Journal of Liquid Chromatography & Related

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A rapid UPLC method for simultaneous separation

and detection of anthocyanidins from Ocimum,
Hibiscus and Syzygium species and estimation of
their antioxidant activity

Biswatrish Sarkar, Pritesh Vyas, Inamul Haque & Kunal Mukhopadhyay

To cite this article: Biswatrish Sarkar, Pritesh Vyas, Inamul Haque & Kunal Mukhopadhyay (2018)
A rapid UPLC method for simultaneous separation and detection of anthocyanidins from Ocimum,
Hibiscus and Syzygium�species and estimation of their antioxidant activity, Journal of Liquid
Chromatography & Related Technologies, 41:10, 658-667, DOI: 10.1080/10826076.2018.1506932

To link to this article: https://doi.org/10.1080/10826076.2018.1506932

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JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES
2018, VOL. 41, NO. 10, 658–667
https://doi.org/10.1080/10826076.2018.1506932

A rapid UPLC method for simultaneous separation and detection of

anthocyanidins from Ocimum, Hibiscus and Syzygium species and estimation
of their antioxidant activity
Biswatrish Sarkara, Pritesh Vyasb
, Inamul Haqueb

, and Kunal Mukhopadhyayb
a
Department of Pharmaceutical Sciences & Technology, Birla Institute of Technology, Mesra, Ranchi, India; bDepartment of Bioengineering,
Birla Institute of Technology, Mesra, Ranchi, India

ABSTRACT ARTICLE HISTORY

Since ages, Ocimum tenuiflorum, Hibiscus rosa-sinensis, Syzygium cumini and Hibiscus sabdariffa are being Received 5 June 2018
used for various health-promoting purposes. Often such beneficial effects have been strongly correlated Accepted 28 July 2018
with flavonoid and anthocyanin contents of these plants. To separate the compounds, present in these spe-
KEYWORDS
cies, an efficient and quick analytical method was developed using an ultra performance liquid chromatog-
UPLC; anthocyanins;
raphy (UPLC) system consisting of a BEH C18 reverse phase column. Separation was achieved by a binary Hibiscus; Ocimum; Syzigium
solvent system of 80/20 acetonitrile and 0.3% phosphoric acid. The developed method simultaneously sepa-
rated all six major anthocyanidins with good resolution, linearity and shorter run time within 6 mins. The
mean recoveries of anthocyanidins after spiking were found to be between 98.082 and 101.988%. This is
one of the few reports of simultaneous separation and identification of anthocyanidins using UPLC in short-
est possible time. The anthocyanin containing extracts showed potential antioxidant activity assessed by in-
vitro hydroxyl radical scavenging assay and 2,2-diphenyl-1-picrylhdrazyl assay. This study generated industri-
ally useful data for efficient recovery and identification of anthocyanidins from a few important ethnophar-
macological plant species which has potential for exploitation as antioxidant rich functional food.

GRAPHICAL ABSTRACT

Introduction phenyl-2-benzopyrylium i.e, the flavylium cations, while

the aglycones are known as anthocyanidins.[1,2] Structural
Anthocyanins impart purple, red and blue colour to
difference in various anthocyanins e.g., Delphindin (Dp),
plants and plant parts. Structurally, anthocyanins are
Cyanidin (Cy), Petunidin (Pt), Pelargonidin (Pg), Peonidin
glycosylated polyhydroxy and polymethoxy derivatives of

CONTACT Biswatrish Sarkar biswatrishsarkar@bitmesra.ac.in Assistant Professor, Department of Pharmaceutical Sciences & Technology, Birla Institute of
Technology, Mesra, Ranchi 835 215, Jharkhand, India.

Present Address: Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib 173101, Himachal Pradesh, India


Present Address: Department of Botany, Derozio Memorial College, Rajarhat Road, P.O. Rajarhat-Gopalpur, Kolkata 700136, India.
Supplemental data for this article can be accessed on the publisher’s website.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljlc.
ß 2018 Taylor & Francis

(Pn) and Malvidin (Mv) arises due to hydroxylation and
methoxylation pattern on flavylium ring of these flavonoids
(Figure S1). Sugars like glucose, galactose, rhamnose, xylose or
arabinose in the side chain confer greater stability and water
solubility to anthocyanidins. Studies revealed flavonoids and
anthocyanins in berries, fruits and vegetables contribute
towards prevention of cardiac and cerebro-vascular diseases,
cancer and diabetes. These beneficial properties have been
strongly correlated with the antioxidant capacities of anthocya-
nins.[3,4] Since natural and plant-derived compounds like
anthocyanins are in prime focus of research around the world,
it is imperative to explore alternative techniques for rapid isola-
tion, identification and characterization of anthocyanins.
Structural diversity of anthocyanins and glycosylation
patterns pose as a challenge for chromatographic analysis.
Moreover, it takes 30–60 mins to achieve complete separ-
ation of anthocyanidins using traditional high performance
liquid chromatography (HPLC).[5,6] In comparison, ultra
performance liquid chromatography (UPLC) enables faster
separation of anthocyanins with better resolution, peak-effi-
ciency and less solvent consumption. In recent times,
marked improvement in the identification of anthocyanins
has been achieved by combining mass spectrometry and
NMR along with liquid chromatography processes. Ocimum
tenuiflorum L. (Lamiaceae), Hibiscus rosa-sinensis L.
(Malvaceae), Syzygium cumini L. (Myrtaceae) and Hibiscus
sabdariffa L. (Malvaceae) (Figure S2), have been reported for
their medicinal properties and other health benefits.[7–11]
The flavonoid and anthocyanin contents in these plants are
quite high and are potential resource as functional food. In
the present study, we have developed a rapid and simultan-
eous separation method for identification of anthocyanidins
from plant extracts using UPLC. The method was optimized
using validated techniques. We also employed different
assays to assess the antioxidant potentials of the anthocyanin
extracts (AEs) of these medicinal plants.
Experimental
Chemicals and Reagents
Analytical reagent grade chemicals were used in all studies.
Primary reference standards of the six anthocyanidins (chlor-
ides of Cy, Dp, Mv, Pt, Pg and Pn) were purchased from
Extrasynthese (Genay, France). Acetonitrile, Methanol, Ortho-
phosphoric acid and Hydrochloric acid (all LC grade) were
purchased from Merck (Darmstadt, Germany). A MilliQ sys-
tem (Millipore, Bedford, MA, USA) was used for purification
of water that was used in all experiments. For antioxidant
studies 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2-deoxy-D-
ribose, thiobarbituric acid (TBA), trichloroacetic acid (TCA)
and quercetin were procured from Sigma-Aldrich, Germany.
Plant materials
Plants used in the study were collected from the Indigenous
Medicinal Plants Garden of Birla Institute of Technology,
Mesra, Ranchi, India (23 240 N, 85 260 E, 619 m asl). Leaves
JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 659
of Ocimum tenuiflorum, petals of Hibiscus rosa-sinensis,
calyx of Hibiscus sabdariffa and fruits of Syzygium cumini
were code named OT, HRS, HS and SC respectively. The
plant materials were identified and authenticated at Central
National Herbarium, Botanical Survey of India, Howrah,
India, (CNH/101/2011/Tech.II/578, Dated: 21.10.2011 and
CNH/30/2012/Tech.II, Dated: 12.04.2012). The voucher
specimens were retained in the Department of
Pharmaceutical Sciences and Technology, Birla Institute of
Technology, Mesra, Ranchi, India for future reference.
Anthocyanin extraction and Standard solution
preparation
Purple fruit peels of Syzygium cumini, red leaves of Ocimum
tenuiflorum, red calyx of Hibiscus sabdariffa and red petals of
Hibiscus rosa-sinensis were flash frozen in liquid nitrogen and
pulverized. Anthocyanins were extracted from 10 g powder
with 50 mL solvent (80/20 acetonitrile/0.3% phosphoric acid),
vortexed for 20s followed by centrifugation at 2500 g for
10 min at room temperature. The supernatants were passed
through a 0.2 mm membrane filters (Ministart Ny25 Sartorius
AG G€ottingen, Germany). The anthocyanin extracts (AEs)
were evaporated to dryness and was used for antioxidant
assays. Anthocyanidin chlorides were obtained after acid
mediated hydrolysis of the filtered sample. The reaction was
carried out in a sealed ampoule containing 1 mL sample with
20 mL of 2M HCl and incubated at 150  C for 30 min.
Respective powders of all six standard anthocyanidins were
dissolved in methanol (0.1 mg/mL) prior to injection.
Ultra performance liquid chromatography (UPLC)
The anthocyanidins were separated using Waters Acquity
UPLC system (Waters Corporation, Milford, MA, USA)
consisting of an Acquity UPLC binary system manager, an
Acquity Tunable UV (TUV) detector, an Acquity UPLC
sample manager and an H/T column heater containing an
UPLC BEH C18 reverse phase column (2.1  50 mm; 1.7 mm
particle size). The binary mobile phase used was: (A) 0.3%
phosphoric acid in water and (B) Acetonitrile. The following
linear gradient elution program was applied to separate the
analytes: Initial: 89% A, 11% B; 4 min: 80% A, 20% B; 5 min:
89% A, 11% B; flow rate was 0.5 mL/min; temperature of
the column and sample manager were set at 42  C and 4  C
respectively. To equilibrate the system, the solvent compos-
ition was held at 89% A and 11% B for an extra 1 min,
yielding a total run time of 6 min. Injection volumes were
2 mL for samples as well as standards. The pressure limits
were set at 0 psi low and 15,000 psi high. The TUV detector
was set at 525 nm. For operation of instrument and process-
ing of all data EmPower2 chromatography data software
(Waters Corporation, Milford, MA, USA) was used.
Method validation
The UPLC method for determination of anthocyanidins
from selected plant extracts was validated after optimization
660 B. SARKAR ET AL.
of the separation process in terms of precision, accuracy,
selectivity, linearity, LOD, LOQ, stability, robustness and
system suitability as per ICH (International Conference on
Harmonisation) guidelines.[12]
Precision
Intraday precision of the anthocyanidins was determined by
injecting the six standards three times in a day. Inter-day
precision was tested on three consecutive days by three sep-
arate analysts. Precision was expressed as percent relative
standard deviation (RSD%).
Accuracy
Accuracy depicts the likelihood between expected and
observed values in any analytical method. In the present
study accuracy was calculated in terms of recovery (R%)
using the following equation: R% ¼ (C2/C1)  100; where, C1
is the known anthocyanidin concentration and C2 is the
calculated anthocyanidin concentration after spiking with
7.6923, 14.8164 and 21.4285 ng of respective anthocyani-
din standards.
Selectivity
The selectivity of the developed method was evaluated by
comparing the peak areas from three injections each of the
blank sample (methanol only), anthocyanindin standard
solutions and respective AEs.
Linearity and detection limit
The calibration curves were plotted with six replicate injec-
tions of reference standard solutions at five different concen-
trations (25, 50, 100, 175, 200 ng/mL) for each of the six
anthocyanidin chlorides. Linearity was determined from
regression analysis of the slope, the intercept and correlation
co-efficient. From the calibration curve, limit of detection
(LOD) and limit of quantification (LOQ) were calculated
based on standard deviation of the response and the slope
i.e., 3.3r/S for LOD and 10r/S for LOQ (where, r¼ stand-
ard deviation of the response and S ¼ slope of the calibra-
tion curve).
Stability test
Stability test of the AEs were assessed by storing them at
4  C for 7, 14, 21 and 28 days as well as freeze thawing three
cycles at 20  C/room temperature. The peak areas were
compared to that of freshly prepared samples.
Robustness
The robustness of the developed method was investigated by
deliberately altering the flow rate of mobile phase and
column temperature, the two major parameters that
govern separation.
Antioxidant properties of anthocyanins
DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay
The free radical scavenging ability of AEs was evaluated by
the DPPH assay with few modifications.[9] The aqueous sol-
utions of AEs (20 mL) at concentrations of 10, 25, 50, 100
and 200 mg/mL was mixed with 3 mL ethanol and 30 mL
10 mM DPPH. The reaction was carried at room tempera-
ture for 30 min. Quercetin in similar concentrations, used as
standard in this experiment, was also processed alike.
Reduction in absorbance (A) of DPPH free radicals, medi-
ated by AEs and standard, was measured at 517 nm. Percent
inhibition (I) was calculated by the following equation and
IC50 was determined by regression analysis.
 
I ð%Þ ¼ ðAcontrol – Asample Þ=Acontrol  100%
Hydroxyl Radical Scavenging Assay
The hydroxyl radical (OH) scavenging capacity of AEs
were assessed by modification of the standard deoxyribose
assay.[13] To ascertain the role of AEs on hydroxyl ion and
on metal chelation, following two experimental variations
(namely non-site-specific assay and site-specific assay)
were performed.
a) Non-site-specific hydroxyl radical-mediated 2-deoxy-D-
ribose degradation:. In a set of test tubes, aqueous solutions
of AEs (100 mL) at a concentration range of 10-200 mg/mL
were mixed with 690 mL of 10 mM of potassium phosphate
buffer (pH 7.4), containing 2.5 mM 2-deoxy-D-ribose. A
premixed solution of ferric ammonium sulfate (1.0 mM) and
1.04 mM EDTA at a volume of 100 mL was added to the
above solutions containing AEs. The reaction mixes were
incubated in a water bath at 37  C for 10 min followed by
addition of 1 mL of 2.8% chilled TCA and 600 mL of 1%
TBA. Thereafter, the test tubes were immersed in a boiling
water bath for 10 min and subsequently cooled down to
room temperature. A series of different concentrations of
Quercetin, which was used as standard, was also processed
alike. Finally, absorbance was recorded at 532 nm and per-
centage scavenging activity was calculated similarly using the
above formula. IC50 was determined by regression analysis.
b) Site-specific hydroxyl radical-mediated 2-deoxy-D-ribose
degradation:. The above-mentioned procedure was followed
in this test, except that 1.0 mM ferric ammonium sulfate was
prepared with 10 mM potassium phosphate buffer (pH 7.4)
without addition of EDTA. IC50 was determined similarly as
mentioned above.
Statistical Analysis
Results of antioxidant studies were expressed as mean ± stan-
dard deviation (n ¼ 3). The comparisons in IC50 values were
ascertained using one-way Analysis of Variance (ANOVA)
followed by Tukey’s multiple comparison tests employing
GraphPad Prism Software (Version 5.01). Differences were
 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 661

Figure 1. Ultra performance liquid chromatogram of simultaneous separation of anthocyanin extracts identified by TUV at 525 nm. (A) Mixture of six anthocyanidin
standards, (B) HS showing presence of delphinidin, cyanidin and peonidin, (C) HRS showing presence of cyanidin and trace amount of peonidin, (D) OT showing
presence of cyanidin and peonidin, (E) SC showing presence of all six anthocyanidin. (HS: Hibiscus sabdariffa HRS: Hibiscus rosa-sinensis, OT: Ocimum tenuiflorum; SC:
Syzygium cumini)
662 B. SARKAR ET AL.
Figure 1. Continued.
considered statistically significant at probability less than
5% (p < 0.05).
Results
Simultaneous separation and identification of
anthocyanins by UPLC
Anthocyanins from the selected plants were isolated using a
combination of acetonitrile: phosphoric acid. Subsequently
the anthocyanidin chlorides were derived after hydrolysis
with HCl. Mobile phases of different compositions were
tested for optimizing the suitable analytical conditions for
rapid separation and simultaneous detection of the antho-
cyanidins. The method was finally optimized using 80/20
acetonitrile containing 0.3% phosphoric acid as binary solv-
ent system. The retention time (RT) for individual
anthocyanidins were 0.937 min, 1.517 min, 1.728 min,
2.197 min, 2.533 min and 2.725 min for Dp, Cy, Pt, Pg, Pn
and Mv respectively. The optimized procedure was then
extended for profiling of anthocyanidins present in these
test plants.
In HRS, Cy (RT: 1.452 min) and modest amount of Pn
(RT: 2.577 min) was detected. Presence of Dp (RT:
0.936 min), Cy (RT: 1.510 min), and trace amount of Pn
(RT: 2.599 min) were found in HS. Cy (RT: 1.497 min)
and Pn (RT: 2.519 min) was found in OT. Whereas, pres-
ence of all the six anthocyanidins were evident in SC
(Figure 1 and Table 1). Identification of the anthocyanidins
in these species was performed by comparing the RTs with
that of standard anthocyanidins. It is evident that, using this
method all the six major anthocyanidin chlorides were
efficiently and simultaneously identified within a total run
 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 663

time of 6 min (which also included 1 min for system

2.518 ± 0.00 2.697 ± 0.00

equilibration).

Mv
–
–
2.519 ±0.001 –
Validation studies

2.560 ± 0.002
2.579 ± 0.003
Pg
Precision and repeatability
The precision of the developed method was ascertained by
conducting the intra-day and inter-day repeatability of the

250983.33 ± 9199.21 66991.66 ± 2541.17 333191.33 ± 14488.75 34022.66 ± 1329.34 34157.66 ± 990.00 452090.33 ± 14855.08 0.927 ± 0.002 1.499 ± 0.001 1.704 ± 0.001 2.132 ± 0.00
Retention time (min)

analytical process (Table 2). The Relative Standard Deviation

HS: Hibiscus sabdariffa; HRS: Hibiscus rosa-sinensis; OT: Ocimum tenuiflorum; SC: Syzygium cumini; Dp: Delphinidin; Cy, Cyanidin; Pt: Petunidin; Pn: Peonidin; Pg: Pelargonidin; Mv: Malvidin.
Pl
–
–
–
(RSD) result of RTs and peak areas were smaller than 1.3%
which indicates suitability of the developed system and the
method has acceptable degree of precision.
Pt
–
–
–

Accuracy
1.453 ± 0.001
0.935 ± 0.003 1.51 ± 0.002

1.49 ± 0.000

On spiking with standard anthocyanidins, the mean recov-

Cy

eries of the analytes were found to vary between 98.082 and

101.929% which was equivalent to ±3.847% of the relative
error (Table 3).
Dp

Selectivity
–
–

Selectivity was examined by viewing the chromatograms to

check the presence/absence of interfering peaks in the AEs.
Mv
–
–
–

It was observed that the AEs did not show any interference
in the chromatograms of standards and samples (Figure 1).
62075.67 ± 541.41
65261.33 ± 371.99
42745 ± 141.74

Linearity and detection limits

Linearity of any analytical procedure is ascertained by
Pg

obtaining a proportional relationship between concentration

of test analytes with working range in the sample [ICH
(International Conference on Harmonisation)-Q2R1].[12]
Thus to establish a standard calibration method, linearity is
tested within a certain range of analyte.
Pn
–
–
–

For quantitative analysis of the hydrolysed anthocyanin

extracts, calibration curves were obtained by injecting the
Area (mV  sec)

Table 1. UPLC profiles of the anthocyanidins extracted from the four species

reference standards of all the six anthocyanidin chlorides at

five different concentrations (25, 50, 100, 175, 200 ng)
(Figure 2). The regression coefficient (R2) higher than 0.99
Pt

for all the curves was evidence of acceptable fit of the

–
–
–

regression data (Figure 2). The correlation coefficient (r) val-

ues found to be >0.99 that indicated good linearity across
all the tested concentrations (Table 2). The limit of detection
7145805.33 ± 16103.31
242171.33 ± 1875.38
1326483 ± 3647.40 119410.33 ± 487.81

(LOD) and limit of quantification (LOQ) were determined

by signal to noise ratio from the calibration curve (Table 2).
Cy

LOD values ranged from 2.8971 to 7.9752 ng/mL and LOQ

values ranged from 8.7790 to 24.1674 ng/mL. These parame-

(Values are mean ± SD for n ¼ 3)

ters are essential for detecting quantitative and qualitative

presence of phyto-chemicals like anthocyanidins co-existing
in a mixture of similar analytes in nature.
Dp

Stability
AEs upon storage at 4  C for 7, 14, 21 and 28 days exhibited
–
–
Sample

degradation after 14 days (Table S1). Whereas degradation

Plant

HRS
OT
HS

SC

was not much evident after freeze thawing. Cyanidin, being

664 B. SARKAR ET AL.

Table 2. Equations for calibration curves, linear ranges, correlation, LOD, LOQ and repeatability.
Repeatability (RSD%)
Samples a
Calibration curve r R 2
LOD (ng/ ml) LOQ (ng/ ml) Intra-day (n ¼ 6) Inter-day (n ¼ 3)
Dp y ¼ 8077xþ 291.2 0.9983 0.9966 5.8428 17.7055 0.5061 0.7500
Cy y ¼ 8707x–26165 0.9977 0.9955 6.7868 20.5661 0.4982 0.9501
Pt y ¼ 16673x þ 11439 0.9993 0.9987 2.8971 8.7790 0.2532 0.7791
Pg y ¼ 7893x– 96083 0.9968 0.9938 7.9752 24.1674 0.3139 0.9320
Pn y ¼ 8988x þ 38701 0.9990 0.9981 3.5224 10.6738 0.6188 1.0772
Mv y ¼ 6481x– 13524 0.9990 0.9982 4.2891 12.9972 0.6784 1.1069
r, correlation coefficient; R2, regression coefficient; LOD, limit of detection; LOQ, limit of quantification; RSD%, relative standard
deviation expressed in percent.
a
Dp: Delphinidin; Cy: Cyanidin; Pt: Petunidin; Pn: Peonidin; Pg: Pelargonidin; Mv: Malvidin.

Table 3. Statistical results of recovery of six anthocyanidins after spiking.

Samplesa Initial amount (ng) Added amount (ng) Detected amount (ng) (mean ± SD) Mean recovery % ± RSD% (n ¼ 3)
Dp 19.3216 7.6923 26.859 ± 0.112 99.426 ± 0.417
18.6056 14.8164 33.052 ± 0.109 98.898 ± 0.330
17.9893 21.4285 39.049 ± 0.286 99.065 ± 0.733

Cy 18.4135 7.6923 25.605 ± 0.109 98.082 ± 0.426

18.1019 14.8147 32.376 ± 0.163 98.358 ± 0.504
17.8125 21.4285 39.629 ± 0.131 100.988 ± 0.33

Pt 11.751 7.6923 19.671 ± 0.016 101.174 ± 0.084

11.3157 14.8147 26.367 ± 0.048 100.906 ± 0.184
10.9116 21.4285 32.407 ± 0.081 100.209 ± 0.250

Pg 23.2562 7.6923 30.767 ± 0.121 99.414 ± 0.393

22.9504 14.8147 38.405 ± 0.033 101.694 ± 0.086
22.6664 21.4285 44.543 ± 0.015 101.017 ± 0.034

Pn 14.5423 7.6923 22.373 ± 0.028 100.625 ± 0.129

14.0037 14.8147 29.371 ± 0.195 101.920 ± 0.665
13.5036 21.4285 35.606 ± 0.167 101.929 ± 0.471

Mv 106.0208 7.6923 113.651 ± 0.066 99.945 ± 0.058

102.0935 14.8147 115.393 ± 0.143 98.704 ± 0.124
98.4473 21.4285 119.420 ± 0.119 99.62 ± 0.100
a
Dp: Delphinidin; Cy: Cyanidin; Pt: Petunidin; Pn: Peonidin, Pg: Pelargonidin; Mv: Malvidin; RSD%: relative standard deviation expressed
in percent.

the predominant anthocyanindin in all the AEs, was chosen
for this study.
Robustness
Robustness tests were performed to find out the effects of
minor changes in the flow rates and column temperature.
Alteration of both the analytical parameters has insignificant
effects on separation of the major component cyanidin chlor-
ide, indicating robustness of the developed method (Table S2).
Antioxidant activity of anthocyanins
The antioxidant activity of the AEs was determined via two
in-vitro assays and comparing their IC50 values with a stand-
ard antioxidant (quercetin). AEs from all the four plant spe-
cies were able to quench DPPH free radicals in a
concentration-dependent manner [Figure 3 (A) and Table
S3]. Comparing the IC50 values among all the species, using
one-way ANOVA followed by Tukey’s multiple comparison
tests, significant difference (p < 0.05) in activities between
different AEs was observed. OT demonstrated the best anti-
oxidant activity with lowest IC50 value (28.880 ± 2.36 mg/mL)
and found to be statistically equivalent in activity with quer-
cetin (20.398 ± 1.89 mg/mL) [Figure 4 (A) and Table S4].
The degradation of hydroxyl radical (OH) mediated 2-
deoxy-D-ribose chromogen was also assessed with AEs.
Similarly, to DPPH assay, all the AEs demonstrated a linear
increment in hydroxyl radical scavenging activity with
increasing concentration [Figure 3(B), 3(C) and Table S3].
SC and HRS both showed excellent results in both site-spe-
cific and non-site-specific hydroxyl radical scavenging assays
and their IC50 values were statistically comparable with
quercetin indicating their high antioxidant potentials
[Figure 4(B), 4(C) and Table S4]. In-fact, the lower IC50
value of SC (26.443 ± 6.42 mg/mL) suggest its better perform-
ance than quercetin (IC50 ¼ 28.581 ± 1.23 mg/mL)
Discussion
Anthocyanins have been classified as a functional food
material possessing several health promoting properties.
Stability and characterization of anthocyanins are the major
concerns of researchers around the world. Various techni-
ques have evolved over the years to characterize anthocya-
nins. Each method has its own suitability in its space of
applicability, but the issue of isolating stable anthocyanin
 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 665

Figure 2. System linearity corresponding to 5 concentration levels of anthocyanin standards. (A) Delphinidin, (B) Cyanidin, (C) Petunidin, (D) Pelargonidin, (E)
Peonidin, (F) Malvidin.

Figure 3. Concentration dependent free radical scavenging activity of anthocyanin extracts. (A) DPPH assay, (B) Non-site-specific OH radical scavenging assay, (C)
Site-specific OH radical scavenging assay (Data presented as mean ± SD, n ¼ 3; HS: Hibiscus sabdariffa, HRS: Hibiscus rosa-sinensis, OT: Ocimum tenuiflorum; SC:
Syzygium cumini)

and characterization is still a challenge for researchers.
UPLC has been employed for separation of anthocyanins in
different plant species but the run time of those methods
were quite long (17–30 min).[14,15] Hence it was imperative
to develop a method which could be applied to resolve
anthocyanin like analytes from different plant species. In the
present study we showed that the developed method is not
only robust and short but can be extrapolated across several
plant species of different families having identi-
cal compounds.
Antioxidant activity of anthocyanins from fruits, vegeta-
bles and wine has been widely reported and reviewed. The
hydroxy groups in flavonoids like anthocyanins transfer
hydrogens to the free radicals. This event give rise to rela-
tively stable phenoxyl radicals of the flavonoid, which in
turn neutralizes the oxidising capacity of the free radicals.[16]
Studies have proven that anthocyanins possesses greater free
666 B. SARKAR ET AL.
Figure 4. Quantitative comparison of IC50 values of in-vitro antioxidant assays of anthocyanin. (A) DPPH free radical scavenging assay, (B) Non-site-specific OH rad-
ical scavenging assay, (C) Site-specific OH radical scavenging assay (Data presented as mean ± SD, for n ¼3); p< 0.05 compared among the test groups as well as
with standard (quercetin).
p< 0.05 indicate no significant difference between the experimental group and standard values (HS: Hibiscus sabdariffa HRS: Hibiscus
rosa-sinensis, OT: Ocimum tenuiflorum; SC: Syzygium cumini).
radical scavenging property than other phenolics.[17] In our
previous study, Hibiscus and Ocimum AEs extracted with
ethanol has demonstrated excellent antioxidant activity and
indicated a strong correlation with anthocyanin contents of
these plants.[9] In the present study the extraction methods
of AEs did not deter the antioxidant capacities of these
plants. Additionally, we found Syzigium fruits contained all
the six major anthocyanins and evidently displayed even bet-
ter antioxidant activities than the other two species
studied here.
Conclusion
To our knowledge, the method is first of its kinds where
analytical prowess of UPLC was utilized for simultaneous
profiling of a mixture of anthocyanidins in more than one
plant species. To our credit, six major anthocyanidins were
separated in shortest possible time reported so far using
UPLC. This validated robust method could be easily adapted
for identification of similar phenolics in another species
also. Free radical scavenging properties of anthocyanins was
demonstrated once again by the antioxidant studies in-vitro.
Further, a correlation study between total anthocyanin con-
tent and radical scavenging capacities will yield valuable
information on the antioxidant properties of these species.
The developed analytical method can be utilized for profil-
ing of other anthocyanins and flavonoid like metabolites in
plants. This simple method of extraction and analysis can be
achieved using lesser amount of organic solvents compared
to traditional chromatography methods. Additionally, using
this analytical platform, stability of anthocyanins in Hibiscus,
Ocimum and Syzigium during storage and seasonal variation
in their concentration could also be investigated.
Funding
This work was supported by the University Grants Commission, New
Delhi, India under Major Research Project Grant [MRP: 34-275/
2008 (SR)].
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