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Characterisation and determination of in vitro antioxidant potential of

betalains from Talinum triangulare (Jacq.) Willd

Article in Food Chemistry · December 2013

DOI: 10.1016/[Link].2013.06.108 · Source: PubMed

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 Food Chemistry 141 (2013) 4382–4390

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Characterisation and determination of in vitro antioxidant potential of

betalains from Talinum triangulare (Jacq.) Willd.
J. Swarna a, T.S. Lokeswari c, M. Smita b, R. Ravindhran a,⇑
a
T.A.L. Samy Centre for Plant Tissue Culture and Molecular Biology, Department of Plant Biology and Biotechnology, Loyola College, Nungambakkam, Chennai 600 034, Tamil
Nadu, India
b
Polymer Science & Engineering Division, National Chemical Laboratory, Dr. Homi Bhaba Road, Pashan, Pune 411 008, Maharashtra, India
c
Department of Biotechnology, Sri Ramachandra University, Porur, Chennai 600 116, Tamil Nadu, India

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

Article history: Talinum triangulare (Jacq.) Willd is an erect, succulent, perennial herb belonging to the family Portulaca-
Received 18 March 2013 ceae. Under extreme environmental conditions, the plant produces betalain pigments which get accumu-
Received in revised form 20 May 2013 lated in the stem region. Hence, in the present study, the betaxanthin and betacyanin patterns from
Accepted 25 June 2013
different samples of T. triangulare have been investigated by applying high-performance liquid chroma-
Available online 8 July 2013
tography photo-diode array detection (HPLC-PDA) coupled with positive ion electro-spray mass spec-
trometry. Two betacyanins and two betaxanthins were identified in aqueous methanolic extract of
Keywords:
flower, stem and leaf. Betanin, isobetanin, immonium conjugates of betalamic acid with dopamine and
Waterleaf
Portulacaceae
tyrosine were elucidated. The total betalain content was estimated by photometric analysis. In vitro anti-
Betalains oxidant activity for the betalain extract determined by various methods revealed potent scavenging abil-
HPLC–MS ity. The current work may possibly be considered beneficial in utilisation of the plant T. triangulare as a
Antioxidant natural colourant in food and beverage industries.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction which is greater than most flavonoids and their superior stability
when compared to anthocyanins (Stintzing & Carle, 2004).
Betalains are a class of nitrogen-containing plant pigments Red beetroots (Beta vulgaris) are the major source of the betacy-
which accumulate in flowers, fruits and occasionally in vegetative anin ‘betanin’ and this crop is exclusively exploited for betalain pro-
tissues of plants belonging to the order Caryophyllales and some duction (Stintzing & Carle, 2004). Despite its high betanin content,
higher fungi belonging to the genera Amanita and Hygrocybe. The B. vulgaris root has several drawbacks, such as: limited pigment
exceptions are the two families Caryophyllaceae and Mollugina- spectrum, carryover of soil microbes leading to microbial contami-
ceae, which accumulate anthocyanins instead. Betalains comprise nation, adverse flavour due to various pyrazines, and an earthy
of the red-violet betacyanins and the yellow betaxanthins that smell, owing to the high levels of the odorant geosmin (Acree, Lee,
are immonium conjugates of betalamic acid with cyclo-dopa and Butts, & Barnard, 1976). These shortcomings promoted a search
amino acids or amines, respectively. Being water-soluble pigments, for other potential betalain-containing sources, such as cactus fruits
they are well suited for colouring low acid foods, as they are stable (Stintzing, Schieber, & Carle, 2002a, 2002b), coloured Swiss chard
over a wide pH range, from pH 3 to pH 7 (Stintzing & Carle, 2004). (Kugler, Stintzing & Carle, 2004), Celosia argentea (Schliemann, Cai,
There is growing interest in the use of natural pigments as food Degenkold, Schmidt, & Corke, 2001) and Bougainvillea sp. (Kugler,
colourants, since artificial colourants are becoming more critically Stintzing, & Carle, 2007). In the Portulacaceae family, betalains have
assessed. Recent reports claim that betalains possess high antioxi- been reported in the common purslane, Portulaca grandiflora
dant capacities (Escribano, Pedreno, Garcia-Carmona, & Munoz, (Adachi & Nakatsukasa, 1983; Trezzini & Zryd, 1991).
1998; Lee, Kim, Kim, & Jang, 2002), anti-fungal, anti-inflammatory Hitherto, no scientific research has been conducted on the beta-
and anti-carcinogenic activities (Stintzing & Carle, 2004). The anti- lain composition of Talinum triangulare, which could be a promis-
oxidant activity of betalains is of significant interest because these ing source as an edible betalain-containing crop. Talinum
pigments are emerging as highly bio-active natural compounds triangulare (Jacq.) Willd., popularly known as waterleaf, is a fleshy,
with potential benefits to human health. The key advantages of saponin-rich, perennial herb accumulating betalain in flowers,
betalains as nutritional antioxidants are their bioavailability, stem and leaves. In traditional medicine, the plant has been impli-
cated in the management of diabetes, cancer, stroke, obesity and
measles (Fontem & Schippers, 2004). Waterleaf is characterised
⇑ Corresponding author. Tel.: +91 44 28178200x330. as tolerant to various soil types, temperatures and moisture levels,
E-mail address: raviloyola1998@[Link] (R. Ravindhran). and grows well under shade. Considering the advantages of

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
[Link]
 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390 4383

continuous crop production of waterleaf and its medicinal value, 2.4. Qualitative chemical tests
the plant can possibly serve as an alternate natural source of beta-
lain. Therefore, the aim of the present study was to investigate the For the identification of betacyanin pigments, certain tests were
betacyanin and betaxanthin patterns of T. triangulare by applying performed as described by Harborne (1998). The plant extracts
high-performance liquid chromatography coupled with mass spec- were separately treated with 2 M HCl at 100 °C and with a few
trometry. The antioxidant capacity of the betalain pigment was drops of 2 M NaOH. The disappearance of pink colour and the
determined by various in vitro assays. Furthermore, quantification change of pink to yellow colour respectively, confirmed the pres-
of betalain content was also carried out by spectrophotometric ence of betacyanin pigment.
analysis.
2.5. Photometric quantification of betalains

2. Materials and methods Contents of betalain pigments, betacyanins and betaxanthins,

were determined using a UV/Vis spectrophotometer (Shimadzu
2.1. Chemicals and reagents UV-1800, Shimadzu Corporation, Kyoto, Japan). Prior to measure-
ments, the sample was diluted using 50% aqueous methanol
De-ionised HPLC-grade water was obtained from Milli-Q Plus (v/v), so that the absorption maxima values fell within the range of
water purification system (Millipore Corp, Bedford, MA, USA) was 0.5 6 A 6 1.0. Measurements were performed in triplicates, and
used in all the chromatography and spectrophotometry experi- the betalain content was quantified based on the spectrophoto-
ments. HPLC-grade methanol and acetonitrile were purchased metric multiple-component method of Nilsson (1970). Determina-
from Qualigens Fine Chemicals (Mumbai, India). Formic acid 90% tion of betalain concentration, i.e., violet and yellow pigments, was
(GPR grade) was obtained from BDH Chemicals Limited (Poole, expressed in terms of betanin and vulgaxanthin I, respectively.
England). All the solvents and reagents prepared were filtered Pigment concentration was calculated based on the absorptivity
using a Millipore Filtration System (Millipore Corp, Bedford, MA, values (A1%), using an extinction coefficient of 1120 for betanin
USA) with a 2.5 lm filter disc, prior to use. (at 538 nm) and 750 for vulgaxanthin I (at 470 nm) (Piattelli &
For the antioxidant activity, standards such as ascorbic acid, gal- Minale, 1964). When calculating contents of red and yellow pig-
lic acid, quercetin and ethylene diammine tetra acetic acid (EDTA) ments, a correction was included, resulting from absorbance of
were purchased from Sigma (St. Louis, MO, USA). 2,2-diphenyl light by impurities present in the sample, on the basis of measure-
1-picryl hydrazyl (DPPH), 2,20 -azino-bis-3-ethylbenzo-thiazoline- ments of extinction at 600 nm. Total pigment content was
6-sulphonic acid (ABTS) and hydroxylamine hydrochloride were expressed as the sum of violent and yellow components.
obtained from Sigma–Aldrich (Steinheim, Germany). Sodium
nitroprusside dihydrate, sulphanilamide, N-(1-naphthyl) ethylene 2.6. High-performance liquid chromatography (HPLC)
diamine dihydrochloride (NEDD), ferric chloride, ferrous chloride,
potassium persulphate (PPS), Folin–Ciocalteu’s reagent and Analysis of betalains was performed on a Waters Alliance HPLC
2-thiobarbituric acid (TBA) were purchased from HiMedia (Mumbai, system (Waters Corp., Milford, MA, USA) equipped with a Waters
India). Trichloro acetic acid (TCA) and ammonium molybdate was e2695 Separations Module in a quarternary gradient mode for sol-
obtained from Fischer Scientific (Mumbai, India). Potassium ferricy- vent delivery, a 2695 micro vacuum degasser, a 2695 thermostated
anide [K3Fe(CN)6] and nitro blue tetrazolium (NBT) were purchased autosampler and a 2695 thermostated column compartment. Data
from SD fine chemicals (Mumbai, India). 2-deoxy-D-ribose was ob- acquisition was performed by MassLynx software (version 4.1) on
tained from SRL (India). Hydrogen peroxide was purchased from a Pentium IV microprocessor. A model 2998 photo diode array
Rankem (Ranbaxy Chemicals Ltd., India). All the other reagents used detector was connected in series possessing a wavelength range
were of analytical grade. of 200 to 600 nm, resolutions of 1.2 nm and a sampling rate of
10 points/s. Chromatographic separation was carried out on an
analytical scale (250  4 mm i.d.) Nucleosil-100 C18 reversed phase
2.2. Plant material
column with a particle size of 5 lm (Macherey–Nagel, Duren,
Germany), operating at a temperature of 25 °C. The betalain compo-
Talinum triangulare plants were collected from the herbal gar-
sition of different samples were studied using acetonitrile (Eluent A)
den at Irula Tribal Women’s Welfare Society, Chengalpattu, Chen-
and 2% formic acid in water (w/w) (Eluent B), at a flow rate of 0.5
nai, India. A herbarium voucher specimen (LCH 42) has been
ml/min. Betalains were separated using the following gradient
deposited in the Loyola College Herbarium. Botanical identification
system: 3% A in B (v/v) at 0 min; gradient to 16% A in B at 17 min;
was carried out by Dr. D. Narasimhan, Centre for Floristic Research,
gradient to 50% A in B at 30 min before re-equilibration to the start-
Department of Plant Biology & Plant Biotechnology, Madras Chris-
ing conditions. The samples were filtered through a 0.45 lm mem-
tian College, Chennai. The plant leaves, epidermal peel of stems
brane filter (Acrodisc 13 CR PTFE, Gelman, MI, USA) before injection
and flower petals were freshly plucked before extraction.
and the injection volume for all extracts was 20 ll. Betaxanthins
and betacyanins were monitored at 480 and 540 nm, respectively.
2.3. Pigment extraction
2.7. High-performance liquid chromatography–mass spectrometry
Extraction of betalains from leaf, stem and flower (1.0 g) was (HPLC–MS)
performed by homogenising in a mortar with liquid nitrogen, to
preclude enzymatic degradation during grinding. The resulting The HPLC–DAD was coupled with a Micromass Quattro micro
powder was extracted with 10 ml of 80% aqueous methanol API™ triple Quadrupole mass spectrometer (Waters Corp., Milford,
(v/v). After continuous stirring for 30 min, the samples were MA, USA) operating in the positive ionisation mode. An external ro-
centrifuged at 10,000 rpm for 10 min at 4 °C in a 5810r Eppendorf tary pump (Edwards model E2m28E vacuum pump) and an inter-
refrigerated centrifuge (Hamburg, Germany). The supernatant nal split-flow turbomolecular pump were combined to create a
was transferred and the pellet was re-extracted twice with 80% vacuum. The conditions used for the electrospray source were as
methanol. All the supernatants were pooled and stored at –20 °C follows: capillary voltage, 3.5 kV; cone voltage, 30 V; extractor,
in the dark until use. 3 V; source temperature, 120 °C; desolvation gas temperature,
4384 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390

350 °C; and unit mass resolution. Nitrogen was used as the drying An increase in absorbance of the reaction mixture indicated an in-
gas at a flow rate of 12 l/min and a pressure of 70 psi. HPLC condi- crease in reducing power.
tions were the same as described above.
2.9.4. Assay of DPPH and ABTS free radical-scavenging activity
2.8. Identification of betalains The effect of BeTT on DPPH radicals was determined using the
method of Blois (1958) with some modifications. A 0.1 mM solu-
For the pigment identification, authentic standards from previ- tion of DPPH was prepared freshly by dissolving in methanol. To
ous betalain studies were used; betanin and its derivatives or 1 ml of BeTT (25–1000 lg/ml), 1 ml of DPPH solution was added,
betaxanthins (Stintzing et al., 2002a, 2002b; Nemzer et al., mixed thoroughly and left in dark at room temperature for
2011); otherwise, the status of the pigment was assigned as un- 30 min. The absorbance was measured at 517 nm. Methanol (80%
known supported by the spectrophotometric and mass spectro- v/v) was used instead of extract in the case of negative control.
metric data. The scavenging capacity of the extract was compared with that
of ascorbic acid, which was utilised as the reference standard.
2.9. In vitro antioxidant activity The ABTS assay was performed as explained by Re, Pellegrini,
Proteggente, Pannala, Yang & Rice-Evans (1999). Stock solutions
2.9.1. Determination of total polyphenol and flavonoid content of ABTS (7 mM) and PPS (2.4 mM) were prepared and stored at
The total polyphenol content of the betalain extract of T. trian- 4 °C in dark. The working solution (ABTS+) was then prepared by
gulare (BeTT) was measured using the Folin–Ciocalteu colorimetric mixing the two stock solutions in equal quantities allowing them
method as described previously by Gao, Ohlander, Jeppsson, Bjork, to react for 12 h at room temperature in dark. This solution was
and Trajkovski (2000), with some slight modifications. Briefly, further diluted by mixing 1 ml of ABTS+ solution with 50 ml of
100 ll of BeTT (100 lg/ml) was mixed with 0.2 ml of Folin–Ciocal- methanol to obtain an absorbance of 0.750 at 734 nm. BeTT
teu’s reagent and made up to 2 ml with distilled H2O. The solution (1 ml) at different concentrations (25–1000 lg/ml) was allowed
was incubated at room temperature for 5 min and 1 ml of 20% to react with 1 ml of the ABTS+ solution. The mixture was incu-
(w/v) sodium carbonate was added to the mixture. The total bated for 10 min in the dark and the absorbance was noted at
polyphenol content was determined after 1 h of incubation at 734 nm. The scavenging capacity of the extract was compared with
room temperature and the absorbance of the resulting blue colour the reference standard, ascorbic acid.
was measured at 765 nm in a spectrophotometer (Shimadzu The ability to scavenge the DPPH/ABTS radicals was expressed
UV-1800, Shimadzu Corporation, Kyoto, Japan). The test was as inhibition percentage of the free radical by the sample and
performed in triplicates with gallic acid as the standard and 80% was calculated as follows:
methanol as negative control. Polyphenol content of BeTT was ex-
pressed as gallic acid equivalents (GAE) (lg/mg of extract) and Radical scavenging capacityð%Þ
quantification was done with respect to the standard curve of gallic
¼ ½ðAcontrol  Asample Þ=ðAcontrol Þ  100 ð1Þ
acid which was linear between 10 and 100 lg concentration.
Estimation of the total flavonoids content in BeTT was estab- where, Acontrol is the absorbance of DPPH/ABTS radicals +0.1% meth-
lished using the method of Ordon, Gomez, Vattuone, and Isla anol and Asample is the absorbance of DPPH/ABTS radicals with sam-
(2006). Briefly, to 1 ml of BeTT (100 lg/ml), 1 ml of 2% (w/v) alu- ple extract or standard.
minium chloride ethanol solution was added. The contents were
incubated for 1 h at room temperature and the absorbance was
measured at 420 nm. Total flavonoid content of BeTT was ex- 2.9.5. Assay of hydroxyl radical (OH) scavenging activity
pressed as quercetin equivalents (QE) (lg/mg of extract) and quan- The hydroxyl radical-scavenging assay was performed by using
tification was done with respect to the standard curve of quercetin a modified method as described previously (Halliwell, Gutteridge,
which was linear between 10 and 100 lg concentration. & Amoma, 1987). The reaction mixture (1 ml) contained 3 mM
2-deoxy-D-ribose (prepared in 20 mM phosphate buffer, pH 7.4),
2.9.2. Determination of total antioxidant capacity 0.2 ml of 0.1 mM EDTA (w/v), 0.2 ml of 0.1 mM (w/v) ascorbic acid,
The assay is based on the reduction of Mo (VI) to Mo (V) by the 0.2 ml of 0.1 mM (w/v) FeCl3 and 0.2 ml of 2 mM (v/v) H2O2, to
extract and subsequent formation of a green phosphate/Mo (V) which, 0.2 ml of BeTT (25–1000 lg/ml) was added. After the sam-
complex at acid pH (Prieto, Pineda, & Aguilar, 1999). The tubes con- ples were incubated at 37 °C for 30 min, 0.2 ml of 15% (w/v) TCA
taining 0.1 ml of different concentrations of BeTT (25, 50, 100, 200, and 0.2 ml of 1% (w/v) TBA were added to the mixture. The tubes
400, 800 and 1000 lg/ml) was combined with 1 ml of reagent solu- were further incubated in a boiling water bath for 30 min for the
tion (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM pink chromogen to develop. The samples were then cooled and
ammonium molybdate). Methanol (80% v/v) was used instead of the absorbance was measured at 532 nm. All the tests were carried
the extract in the case of negative control. The tubes were incu- out in triplicate with ascorbic acid as positive control and 80% (v/v)
bated in a boiling water bath at 95 °C for 90 min. After the samples methanol as negative control. The hydroxyl radical-scavenging
had cooled to room temperature, the absorbance of each solution activity of the extract was measured as the percentage of inhibition
was measured at 695 nm. Ascorbic acid was used as the standard of deoxyribose degradation and was calculated according to Eq. (1).
and the experiments were performed in triplicates.
2.9.6. Assay of nitric oxide radical (NO) scavenging activity
2.9.3. Reducing power assay In this experiment, 1 ml of sodium nitroprusside (10 mM w/v)
The reducing power of BeTT was determined according to the in phosphate buffer (pH 7.4) was added to 0.5 ml of BeTT
method of Yen and Chen (1995). BeTT (25–1000 lg/ml) were (25–1000 lg/ml) and incubated at room temperature for 4 h. After
mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml the incubation period, 0.25 ml of Griess reagent (1% sulfanilamide,
of 1% (w/v) K3Fe(CN)6. The contents were incubated at 50 °C for 2% O-phosphoric acid and 0.1% NEDD) was added. The absorbance
30 min. 2.5 ml of 10% (w/v) TCA was added to the mixture and cen- of the pink chromophore formed during diazotisation of nitrite
trifuged at 3000 rpm for 10 min. The upper layer of the solution with the sulphanilamide and its subsequent coupling with NEDD
(2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of was read at 546 nm (Shukla, Mehta, Mehta, & Bajpai, 2012). Ascor-
0.1% (w/v) FeCl3 and the absorbance was measured at 700 nm. bic acid was used as standard and 80% (v/v) methanol as negative
 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390 4385

control. The percentage nitric oxide-scavenging activity was calcu-

lated using the formula (1).

2.9.7. Assay of superoxide radical (O2) scavenging activity

The superoxide-scavenging activity of BeTT was measured
using the NBT method as described by Shukla et al. (2012). In this
experiment, the reaction mixture consisted of 1 ml of 50 mM (w/v)
sodium carbonate, 0.4 ml of 24 mM (w/v) NBT and 0.2 ml of
0.1 mM (w/v) EDTA with BeTT in different concentrations
(25–1000 lg/ml). The absorbance was noted immediately at
560 nm. To the mixture, 0.4 ml of 1 mM (w/v) hydroxylamine
hydrochloride was added to initiate the reaction. The samples were
incubated at 25 °C for 15 min and the reduction of NBT was mea-
sured at 560 nm. Ascorbic acid was used as the reference com-
pound. Decreased absorbance of the reaction mixture indicated
increased superoxide anion scavenging activity. The percentage Fig. 1. Qualitative tests for confirmation of betacyanin pigment in T. triangulare
superoxide-scavenging activities of the extracts were calculated extract. (a) Aqueous methanolic extract from fresh flower samples. (b) Colour
change from pink to yellow after addition of NaOH. (c) Destruction of pink colour on
according to Eq. (1).
addition of HCl. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
2.9.8. Metal chelating activity
The chelating ability of ferrous ions by the BeTT was deter-
mined by the method of Dinis, Madeira, and Almeida (1994). higher than in the flower and stem samples (0.857 ± 0.084 and
Briefly, BeTT (25–1000 lg/ml) was added to a solution of 2 mM 0.546 ± 0.120 mg/gm FW respectively). The concentration of the
(w/v) FeCl2 (0.05 ml). The reaction was initiated by the addition total betalains was 4.993 ± 0.131 mg/gm FW in floral extract,
of 0.2 ml of 5 mM (w/v) ferrozine (prepared in 95% ethanol). The 2.778 ± 0.138 mg/gm FW in stem sample while in leaves it was re-
mixture was then vortexed and left to stand at room temperature corded as 1.097 ± 0.175 mg/gm FW. The flowers of T. triangulare
for 10 min. Absorbance of the solution was then measured spectro- are characterised by an intense pink colour caused by the presence
photometrically at 562 nm. The percentage inhibition of ferrozine– of betacyanin pigments. These quantitative determinations dem-
Fe2+ complex formation was determined using the formula (1). onstrated that the betacyanin content that was predominant in
EDTA was used as the positive control. the samples were responsible for the purple–pink colouration.

2.10. Statistical analysis

3.3. Separation and identification of betalains by HPLC–DAD MS
analysis
Antioxidant activity data were analysed statistically using IBM
SPSS statistics 19 (SPSS Inc., Chicago, USA) and the experimental
Betalain patterns of T. triangulare samples have not been
results were expressed as mean ± SE of three parallel measure-
described previously. Therefore, in the present study, the charac-
ments. The significance of differences among means was carried
terisation of betalains was performed on the basis of UV/Vis
out at 5% probability level using Duncan’s Multiple Range Test
spectrometry, retention times (tR) and mass spectrometry. The
(DMRT). The half maximal inhibitory concentration (IC50 value–
acetonitrile – 2% formic acid solvent system was effective in
the antiradical dose required to cause a 50% inhibition) was calcu-
separating the betalain components.
lated in lg/ml from the logarithmic regression equation between
The HPLC chromatographic pattern of the aqueous methanolic
sample concentration and percentage of inhibition using Microsoft
extract of flower, stem peels and leaf samples for betaxanthin
Excel software (MS Office).
and betacyanin pigments, monitored at 480 nm and 540 nm,
respectively, is shown in Fig. 2. The various patterns of visible light
3. Results and discussion absorption spectra obtained for the pigment extracts revealed dif-
ferences among the three samples tested. In flower, three promi-
3.1. Basic qualitative tests nent peaks (Peak 1, 2 3) were detected at both 480 and 540 nm
having tR of 19.0, 20.3 and 21.5 min respectively (Table 1). In the
Aqueous methanolic extracts of flower, stem and leaf of stem sample, the major peaks 2 and 3 were noted. While in the leaf
T. triangulare were subjected to qualitative chemical tests to extract, peak 2 and 3 were slightly diminished when compared to
confirm the presence of the pink-purple betacyanin pigment the other two samples. Peak 4 at tR 27.9 min was recorded in all
(Fig. 1a). Addition of a few drops of NaOH converted the pink col- three samples (Table 1).
oured pigment to a dull yellow colour (Fig. 1b) When the samples The identity of these compounds was confirmed by HPLC cou-
were treated with hot aqueous HCl it led to the destruction of the pled with electrospray mass spectrometry in the positive ionisa-
pink colour (Fig. 1c). Thus, these tests validated the presence of tion mode, which provided molecular mass and structural
betalains as the major pigments in this plant and not anthocyanins. information. A tentative identification of the bands was performed
according to the visible spectra and by comparison with previous
3.2. Photometric betalain quantification data published by other authors (Stintzing et al., 2002a, 2002b;
Nemzer et al., 2011). Accordingly, the 4 peaks that were obtained
Betacyanin and betaxanthin contents of flower, stem and leaf in the different samples were assigned to two betacyanins and
were determined spectrophotometrically. The highest betacyanin two betaxanthins, namely portulacaxanthin II [tyrosine-betaxan-
content was recorded in flower sample (4.136 ± 0.047 mg/gm fresh thin] (peak 1), betanin (peak 2), isobetanin (peak 3) and miraxan-
weight; FW) followed by stem (2.232 ± 0.018 mg/gm FW) and with thin V [dopamine-betaxanthin] (peak 4) (Table 1). The principal
the lowest content in leaves (0.065 ± 0.005 mg/gm FW). The beta- betacyanins, namely betanin and isobetanin, were present in high
xanthin quantity in leaves (1.032 ± 0.170 mg/gm FW) was much quantities. Flower extract showed the highest level of betacyanin
4386 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390

Fig. 2. HPLC elution profiles of betalains from flower (a,b), stem (c,d) and leaf (e,f) samples of T. triangulare monitored at 480 and 540 nm respectively.

pigmentation with a betanin: isobetanin ratio of 4:1 followed by Mass spectral data of peaks 2 and 3 are presented in Fig. 3a
stem (2:1) and leaf (2:1) samples. Miraxanthin V was detected in and b. The presence of betanin (peak 2) and isobetanin, its
minor quantities in all samples while portulacaxanthin II was 15R-isoform (peak 3), was confirmed first by their identical spec-
recorded only in flower sample. tral properties (kmax 540 nm) by the presence of the protonated
 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390 4387

Table 1
Retention times, HPLC-PDA and mass spectrometric data of betalains from different extracts of T. triangulare.

Peak Retention time (tR) (mins) Betacyanin Betaxanthin kmax (nm) m/z [M+H]+(Daughter ions) Plant sample
Trivial name (Amino acid/amine) F S L
1 19.0 Portulacaxanthin II Tyrosine-bx 472 375 (303) + – –
2 20.3 Betanin 538 551 (389) ++ ++ +
3 21.5 Isobetanin 538 551 (389) + ++ +
4 27.9 Miraxanthin V Dopamine-bx 461 347 () + + +

F: Flower, S: Stem, L: Leaf, bx: betaxanthin. ++ Present in high amounts; +: present in moderate amounts; : present in traces or not detectable. Peak assignment and
retention times refer to Fig. 2.

Fig. 3. Antioxidant activities of BeTT as estimated by total antioxidant capacity (a) and reducing power (b). Means followed by the same letter within columns are not
significantly different at 5% probability level using Duncan’s Multiple Range Test (DMRT).

molecular ions [M + H]+ with m/z 551. The secondary daughter Mass spectral data revealed m/z 375, which supported the elucida-
ions were noted at m/z 389 due to the presence of protonated tion as portulacaxanthin II. This tyrosine-immonium conjugate of
aglycones [betanidin + H]+ or [isobetanidin + H]+ for betanin or betalamic acid was first reported in Portulaca grandiflora (Trezzini
its C-15 epimer, isobetanin respectively. Both the compounds & Zryd, 1991). It has also been detected in members of other fam-
differ only in the absolute configuration of their C-15 chiral ilies such as Bougainvillea sp. (Kugler et al., 2007) and Beta vulgaris
centre. (Nemzer et al., 2011). The presence of miraxanthin V was recorded
Two betaxanthins were detected in the plant samples, viz., in all the three samples yielding m/z 347. This dopamine based
tyrosine-bx (kmax 472 nm) and dopamine-bx (kmax 461 nm). The betaxanthin has been reported to be the major betaxanthin com-
hydrophobic betaxanthin, portulacaxanthin II, possessing tyrosine ponent in plants such as Celosia argentea (Schliemann et al.,
as the amino acid moiety, was found in minor quantities only in 2001), Swiss chard (Kugler, Stintzing, & Carle, 2004) and Beta vul-
the flower sample and was absent in the stem and leaf extracts. garis (Nemzer et al., 2011).
4388 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390

3.4. In vitro antioxidant activity betalamic acid moiety of the betalain structure enhances the
antiradical activity. The aromatic ring in resonance with the main
3.4.1. Total polyphenol and flavonoids content electron resonance system and the hydroxyl groups in the mole-
Many phenolic compounds derived from plant sources act as cule increases the antioxidant and antiradical capacity of the
powerful natural antioxidants and may contribute directly to its pigment (Gandia-Herrero et al., 2010).
antioxidative action. Flavonoids are natural phenolic compounds
and well known antioxidants in terms of their ability to act as effi- 3.4.4. Hydroxyl radical-scavenging assay
cient radical scavengers. The interest in phenolics of the food The results for hydroxyl scavenging assay is shown in Fig. 4c.
industry is increasing, because they retard oxidative damage of lip- The concentrations for 50% inhibition were found to be 167.31
ids and thereby improve the quality and nutritional value of food. and 53.94 lg/ml for BeTT and ascorbic acid, respectively. A steady
In the present study, the total polyphenolic and flavonoids content increase in the hydroxyl radical-scavenging capability of the
in BeTT was estimated to be 21.61 ± 0.86 lg GAE/mg of extract and extract with respect to concentration was observed. The hydroxyl
9.61 ± 1.15 lg QE/mg of extract respectively. These results suggest radical has the ability to join nucleotides in DNA and cause strand
that high antioxidant capacity could be attributed to the total phe- breakage, leading to mutagenesis, carcinogenesis and cytotoxicity.
nol content. In this assay, the hydroxyl radical generated by the Fenton reaction
was estimated with deoxyribose as the detector molecule, to
3.4.2. Total antioxidant capacity and reducing power determination detect the damage by OH radicals in the presence or absence of
The grouped bar chart (Fig. 3a) represents the total antioxidant EDTA (Halliwell et al., 1987). In earlier studies, it was reported that
capacity of BeTT and ascorbic acid at different concentrations. The betanidin (betacyanin) and miraxanthin V (betaxanthin) when
reducing capability of a compound is associated with its electron monitored spectrophotometrically resulted in the reduction of Fe
donating capacity and serves as an indicator of antioxidant activity (III) to Fe (II) (Gandía-Herrero et al., 2013). In this experiment,
(Prieto et al., 1999). Phosphomolybdenum-reducing ability in- the values observed for BeTT exhibited pronounced effects in scav-
creased with the concentration of BeTT. A direct relationship be- enging the hydroxyl radical in free solution, thus preventing the
tween the dose and absorbance was observed for both BeTT and degradation of deoxyribose sugar.
ascorbic acid.
Fig. 3b shows the reductive capabilities of sample extract in 3.4.5. Nitric oxide radical-scavenging activity
comparison with ascorbic acid (25–400 lg/ml). The reducing The extract exhibited good nitric oxide scavenging activity
power of betalain was based on the Fe3+–Fe2+ transformation in between 50 and 400 lg/ml in a dose-dependent manner
the presence of BeTT. Earlier reports state that a direct correlation (IC50=60.81 lg/ml). The percentage inhibition was increased with
between antioxidant activity and reducing power of the plant ex- increasing concentration of the extract (Fig. 4d). The IC50 value of
tracts has been observed (Pin-Der-Duh, 1998). The reduction of ascorbic acid (IC50=36.84 lg/ml) was comparatively lower than
Fe3+/ferricyanide complex to Fe2+ may be due to the occurrence BeTT. The extract was found to be a moderate scavenger of NO,
of antioxidants (reductants) in the extract (Pin-Der-Duh, 1998). which has been implicated in inflammation, cancer and other path-
The current data on the reducing power of BeTT increased with ological conditions (Moncada, Palmer, & Higgs, 1991). The proce-
increasing concentration of sample, suggesting that BeTT consider- dure is based on the principle that, nitric oxide generated
ably contributed towards the observed antioxidant effect. spontaneously from sodium nitroprusside in aqueous solution at
physiological pH interacts with oxygen to produce nitrite ions that
3.4.3. DPPH and ABTS radical-scavenging activity can be estimated by using Griess reagent. Scavengers of nitric
DPPH is often used as a substrate to evaluate the antiradical oxide compete with oxygen, leading to reduced production of ni-
activity of antioxidants. This method is based on the reduction of trite ions. Plant extracts may have the property to counteract the
methanolic DPPH solution in the presence of a hydrogen effect of NO formation and in turn may be of considerable interest
donating antioxidant, due to the formation of the non-radical form in preventing the ill effects of excessive NO generation in the hu-
[DPPH–H] by the reaction. Glutathione, ascorbic acid, tocopherol, man body.
polyhydroxy aromatic compounds and aromatic amines reduce
and decolourise DPPH by their hydrogen donating ability (Blois, 3.4.6. Superoxide radical-scavenging activity
1958). In the present study, BeTT was able to reduce the stable Fig. 4e depicts the superoxide radical scavenging activity of the
radical DPPH to the yellow-coloured diphenylpicrylhydrazine. extract, as measured by the NBT system in vitro. The principle
The BeTT and ascorbic acid showed a concentration-dependent behind this method is based on the generation of superoxide rad-
antioxidant activity by inhibiting the DPPH radical with an IC50 ical (O2) by auto oxidation of hydroxylamine hydrochloride in
value of 144.82 lg/ml and 53.94 lg/ml respectively (Fig. 4a). How- presence of NBT, which gets reduced to nitrite. Superoxide radical
ever, scavenging activity of ascorbic acid, used as positive control, is known to be very harmful to cellular components as a precursor
was relatively more pronounced than that of BeTT. of more reactive oxygen species (Halliwell & Gutteridge, 1985).
In this study, the suppression of the absorbance of ABTS+ in a Our results indicated that the tested extract (IC50=189.65 lg/ml)
concentration-dependent manner is shown in Fig. 4b. The IC50 had a notable effect on the scavenging of superoxides when com-
value for BeTT (133.72 lg/ml) was appreciable when compared pared with ascorbic acid (IC50=103.91 lg/ml). When increasing
to ascorbic acid (66.43 lg/ml). Phenolic antioxidants have been concentration of BeTT or ascorbic acid was added to the radical
reported to scavenge ABTS+ through hydrogen atom donation (Re mixture, a linear effect was observed on the removal of the radical.
et al., 1999). This elucidates the current interest in the applicability
of the ABTS+ assay in determining the radical scavenging activities 3.4.7. Metal chelating activity
of plant extracts. Similar to the present study, miraxanthin V The chelating of ferrous ions by the extract and EDTA is shown in
isolated from Lampranthus productus exhibited a high free radi- Fig. 4f. Ferrozine can quantitatively form complexes with Fe2+. In the
cal-scavenging capacity (Gandía-Herrero, Cabanes, Escribano, Gar- presence of other chelating agents, the complex formation is dis-
cía-Carmona, & Jimenez-Atienzar, 2013) rupted with a decrease in the formation of the red colour. Therefore,
Structure–activity relationships for betalain-antioxidant activ- measurement of the rate of colour reduction allows the estimation of
ity have been studied earlier (Gandia-Herrero, Escribano, & Gar- the chelating activity of the coexisting chelator. In this assay, both
cia-Carmona, 2010). The presence of aromatic compound in the extract (IC50=192.73 lg/ml) and EDTA (IC50=78.24 lg/ml) interfered
 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390 4389

a b

c d

e f

Fig. 4. Antioxidant activities of different concentrations of BeTT (25–1000 lg/ml) as determined by DPPH free radical-scavenging assay (a), ABTS free radical-scavenging
assay (b), hydroxyl radical-scavenging assay (c), nitric oxide radical-scavenging assay (d), superoxide radical-scavenging assay (e) and metal chelating activity (f).

with the formation of ferrous–ferrozine complex, suggesting Photometric quantification revealed that the betacyanin pigment
that it has chelating activity by capturing the ferrous ion before was prominent in the flower and stem samples which could be
ferrozine. The absorbance of Fe2+–ferrozine complex decreased responsible for the purple–pink colouration. Reversed-phase
dose-dependently signifying that the activity of BeTT increased on HPLC-photodiode array detection coupled with mass spectrometry
increasing concentrations. was able to determine that the betacyanin patterns in flower and
stem sample was dominated by betanin and the C15 diastereomer
4. Conclusion isobetanin as the principle red pigment. Various in vitro antioxi-
dant assays revealed that betalain extracts possessed potent anti-
Betalain profiling of T. triangulare for estimation and detection oxidant abilities which might be helpful in preventing or slowing
of betacyanins and betaxanthins was attempted for the first time. the progress of various oxidative stress-related disorders. Thus
4390 J. Swarna et al. / Food Chemistry 141 (2013) 4382–4390

the present study may prove its suitability for screening of beta- Harborne, J. B. (1998). Phytochemical methods: A guide to modern techniques of plant
analysis. London: Chapman A & Hall.
lain-containing plants as potential sources for natural food colours.
Kugler, F., Stintzing, F. C., & Carle, R. (2004). Identification of betalains from
differently coloured Swiss chard (Beta vulgaris ssp. cicla [L.] Alef. Cv. Bright
Acknowledgements Lights) by high-performance liquid chromatography electrospray ionization
mass spectrometry. Journal of Agricultural and Food Chemistry, 52,
2975–2981.
The authors are very grateful to Dr. Surendra Ponrathnam and Kugler, F., Stintzing, F. C., & Carle, R. (2007). Characterization of betalain patterns
Dr. Sunil Bhongale, Polymer Science and Engineering Division, Na- of differently coloured inflorescences from Gomphrena globosa L. and
Bougainvillea sp. by HPLC–DAD–ESI–MS. Analytical and Bioanalytical
tional Chemical Laboratory, Pune, for their assistance with the LC–
Chemistry, 387, 637–648.
MS instrumentation and providing the facilities to carry out a part Lee, J. C., Kim, H. R., Kim, J., & Jang, Y. S. (2002). Antioxidant activity of an ethanol
of the research work. The authors are obliged to Dr. T. S. Lokeswari, extract of the stem of Opuntia ficus-indica var. saboten. Journal of Agricultural and
Head, Department of Biotechnology, Sri Ramachandra University, Food Chemistry, 50, 6490–6496.
Moncada, A., Palmer, R. M. J., & Higgs, E. A. (1991). Nitric oxide: Physiology,
Porur, Chennai- 600116, Tamil Nadu, India, for her valuable advice pathophysiology and pharmacology. Pharmacological Reviews, 43, 109–142.
and guidance. Nemzer, B., Pietrzkowski, Z., Sporna, A., Stalica, P., Thresher, W., Michalowski, T.,
et al. (2011). Betalainic and nutritional profiles of pigment enriched red beet
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