensive chemical and biological profiling of dif-
After immersion as discussed, plates were dried followed by ferent Juniperus fractions was the target of this study. In this
its scanning using a flatbed scanner Canon-MF 4750. regard, a novel bioautographic method coupled to a validated

Fig. 2 TLC chromatograms of methylene chloride fractions of Juniperus and 15, 16) and Juniperus chinensis (tracks 11, 12 and 17, 18) were
communis (tracks 1, 2), Juniperus horizontalis (tracks 3, 4) and Juniperus developed in system II, under UV lamp 254 nm (a) and (c), after
chinensis (tracks 5, 6) were developed in system I and TLC dipping in peroxidase enzyme followed by dipping in H2O2 solution
chromatograms of ethyl acetate and butanol fractions Juniperus and benzidine solution, separately and respectively (b) and (d)
communis (tracks 7, 8 and 13, 14), Juniperus horizontalis (tracks 9, 10
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 SAC ¼ MS =ME ð1Þ
considered as the sum of antiperoxidase activity of all extract
ingredients. The TEA value is compared to areas obtained for Where MS and ME are the quantities of standard and ex-
standards, and then calculated using the experimentally deter- tract/fraction, respectively, in μg/spot; MS and ME are in the
mined Eqs. (1) and (2). The only requirement is working inside linear range, and both have the same antiperoxidase activity
the linear range of the peak area response, both for the standard (TEA). The standards and crude extracts/fractions cannot be
and extract/fraction measured [3, 24]. Having the linear cali- applied at levels resulting in the same TEA, because of differ-
bration curve for a reference compound, one can compute the ent linearity ranges. Additionally, at lower concentrations,
equivalent amount of the standard, giving the same some compounds can be applied below the detection limit.
antiperoxidase activity (TEA) as the extract/fraction. This can Furthermore, both samples are spotted in the middle of their
be called the standard activity coefficient (SAC). For example linearity range and the SAC is calculated from the formula
if the SAC equals 2, this mean that the dry extract/fraction is containing the TEA of an extract/fraction and calibration
twice as active as the pure reference compound. 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 ide scavengers. J Pharm Biomed Anal 150:87–94
6. Simões-Pires CA, Hmicha B, Marston A, Hostettmann K (2006) A
Where TEA is total extract or fraction area; I is intercept TLC bioautographic method for the detection of α- and β-glucosi-
dase inhibitors in plant extracts. Phytochem Anal 20:511–515
from the standard’s curve equation; 7. Hassan AMS (2012) TLC bioautographic method for detecting
S-slope from the standard’s curve equation; and ME is ap- lipase inhibitors. Phytochem Anal 23:405–407
plied microgram of extract or fraction. Due to introduction of 8. Hsu KD, Chan YH, Chen HJ, Lin SP, Cheng KC (2018)
parameters from the standard’s curve equation, the SAC be- Tyrosinase-based TLC Autography for anti-melanogenic drug
screening. Sci Rep 8:1–10
comes independent of the applied amount of extract or fraction. 9. Khan AA, Rahmani AH, Aldebasi YH, Aly SM (2014)
Studying SAC values of the different fractions (Table 4, Biochemical and Pathological Studies on Peroxidases –An
Fig. 2a–d) revealed that fractions of the highest peroxidase Updated Review. Glob J Health Sci 6:87–98
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Active Disease Biomarker: Recent Biochemical and Pathological
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with SAC value of 0.218 (Table 4). peroxidase activity and scavenging of reactive oxygen species by
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This study presented the development and validation of a novel reactive oxygen species to cartilage degradation in rheumatic dis-
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