ORIGINAL  RESEARCH

New  insights  on  effects  of  a  dietary  supplement  on

oxidative  and  nitrosative  stress  in  humans

Boris  V.  Nemzer^1,2 ,  Nelli  Fink³  &  Bruno  Fink³

¹VDF  FutureCeuticals  Inc.,  2692  N  State  Rt.  1-17,  Momence,  Illinois,  60954

²University  of  Illinois  at  Urbana-Champaign,  1201  W.  Gregory  Dr,  Urbana,  Illinois,  61801

³Noxygen  Science  Transfer  &  Diagnostics  GmbH,  Lindenmatte  42,  79215,  Elzach,  Germany

Keywords

Abstract

Dietary  supplement,  EPR,  inflammatory

response,  nitric  oxide,  oxidative  stress,  RONS,

The  research  community  is  generally  agreed  that  maintenance  of  healthy  levels

SPECTRA^TM,  vitality  test

of  free  radicals  and  related  oxidants  are  important  for  good  health.  However,

utilization  of  the  “redox  stress  hypothesis”  can  provide  us  with  concrete  nutri-

Correspondence

tional  targets  in  order  to  better  support  and  maintain  “optimal  health.”  Follow-

Boris  V.  Nemzer,  Department  of  Food

ing  this  hypothesis  we  performed  a  crossover,  double-blind,  placebo-controlled,

Science  and  Human  Nutrition,  University  of

single-dose  study  on  the  effects  of  SPECTRA^TM,  a  dietary  supplement,  on  oxida-

Illinois  at  Urbana-Champaign  and

FutureCeuticals  Inc.,  2692  N  State  Rt.  1-17,

tive  stress  markers  (OSM)  in  human  participants  (n  =  22).  The  measurement

Momence,  IL  60954.  Tel:  +1-815-507-1427;

of  OSM  (ex  vivo  intra-  and  extracellular  formation  of  reactive  oxygen  species

Fax:  +1-815-550-0013;

(ROS,  O   ^*

₂    ,  H₂O₂  ,  OH^*)  in  whole  blood,  respiratory  activity  of  blood  cells,  as

E-mail:  bnemzer@futureceuticals.com

well  as  mitochondrial-dependent  ROS  formation,  and  respiratory  activity),  was

performed  using  EPR  spectrometer  nOxyscan,  spin  probe  CMH,  and  oxygen

Funding  Information

label  NOX-15.1,  respectively.  Furthermore,  we  investigated  the  ability  of  SPEC-

This  study  was  supported  by  VDF

TRA^TM  to  modulate  ex  vivo  cellular  inflammatory  responses  induced  by  stimu-

FutureCeuticals,  Inc.

lation  with  exogenous  TNF-a  and  also  followed  changes  in  bioavailable  NO

Received:  15  July  2014;  Revised:  1

concentrations.  In  this  clinical  study,  we  demonstrated  that  administration  of

September  2014;  Accepted:  2  September

SPECTRA^TM   resulted   in   statistically   significant   long-term   inhibition   of   mito-

2014

chondrial  and  cellular  ROS  generation  by  as  much  as  17%  as  well  as  3.5-times

inhibition  in  extracellular  NADPH  system-dependent  generation  of  O   ^*

₂   ,  and

Food  Science  &  Nutrition  2014;  2(6):  828–

nearly  complete  inhibition  of  extracellular  H₂O₂  formation.  This  was  reflected

839

in  more  than  two  times  inhibition  of  ex  vivo  cellular  inflammatory  response

and  also  increases  in  bioavailable  NO  concentration.  For  the  first  time,  we  have

doi: 10.1002/fsn3.178

measured  synergetic,  biological  effects  of  a  natural  supplement  on  changes  in

OSM   and   cellular   metabolic   activity.   The   unique   design   and   activity   of   the

plant-based  natural  supplement,  in  combination  with  the  newly  developed  and

extended  Vitality  test,  demonstrates  the  potential  of  using  dietary  supplements

to  modulate  OSM  and  also  opens  the  door  to  future  research  into  the  use  of

natural  supplements  for  supporting  optimal  health.

Introduction

eficial  and  harmful  effects  caused  by  reactive  oxygen  and

nitrogen  species  (RONS)  is  an  important  aspect  of  living

During  the  last  four  decades,  the  research  community  has

organisms.  Emerging   research  suggests   that  this   balance

generally   agreed   that   a   dynamic,   appropriately   reactive,

may  be  achieved  by  a  mechanism  called  “redox  regula-

and   healthy   balance   between   levels   of   free   radicals   and

tion.”  This  theory  contends  that  the  process  of  redox  reg-

levels   of   related   oxidants   is   important   for   “optimal

ulation  protects  living  organisms  from  various  oxidative

health.”    Imbalances    of  free    radicals,    and    potentially

stresses  and  maintains  “redox  homeostasis”  by  controlling

unhealthy  levels  of  oxidants  versus  antioxidants,  are  col-

the  redox  status  in  vivo  (Droge€     2002).  An  exciting  discov-

lectively  defined  by  the  scientific  community  as  “oxidative

ery  (Sohal  and  Orr  2012)  has  refocused  and  refined  this

and  nitrosative  stress.”  The  delicate  balance  between  ben-

theory  into  the  “redox  stress  hypothesis”  of  aging.  In  this

828

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.  This  is  an  open  access  article  under  the  terms  of

the  Creative  Commons  Attribution  License,  which  permits  use,  distribution  and  reproduction  in  any  medium,

provided  the  original  work  is  properly  cited.

B.  V.  Nemzer  et  al.

New  Insights  on  Effects  of  a  Dietary  Supplement

new  view,  aging  is  the  result  of  functional  losses  that  are

these  weaknesses,  Noxygen  Science  Transfer  &  Diagnostics

primarily  caused  by  a  progressive  pro-oxidizing  shift  in

GmbH   (Elzach,   Germany)   designed   a   bench-top   EPR

the  redox  status  of  cells  and  tissues.  This  in  turn  leads  to

spectrometer  “nOxyscan.”  These  advances  in  instrumenta-

the   overoxidation   of   redox-sensitive   protein   thiols   and

tion  provided  us  with  an  opportunity  to  perform  a  pilot

the   consequent   disruption   of   normal   cellular   functions.

study  to  investigate  the  bioactivity  of  a  nutritional  supple-

The  “redox  stress  hypothesis”  is  based  upon  the  status  of

ment  SPECTRA^TM,  a  formulation  consisting  of  high  anti-

the  redox  buffers  of  cells,  tissue,  and  organisms.  Accord-

oxidant  activity  fruit,  vegetable  concentrates,  and  herbal

ing  to  this  theory,  many  of  the  components  of  our  redox

extracts,   manufactured   by   FutureCeuticals,   Inc.   (Mo-

buffers  are  fundamental  species  of  our  antioxidant  net-

mence,  IL)  and  standardized  to  a  minimum  of  total  anti-

work.  Just  as  it  is  essential  to  maintain  our  pH  buffers,

oxidant  capacity  (TAC)  as  measured  by  a  series  of  oxygen

we   must   also   maintain   a   healthy,   and   appropriately

radical   absorbance   capacity   (ORAC)—based   assays   col-

reduced  oxidative  state  for  our  redox  buffers.  The  effect

lected   under   the   name   ORAC   5.0,   including   ORAC,

of  reactive  oxygen  species  (ROS)  that  may  cause  potential

HORAC,   NORAC,   SORAC,   and   SOAC   (Mullen   et  al.

biological  damage  has  been  termed  “oxidative  stress”  and

2011).

the   effect   of   reactive   nitrogen   species   (RNS)   has   been

termed   “nitrosative   stress”   (Kovacic   and   Jacintho   2001;

Valko  et  al.  2001;  Ridnour  et  al.  2005).  This  model  pre-

Material  and  Methods

sents   us   with   concrete   targets   for   potential   nutritional

intervention  in  order  to  maintain  optimal  health  and  sup-

Natural  SPECTRA^TM  total  ORAC  5.0  blend

port  healthy  aging  and  provides  a  means  to  investigate

This  full-spectrum  antioxidant  activity  product  is  a  pro-

the  direct  effects  of  nutritional  materials  on  biomarkers  of

prietary  combination  of  fruit,  vegetable,  and  herb  extracts

significance   for   healthy   aging   (Broedbaek   et  al.   2013).

and  concentrates:   broccoli   powder  and   broccoli   sprouts

The   healthy   balance   in   the   body   is   comprised   of   four

concentrate,  onion  extract,  tomato  concentrate,  dried  car-

components:

rot,  spinach,  kale  concentrate,  brussel  sprout  concentrate,

1   An  appropriately  modulated,  healthy  flux  of  free  radi-

whole   coffee   fruits   extract,   acerola   extract,   camu   camu

cals  and  oxidants.

powder,  acai  berry  concentrate,  mangosteen  concentrate,

2   An  appropriate  level  of  antioxidants  coupled  with  fully

green  tea  extract,  apple  extract,  turmeric  concentrate,  gar-

functional  systems  to  recycle  these  antioxidants.

lic,    basil   concentrate,   oregano,   cinnamon   concentrate,

3   Robust  nutritional  support  that  helps  to  maintain  opti-

elderberry    concentrate,    blackcurrant    extract,    blueberry

mal  levels  of  supportive  antioxidants  and  cofactors.

extract,  sweet  cherry  powder,  blackberry  powder,  choke-

4   Fully    functioning    enzyme    systems    that    repair    or

berry,  raspberry  powder,  and  bilberry  extract.  The  ORAC

recycle   and   replace   damaged   cellular   materials,   for

5.0  assay  measures  antioxidant  activities  against  hydroxyl,

example,  DNA,  RNA,  enzymes,  proteins,  and  endoge-

peroxyl,   peroxynitrite,   singlet   oxygen,   and   superoxide

nous  redox  molecules  (glutathione,  vitamin  C,  vitamin

anion.   SPECTRA^TM   is   standardized   to   minimum   40,000

E  etc.).

lmol  trolox  equivalent  (TE)  per  gram  of  ORAC  5.0  assay

Electron  paramagnetic  resonance  (EPR)  spectroscopy  is

(Nemzer et  al. 2014).

a  technique  that  is  recognized  in  the  scientific  community

as  a  gold  standard  methodology  (Dikalov  et  al.  2007)  for

direct  observation  ex  vivo  or  in  vivo  of  the  formation  of

Sample  preparation  for  antioxidant

RONS.  Recently,  we  also  published  observation  of  imag-

measurements

ing  of  ROS  (Ji  et  al.  2012)  performed  in  living  animals.  It

The   sample   preparation   was   conducted   following   the

has  been  shown  that  for  studies  of  intact  tissues  and  cells,

previous   protocol   (Mullen   et  al.   2011;   Nemzer   et  al.

the   cyclic   hydroxylamine   spin   probes   offer   a   distinct

2014).     Approximately     20  mg     of     SPECTRA^TM     was

advantage  over  nitrone  spin  traps  to  measure  the  produc-

extracted   with   20  mL   of   ethanol/water   (70:30   v/v)   for

tion  of  superoxide  anion  and  other  radicals  due  to  the

1  h   at   room   temperature   on   an   orbital   shaker.   After

fact  that  they  yield  very  stable  products  and  strong  EPR

centrifugation   at   4164g,   the   supernatant   of   the   extract

signals.   Cyclic   hydroxylamine   spin   probes   such   as   1-

was  subjected  to  the  TAC  assay.  The  TAC  includes  the

hydroxy-3-carboxy-pyrrolidine  and  1-hydroxy-3-methoxy-

determination   of   radical   scavenging   capacities   against

carbonyl-2.2.5.5-tetramethylpyrrolidine   are   very   effective

five   free   radicals,   namely,   peroxyl,   hydroxyl,   peroxyni-

scavengers   of   superoxide   radicals   (Dikalov   et  al.   1998;

trite,  superoxide  anions,  and  singlet  oxygen  radicals.  All

Fink   et  al.   2000;   Dikalov   and   Fink   2005).   The   major

results   were   expressed   as   Trolox   equivalent   per   gram

weaknesses   of   EPR   are:   (1)   it   is   expensive   and   (2)   it

(lmol  TE/g)  and  the  TAC  was  the  sum  of  the  five  indi-

requires   a   substantial   amount   of   space.   To   overcome

vidual  results.

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

829

New  Insights  on  Effects  of  a  Dietary  Supplement

B.  V.  Nemzer  et  al.

Peroxyl  radical  scavenging  capacity  (ORAC

Superoxide  anion  scavenging  assay  (SORAC

assay)

assay)

The  ORAC  assay  was  measured  according  to  a  previous

The  SORAC  assay  was  conducted  following  the  previously

report  by  Ou  et  al.  (2002)  and  Huang  et  al.  (2002)  with

described  method  by  Zhang  et  al.  (2009).  Hydroethidine

modification.   The   FL600   microplate   fluorescence   reader

(HE)  was  used  to  measure  O   ^*

₂     scavenging  capacity.  The

(Bio-Tek  Instruments,  Inc.,  Winooski,  VT)  was  used  with

mixture   of   xanthine   and   xanthine   oxidase   was   used   to

an   excitation   wavelength   of   485   (20  nm)   and   emission

generate   O   ^*

₂     radicals.   Nonfluorescent   HE   was   oxidized

wavelength  of  530  (25  nm).  About  2,20-Azobis(2-amidi-

by  O   ^*

₂    to  form  a  species  of  unknown  structure  that  emits

nopropane)  dihydrochloride  (AAPH)  was  used  to  generate

fluorescence   signal   at   586  nm.   Addition   of   superoxide

peroxyl  radical.  Fluorescein  (FL)  was  used  as  a  fluorescent

dismutase  (SOD)  inhibits  the  HE  oxidation.

probe  to  indicate  the  extent  of  damage  from  its  reaction

with  the  peroxyl  radical.  The  antioxidant  effect  was  mea-

sured  by  comparing  the  fluorescence  time/intensity  area

Singlet  oxygen  scavenging  assay  (SOAC

under  the  curve  of  the  sample  to  that  of  a  control  with  no

assay)

antioxidant.  Trolox  was  prepared  as  the  standard  solution.

The  SOAC  assay  was  modified  based  on  the  previously

Fluorescence  was  measured  every  min  for  up  to  35  min.

described  method  by  Zhao  et  al.  (2003).  HE  was  used  as

a  probe  to  measure  singlet  oxygen.  The  mixture  of  H₂O₂

Hydroxyl  radical  scavenging  capacity

and  MoO   ^2*

₄      was  used  to  generate  singlet  oxygen.  About

(HORAC  assay)

40  lmol/L  solution  of  HE,  2.635  mmol/L  Na2MoO4,  and

13.125  mmol/L  H₂O₂  working  solutions  were  prepared  in

The  assay  was  modified  according  to  a  report  by  Ou  et  al.

N,N-dimethylacetamide   (DMA).   HE   solution   (125  lL)

(2002).  Fluorescein  (FL)  was  used  as  a  fluorescent  probe.

was   added   to   a   well   followed   by   addition   of   25  l   L   of

The   antioxidant   effect   was   measured   by   comparing   the

2.635  mmol/L   Na₂MoO₄   and   25  lL   of   13.125  mmol/L

fluorescence   time/intensity   area   under   the   curve   of   the

H₂O₂,     respectively.     Singlet     oxygen     scavenging     was

sample  to  that  of  a  control  with  no  antioxidant.  Trolox

measured   in   a   fluorescence   reader   with   an   excitation

was  used  as  the  standard  for  calibration.

wavelength    of    530  nm    and    emission    wavelength    of

620  nm.

Peroxynitrite  scavenging  capacity  (NORAC

assay)

Study  design

Peroxynitrite   (ONOO_*)   scavenging   values   were   deter-

Twenty-two  healthy  participants  (13  females,  nine  males)

mined   by   monitoring   the   oxidation   of   DHR-123   based

with  a  mean  age  of  41  years  (range  21–59),  and  a  mean

on  a  protocol  by  Chung  et  al.  (2001).  A  stock  solution  of

body  weight  of  77  kg  (range  62–92)  entered  this  study.

DHR-123  (5  mM)  was  prepared  in  dimethylformamide,

The  study  was  carried  out  according  to  the  recommenda-

purged  with  nitrogen,  and  stored  at  *80°C.  A  working

tions  for  clinical  trials  in  humans,  declaration  of  Helsinki.

solution  of  DHR-123  (final  concentration,  fc,  5  lmol/L)

All  subjects  were  in  generally  good  health  as  confirmed  by

diluted  from  the  stock  solution  was  placed  on  ice  in  the

physical   examination   and   laboratory   tests   investigating

dark  before  the  experiment  started.  The  reaction  buffer

lipid,  carbohydrate,  and  inflammatory  profiles  (see  sum-

consisting   of   90  mmol/L   sodium   chloride,   50  mmol/L

mary  Table  in  section  Results).  The  study  was  performed

sodium   phosphate   (pH   7.4),   and   5  mmol/L   potassium

in  double-blind,  single-dose,  crossover,  placebo-controlled

chloride   with   100  lmol/L   (fc)   diethylenetriaminepenta-

fashion.  Generally  accepted  contraindications  to  physical

acetic  acid  (DTPA)  was  purged  with  nitrogen  and  placed

exercise;  previously  diagnosed  type  1  and  2  diabetes;  fast-

on  ice  before  use.  ONOO^*  scavenging  was  measured  in  a

ing  glucose  >110;  C-reactive  proteins  >3;  liver  and  kidney

fluorescence  reader  with  an  excitation  wavelength  of  485

impairments;   psychiatric   disorders,   other   disorders   of

(20  nm)  and  emission  wavelength  of  530  (25  nm).  Five

acute   or    chronic   nature   (gastrointestinal,    pulmonary,

minutes  after  treating  with  or  without  SIN-1  (fc  10  lmol/

renal,   cardiac,   neurological,    or   psychiatric    disorders),

L)  or  authentic  ONOO^*  (fc  10  lmol/L)  in  0.3  N  sodium

known   allergies   to   foods   or   their   ingredients,   use   of

hydroxide,   the   background   and   final   fluorescent   signals

weight   reducing   preparations   or   appetite   suppressants,

were   measured.   Oxidation   of   DHR-123   increased   by

b-blockers,  ACE  inhibitors,  statins,  insulin,  NSAID,  pain

decomposition   of   SIN-1   gradually,   whereas   authentic

medications,  participation  in  a  clinical  study  within  the

ONOO^*  rapidly  oxidized  DHR-123  with  its  final  fluores-

last  30  days  prior  to  the  beginning  of  this  study  or  during

cent  signal  being  stable  over  time.

this     study     as     well     as     intake     of     vitamins/dietary

830

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

B.  V.  Nemzer  et  al.

New  Insights  on  Effects  of  a  Dietary  Supplement

supplements  2  weeks  prior  to  the  start  or  during  the  trial

were  exclusion  criteria  for  participation  in  this  study.

Cellular  TNF-a  response  assay

In  addition  to  H₂O₂  detection,  Noxygen  Science  Transfer

Study  protocol

&  Diagnostics  GmbH  has  developed  and  validated  an  ex

vivo  cellular  inflammatory  response  assay.  Application  of

All   22   participants   were   separated   into   the   two   groups

this  assay  provides  results  describing  changes  in  ROS  gen-

and  were  matched  for  age  and  gender  to  the  best  practi-

eration   by   blood   cells   after   stimulation   with   external

cal   and/or   possible   degree.   They   received   an   emotional

(nonendogenous)   TNF-a.   TNF-a   had   previously   been

and   general   health   evaluation   questionnaire.   At   day   0

reported  to  be  a  key  factor  of  inflammation  (Feuerstein

(requirement),  blood  was  drawn  after  12  h  of  fasting  per-

et  al.  1994).  The  assay  was  performed  using  blood  sam-

iod  for  performance  of  laboratory  tests  and  for  analysis

ples   from   each   of   the   study   subjects   (20  lL).   Samples

of  glucose  as  well  as  extended  Vitality  test.  Standardized

were  not  analyzed  for  changes  in  TNF-a  concentration,

breakfast   (one   bread   roll   with   a   glass   of   water)   was

but   rather   for   changes   in   downstream   effects   resulting

served  at  day  0.  Standardized  breakfast  was  also  served

from   exogenous   TNF-a   challenge.   Samples   were   mixed

on  day  1  and  day  2  along  with  placebo  or  SPECTRA^TM

with     20  lL     solution     of     human     TNF-a     (#T6674;

100  mg   capsule,   respectively.   Capillary   blood   was   col-

Sigma-Aldrich,    St.    Louis,    MO)    and    spin    probe    1-

lected   for   performance   of   extended   Vitality   test   at   the

Hydroxy-4-phosphono-oxy-2,2,6,6-tetramethyl-piperidine

time  0,  and  also  immediately  prior  to  the  standardized

(PPH,  Noxygen  GmbH,  #  NOX-4.1)  solved  in  Krebs  He-

breakfast  on  day  0,  and  prior  to  the  standardized  break-

pes   buffer   (20  mmol/L,   pH   7.4).   Final   concentrations

fast  and  treatment  on  days  1  and  2,  as  well  as  after  1,  2,

were  40  ng/mL  TNF-a  and  500  lmol/L  PPH,  respectively.

and    3  h    after    capsule    administration.    Cardiovascular

The  mixture  filled  in  a  teflon,  oxygen  permeable  capillary

parameters   and   blood   pressure   were   recorded   using   a

tube   was   placed   in   the   resonator   of   EPR   spectrometer

Dinamap    XL    (Johnson    &    Johnson    Medical    GmbH,

(nOxyscan,   Noxygen   GmbH)   equipped  with   a   tempera-

Norderstedt,  Germany).

ture  and  gas  controller  BIO-III  (TGC,  Noxygen  GmbH)

for  monitoring  of  EPR  signal  within  60  min.

Detection  of  ROS  in  human  blood

The   TGC   setting   was   as   follows:   temperature   37°C,

10  mmHg  pressure,  and  4%  oxygen  concentration.  EPR

The   extended   “Vitality”   test   that   we   employed   in   this

settings:   center   field:   3472  G;   sweep   width:   60  G;   static

pilot  study  was  developed  by  Noxygen  Science  Transfer  &

field:   3458  G;   frequency:   9.76  GHz;   attenuation:   4.0  dB;

Diagnostics  GmbH  (Elzach,  Germany).  The  principle  of

microwave   power:   20  mW;   gain:   1  9  10³;   modulation

the  method  is  based  upon  the  monitoring  of  ESR  signal

frequency:  86.00  kHz;  modulation  amplitude:  2.2  G;  time

of   spin   probe   (CMH,   200  lmol/L)   oxidation   that   has

constant:  40.96  msec;  conversion  time:  10.24  msec;  sweep

been  mixed  with  freshly  drawn  blood.  During  the  pro-

time:  5.24  sec;  number  of  scans:  10;  number  of  points:

cess,  the  blood  cells  stand  under  the  original  physiological

46;   experimental   time:   60  min.   A   kinetic   curve   slope

environment   (t  =  37°C,   pO₂  =  110  mmHg)   and   remain

(EPR  signal  amplitude  vs.  time)  for  the  60  min  was  inte-

surrounded   by   blood   plasma   that   releases   biologically

grated   and   expressed   as   formation   of   ROS   lmol/L   per

available  ROS  that  interacts  in  intracellular  and  extracel-

min.

lular   space   with   CMH   to   form   a   stable   radical   CM°

(Bassenge   et  al.   1998;   Fink   et  al.   2000;   Mrakic-Sposta

et  al.   2012).   Addition   of   oxygen-sensitive   label   (NOX-

Bioavailable  NO  concentration  assay

15.1–5  lmol/L)  to  the  blood  sample  allows  us  to  monitor

Analysis    of    circulating    NO    concentration    in    human

oxygen  concentrations  and  cellular  as  well  as  mitochon-

blood,  second  key  signaling  molecule  of  vascular  physiol-

drial  oxygen  consumption  (Bobko  et  al.  2009;  Komarov

ogy  and  an  in  vivo  antioxidant,  was  performed  in  previ-

et  al.   2012).   Bench-top   EPR   spectrometer   “nOxyscan”

ous   studies   (Dikalov   and   Fink   2005;   Pisaneschi   et  al.

was    used    with    the    following    settings:    center    field:

2012).

g  =  2.011,    sweep    width:    60  G,    frequency:    9.76  GHz,

power:   20  mW,   gain:   1  9  10³,   modulation   amplitude:

1.2  G,  sweep  time:  5.24  sec,  number  of  scans:  10,  number

Chemicals

of  points:  512,  total  experimental  time:  5  min.  Calibra-

The  spin  probes  1-hydroxy-3-methoxycarbonyl-2.2.5.5-te-

tion  of  EPR  signal  was  performed  using  calibration  solu-

tramethylpyrrolidine    (CMH),    1-hydroxy-4-phosphono-

tion  with  standard  concentration  of  CM°  (10  lmol/L)  or

oxy-2.2.6,  6-tetramethylpiperidine  (PPH),  the  metal  chela-

oxygen  label  NOX-063  (5  lmol/L)  filled  in  to  50  lL  glass

tors    defferoxamine    (DF),    and    diethyldithiocarbamate

capillary.

(DETC).  Krebs–Hepes  buffer  (KHB),  and  the  oxygen  label

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

831

New  Insights  on  Effects  of  a  Dietary  Supplement

B.  V.  Nemzer  et  al.

NOX-15.1  were  obtained  from  Noxygen  Science  Transfer

pressure   123  ♥  4  mmHg,   did   not   exceed   the   values   of

&   Diagnostics,   CuZn   superoxide   dismutase   (SOD)   was

healthy   persons.   Mean   values   of   laboratory   parameters

obtained  from  Sigma–Aldrich  (St.  Louis,  MO).  All  other

from  all  study  participants  at  day  “0”  are  depicted  below

chemicals  and  reagents  used  were  of  analytical  grade  and

in  the  Table  2.

were   purchased   from   Sigma–Aldrich   unless   otherwise

Detection  of  ROS  was  performed  with  extended  Vitality

specified.

test  protocol  which  allows  analysis  for:

1  “total”—(extracellular/intracellular)  generation  of  ROS/

Statistical  analysis

cellular  oxygen  consumption;

2  “extracellular”—generation  of  O   ^*

₂   /cellular  oxygen  con-

All  statistical  analyses  were  performed  with  the  SigmaPlot

sumption  by  addition  of  SOD  (50  U/mL);

11.0  (Chicago,  IL).  P  value  was  calculated  using  one-way

3  “extracellular”—generation    of    H₂O₂/cellular    oxygen

ANOVA   with   Holm-Sidak   method,   and   P  <  0.05   was

consumption  by  addition  of  catalase  (50  U/mL);

considered  as  statistically  significant.

4  “mitochondrial”—generation    of    mitochondrial    O   ^*

2   /

mitochondrial   oxygen   consumption   by   addition   of

Results

Antimycin  A  (10  lmol/L).

As    Figure  1    immediately    below    demonstrates,    at

The   biological   active   phytochemical   compounds,   vita-

60  min  after  100  mg  single-dose  SPECTRA^TM  administra-

mins,  minerals,  and  TAC  per  100  mg  serving  size  of  the

tion  we  observed  a  significant  decrease  in  total  generation

SPECTRA^TM  are  summarized  in  Table  1.

of  ROS/metabolic  activity  of  blood  cells  in  human  volun-

According   to   the   study   protocol,   we   recruited   nine

teers.  These  effects  persisted  for  another  2  h  followed  by  a

male  and  13  female  generally  healthy  participants  between

significant  trend  toward  baseline  3  h  after  administration.

the   ages   of   21   and   59  years   and   with   body   weight   of

77.4  ♥  10.2  kg   and   BMI   of   26.3  ♥  2.2   (typical   for   an

industrial  country  like  Germany).  Cardiovascular  parame-

Table  2.   Laboratory   parameters—hemogram,   metabolic-,   inflamma-

ters    such    as    heart    rate    67  ♥  8  bit/min,    and    blood

tory-,  and  lipid-profile.

Inflammatory

Table  1.   Biological   active   compounds   and   TAC   for   SPECTRA^TM   per

Hemogram

parameters

serving  size  100  mg.

Hemoglobin

14.1  ♥  1.3

Leukocytes  (tsd/lL)

5.7  ♥  1.6

Units

Result

(g/dL)

Erythrocytes

4.7  ♥  0.4

Neutrophils

3.0  ♥  1.0

Phytochemical  compound

(mio/lL)

absolute  (tsd/lL)

Glucosinolates

mg

0.1

Hematocrit  (%)

40.9  ♥  3.6

Neutrophils  (%)

52.9  ♥  7.5

Quercetin

mg

10.8

MCH  (pg)

30.1  ♥  1.2

Eosinophil

0.15  ♥  0.10

Lycopene

l

g

43

absolute  (tsd/lL)

Chlorogenic  acids

mg

6.7

MCV  (fL)

87.1  ♥  3.0

Eosinophil  (%)

2.5  ♥  1.2

Vitamin  C

mg

1.2

MCHC  (g/dL)

34.5  ♥  0.8

Lymphocytes

2.0  ♥  0.6

Catechins

mg

10.3

absolute  (tsd/lL)

Allicin

l

g

10Immature

0.3  ♥  0.1

Lymphocytes  (%)

35.0  ♥  6.9

Alliin

l

g

20granulocytes

Anthocyanins

mg

0.5

(%)

Vitamin  E

lg

13.4

Thrombocytes

218.0  ♥  52.2      CRP  (mg/L)

2.7  ♥  2.3

Beta-carotene

lg

36.4

(tsd/lL)

Folate

lg

1.2

Vitamin  K

lg

3.1

Calcium

mg

0.77

Metabolic-profile

Lipid-profile

Magnesium

mg

0.53

Insulin  (mU/L)

10.6  ♥  6.5

Triglyceride/neutral      124.8  ♥  72.3

Potassium

mg

4.3

fat  (mg/dL)

Activity  against  individual  radicals

Glucose  in

90.7  ♥  10.2      Cholesterol

217.7  ♥  54.7

Activity  against  peroxyl  radicals

lmol  TE

1070

serum  (mg/dL)

Activity  against  hydroxyl  radicals

lmol  TE

1511

Glucose/whole

73.2  ♥  9.7

HDL  cholesterol

51.7  ♥  22.6

Activity  against  peroxynitrite

lmol  TE

110

blood  (mg/dL)

(mg/dL)

Activity  against  superoxide  anion

lmol  TE

1337

Homa-index

2.3  ♥  1.7

VLDL  cholesterol

24.2  ♥  14.4

Activity  against  singlet  oxygen

lmol  TE

417

(mg/dL)

(mg/dL)

Total  activity

lmol  TE

4445

LDL  cholesterol

141.8  ♥  40.1

(mg/dL)

TAC,  total  antioxidant  capacity.

832

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

B.  V.  Nemzer  et  al.

New  Insights  on  Effects  of  a  Dietary  Supplement

Figure  1.   Effect   of   SPECTRATM

Figure  2.   Effect   of   SPECTRA^TM   on   cellular   oxygen   consumption   of

on   cellular   “total”   ROS  generation/

blood   cells   collected   from   human   volunteers.   Oxygen   consumption

metabolic  activity  in  human  participants.  Detection  of  reactive  oxygen

analysis  was  performed  with  the  same  blood  samples  using  spin  label

species   was   performed   using   spin   probe   CMH   (200  lmol/L)   and

NOX-15.1  (5  lmol/L)  and  bench-top  EPR  spectrometer  nOxyscan  in  22

bench-top  EPR  spectrometer  nOxyscan  in  22  generally  healthy,  fasted

generally  healthy,  fasted  (minimum  12  h)  participants.  Blue  columns

(minimum  12  h)  participants.  Blue  columns  (control):  prior  to  and  60,

(control):   prior   to   and   60,   120,   180  min   after   consumption   of

120,   180  min   after   consumption   of   standard   breakfast   (bread   roll

standard   breakfast   (bread   roll   with   glass   of   water);   red   columns

with   glass   of   water);   red   columns   (placebo):   after   consumption   of

(placebo):   after   consumption   of   standard   breakfast   and   placebo

standard    breakfast    and    placebo    capsule;    and    green    columns

(SPECTRATM):  after  consumption  of  standard  breakfast  and  SPECTRATM

capsule;   and   Green   columns   (SPECTRA^TM):   after   consumption   of

standard   breakfast   and   SPECTRA^TM   capsule.   Observation   of   oxygen

capsule.  For  EPR  settings,  please  refer  to  material  and  methods.  Data

concentration     changes     was     possible     due     to     optimization     of

are  mean  (n  =  22)  ♥  SEM,  *P  <  0.05  versus  value  “before.”  CMH,  1-

modulation   amplitude   1  G,   which   makes   it   possible   to   follow   EPR

hydroxy-3-methoxycarbonyl-2.2.5.5-tetramethylpyrrolidine; EPR, electron

amplitude  of  separately  appearing  CM-radical  and  oxygen  label  EPR

paramagnetic resonance.

lines.    Data    are    mean  ♥  SEM    (n  =  22),    *P  <  0.05    versus    value

“before.”  EPR,  electron  paramagnetic  resonance.

Under  pathological  conditions,  increased  oxidative  stress

itself   can   alter   oxygen   levels.   Changes   in   oxygen   levels

may  subsequently  affect  mitochondrial  oxygen  consump-

tion.  In  order  to  circumvent  this  problem,  EPR  method

^0.20

Control    Placebo     SpectraTM

has  been  developed  for  measuring  superoxide  and  oxygen

consumption    in    mitochondrial    respiratory    complexes

_0.15

using   the   oxygen   labels   such   as   NOX-15.1   (Mariappan

et  al.  2009).  By  using  a  gas  controller  and  nontoxic  spin

label   NOX-15.1,   we   were   able   to   measure   oxygen   con-

^0.10

sumption   simultaneously   during   detection  of  ROS.   The

merit   of   this   method   is   that   it   allows   us   to   measure

0.05

superoxide  production  and  oxygen  consumption  in  paral-

lel  using  the  same  incubation  medium  and  substrate  con-

centration    in    each    blood    sample.    Simultaneously    to

^0.00

Before

1 h after

2 h after

3 h after

changes   in   levels   of   ROS,   we   observed   a   significant

increase  in  cellular  oxygen  consumption  (Fig.  2)  as  well

Figure  3.   Influence    of    SPECTRA^TM     on    “mitochondrial”    oxygen

as  mitochondrial  oxygen  consumption  (Fig.  3)  after  1  h

consumption    of    blood    cells    collected    from    human    participants.

of   SPECTRA_TM

Mitochondrial  oxygen  consumption  was  performed  in  the  same  blood

ingestion.   These   levels   continued   to   be

samples   using   spin   label   NOX-15.1   (5  lmol/L)   and   bench-top   EPR

slightly  elevated  during  the  next  2  h.  These  findings  sug-

spectrometer   nOxyscan   after   addition   of   Antimycin   A   (10  lmol/L)

gest  optimization  of  redox  balances  and  optimization  of

prior   to   and   at   60,   120,   180  min   after   consumption   of   standard

respiratory    activity    of    mitochondria    factors    that    are

breakfast  (bread  roll  with  glass  of  water).  Blue  columns  (control):  prior

important  for  healthy  aging  (Dikalova  et  al.  2010;  Naza-

to   and   60,   120,   180  min   after   consumption   of   standard   breakfast

rewicz  et  al.  2013).  The  same  pattern  as  cellular  ROS  gen-

(bread    roll    with    glass    of    water);    red    columns    (placebo):    after

eration  was  recognized  in  the  mitochondrial  generation  of

consumption  of  standard  breakfast  and  placebo  capsule;  and  green

ROS  after  SPECTRATM

columns  (SPECTRA^TM):  after  consumption  of  standard  breakfast  and

administration.  It  decreased  signif-

SPECTRA^TM    capsule.    The    values    of    oxygen    consumption    were

icantly  after  1  h  and  continued  to  decrease  for  the  next

calculated  as  delta  value  between  “total”  and  “Antimycin  A”  sample.

2  h  (Fig.  4).  Although  generation  of  ROS  in  the  control

Data  are  mean  ♥  SEM,  P  <  0.05  versus  value  “before.”  EPR,  electron

and    placebo    group    showed    nonsignificant    tendencies

paramagnetic  resonance.

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

833

New  Insights  on  Effects  of  a  Dietary  Supplement

B.  V.  Nemzer  et  al.

0.60

Control     Placebo     SpectraTM

1.25

Control     Placebo     SpectraTM

0.50

1.10

0.40

0.95

0.30

0.20  

0.80

Before

1 h after

2 h after

3 h after

Before

1 h after

2 h after

3 h after

Figure  4.   Effect  of  SPECTRA^TM   on  “mitochondrial”  ROS  generation

Figure  5.   Influence    of    SPECTRA^TM    on    “extracellular”    superoxide

in    blood    cells    collected    from    human    volunteers.    Detection    of

(O   ^*

₂   )   formation   in   blood   cells   collected   from   human   volunteers.

mitochondrial  ROS  generation  was  performed  using  spin  probe  CMH

Superoxide   formation   was   analyzed   in   human   blood   using   EPR

(200  lmol/L)    and    bench-top    EPR    spectrometer    nOxyscan    after

spectrometer  nOxyscan,  spin  probe  CMH  (200  lmol/L)  after  addition

addition  of  Antimycin  A  (10  lmol/L)  in  the  blood  samples  taken  prior

of  SOD  (50  U/mL)  in  the  blood  samples  taken  prior  to  and  at  60,  120,

to  and  at  60,  120,  180  min  after  consumption  of  standard  breakfast

180  min   after   consumption   of   standard   breakfast   (bread   roll   with

(bread  roll  with  glass  of  water).  Blue  columns  (control):  prior  to  and

glass  of  water).  Blue  columns  (control):  prior  to  and  60,  120,  180  min

60,  120,  180  min  after  consumption  of  standard  breakfast  (bread  roll

after   consumption   of   standard   breakfast   (bread   roll   with   glass   of

with   glass   of   water);   red   columns   (placebo):   after   consumption   of

water);    Red    columns    (placebo):    after    consumption    of    standard

standard    breakfast    and    placebo    capsule;    and    green    columns

breakfast   and   placebo   capsule;   and   Green   columns   (SPECTRA^TM):

(SPECTRA^TM):     after     consumption     of     standard     breakfast     and

after  consumption  of  standard  breakfast  and  SPECTRA^TM  capsule.  The

SPECTRA^TM   capsule.   The   values   of   ROS   generation   were   calculated

values of superoxide generation were calculated as delta value between

as   delta   value   between   “total”   and   “Antimycin   A”   sample.   Data

“total”  and  “SOD”  sample.  Data  are  mean  ♥  SEM  (n  =  22),  *P  <  0.05

are     mean  ♥  SEM     (n  =  22),     *P  <  0.05     versus     value     “before.”

versus   value   “before.”   CMH,   1-hydroxy-3-methoxycarbonyl-2.2.5.5-

CMH,  1-hydroxy-3-methoxycarbonyl-2.2.5.5-tetramethylpyrrolidine;  EPR,

tetramethylpyrrolidine; EPR, electron paramagnetic resonance.

elec-tron  paramagnetic  resonance;  ROS,  reactive  oxygen  species.

toward   depletion   of   ROS   formation,   this   is   consistent

with  previous  observations  collected  from  nonathletically

^1.40

Control     Placebo     SpectraTM

trained  participants  (B.  Fink,  unpubl.  data).  In  addition

to   the   decrease   in   “total”   ROS   generation   and   oxygen

consumption,   the   administration   of   a   single   dose   of

^1.20

SPECTRA^TM  significantly  inhibited  generation  of  extracel-

lular  NADPH  oxidase-dependent  superoxide  (O   ^*

₂   ,  Fig.  5)

and    peroxidase-dependent    hydrogen    peroxide    (H₂O₂,

_1.00

Fig.  6).   The   possibility   for   such   regulatory   effects   on

NADPH-oxidases  activity  suggests  that  SPECTRA^TM   may

help  support  cardiovascular  health  in  healthy  aged  sub-

jects  (Wyche  et  al.  2004;  Dikalov  et  al.  2012).  Compared

^0.80

Before

1 h after

2 h after

3 h after

to  the  “total”  ROS  generation,  the  values  of  both  super-

oxide  and  hydrogen  peroxide  generation  in  the  control  as

Figure  6.   Influence  of  SPECTRA^TM  on  extracellular  H2O2  formation  in

well  as  in  the  placebo  group  showed  nonsignificant  deple-

blood   cells   collected   from   human   volunteers.   H²O²   formation   was

analyzed  in  human  blood  using  EPR  spectrometer  nOxyscan,  spin  probe

tion  tendencies  over  the  observation  period.

CMH  (200  lmol/L)  after  addition  of  catalase  (50  U/mL)  in  the  blood

In   order   to   provide   more   robust   scientific  proof   on

samples  taken  prior  to  and  at  60,  120,  180  min  after  consumption  of

inhibition   of   peroxidase   activities,   which   are   linked   to

standard   breakfast   (bread   roll   with   glass   of   water).   Blue   columns

inflammatory  response,  we  performed  analysis  of  ex  vivo

(control):  prior  to  and  60,  120,  180  min  after  consumption  of  standard

changes   in   cellular   ROS   (almost   hydrogen   peroxide,

breakfast  (bread  roll  with  glass  of  water);  red  columns  (placebo):  after

H₂O₂)   formation   after   a   challenge   by   stimulation   with

consumption  of  standard  breakfast  and  placebo  capsule;  and  green

externally  introduced  TNFa.  TNFa  is  recognized  as  one

columns  (SPECTRA^TM):  after  consumption  of  standard  breakfast  and

SPECTRA^TM  capsule.  The  values  of  H

of   the   key   mediators   of   inflammation   that   is   directly

²O²  generation  were  calculated  as

delta    value    between    “total”    and    “catalase”    sample.    Data    are

linked   to   ROS   generation   and   apoptosis.   We   demon-

mean  ♥  SEM   (n  =  22),   *P  <  0.05   versus   value   “before.”   CMH,   1-

strated   significant   inhibition   of   cellular   response   after

hydroxy-3-methoxycarbonyl-2.2.5.5-tetramethylpyrrolidine; EPR, electron

administration  of  SPECTRA^TM   (Fig.  7).  Another  example

paramagnetic resonance.

834

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

B.  V.  Nemzer  et  al.

New  Insights  on  Effects  of  a  Dietary  Supplement

0.12

30.00

Control      Placebo      SpectraTM

Placebo

Spectra-TM

0.09

20.00

0.06

10.00

0.03  

0.00

Figure  7.   Inhibition      of      TNFa-induced      “cellular      inflammatory

Figure  8.   Influence  of  SPECTRA^TM  on  circulating  NO  concentration  in

response”   after   single   dose   of   SPECTRA^TM   in   blood   cells   collected

blood   of   human   volunteers.   Bioavailable   NO   level   was   analyzed   in

from  human  volunteers.  This  testing  measured  response  of  blood  cells

human   blood   according   to   material    and   methods   described   by

after   chemical   insult   by   stimulation   with   40  ng/mL   of   exogenous

protocol    detection    of    circulating    NOHb    concentration    in    blood

human  TNFa.  As  expected,  this  stimulation  subsequently  induced  ROS

samples.   Green   column   (placebo):   180  min   after   consumption   of

(H₂O₂)  formation.   Levels   of   H₂O₂   in  blood   samples   from   the   study

standard     breakfast     and     placebo     capsule;     and     Blue     column

subjects     were     analyzed     using     EPR     spectrometer     nOxyscan,

(SPECTRA^TM):  180  min  after  consumption  of  standard  breakfast  and

nonmembrane  permeable  spin  probe  PPH  (500  lmol/L).  Blue  column

SPECTRA^TM    capsule.    Data    are    mean  ♥  SEM    (n  =  22),    *P  <  0.01

(control):  180  min  after  consumption  of  standard  breakfast  (bread  roll

versus  placebo.

with     glass     of     water);     red     column     (placebo):     180  min     after

consumption  of  standard  breakfast  and  placebo  capsule;  and  Green

column   (SPECTRA^TM):   180  minutes   after   consumption   of   standard

The   previously   reported   cardiosupportive   action   of

breakfast  and  SPECTRA^TM   capsule.  The  accumulation  of  oxidized  PP-

quercetin,  one  of  the  major  active  compounds  in  SPEC-

radical   was   observed   during   1  h   incubation   at   37C   and   40  mmHg

TRA^TM,  was  described  as  a  compound  attenuating  oxida-

oxygen   partial   pressure.   Data   are   mean  ♥  SEM   (n  =  22),   P  <  0.01

tive    stress    by    depletion    of    serum    and    tissue    MDA

versus  placebo.  Baseline  and  posttreatment  levels  of  TNF-a  were  not

measured.  ROS,  reactive  oxygen  species.

(malondialdehyde)  formation  and  moderate  incrementa-

tion  of  antioxidant  reserves  (Annapurna  et  al.  2009).  Such

cardiosupportive  effects  may  be  caused  by  inhibition  of

of   the   multifaceted   effect   of   SPECTRA^TM,   especially   in

mitochondrial  ROS  generation,  which  have  been  demon-

terms  of  potential  for  support  of  cardiovascular  health,  is

strated  in  this  clinical  study  (Fig.  4).  Possible  explanation

normalization  of  “nitrosative  stress,”  which  may  be  evalu-

of  that  mechanism  was  proposed  by  Chen  et  al.  (2013)

ated  based  on  analysis  of  bioavailable  circulating  NO  con-

wherein  it  was  reported  that  inhibition  of  doxorubicin-

centration  in  vivo  (Pisaneschi  et  al.  2012).  Detection  of

dependent   cardiomyocyte   oxidative   damage   was   caused

circulating  NO  concentration  in  whole  blood  of  partici-

by  uncoupling  of  mitochondria.  Another  cardiosupportive

pants  after  administration  of  SPECTRA^TM  showed  signifi-

mechanism   of   quercetin   has   been   suggested   in   recent

cant  increase  in  the  level  of  NO  (Fig.  8).

studies  that  reported  the  inhibitory  effects  of  quercetin  on

inducible  NO  synthase  over  TNF-a  and  on  inflammatory

Discussion

gene  expression  (Wadsworth  and  Koop  2001;  Wadsworth

et  al.  2001;  Boesch-Saadatmandi  et  al.  2011).  It  has  been

In  this  pilot  study,  we  delivered  evidence  expanding  on

shown   that   these   effects   as   well   as   predisposition   of

the  introduction  of  the  free  radical  theory  of  aging  pro-

inflammatory  cascade  components  begin  with  phenotypic

posed  by  Harman  (1956).  Thereafter,  in  1969,  the  discov-

differences   in   redox-enzymes   such   as   NADPH   oxidase

ery  of  the  enzyme  superoxide  dismutase  (SOD),  provided

(Wyche  et  al.  2004),  GSH  reductase  (Bailey  et  al.  2014),

further  convincing  evidence  suggesting  the  importance  of

catalase   (Suvorava   et  al.   2005),   heme   oxygenase   (Seo

healthy  levels  of  free  radicals  in  living  systems  (McCord

et  al.  2013)  and  play  an  important  role  in  inflammatory

and  Fridovich  1969),  and  the  possible  use  of  nutritional

responses.   In   this   study,   we   observed   the   modulatory

supplements  to  maintain  “optimal  health”  by  modulating

effect   of   SPECTRA^TM   on   increases   in   ROS   generation

the   extent   of   “oxidative   and   nitrosative   stress.”   The

brought  about   due  to  challenge   with   exogenous  TNF-a

observed  multifaceted  biological  effects  of  SPECTRA^TM  on

(Fig.  7)  as  well  as  on  preservation  of  bioavailable  NO  by

“oxidative  and  nitrosative  stress”  may  be  directly  attribut-

reduction  in  cellular  and  mitochondrial  ROS  formation

able  to  the  supplement’s  biologically  active  compounds  as

(Fig.  8).

well    as    substrates    required    for    healthy    function    of

Tea  polyphenols  known  as  catechins  are  present  in  two

enzymes  involved  in  redox  regulation  reported  in  Table  1.

SPECTRA^TM   components—green   tea   extract   and   apple

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

835

New  Insights  on  Effects  of  a  Dietary  Supplement

B.  V.  Nemzer  et  al.

extract.  Previous  studies  (Yamamoto  et  al.  2004;  Manach

to  measure  the  influence  of  SPECTRA^TM  on  all  four  com-

et  al.  2005)  reported  that  catechins  may  increase  the  anti-

ponents  of  the  “healthy  aging  hypothesis.”  The  strength

oxidant  capacity  of  human  plasma,  which  in  turn  could

of  the  signals  generated  by  cyclic  hydroxylamines  and  the

support  cardiovascular  health,  improvement  of  processes

ability  to  use  the  technology  to  study  changes  that  occur

associated  with  lipoprotein  oxidation,  blood  aggregation,

at  the  level  of  cellular  components  such  as  mitochondria,

and   changes   in   lipid   profiles.   Other   studies   (Imai   and

vessels,  cells,  and  human  blood  (Fink  et  al.  2000;  Mrakic-

Nakachi  1995;  Sesso  et  al.  1999;  Nakachi  et  al.  2000;  Sas-

Sposta  et  al.  2012)  allow  us  to  follow  quantifiable  biologi-

azuki  et  al.  2000;  Sano  et  al.  2004;  Kuriyama  et  al.  2006;

cal  effects  in  healthy  human  subjects.  CMH  was  adopted

Kuriyama  2008)  also  suggested  that  tea  polyphenols  con-

due  to  the  fact  that  it  is  a  molecule  capable  of  diffusion

sumption  may  promote  cardiovascular  health  due  to  acti-

in   cell   compartments,   including   mitochondria   (Dikalov

vation  of  CuZn-SOD  activity  and  by  increasing  enzyme

et  al.  2011).  Indeed,  due  to  its  particular  physical-chemi-

expression  a  parameter  also  observed  by  SOD-dependent

cal  properties,  the  CMH  probe  is  able  to  cross  biological

extracellular  O   ^*

₂    generation  (Fig.  5)  in  this  current  study.

membranes,  thereby  detecting  ROS  both  in  plasma  and

Earlier   science   has   suggested   that   dietary   chlorogenic

intracellular   compartments.   In   this   way,   EPR   measure-

acids  (CGA)—the  major  group  of  coffee   polyphenols—

ments  enable  us  to  make  relative  quantitative  determina-

may  reduce  the  oxidative  stress  and  improve  nitric  oxide

tions  of  ROS  production  rates  in  human  blood  samples.

bioavailability  by  inhibiting  excessive  production  of  reac-

Additionally,  owing  to  its  high  efficiency  in  radical  detec-

tive   oxygen   species   in   the   vasculature,   and   lead   to   the

tion,  CMH  probe  can  be  used  at  very  low  concentrations

attenuation  of  endothelial  dysfunction  (Ohga  et  al.  2009;

(0.2  mmol/L)   compared   to   spin   traps   (10–50  mmol/L),

Yan   et  al.   2013).   Others   reported   that   the   initial   CGA

an  attribute  that  minimizes  side-effects  of  the  probes  on

metabolite,  caffeic  acid,  significantly  increased  superoxide

cell  physiology.  Moreover,  CMH  rapidly  reacts  and  allows

dismutase,   catalase,   and   glutathione   peroxidase   activity

radical   detection   via   a   single   chemical   reaction,   while

and  lowered  plasma  glucose  concentration  (Rustan  et  al.

other  probes  require  at  least  two  reactions  that  may  cause

1997;  Jung  et  al.  2006).  CGA  has  also  been  examined  in

artifacts  by  interaction  of  two  byproducts  (Zielonka  et  al.

human  studies  for   possible   effects  upon   blood   pressure

2005).

and  vasoreactivity  effects  (Watanabe  et  al.  2006).

In  addition  to  the  above-reported  biological  effects  on

Observation   of   allicin   showed   spontaneous   inhibition

oxidative   and   nitrosative   parameters,   we   observed   an

and  TNF-a  induced  secretion  of  proinflammatory  cyto-

additional  effect  of  lowering  of  glucose  concentrations  in

kines   and   chemokines   from   intestinal   epithelial   cells

the  participants’  blood  (Fig.  9)  that  may  have  been  asso-

(Lang  et  al.  2004).  Allicin  can  permeate  epithelial  and  red

ciated  with  increases  of  mitochondrial  oxygen  consump-

blood   cells   membranes   of   phospholipids   bilayers,   carry

tion  as  well  as  metabolic  activity  of  cells.  Such  possible

out  its  activity  intracellularly,  and  interact  with  SH  groups

effect  of  SPECTRA^TM  is  worthy  of  further  investigation.

(Miron  et  al.  2000).

Biologically   active   compounds   as   well   as   microele-

ments,   vitamins,   and   enzymes   in   natural   supplement

130

Control

Placebo

SpectraTM

SPECTRA^TM   may  participate  and  support  the  regulation

of  degree  from  “oxidative  and  nitrosative  stress.”  Previous

clinical  studies  have  reported  that  administration  of  vita-

115

min  C  (Bassenge  et  al.  1998)  or  vitamin  E  (Mah  et  al.

2013)  in  low  dosages  were  able  to  inhibit/restore  endothe-

lial   dysfunction,   a   consequence   of   excessive   “oxidative

100

and  nitrosative  stress.”  Therefore,  it  may  be  possible  that

components  of  SPECTRA^TM   may  contribute  in  unfolding

activity  of  biologically  active  enzymes.

Initially,  the  Total  ORAC_FN   assay  was  used  to  deter-

85

mine  SPECTRA^TM’s  ability  to  modulate  the  in  vitro  anti-

oxidant   scavenging   capacity   of   five   major   free   radicals

(peroxyl,  superoxide  anion,  hydroxyl,  singlet  oxygen,  and

70

peroxynitrite)   that   are   naturally   produced   in   the   body.

Fasting

1 h after

2 h after

3 h after

This   product   has   been   standardized   to   deliver   a   total

Figure  9.   Changes

in

blood

glucose

concentration

after

minimum   ORACFN   of   40,000  lmol  TE/g.   In   order   to

supplementation   of   standard   breakfast   with   or   without   placebo/

confirm  that  SPECTRA^TM  could  exert  any  activity  in  vivo,

SPECTRA^TM.    Data    are    mean  ♥  SEM    (n  =  22),    *P  <  0.025    versus

we  employed  the  extended  “Vitality”  test,  which  allows  us

placebo.

836

ª  2014  VDF  FutureCeuticals,  Inc.  Food  Science  &  Nutrition  published  by  Wiley  Periodicals,  Inc.

B.  V.  Nemzer  et  al.

New  Insights  on  Effects  of  a  Dietary  Supplement

Conclusions

Broedbaek,  K.,  V.  Siersma,  T.  Henriksen,  A.  Weimann,  M.

Petersen,  J.  Andersen,  et  al.  2013.  Association  between

For  the  first  time,  we  were  able  to  measure  the  biological

urinary  markers  of  nucleic  acid  oxidation  and  mortality  in

effects   of   a   natural   dietary   supplement   on   changes   of

type  2  diabetes:  a  population-based  cohort  study.  Diabetes

“oxidative   and   nitrosative   stress   markers”   and   cellular

Care  36:669–676.

metabolic  activity  through  the  use  of  the  extended  “Vital-

Chen,  J.,  R.  Hu,  and  H.  Chou.  2013.  Quercetin-induced

ity  Test.”  Unique  activity  of  SPECTRA^TM  suggests  poten-

cardioprotection  against  doxorubicin  cytotoxicity.  J.

tial   for   the   use   of   the   supplement   in   modulation   of

Biomed.  Sci.  20:95.

oxidative     stress,     NO     bioavailability,     inflammatory

Chung,  H.,  H.  Choi,  J.  Park,  J.  Choi,  and  W.  Choi.  2001.

response,  blood  glucose  levels,  and  ultimately  supporting

Peroxynitrite  scavenging  and  cytoprotective  activity  of

“optimal  health.”

2,3,6-tribromo-4,5-dihydroxybenzyl  methyl  ether  from  the

marine  alga  Symphyocladia  latiuscula.  J.  Agric.  Food  Chem.

49:3614–3621.

Acknowledgments

Dikalov,  S.,  and  B.  Fink.  2005.  ESR  techniques  for  the

Herewith  we  thank  Dr.  V.  Kagan  (Director  of  the  Center

detection  of  nitric  oxide  in  vivo  and  in  tissues.  Methods

for  Free  Radical  and  Antioxidant  Health,  Vice-Chairman

Enzymol.  396:597–610.

of  the  Environmental  and  Occupational  Health  Depart-

Dikalov,  S.,  M.  Skatchkov,  B.  Fink,  O.  Sommer,  and  E.

ment  at  the  University  of  Pittsburgh)  and  Dr.  A.  M.  Za-

Bassenge.  1998.  Formation  of  reactive  oxygen  species  in

fari  (Associate  Professor  of  Medicine  and  Director  of  the

various  vascular  cells  during  glyceryltrinate  metabolism.

Cardiovascular   Training   Program   at   Emory   University

J.  Cardiovasc.  Pharmacol.  Ther.  3:51–62.

School   of   Medicine),   John   M.   Hunter,   and   Brad   Evers

Dikalov,  S.,  K.  K.  Griendling,  and  D.  G.  Harrison.  2007.

(FutureCeuticals,  Inc.)  for  review  and  helpful  discussion

Measurement  of  reactive  oxygen  species  in  cardiovascular

during  editing  of  this  manuscript.  The  authors  also  thank

studies.  Hypertension  49:717–727.

Dr.  Luis  Valera  for  his  independent  statistical  analysis  of

Dikalov,  S.,  I.  Kirilyuk,  M.  Voinov.  and  I.  A.  Grigor’ev.  2011.

EPR  detection  of  cellular  and  mitochondrial  superoxide

data  reported  in  this  article.

using  cyclic  hydroxylamines.  Free  Radic.  Res.  45:417–430.

Dikalov, S., W. Li, A. Doughan, R. Blanco, and A. Zafari. 2012.

Conflict  of  Interest

Mitochondrial reactive oxygen species and calcium uptake

regulate activation of phagocytic NADPH oxidase. Am. J.

None  declared.

Physiol. Regul. Integr. Comp. Physiol. 302:R1134–R1142.

Dikalova,  A.,  A.  Bikineyeva,  K.  Budzyn,  R.  Nazarewicz,  L.

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