Chlorination of Betacyanins in Several Hypochlorous Acid Systems

Article abstract of DOI:10.1021/acs.jafc.5b06020

This study presents a comparative evaluation of chlorination of betanin, betanidin, and neobetanin exposed to sodium hypochlorite and myeloperoxidase (MPO)/H₂O₂/Cl^- systems. For betanin/betanidin, the chlorination takes place at the aglycone unit, but for neobetanin, no chlorinated products in the reaction mixtures can be detected. In the RP-HPLC system, monochloro-betanin/-betanidin were eluted earlier than their corresponding nonchlorinated substrates. An influence of Cl^- concentration on betanin/betanidin chlorination efficiency in sodium hypochlorite and MPO systems was investigated. At pH 3-5, the yields of formed monochloro-betanin/-betanidin decrease dramatically at higher Cl^- concentrations, indicating that generated Cl₂ is not the chlorinating agent in the presence of sodium hypochlorite. The intriguing low activity of Cl₂ in betanin/betanidin chlorination compared to HOCl and/or Cl₂O can be explained by a special position of the attack by molecules of HOCl and/or Cl₂O. In the MPO/H₂O₂/Cl^- system, the highest efficiency of monochloro-betanin/-betanidin generation is observed at pH 5.

Full text of DOI:10.1021/acs.jafc.5b06020

Article
pubs.acs.org/JAFC
Chlorination of Betacyanins in Several Hypochlorous Acid Systems
Sławomir Wybraniec,*,† Karolina Starzak,† and Zbigniew Pietrzkowski§
†Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of
Technology, ul. Warszawska 24, Cracow 31-155, Poland
§FutureCeuticals, Inc., 16259 Laguna Canyon Road, Irvine, California 92618, United States
ABSTRACT: This study presents a comparative evaluation of chlorination of betanin, betanidin, and neobetanin exposed to
sodium hypochlorite and myeloperoxidase (MPO)/H2O2/Cl− systems. For betanin/betanidin, the chlorination takes place at the
aglycone unit, but for neobetanin, no chlorinated products in the reaction mixtures can be detected. In the RP-HPLC system,
monochloro-betanin/-betanidin were eluted earlier than their corresponding nonchlorinated substrates. An influence of Cl−
concentration on betanin/betanidin chlorination efficiency in sodium hypochlorite and MPO systems was investigated. At pH 3−
5, the yields of formed monochloro-betanin/-betanidin decrease dramatically at higher Cl− concentrations, indicating that
generated Cl2 is not the chlorinating agent in the presence of sodium hypochlorite. The intriguing low activity of Cl2 in betanin/
betanidin chlorination compared to HOCl and/or Cl2O can be explained by a special position of the attack by molecules of
HOCl and/or Cl2O. In the MPO/H2O2/Cl− system, the highest efficiency of monochloro-betanin/-betanidin generation is
observed at pH 5.
KEYWORDS: betanin, betanidin, neobetanin, betacyanins, betalains, myeloperoxidase, MPO, inflammation, neutrophils, chlorination,
hypochlorite
betanidin) is presumably oxidized solely via generation of a
quinone methide intermediate that rearranges to 2,3-dehydro-
or neo-derivatives.19 Further comprehensive nonenzymatic
studies on the oxidation mechanism in neobetanin and betanin
as well as its decarboxylated derivatives in the presence of
ABTS cation radicals as a commonly used diagnostic oxidation
agent were accomplished recently.20
INTRODUCTION
■
Betalains are water-soluble plant pigments commonly used as
food colorants.1,2 A remarkable betalain subgroup includes
betacyanins, which are primarily immonium conjugates of
betalamic acid with glycosylated cyclo-DOPA.1,2 Betanin
(Figure 1), the principal pigment of red beet root (Beta
vulgaris L.), is the first and most frequently studied betalain for
its antioxidant activity.3−10
Products containing betalains have more often been used for
modulation of various health conditions.3−7 Moreover, a
betalain-rich concentrate (BRC) derived from B. vulgaris
roots standardized to a minimum content of 25% betalains
and substantially free of sugars and nitrates was tested in a pilot
clinical study, which showed that short-term treatment with
BRC improved the function and comfort of knee joints in
individuals with knee distress.11,12
Except for the paper of Allegra et al.18 no studies on betalain
chlorination were performed that would clarify a role of these
potent antioxidants in hypochlorite scavenging. Similarly, no
chlorination/oxidation products of betalains were detected or
identified. Therefore, a comprehensive study of betanin,
betanidin, and neobetanin (partially oxidized betanin) reacting
with hypochlorous acid and a comparative evaluation of the
products based on the LC-DAD-MS/MS results were
performed. In addition, the chromatographic properties of the
generated chloro-betalains were also studied.
The above-referenced studies show that hypochlorous acid
may trigger several detrimental processes in the human body.
Allegra et al.13 reported the effectiveness of the betalains,
betanin and indicaxanthin, in scavenging of hypochlorous acid.
In addition, both of these betalains have been reported to
reduce myeloperoxidase activity and oxidation, two key
components of the inflammatory process,13−17 which also
results in a reduction of hypochlorous acid generation.14,18 In
recent studies on enzymatic oxidation of betanidin with
horseradish peroxidase,19 a formation of oxidation products 2-
decarboxy-2,3-dehydro-betanidin and 2,17-bidecarboxy-2,3-de-
hydro-betanidin indicated their generation via two possible
reaction paths with two different quinonoid intermediates:
dopachrome derivative and quinone methide. Both reaction
pathways lead to a decarboxylative dehydrogenation of
betanidin. Subsequent oxidation and rearrangement of the
conjugated chromophoric system results in the formation of
14,15-dehydrogenated derivatives. Betanin (5-O-glucosylated
MATERIALS AND METHODS
■
Reagents. Formic acid, LC-MS grade methanol, and water were
obtained from Sigma Chemical Co. (St. Louis, MO, USA). NaOCl
solution, H2O2, NaCl, horseradish peroxidase, and myeloperoxidase
from human leukocytes (MPO) were also obtained from Sigma
Chemical Co.
Preparation of Natural and Hydrolyzed Analytes. Betanin and
neobetanin were isolated from a betalain-rich red beet root extract
obtained from FutureCeuticals, Inc. (Momence, IL, USA).21 The
extract was dissolved in water and was separated on Sephadex DEAE
A-25 gel and by solid phase extraction on C18 cartridges before HPLC
preparative fractionation.22,23 For betanidin preparation, purified
Received: December 21, 2015
Revised: March 3, 2016
Accepted: March 6, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 1. Chemical stuctures of the studied betalains in the hypochlorous systems.
betanin was subjected to enzymatic hydrolysis catalyzed by almond β-
glycosidase24 and the same cleanup as in the case of the natural
pigments. The eluates were concentrated under reduced pressure at 25
°C.
Chromeleon 4.32 (Gynkotek Separations) software package. Samples
were eluted through a 250 mm × 3 mm i.d., 5 μm, Luna C18(2)
column preceded by a 4 mm × 2 mm i.d. guard column of the same
material (Phenomenex). The injection volume was 10 μL, and the flow
rate was 0.5 mL/min. The column was thermostated at 35 °C. For the
separation of the analytes, a gradient system was used (system 2): 93%
A in B at 0 min; gradient to 80% A in B at 35 min (A, 2% formic acid
in water; B, methanol). Online UV/vis spectra acquisition was
performed using the diode array detection (DAD) mode typically at
538, 505, 480, and 440 nm. The same chromatographic conditions
were applied to LC-DAD-ESI-MS/MS analyses.
LC-ESI-MS/MS Analysis. The positive ion electrospray mass
spectra were recorded on an LCMS-8030 mass spectrometry system
(Shimadzu, Kyoto, Japan) coupled to a LC-20ADXR pump utilizing
the HPLC gradient system 2. The LC-MS system was controlled with
LabSolutions software (Shimadzu), which recorded total ion
chromatograms and mass spectra (electrospray voltage, 4.5 kV;
capillary, 250 °C; sheath gas, N2). Argon was used to improve trapping
efficiency and as the collision gas for CID experiments. The relative
collision energies for MS/MS analyses were set at −35 V.
Semipreparative HPLC. For the semipreparative isolation of
betanin and neobetanin from the purified red beet root extract as well
as betanidin, an HPLC system with a UVD170S detector, a series
P580 pump, and a thermostat (Gynkotek Separations, H. I. Ambacht,
The Netherlands) was used. The semipreparative column used was a
250 mm × 10 mm i.d., 10 μm, Luna C18(2), with a 10 mm × 10 mm
i.d. guard column of the same material (Phenomenex, Torrance, CA,
USA) under the following gradient system (system 1): 6% A in B at 0
min; gradient to 10% A in B at 30 min (A, acetonitrile; B, 4% (v/v)
HCOOH in H2O). In each case, the injection volume was 100 μL and
the flow rate was 3 mL/min. Detection was generally performed at
538, 505, 480, and 310 nm with a DAD UV/vis detector. The columns
were thermostated at 30 °C. All fractions obtained were diluted with
water and submitted to freeze-drying and analysis.
Spectrophotometric Monitoring of Chlorination Kinetics.
The main betalain chlorination experiments in the Cl2/HOCl/OCl−
systems were performed by their reaction with NaOCl aqueous
solutions buffered with 25 mM acetate (pH 3−5) and phosphate (pH
6−8) buffers in 96-well plates of an Infinite 200 microplate reader
RESULTS AND DISCUSSION
■
The possibility of a chlorination of betacyanins as well as
detection of the chlorinated pigments was studied on the
closely related compounds betanin, 1; betanidin, 2; and
neobetanin, 3 (Figure 1). Betanin is a glucosylated derivative
of the chromophoric structure of betanidin. The chromophoric
unit of betanidin is the only one with the 5,6-dihydroxy
(catechol) moiety, and consequently it possesses a high
antioxidant activity.6,9,10 Recent enzymatic oxidation experi-
ments on betanidin at pH 3−8 confirmed the prevailing effect
of the o-diphenol group on the oxidation pathway.19 However,
taking into account that betanin also exhibits antioxidative
properties,6,9,10 the possibility of a formation of oxidized
products during betanin exposure to hypochlorous acid was
investigated and verified. In addition, neobetanin, partially
oxidized betanin, represents another reactive derivative, which
arises during numerous oxidation processes and is a key
diagnostic intermediate in the oxidation pathways.1
(Tecan Austria GmbH, Grodig/Salzburg, Austria). The concentration
̈
of the NaOCl solution was determined spectrophotometrically at pH
12 (ε292 = 350 M/cm). Just before the measuring step, 20 μL of
NaOCl solution was dispensed to each well containing 40 μM
dissolved pigment, bringing the volume to 200 μL. The final
concentration of the NaOCl solution in the wells was in the range
of 40−120 μM for all of the tested pigments except neobetanin (10−
40 μM). For the experiments with influence of NaCl on the tested
reactions, the salt was added to the wells to reach the final
concentration of 20−1500 mM. The mixture was then shaken for
20 s by a shaker within the reader. The spectra were collected over 30
min at a temperature of 25 °C by spectrophotometric detection at the
wavelength range of 350−550 nm. For the chromatographic analyses,
separate experiments were performed, and 20 μL samples after 5 min
of reaction were injected directly to the LC-DAD or LC-DAD-ESI-
MS/MS system without further purification. The measurements were
performed in triplicate.
Spectrophotometric Monitoring of Enzymatic Chlorination
Kinetics. The enzymatic chlorination experiments on 40 μM betalains
in the MPO/H2O2/Cl− system were performed in aqueous solutions
(buffered with 25 mM acetate (pH 3−5) and phosphate (pH 6−8)
buffers) during reaction catalyzed by 1 μM MPO in the presence of 50
μM H2O2 and 150 mM NaCl in 96-well plates of the microplate
reader. Just before the measuring step, 20 μL of H2O2 solution was
dispensed to each well containing all of the components, bringing the
volume to 200 μL. For the experiments with the influence of NaCl on
the tested reactions, the salt was added to the wells to reach the final
concentration of 20−600 mM. The mixture was then shaken for 20 s
by a shaker within the reader, and the spectra were collected over 120
min of the experiment. For the chromatographic analyses, separate
experiments were performed, and each injection to the LC-DAD-ESI-
MS/MS system was performed after 120 min of the reaction. The
measurements were performed in triplicate.
Chlorination of Betanin by Sodium Hypochlorite. The
spectrophotometric monitoring of betanin, 1, reaction induced
by NaOCl at pH 3, 5, and 7.4 was performed in dependence on
different NaOCl concentrations. The highest oxidation activity
of the reagent is observed at pH 7.4, and it is in contrast to the
results of enzymatic oxidation of betanin that indicated the
highest rate of oxidation at pH 3.19 However, analysis of the
chlorination UV/vis spectra suggests completely different
mechanisms of betanin reaction in acidic and close to neutral
media. At pH 7.4, the main absorption band of betanin (λmax
538 nm) is slightly shifted during the chemical changes (λmax
534 nm), whereas at pH 3, a hypsochromic shift of λmax is
observed, reaching 522−524 nm at NaOCl concentration of 40
μM after 5 min of the experiment. At pH 5, a medium point of
the shift is attained (λmax 526 nm) at higher NaOCl
Chromatographic Analysis. The equipment was the same as that
used for the semipreparative HPLC. Data were acquired with the
B
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 2. Chromatographic traces of the products of 40 μM betanin 1 (A), betanidin 2 (B), and neobetanin 3 (C) oxidation at pH 5 (pH 7.4 for
neobetanin) by 1 μM MPO in the presence of 50 μM H2O2 and 150 μM NaCl and monitored at 522 nm (480 nm for neobetanin) and 25 °C. Peak
numbering is presented in Table 1.
concentrations. The same results were obtained for the
chlorination of isobetanin 1′ (the diastereomeric form 15R)
evidently with a chlorinated diastereomer 1a′.
Because the chlorination of betanin is especially favored in
acidic conditions, the high activity of molecular chlorine (Cl2)
generated from hypochlorous acid toward betanin at pH 3−5
was suspected to be the chlorinating factor. HOCl generated in
aqueous solutions is in equilibrium with Cl2:25
These data are confirmed by the HPLC-DAD analysis of the
reaction mixtures revealing the formation of a main product
absorbing light at λmax 522 nm. In Figure 2A,B, typical
chromatographic profiles of chlorinated betacyanins together
with their nonchlorinated precursors obtained for enzymatic
chlorination (discussed in the next section) at pH 5 are
presented. The chlorinated derivatives formed were eluted from
the column earlier than their precursors.
HOCl + Cl− + H+ ⇌ Cl2 + 2H2O
(1)
Therefore, the influence of Cl− concentration on the reaction
progress would result in the detection of higher concentrations
of the chlorinated products, especially at higher acidity (pH 3−
5). Surprisingly, this was not the case shown in the betanin
chlorination experiments with increasing NaCl concentration
(Figure 3A). At pH 3−5, the yield of monochlorinated betanin
decreases dramatically at higher Cl− concentrations, indicating
that the generated Cl2 is not the chlorinating agent. At pH 6−8,
the yield of formed monochloro-betanin is much lower than at
pH 3−5; however, it is almost independent of Cl−
concentration. At pH 3, a decreased yield of monochloro-
betanin formation in comparison to pH 4−5 is also seen in the
whole Cl− concentration range.
Further LC-MS/MS analytical results indicate a chlorination
of betanin (characterized by λmax 538 nm and m/z 551) due to
the fact that the new chromatographic peak formed with λmax
522 nm and m/z 585 (protonated molecular ion) suggests the
substitution of a hydrogen in the molecule by one chlorine
atom (m/z difference 585 − 551 = 34). The characteristic
isotopic profile of the protonated molecular ions containing
35Cl or 37Cl isotopes confirms the presence of one chlorine
atom in the molecule. A fragmentation experiment of the [M +
H]+ ion results in a subtraction of one glucose unit and the
formation of a protonated monochloro-betanidin/isobetanidin
ion (m/z difference 585 − 162 = 423). This confirms that the
chlorination takes place at the aglycone unit (betanidin).
In Figure 3B, the remaining substrate (betanin) fraction (%)
analyzed by LC-MS in the reaction mixtures is presented in
dependence on Cl− concentration and pH. The highest
reactivity of betanin is observed at pH 3−5 with the lowest
Cl− concentration when only <6% of unreacted betanin is
C
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
In more acidic media, HOCl is partially converted to chlorine
monoxide Cl2O, which is a more potent chlorinating agent even
at lower concentrations:25,27−29
2HOCl ⇌ Cl2O + H2O
(3)
Therefore, the influence of pH on the formation of chlorinated
betanin can be explained by two factors: diminished
dissociation of HOCl in acidic media and increased generation
of Cl2O from HOCl instead of chlorine in acidic media.
Both of the compounds, HOCl and Cl2O, can have an
impact on the chlorination of betanin via electrophilic
substitution. As mentioned in recent literature,25,27−29 the
potency of Cl2O in chlorination and oxidation has been
recognized for over 170 years. Aqueous free chlorine
equilibrium speciation for HOCl, OCl−, Cl2, and Cl2O as
potential chlorinating and oxidizing agents has been presented
and kinetically confirmed in selected reactions.25,27−29 The
intriguing low activity of Cl2 in betanin chlorination compared
to HOCl and Cl2O can be explained by a special position of the
attack by a molecule of HOCl and Cl2O, which is not in the
aromatic ring of betanin (typical Cl2 target) but in the carbon
position C-18 with acidic hydrogen.30 This is a carbon of
enhanced negativity, frequently noted to rapidly exchange
hydrogen in deuterium dioxide;30 therefore, reactions with
HOCl and Cl2O are proposed according to a mechanism based
on leaving group ability from Cl+ in HOCl (−OH−) and in
Cl2O (−OCl−) (Figure 4).25,27−29 This is also supported by the
inactivity of neobetanin toward chlorination. Neobetanin is an
aromatized betanin with a pyridine ring resulting from betanin
oxidation. The fact that this ring cannot be chlorinated
confirms that in betacyanins, only the unsaturated bond is
attacked, preferably at C-18 because of its partial negative
charge.
Because the leaving group ability from Cl2O is much higher
than in HOCl,25,27−29 the chlorinating power of Cl2O is much
stronger even at lower concentration than that of HOCl. But
for HOCl, an additional mechanism of chlorination based on
the formation of hypochlorous acidium ion in acidic media can
be proposed:25,31
HOCl + H+ ⇌ H2OCl+
This would support the chlorinating ability of HOCl toward
betanin (Figure 4) and betanidin and also the oxidative
properties toward betanidin.
Chlorination of Betanin by MPO System. According to
the above results, hypochlorous acid generated by myeloper-
oxidase in the presence of chloride anions and hydrogen
peroxide (reaction 5) should also chlorinate betanin throughout
the entire tested pH range and with the highest efficiency at pH
3−6. However, the most optimal pH increasing the MPO
activity toward HOCl formation that may occur in the
phagosome is close to 5:26
(4)
Figure 3. Influence of NaCl concentration on efficiency of betanin 1
chlorination by NaOCl (A) and stability of betanin (B) as well as
myeloperoxidase-catalyzed chlorination (C) in dependence on pH. At
high NaCl concentration (increased Cl2 delivery), a diminished
chlorination efficiency is noted for pH 3−5 (A, B) or in all of the MPO
reaction media (C).
detected. At high Cl− concentration (1.5 M), unreacted betanin
is detected at a level of 18−28%. In contrast, the levels of
remaining unreacted betanin seen at pH 6−8 do not change
meaningfully with the increase of Cl− concentration, confirming
that the generation of Cl2 is low at pH 6−8 and is not changing
meaningfully. Interestingly, at pH 3, the highest concentration
of betanin is detected, indicating that the reaction progress is
decreased in comparison to pH 4−5.
HOCl is a weak acid (pKa = 7.54); therefore, at physiological
pH, it is present at a comparable concentration with its
dissociated form OCl−, which is presumably acting as a direct
oxidant for betanin:25
Cl− + H2O2 + H+ ⇌ HOCl + H2O
(5)
Because the activity of MPO strongly depends on the pH of
the reaction medium and the enzymatic process is much slower
than direct chlorination by NaOCl, a series of experiments was
performed on the pH influence on the generation of
chlorinated betanin and kinetic monitoring of the reaction
progress. Anticipated detection of the chlorinated betanin in
the MPO system at pH ≥5 was supposed to confirm the
possibility of its formation during a hypochlorite scavenging
process, which possibly may take place under in vivo
HOCl ⇌ OCl− + H+
(2)
D
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 4. General reaction mechanism proposed for the chlorination of betanin 1 by hypochlorous acid (HOCl) or dichlorine oxide (Cl2O). For
HOCl, two alternative chlorination pathways are considered.24,26−28 A similar mechanism can be assigned for betanidin 2.
conditions. In addition, the strong oxidizing activity of HOCl
could be still observable at physiological pH levels.26
The chromatographic traces of the reaction mixtures
recorded after 2 h of incubation of MPO and H2O2 with the
pigment at pH 5 are depicted in Figure 2. The chromatogram
confirms a partial transformation of the pigment into the
chlorinated derivative.
In the enzymatic system, the chlorination progress is
controlled by a continuous generation of HOCl; therefore, a
continuous absorption maximum shift is observed. This is in
contrast to the rapid chlorination of the pigments by NaOCl at
a defined reagent concentration. The observed directions of
changes in the spectra are a result of a superposition of the
strong betanin tendency to be chlorinated at more acidic media
(pH 3−5) and the highest MPO activity at pH 5−5.5 toward
the generation of HOCl. In the course of spectrophotometric
changes, the main absorption maximum of betanin at pH 5
shifts toward the characteristic maximum of chlorinated betanin
(522 nm) observed formerly during the direct chlorination by
NaOCl.
Further experiments on MPO-mediated chlorination of
betanin with increasing NaCl concentration (Figure 3C) led
also to the conclusion that an overdose of NaCl (up to 600
mM) hampers the generation of monochloro-betanin as a result
of Cl2 formation (reaction 1), which is not the chlorinating
agent according to the results of the above nonenzymatic
chlorination experiments (Figure 3A). However, at lower NaCl
concentrations, this effect is overwhelmed by a continuous
production of HOCl from Cl− catalyzed by MPO, which reacts
with betanin to produce the highest quantities of chlorinated
betanin within a concentration range of NaCl at 50−150 mM
(Figure 3C).
In Figure 3C, as a result of the chromatographic analyses, the
highest efficiency of monochloro-betanin generation is clearly
indicated at pH 5. The efficiency at pH 3 is significantly
diminished in contrast to the nonenzymatic chlorination.
Interestingly, the MPO-mediated chlorination is still observed
at pH 6−7.4 at the physiological levels of the reagents (Figure
3C), confirming that HOCl is still generated by MPO activity
even at pH 7.4. In addition, the lack of the diagnostic betanin
dehydrogenated and decarboxylated derivatives (possessing the
characteristic chromophoric systems) in the chromatograms at
any applied pH (Figure 2) suggests that no evidence of betanin
oxidation can be obtained. Therefore, no peroxidase activity of
MPO toward betanin is confirmed, which is in contrast to the
previously observed activity of horseradish peroxidase,
especially in acidic media.19
Chlorination of Betanidin by Sodium Hypochlorite.
For betanidin, 2, which is the only nonglucosylated betacyanin,
characterized by the absorption maximum at λmax 540 nm,1,2
the analogous strong hypsochromic shifts in the spectra (down
to 528 nm) are observed at pH 3. In addition, at higher NaOCl
concentrations (>100 μM), the main absorption band is almost
undiminished but shifted to the characteristic λmax of
monochloro-betanidin, whereas at pH 7.4, it gradually
disappears with additional shift toward 528 nm. For pH 7.4,
longer hypsochromic shifts in the spectra of the pigment are
observable, starting from an NaOCl concentration of 100 μM,
suggesting a substantial formation of dehydrogenated deriva-
tives rather than chlorinated ones. This is in accordance with a
high betanidin activity against various oxidation agents,
especially in neutral media.6,9,10 Therefore, except for the
formation of a chlorinated derivative, the bulk of oxidized
products can be expected to be generated in the reaction
mixture especially in neutral media.19
The formation of monochloro-betanidin was confirmed by
detection of a principal reaction product characterized by m/z
423 (423 − 389 = 34), suggesting the substitution of a
hydrogen by chlorine in betanidin. In addition, the character-
istic isotopic profile of 35Cl and 37Cl isotopes is observed for
the protonated molecular ion, and analogous results are
obtained for isobetanidin, 2′. In addition, the generated
chlorinated betanidin/isobetanidin moieties are eluted earlier
than their precursors (Figure 2B). Monochloro-isobetanidin,
2a′, exhibits a positive shift in the retention time in relation to
2a. In addition, two chlorinated derivatives in the reaction
E
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
pathway are detected (Table 1), monochloro-17-decarboxy-
betanidin, 2d, and monochloro-2,17-bidecarboxy-2,3-dehydro-
is observed for the lowest Cl− concentration (at pH 3−5). At
pH 6−7.4, the yield of formed monochloro-betanidin is almost
independent of Cl− concentration and also much lower than at
pH 3−5. Similar to betanin chlorination, at pH 3, a decreased
yield of monochloro-betanidin formation in comparison to that
at pH 4 is visible at higher Cl− concentration range.
Table 1. Chromatographic, Spectrophotometric, and Mass
Spectrometric Data of the Analyzed Betalains and Their
Products of Chlorination by NaOCl as well as the MPO
System
Therefore, for betanidin and betanin, a similar mechanism of
chlorination can be suggested, indicating that the generated Cl2
is not the chlorinating agent but rather HOCl and/or Cl2O.
This is partially confirmed by the reactivity test on betanidin
stability, which is highest at pH 3−6 with the lowest Cl−
concentration when ca. 17−20% of unreacted betanidin is
detected. However, the levels of remaining unreacted betanidin
at pH 7−7.4 decrease meaningfully with the increase of Cl−
concentration to 0.5 M, which should be rather a result of
increased oxidation (Figure 5) effect than of chlorination.
Chlorination of Betanidin by MPO System. In the case
of betanidin myeloperoxidase-assisted chlorination, the chlori-
nating activity of generated hypochlorous acid by MPO in the
presence of chloride anions and hydrogen peroxide (reaction 3)
is strongly modulated by the oxidizing activity of HOCl (as
indicated in the above NaOCl tests) as well as the peroxidase
function of MPO. Both oxidizing effects were observed to
prevail in neutral media.
The spectrophotometric monitoring of MPO-mediated
chlorination of betanidin was also performed. As in the case
of betanin, a continuous shift of the absorption maximum
toward λmax of the chlorinated betanidin (528 nm), as well as
the highest MPO activity at pH 5, is observed. The similarity is
also visible in the high reaction rate at neutral media, which
should be assigned mainly to the oxidation of betanidin.
The chromatographic trace of the reaction mixture recorded
after 2 h of incubation of MPO and H2O2 with betanidin at pH
5 is depicted in Figure 2B. The chromatogram confirms not
only the partial transformation of the pigment into the
chlorinated derivative but also the generation (Figure 5) of a
series of the oxidized derivatives as in the case of the
nonenzymatic oxidation. The presence of all of the derivatives
2a−2f (Table 1) known from the previous NaOCl study was
observed.
Further experiments were performed on MPO-catalyzed
chlorination of betanidin with increasing NaCl concentration.
As in the case of betanin, the overdose of NaCl also hampers
the generation of the chlorinated pigment as a result of Cl2
formation (reaction 1), and the highest MPO chlorinating
activity is documented at pH 5. However, the chlorination of
betanidin at pH 7.4 is much less efficient than that of betanin
even at the physiological concentration of NaCl. This is
evidently a result of the high oxidizing activity of MPO (acting
at the peroxidase mode) as well as HOCl. The sole oxidizing
MPO activity is also observed in the case of the lack of NaCl
(no production of HOCl) in the reaction mixture.
The generation of betanidin oxidation and chlorination
products at 150 mM NaCl in dependence on pH was also
studied. The highest efficiency of MPO-mediated monochloro-
betanidin generation at pH 5 is indicated; however, it is
overwhelmed by the presence of the most indicative oxidation
(2,17-bidecarboxy-2,3-dehydro-betanidin, 2b, and 2-decarboxy-
2,3-dehydro-betanidin, 2c) and double-oxidation (2,17-bide-
carboxy-2,3-dehydro-neobetanidin, 2f) products. Furthermore,
these compounds are generated over the whole tested pH range
of 3−7.4 in substantial quantities, which contrasts with the
nonenzymatic activity of NaOCl but is similar to the overall
m/z from
tR
λmax
m/z
MS/MS of
no.
compound
(min) (nm) [M + H]+
[M + H]+
a
1a
1
monochloro-betanin
8.4
9.7
522
538
522
538
528
541
528
585
551
585
551
423
389
423
423; 379
389
betanin
a
1a′ monochloro-isobetanin
9.9
423; 379
389
1′
2a
2
isobetanin
10.7
10.8
12.6
12.8
a
monochloro-betanidin
betanidin
379
345
2a′ monochloro-
379
a
isobetanidin
2′
2b
isobetanidin
13.6
16.2
541
472
389
299
345
2,17-bidecarboxy-2,3-
dehydro-betanidin
255; 253
a,b
2c
2d
2e
2-decarboxy-2,3-
dehydro-betanidin
16.4
16.6
17.6
498
525
465
343
379
333
299; 255
335
ab
,
monochloro-17-
decarboxy-betanidin
monochloro-2,17-
bidecarboxy-2,3-
dehydro-betanidin
289
2f
2,17-bidecarboxy-2,3-
dehydro-
17.7
415
297
253
ab
,
neobetanidin
3
neobetanin
14.8
17.3
466
420
549
503
387
3a
2-decarboxy-2,3-
dehydro-neobetanin
341; 297
ac
,
a
b
Tentatively identified. Oxidation products identified previously in
c
ref 19. Oxidation product identified previously in ref 20.
betanidin, 2e. The formation of 2d in the reaction mixtures is
evidently a result of a spontaneous decarboxylation of
monochloro-betanidin, 2a, in the presence of HOCl, whereas
the formation of 2e can be explained in two parallel ways:
decarboxylative oxidation of monochloro-17-decarboxy-betani-
din, 2d, and/or chlorination of the already oxidized compound,
2,17-bidecarboxy-2,3-dehydro-betanidin, 2b (Figure 5).
The influence of pH on the formation of the oxidized/
chlorinated betanidin derivatives analyzed by LC-MS system
was also studied. The contradictory effect of pH on the
presence of the main chlorinated product 2a in the reaction
mixtures (prevailing at more acidic conditions) as well as the
oxidized and decarboxylated compound 2b (2,17-bidecarboxy-
2,3-dehydro-betanidin) and monochloro-2,17-bidecarboxy-2,3-
dehydro-betanidin, 2e (both increasing their concentration
toward more neutral media), indicates the highest chlorination
efficiency of NaOCl at more acidic conditions, as in the case of
betanin, as opposed to the oxidation in more neutral
conditions. The direct betanidin oxidation product, 2-
decarboxy-2,3-dehydro-betanidin, 2c, is evidently less stable
than 2b because it converts spontaneously to 2b over the whole
pH range, whereas a further oxidation of 2b is again observed at
more neutral pH, resulting in the formation of 2,17-
bidecarboxy-2,3-dehydro-neobetanidin, 2f.
As in the case of betanin, the chlorination experiments on
betanidin with increasing NaCl concentration confirmed that
the highest efficiency of monochlorinated betanidin generation
F
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 5. Mechanism of betanidin 2 oxidation by hypochlorous acid (HOCl) generated in the MPO or NaOCl system. Oxidation proceeds via a
quinone methide intermediate19 and is accompanied by a chlorination of the resulting product 2f.
activity of horseradish peroxidase over the whole pH range.19 In
addition, the oxidation efficiency of betanidin at pH 7.4 is so
high that even the oxidation products are converted to other
nondetectable compounds not possessing the characteristic
chromophoric systems.
Oxidation of Neobetanin by Sodium Hypochlorite.
Further chlorination experiments were performed on neo-
betanin, 3, (Figure 1), which is the only partially oxidized
betanin derivative known in the plant kingdom.1,2 Because of
the dehydrogenation of the dihydropyridine ring in betanin, 1,
the chiral center at carbon C-15 disappears in the resulting
neobetanin. Its chemical structure is different from that of
betanin by the presence of a pyridine ring that completely
changes the chemical properties of the compound. This is also
observed in the obtained results within which no chlorinated
products in the reaction mixtures can be detected by mass
spectrometry for any pH values. In addition, the applied NaOCl
at concentrations of 80−120 μM completely bleaches the
pigment in the whole pH range tested. Therefore, we reduced
the experimental concentration range of NaOCl a few times (to
5−40 μM) to enable the detection of any intermediate of the
reaction. Under these conditions, a gradual decrease of the
main absorption bands can be observed in all of the tested
samples. The highest reaction rate is noted at pH 3−4 as well as
pH 7−7.4 and the lowest rate at pH 5−5.5. The LC-MS
analyses resulted in the detection of minute amounts of 2-
decarboxy-2,3-dehydro-neobetanin, 3a, at pH 7−7.4 (a
chromatogram after enzymatic oxidation is presented in Figure
G
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 6. Mechanism of neobetanin, 3, oxidation by hypochlorous acid (HOCl) generated in the MPO or NaOCl systems. The oxidation proceeds
via a quinone methide intermediate.19,20
2C), a reaction product detected in previous studies on
enzymatic and chemical oxidation of betalains.20 Presumably,
this compound is directly generated from neobetanin, 3,
through a quinone methide intermediate and its subsequent
rearrangement with decarboxylation at carbon C-2 (Figure 6).20
However, the high bleaching rate of neobetanin indicates that
other undetected compounds are being generated that do not
possess the characteristic chromophoric system. In addition, the
only detectable product, 3a, presumably reacts further with the
hypochlorite reagent; therefore, it is present at low concen-
tration levels in the reaction mixtures.
The bleaching experiments on neobetanin with increasing
NaCl concentration revealed an interesting pH influence on the
reactivity of the pigment. In the absence of NaCl, the highest
rate of reaction is observed at pH 3, whereas the addition of
NaCl to the reaction mixture results in a meaningful decrease of
the reaction rate at pH 3 (the highest level of unreacted
neobetanin). Instead, at pH 7−7.4, increasing NaCl concen-
tration results in an acceleration of the bleaching rate. This
suggests that in more acidic media the main reacting form is
HOCl, as well as Cl2O, because at higher NaCl concentrations
generated Cl2 is not reactive toward neobetanin. At neutral pH,
generation of Cl2 is diminished anyway; therefore, increasing
the bleaching rate is not an effect of the generation of Cl2.
Chlorination of Neobetanin by the MPO System. As in
the case of NaOCl, no chlorinated products were detected in
any media during MPO-catalyzed bleaching of neobetanin.
Even if at pH 3 a fast decay of the substrate is recorded, no shift
of the main absorption maximum occurs, which would indicate
a new chlorinated compound. The results are confirmed by
chromatography of the reaction products. A chromatogram
obtained at pH 7.4 (Figure 2C) indicates the formation of a
minute amount of oxidation product 2-decarboxy-2,3-dehydro-
neobetanin,20 as in the case of NaOCl chlorination, but no
other bulk products of the bleaching reaction can be detected.
Increasing NaCl concentration significantly enhances the
reactivity of neobetanin at pH 7−7.4 over almost the whole
NaCl concentration range, whereas at pH 3−5, where a higher
bleaching rate is observed in the absence of NaCl, the influence
is manifested only at lower concentrations of NaCl (25−50
mM). At a higher dose of NaCl, an increase of neobetanin
stability is noted (higher concentration of less reactive Cl2,
similar to betanin and betanidin). In any case, the increase of
the pigment reactivity at low NaCl concentration can be
explained by the action of MPO.
H
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
properties of its betalains: betanin and indicaxanthin. J. Agric. Food
Chem. 2002, 50, 6895−6901.
This is the first report on chlorinated betalains that may be
generated by in vivo chlorination of these ingested pigments as
a result of inflammatory processes. In particular, it explains for
the first time a possible function of betalains in inhibiting the
effects of chlorination in the blood and increasing the comfort
of joints in individuals, as had been observed recently.11,12
The mechanism of betacyanin chlorination and oxidation is
of significant interest because of recent appreciation of these
pigments as highly active natural compounds with antioxidative
properties and their potential benefits to human health as well
as a lack of toxicity, which suggests that they may function well
as candidates for use in numerous health applications.
In addition, betalains can presumably serve as indicators of
hypochlorite in inflamed tissues because their chlorination can
be confirmed by the applied analytical methods. From the first
chlorination experiments, it can be deduced that monochloro-
betanin is more stable than its precursor and should be easily
monitored in blood because it retains its chromophoric
structure after chlorination and does not degrade in the
reaction media. Further bioavalaibility studies should confirm
the formation of chlorinated betalains in humans after the
consumption of betalains, especially under pro-inflammatory
conditions.
(8) Cai, Y.; Sun, M.; Corke, H. Antioxidant activity of betalains from
plants of the Amaranthaceae. J. Agric. Food Chem. 2003, 51, 2288−
2294.
(9) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Structural
implications on color, fluorescence, and antiradical activity in betalains.
Planta 2010, 232, 449−460.
(10) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. The role
of phenolic hydroxy groups in the free radical scavenging activity of
betalains. J. Nat. Prod. 2009, 72, 1142−1146.
(11) Pietrzkowski, Z.; Nemzer, B.; Michałowski, T.; Wybraniec, S.
Influence of betalain-rich extract on reduction of discomfort associated
with osteoarthritis. New Med. 2010, 1, 12−17.
(12) Pietrzkowski, Z.; Argumedo, R.; Shu, C.; Nemzer, B.;
Wybraniec, S.; Reyes-Izquierdo, T. Betalain-rich red beet concentrate
(BRC) improves knee discomfort and function: a double blind,
placebo-controlled clinical study. Nutr. Diet. Suppl. 2014, 6, 9−13.
(13) Allegra, M.; Tesoriere, L.; Livrea, M. A. Betanin inhibits the
myeloperoxidase/nitrite-induced oxidation of human low-density
lipoproteins. Free Radical Res. 2007, 41, 335−341.
(14) Zielinska-Przyjemska, M.; Olejnik, A.; Dobrowolska-Zachwieja,
A.; Grajek, W. In vitro effects of beetroot juice and chips on oxidative
metabolism and apoptosis in neutrophils from obese individuals.
Phytother. Res. 2009, 23, 49−55.
(15) Gentile, C.; Tesoriere, L.; Allegra, M.; Livrea, M. A.; D’Alessio,
P. Antioxidant betalains from cactus pear (Opuntia ficus-indica) inhibit
endothelial ICAM-1 expression. Ann. N. Y. Acad. Sci. 2004, 1028, 481−
486.
(16) Netzel, M.; Stintzing, F.; Quaas, D.; Straß, G.; Carle, R.; Bitsch,
R.; Bitsch, I.; Frank, T. Renal excretion of antioxidative constituents
from red beet in humans. Food Res. Int. 2005, 38, 1051−1058.
(17) Stintzing, F. C.; Herbach, K. M.; Mosshammer, M. R.; Carle, R.;
Yi, W.; Sellappan, S.; Akoh, C. C.; Bunch, R.; Felker, P. Color, betalain
pattern, and antioxidant properties of cactus pear (Opuntia spp.)
clones. J. Agric. Food Chem. 2005, 53, 442−451.
(18) Allegra, M.; Furtmuller, P. G.; Jantschko, W.; Zederbauer, M.;
Tesoriere, L.; Livrea, M. A.; Obinger, C. Mechanism of interaction of
betanin and indicaxanthin with human myeloperoxidase and
hypochlorous acid. Biochem. Biophys. Res. Commun. 2005, 332, 837−
844.
(19) Wybraniec, S.; Michałowski, T. New pathways of betanidin and
betanin enzymatic oxidation. J. Agric. Food Chem. 2011, 59, 9612−
9622.
AUTHOR INFORMATION
Corresponding Author
*(S.W.) Phone: +48-12-628-3074. Fax: +48-12-628-2036.E-
mail: swybran@chemia.pk.edu.pl.
■
Funding
This research was financed by the Polish National Science
Centre for years 2015−2018 (Project UMO-2014/13/B/ST4/
04854).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We express our gratitude to John Hunter (FutureCeuticals,
Inc.) for his comments and suggestions in the preparation of
this paper.
́
(20) Wybraniec, S.; Starzak, K.; Skopinska, A.; Nemzer, B.;
REFERENCES
■
Pietrzkowski, Z.; Michałowski, T. Studies on nonenzymatic oxidation
mechanisms in neobetanin, betanin, and decarboxylated betanins. J.
Agric. Food Chem. 2013, 61, 6465−6476.
(1) Khan, M. I.; Giridhar, P. Plant betalains: chemistry and
biochemistry. Phytochemistry 2015, 117, 267−295.
(2) Azeredo, H. M. C. Betalains: properties, sources, applications,
and stability − a review. Int. J. Food Sci. Technol. 2009, 44, 2365−2376.
(3) Tesoriere, L.; Butera, D.; Allegra, M.; Fazzari, M.; Livrea, M. A.
Distribution of betalain pigments in red blood cells after consumption
of cactus pear fruits and increased resistance of the cells to ex vivo
induced oxidative hemolysis in humans. J. Agric. Food Chem. 2005, 53,
1266−1270.
́
(21) Nemzer, B.; Pietrzkowski, Z.; Sporna, A.; Stalica, P.; Thresher,
W.; Michałowski, T.; Wybraniec, S. Betalainic and nutritional profiles
of pigment-enriched red beet root (Beta vulgaris L.) dried extracts.
Food Chem. 2011, 127, 42−53.
(22) Wybraniec, S.; Nowak-Wydra, B. Mammillarinin − a new
malonylated betacyanin in fruits of Mammillaria. J. Agric. Food Chem.
2007, 55, 8138−8143.
(4) Kapadia, G. J.; Azuine, M. A.; Sridhar, R.; Okuda, Y.; Tsuruta, A.;
Ichiishi, E.; Mukainake, T.; Takasaki, M.; Konoshima, T.; Nishino, H.;
Tokuda, H. Chemoprevention of DMBA-induced UV-B promoted,
NOR-1-induced TPA promoted skin carcinogenesis, and DEN-
induced phenobarbital promoted liver tumors in mice by extract of
beetroot. Pharmacol. Res. 2003, 47, 141−148.
(5) Kanner, J.; Harel, S.; Granit, R. Betalains − a new class of dietary
cationized antioxidants. J. Agric. Food Chem. 2001, 49, 5178−5185.
(6) Escribano, J.; Pedreno, M. A.; García-Carmona, F.; Munoz, R.
(23) Wybraniec, S. Effect of tetraalkylammonium salts on retention
of betacyanins and decarboxylated betacyanins in ion-pair reversed-
phase high-performance liquid chromatography. J. Chromatogr. A
2006, 1127, 70−75.
(24) Gandía-Herrero, F.; Escribano, J.; García-Carmona, F.
Characterization of the activity of tyrosinase on betanidin. J. Agric.
Food Chem. 2007, 55, 1546−1551.
(25) Kettle, A. J.; Albrett, A. M.; Chapman, A. L.; Dickerhof, N.;
Forbes, L. V.; Khalilova, I.; Turner, R. Measuring chlorine bleach in
biology and medicine. Biochim. Biophys. Acta, Gen. Subj. 2014, 1840,
781−793.
̃
̃
Characterization of the antiradical activity of betalains from Beta
vulgaris L. roots. Phytochem. Anal. 1998, 9, 124−127.
(7) Butera, D.; Tesoriere, L.; Di Gaudio, F.; Bongiorno, A.; Allegra,
M.; Pintaudi, A. M.; Kohen, R.; Livrea, M. A. Antioxidant activities of
Sicilian prickly pear (Opuntia ficus-indica) fruit extracts and reducing
(26) Klebanoff, S. J. Myeloperoxidase: friend and foe. J. Leukocyte
Biol. 2005, 77, 598−625.
I
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
(27) Sivey, J. D.; McCullough, C. E.; Roberts, A. L. Chlorine
monoxide (Cl2O) and molecular chlorine (Cl2) as active chlorinating
agents in reaction of dimethenamid with aqueous free chlorine.
Environ. Sci. Technol. 2010, 44, 3357−3362.
(28) Cai, M. Q.; Feng, L.; Jiang, J.; Qi, F.; Zhang, L. Q. Reaction
kinetics and transformation of antipyrine chlorination with free
chlorine. Water Res. 2013, 47, 2830−2842.
(29) Cheng, H.; Song, D.; Chang, Y.; Liu, H.; Qu, J. Chlorination of
tramadol: reaction kinetics, mechanism and genotoxicity evaluation.
Chemosphere 2015, 141, 282−289.
(30) Strack, D.; Steglich, W.; Wray, V. Betalains. In Methods in Plant
Biochemistry; Dey, P. M., Harborne, J. B., Waterman, P. G., Eds.;
Academic Press: London, UK, 1993; Vol. 8, pp 421−450.
(31) Swain, C. G.; Crist, D. R. Mechanisms of chlorination by
hypochlorous acid. The last of chlorinium ion, Cl+. J. Am. Chem. Soc.
1972, 94, 3195−3200.
J
DOI: 10.1021/acs.jafc.5b06020
J. Agric. Food Chem. XXXX, XXX, XXX−XXX