FACULTY OF SCIENCE

UNIVERSITY OF COPENHAGEN
 

PhD thesis

Dorota Kotowska

Department of Biology

Copenhagen University

Health promoting effects of bioactive compounds in

plants
 Dissertation  for  the  degree  of  Doctor  of  Philosophy  
Copenhagen  University,  Department  of  Biology  
Copenhagen,  Denmark  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Author:  Dorota  Kotowska  
Title:  Health  promoting  effects  of  bioactive  compounds  in  plants.  
Academic  advisors:    
Karsten  Kristiansen,  Department  of  Biology,  Copenhagen  University,  Copenhagen,  
Denmark  
Submitted:  June  2013  
 
English  abstract  
  While  type  2  diabetes  is  an  increasing  problem  worldwide,  there  is  still  no  cure  
and  therefore  search  for  the  new  insulin  sensitizer  continues.  Plants  are  a  natural  source  
of   bioactive   compounds   and   have   been   used   to   improve   human   health   and   wellbeing  
for  centuries.  Today,  several  studies  concentrate  on  screening  plant  extracts  commonly  
used   in   folk   medicine   for   pure   compounds,   exploiting   promising   results   in   treatment   of,  
among   others,   type   2   diabetes.   Another   area   of   diabetes   research,   focused   on   the  
complex   biology   of   adipose   tissue   and   its   influence   on   the   development   of   insulin  
resistance.   Moreover,   the   crosstalk   between   gut   bacteria   and   insulin   sensitive   tissues  
like   fat,   pancreas   and   skeletal   muscle   has   received   much   attention   and   all   aspects   are  
important  in  order  to  better  understand  the  basics  of  this  disease.    
  This   PhD   thesis   is   based   on   5   scientific   papers   focusing   on   plant   derived  
compounds   and   their   influence   on   adipocyte   differentiation,   lipid   storage,   glucose  
uptake  and  gut  microbiota.    
 

Dansk  resumé  
  Selv   om   type   2   diabetes   er   et   stigende   problem   på   verdensplan,   er   der   stadig  
ingen   kur   og   der   søges   til   stadighed   efter   nye   måder   at   øge   sensitiviteten   for   insulin.    
Planter   er   en   naturlig   kilde   til   bioaktive   stoffer   og   har   været   brugt   til   at   forbedre  
menneskers  helbred  i  århundreder.  Nu  til  dags  er  de  fleste  studier  koncentreret  om  at  
screene   planter,   almindeligt   brugt   i   folkemedicin,   for   de   rene   stoffer.   Det   udnyttes,   at  
visse  planter  udviser  lovende  resultater  i  behandlingen  af  bl.a.  type  2  diabetes.  Diabetes  
forskningen   fokuserer   også   på   den   komplekse   biologi   i   fedtvæv   og   dets   indflydelse   på  
udviklingen   af   insulinresistens.   Kommunikationen   mellem   tarmbakterier   og   insulin-­‐
sensitive   væv   som   fedt,   bugspytkirtel   og   skeletmuskulatur,   er   også   blevet   et  
fokusområde,   og   alle   er   vigtige   for   bedre   at   forstå   sygdomsmekanismen   i   type   2  
diabetes.  
  Denne  ph.d.-­‐afhandling  er  baseret  på  5  videnskabelige  artikler,  der  fokuserer  på  
stoffer   baseret   på   planteudtræk   og   deres   indflydelse   på   fedtcelle   differentiering,   lipid  
opbevaring,  glucose-­‐optagelse  og  tarmflora.  
 
 
 
 
 
 
 
 
 
 
  
 

List  of  papers  included  in  the  thesis  

  1.  K.B.  Christensen,  M.  Jørgensen,  D.  Kotowska,  R.K.  Petersen,  K.  Kristiansen,  L.P.  
Christensen,  2010,  Activation  of  the  nuclear  receptor  PPARγ  by  metabolites  isolated  
from  sage  (Salvia  officinalis  L.),  J.  Ethnophamacology,  132(1):127-­‐33
  2.  R.  B.  El-­‐Houri,  D.  Kotowska,  L.  C.  B.  Olsen,  S.  Bhattacharya,  L.  P.  Christensen,  K.  
Grevsen,   N.   Oksbjerg,   N.   Færgeman,   K.   Kristiansen,   K.   B.   Christensen,   Screening   for  
Bioactive   Metabolites   in   Plant   Extracts   Modulating   Glucose   Uptake   and   Fat  
Accumulation,  manuscript  
  3.   C.   Andersen,   D.   Kotowska,   C.   G.   Tortzen,   K.   Kristiansen,   J.   Nielsen,   R.   K.  
Petersen,   2-­‐(2-­‐bromophenyl)-­‐formononetin   and   2-­‐heptyl-­‐formononetin   are   PPARγ  
partial  agonists  and  reduce  lipid  accumulation  in  3T3-­‐L1  adipocytes,  manuscript  
  4.  D.  Kotowska,  J.  Olesen,  M.  Hansen,  R.  S.  Bienso,  C.  M.  Kristensen,  N.  Brandt,  S.  
A.  B.  Larsson,  W.  A.  Al-­‐Soud  ,L.  Hansen,  K.  Kristiansen,  H.  Pilegaard,  Resveratrol,  exercise  
and  gut  microbiota,  manuscript  
  5.   D.   Kotowska,   R.   B.   El-­‐Houri,   K.   B.   Christensen,   X.   Frette,   K.   Grevsen,   L.   P.  
Christensen,   K.   Kristiansen,   Novel   PPARγ   partial   agonist   isolated   from   Echinacea  
purpurea  exploits  insulin  sensitizing  effect  in  3T3-­‐L1  adipocytes,  manuscript  
  6.  E.  Sokol,  R.  Almeida,  H.  K.  Hannibal-­‐Bach,  D.  Kotowska,  J.  Vogt,  J.  Baumgart,,  R.  
Nitsch,  O.  N.  Jensen,  J.  Knudsen,  C.  S.  Ejsing1,  Quantitative  profiling  of  lipid  species  by  
normal-­‐phase  liquid  chromatography,  chip-­‐based  nanoelectrospray  ionization  and  
hybrid  ion  trap-­‐orbitrap  mass  spectrometry,  manuscript  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
Table  of  contents  
 
English  abstract  ..................................................................................................  3  
Dansk  resumé  .....................................................................................................  3  
List  of  papers  included  in  the  thesis  ...........................................................  4  
List  of  abbreviation  ...........................................................................................  6  
1.  Introduction  ....................................................................................................  7  
1.1  Obesity,  insulin  resistance  and  pharmacology  .................................................................  8  
1.2  Plant  derived  compounds  ..........................................................................................................  9  
1.3  Adipocytes  and  3T3-­‐L1  model  ..............................................................................................  18  
1.4  New  player:  microbiota  ...........................................................................................................  20  
2.  Discussion  of  the  papers  ..........................................................................  22  
PAPER  I  ...................................................................................................................................................  23  
PAPER  II  .................................................................................................................................................  24  
PAPER  III  ................................................................................................................................................  25  
PAPER  IV  ................................................................................................................................................  26  
PAPER  V  ..................................................................................................................................................  28  
OTHER  PAPERS  ...................................................................................................................................  29  
2.1  Concluding  remarks  ..................................................................................................................  30  
Literature  ..........................................................................................................  31  
Annex  ..................................................................................................................  39  

 
 
 
 
 
 
  
List  of  abbreviation    
  TZDs     Thiazolidinediones  
  PPARγ     Peroxisome-­‐proliferator  activated  receptor  gamma  
  EGCG     Epigallocatechin  gallate  
  ERK1/2     Extracellular-­‐signal-­‐regulated  kinase
  AMPK     AMP-­‐activated  kinase  
  Irs     Insulin  receptor  substrate
  Glut     Glucose  transporter  
  Akt     Protein  kinase  B  
  Sirt2     NAD-­‐dependent  deacetylase  sirtuin-­‐2
  LDL     Low  density  lipoprotein  
  2BrPhF     2-­‐(2-­‐bromophenyl)-­‐formononetin  
  C7F     2-­‐heptyl  formononetin  
  MSC     Mesenchymal  stem  cell  
  C/EBP     CCAAT/enhancer  binding  protein  
  SREBP     Sterol  regulatory  element  binding  protein  
  aP2     Fatty  acid  binding  protein  2  
  IR     Insulin  receptor  
  FAS     Fatty  acid  synthase  
  ACC     Acetyl-­‐CoA  carboxylase  
  PI3K     Phophatidyloinositol  3-­‐kinase  
  Fiaf     Fasting-­‐induced  adipocyte  protein  
  LPL     Lipoprotein  lipase  
  SCFA     Short  chain  fatty  acid  
  LXR     Liver  X  receptor  
  MAPK       Mitogen-­‐activated  protein  kinase  
  TIMP1     Tissue  inhibitor  of  metalloproteinases  1  
 
 
 
 
 
 
 
 
 
 
 
 
  
 

1.  Introduction    
Plants   are   the   natural   source   of   compounds   and   have   been   used   to   maintain  
human   health   and   improve   the   quality   of   life   for   hundreds   of   years.   Traditional  
medicine,   using   plant   extracts,   herbal   mixtures   and   natural   compounds   isolated   from  
various   species   of   plants,   is   still   used   for   primary   health   care,   despite   the   growth   of  
pharmaceutical   industry   (Egan,   C.D.,  2002).   At   the   same   time,   WHO   estimates,   that  
approximately  two  thirds  of  modern  drugs  originate  from  plants.  
Apart   from   being   the   great   source   of   bioactive   compounds   and   drugs,   plants   are  
also  important  sources  of  food.  It  is  well  known  that  intake  of  various  types  of  fruits  and  
vegetables   has   health   promoting   effect   and   may   prevent   modern   life   style   associated  
diseases   such   as   obesity,   diabetes,   atherosclerosis   and   metabolic   syndrome.   Indeed,  
thorough  and  careful  exploration  for  novel,  natural  compounds  has  attracted  renewed  
interest   worldwide,   especially   in   case   of   search   for   new   insulin   sensitizers   and   anti-­‐
obesity   agents.   The   era   of   TZDs   as   potent   insulin   sensitizers   and   full   PPARγ   agonists  
seem   to   come   to   the   end,   mostly   because   TZDs’   unwanted   side   effects   like   weight   gain,  
edema  or  fluid  retention.  Therefore  new  compounds,  able  to  increase  insulin  sensitivity,  
prevent   development   of   type   2   diabetes   and   avoid   causing   unwanted   side   effects   are  
needed,  and  plants  may  be  excellent  sources  of  new,  health  promoting  compounds.  
First   line   of   therapy   for   managing   obesity   and   the   related   metabolic   syndrome   is  
to  modify  patient’s  lifestyle.  However,  while  a  Mediterranean  type  of  diet,  rich  in  fruits  
and   vegetables,   is   considered   first   line   treatment   to   improve   one’s   health,   change   to  
more  active  lifestyle  is  also  needed.  It  is  known,  that  exercise  has  a  beneficial  effect  in  
both   animal   models   and   human.   Exercise   promotes   weight   loss   and   improves   insulin  
sensitivity   and   therefore   physical   activity   may   be   considered   an   environmental   factor  
influencing  health  and  welfare  together  with  a  diet.  
The   work   presented   in   this   thesis   was   initiated   as   part   of   collaboration   between  
three   Danish   universities   with   a   goal   of   identifying   new   plant   compounds   exhibiting  
health  promoting  effects.  In  order  to  address  this  broad  topic  we  focused  our  attention  
on   insulin   resistance   and   the   development   of   adipocytes   based   on   established   cell  
culture   model.   By   using   unique   rational   combination   of   screening   modalities   we   were  
able   to   identify   two   polyacetylens   (falcarinol   and   falcarindiol)   isolated   from   Daucus  
carrota   and   dodeca-­‐2E,4E,8Z,10E/Z   tetraenoic   acid-­‐   2-­‐   methylbutylamides-­‐   novel  
compound   isolated   from   Echinacea   purpurea.   Both   polyacetylenes   and   Echinacea  
alkamide   show   promising   insulin   sensitizing   effects   and   may   serve   as   leads   for   the  
development   of   the   next   generation   of   pharmaceuticals   targeting   obesity   and   obesity  
related  disorders.    
 
 
 
1.1  Obesity,  insulin  resistance  and  pharmacology  
Obesity   is   now   considered   major   problem   for   public   health   in   developing   and  
developed  countries.  According  to  WHO  over  four  hundred  million  adults  are  considered  
obese   and   approximately   1,6   billion   are   overweight   (http://www.who.int).  
Corresponding  with  an  increased  number  of  obese  individuals  is  an  increasing  number  
of   people   suffering   from   obesity   related   disorders   such   as   type   2   diabetes   (T2D),   cancer  
and   hypertension   (Rappange,   D.   R.,   et   al.,   2009).   This   rapid   growth   in   the   number   of  
obese   individuals   is   most   probably   connected   to   altered   dietary   intake   versus   energy  
expenditure  on  top  of  a  genetic  makeup  responsible  for  adipose  tissue  development.    
Adipocytes   play   important   role   in   metabolic   regulation   by   storing   lipids   (and  
releasing   fatty   acids)   and   secreting   adipokines,   which   regulate   insulin   sensitivity   and  
therefore  regulate  systemic  energy  balance.  In  the  normal  healthy  person,  energy  intake  
is  balanced  by  energy  expenditure.  Excess  energy  is  stored  in  adipocytes  in  form  of  fat  
droplets   and   released   as   fatty   acids   when   energy   intake   is   scarce.   However,   when  
energy   intake   continuously   exceed   energy   expenditure   adipocyte   tissue   expands   first  
due   to   hypertrophy   and   later   due   to   hyperplasia.   Hypertrophied   adipocytes   secrete  
large   amounts   of   adipokines,   which   are   known   to   not   only   interfere   with   insulin  
signaling   pathways   but   also   recruit   macrophages.   In   turn,   recruited   macrophages  
secrete  pro-­‐inflammatory  cytokines,  inducing  an  inflamed  state  in  the  tissue  and  further  
increase  the  level  of  insulin  resistance  in  the  adipocytes.  Moreover,  secreted  cytokines  
cause   a   decrease   in   the   net   flux   of   fatty   acids   into   the   adipocytes   and,   as   a   result,  
increase   deposition   of   fatty   acids   in   other   tissues   such   as   liver,   muscle   and   pancreas  
(Siersbaek,  R.  et  al.,  2010).  The  ectopic  fatty  acid  deposition  leads  to  insulin  resistance  in  
these  tissues  and  further,  to  systemic  insulin  resistance  and  type  2  diabetes,  if  need  for  
insulin  exceed  the  secretory  capacity  of  the  beta  cells  (Lehrke,  M.  et  al.,  2005).    
Before  insulin  therapy  in  1922,  starvation  and  traditional,  herbal  medicine  were  the  
only  treatments  for  diabetic  patients  but  as  soon  as  insulin  was  introduced  to  broad  use,  
traditional   treatments   were   forgotten.   However,   insulin   is   a   life-­‐   saver,   but   it   is   not   a  
cure  and  therefore  other  kind  of  drugs  was  needed.  In  the  late  1990s  TZDs  (glitazones)  
were   introduced   as   a   new   class   of   anti-­‐diabetic,   insulin   sensitizers.   Thiazolidinediones  
are   selective   agonists   of   peroxisome   proliferated-­‐   receptor   gamma.   When   PPARγ   is  
activated  by  TZDs,  differentiation  of  non-­‐  visceral  adipose  depots  is  initiated.  Fatty  acid  
storage   in   peripheral   tissues   is   increased,   creating   “lipid   steal”   phenomenon,   which   in  
turn   leads   to   lower   levels   of   circulating   fatty   acids   and   reduced   concentration   of  
triglycerides   in   muscles   and   liver.     At   the   same   time   thiazolidinediones   acutely   reduce  
insulin  levels  and  arrest  the  decline  in  beta-­‐cell  function  in  the  long  run.    
Promotion   of   peripheral   tissue   adipogenesis   is   only   one   of   the   ways   that   TZDs   are  
working.  Adipocytes  per  se  are  endocrine  organ  secreting  adipokines  (including  leptins,  
adiponectin,  resistin  and  TNFα)  and  TZDs,  via  PPARγ  pathway,  increase  the  production  
of   adiponectin   and   reduce   secretion   of   TNFα   and   resistin,   responsible   for   impaired  
insulin  actions  (Gurnell,  M.  et  al.,  2003,  Mudaliar,  S.  et  al.,  2001,  Greenfield,  J.  R.,  2004,  
Tang,  W.  et  al.,  2011,  Cariou,  B.,  2012).  
 Despite   their   therapeutic   abilities,   exploit   of   TZDs   was   challenged   by   observed   in  
clinical   use   side   effects,   which   include   severe   weight   gain,   edema,   fluid   retention,  
cardiac   failure   and   bone   fractures.   Recently,   two   drugs,   rosiglitazone   and   troglitazone  
were  withdrawn  from  the  market  due  to  observed  hepatotoxicity  and  a  remaining  drug,  
pioglitazone,  has  been  reassessed  in  light  of  increased  risk  of  bladder  cancer  (Cariou,  B.  
et  al.,  2012,  Kahn,  B.  B.  et  al.,  2010).    
Besides   TZDs,   other   anti-­‐obesity   and   anti-­‐diabetes   therapies   are   introduced,   but  
there  is  still  no  ideal  drug  on  the  market.  The  only  drug  approved  in  Europe  is  orlistat  
(Baretic,  M.,  2012),  working  as  pancreatic  lipase  inhibitor,  which  results  in  secretion  of  
undigested   fatty   acids   through   feces.   Until   recently   sibutramine,   an   appetite  
suppressant,  working  via  inhibition  of  reuptake  of  serotonin  and  norepinephrine,  was  an  
alternative  to  orlistat,  but  was  withdrawn  due  to  cardiovascular  side  effects  (James,  W.  
P.  et  al.,  2010).    
Taken  together,  novel  compounds  able  to  increase  insulin  sensitivity  without  causing  
unwanted   side   effects   are   needed.   As   mentioned   before,   plants   have   long   history   of  
preventing   diseases   and   improving   human   health   and   they   are   usually   considered   less  
toxic   and   elucidate   less   side   effects   compared   to   synthetic   drugs   (Yeh,   G.   Y.   et   al.,   2003)  
therefore  can  be  considered  excellent  source  of  bioactive  agents.    
 

1.2  Plant  derived  compounds  

Phytochemicals   are   reported   to   have   beneficial   effect   on   prevention   of  
cardiovascular   diseases,   inflammation,   glucose   intolerance   and   obesity   and   plant  
extracts   and   purified   plant   compounds   are   gaining   more   and   more   interest   since   they  
are   components   of   the   every-­‐   day   diet.   In   vivo,   they   work   via   different   pathways  
causing,   among   others,   inhibition   of   nutrient   absorption,   appetite   suppression,   increase  
of  energy  expenditure  and  modulation  of  fat  storage.    Below,  there  are  few  examples  of  
plants   derived   compounds   and   extracts,   showing   promising   effects   in   case   of   treating  
obesity  and  type  2  diabetes.  
Catechins   are   main   compounds   of   green   tea,   which   is   one   of   the   most   popular  
beverages   in   the   world   (right   after   water).   Green   tea   contains   five   major   catechins,  
including:   catechin,   epicatechin,   epicatechin   gallate,   epigallocatechin   and  
epigallocatechin  gallate  (EGCG),  and  the  anti-­‐obesity  effect  is  mostly  attributed  to  EGCG.  
The   mechanism   of   action   of   EGCG   involves   inhibition   of   adipocyte   differentiation   and  
proliferation,   inhibition   of   fat   absorption   from   gut   and   inhibition   of   fatty   acids   oxidation  
in  brown  adipose  tissue  (Hursel,  R.  et  al.,  2010,  Lin,  J.  K.,  2006,  Ikeda,  I.,  2005).    In  3T3-­‐L1  
cells,   EGCG   inhibited   adipocyte   proliferation   by   decreasing   levels   of   phosphorylated  
ERK1/2   and   inducing   apoptosis   in   mature   cells   (Lin,   J.,   et   al.,   2005)   and   increasing  
phosphorylation  of  AMPK  (Murase,  T.,  et  al.,  2009).  
Green   tea   is   only   one   of   the   extracts   exhibiting   beneficial   effects   in   anti-­‐obesity  
treatment.   Another   example   is   an   extract   of   chokeberry   (Aronia   melanocarpa),   rich   in  
anthocyanins.   In   rats,   consumption   of   chokeberry   extract   together   with   high   fructose  
diet,  improved  insulin  sensitivity  in  epididymal  adipose  tissue  via  Irs1  and  Irs2  pathway  
and   increased   expression   of   Glut1   and   Glut4   (Qin,   B.,   et   al.,   2011).   Therefore,   it   was  
concluded   that   chokeberry   extract   may   be   promising   diet   supplement,   but   human  
studies  are  still  needed  to  assess  the  beneficial  effects  vs.  risk  factors.  
 Moderate   red   wine   intake   was   positively   correlated   with   lower   occurrence   of  
cardiovascular  and  metabolic  diseases,  including  obesity  and  type  2  diabetes  (Zoechling,  
A.,   et   al.,   2011).   A   grape   skin   ethanol   extract   was   also   found   to   decrease   adipogenic  
transcription   factor   gene   expression   and   therefore   inhibit   triglyceride   accumulation   in  
3T3-­‐L1   adipocytes   mostly   via   PPARγ   signaling   pathway   (Jeong,   Y.   S.,   et   al.,   2012),   and  
grape  seed  extract  was  reported  to  lower  triglyceride  and  total  cholesterol  levels  in  the  
plasma  of  rats  fed  high-­‐fat  diet  (Charrad,  K.,  et  al.,  2011).  These  studies  correlate  with  
reports   of   the   pure   compound,   resveratrol,   found   in   grapes,   pistachios,   red   wine   and  
berries.   Resveratrol   is   well-­‐   studied   polyphenol   and   has   been   suggested   to   have  
beneficial  effect  in  preventing  several  diseases,  including  cancer,  cardiovascular  disease,  
obesity  and  diabetes  (Hung,  L.  M.  et  al.,  2000,  Atten,  M.  J.,  et  al.,  2005,  van  der  Spuy,  W.  
J.   et   al.,   2009).   In   rodents   fed   high   fat   diet   supplemented   with   resveratrol,   significant  
reduction  of  body  fat  depots  was  observed  (Ahn,  J.  et  al.,  2008,  Macarulla,  M.  T.,  et  al.,  
2009,  Shang,  J.,  et  al.,  2008,  Lagouge,  M.,  et  al.,  2006,  Baile,  C.  A.,  et  al.,  2011).  Also,  our  
own   study   confirms   these   findings.   In   our   setup,   mice   fed   high   fat   diet   supplemented  
with   resveratrol   at   dose   4g/kg   food   at   libitum   feeding   were   protected   against   diet  
induced  obesity  and  their  body  weight  was  not  significantly  different  from  body  weight  
of  mice  from  control  group  fed  with  standard,  chow  diet  (PAPER  IV).    
Resveratrol’s   beneficial   effects   on   obesity   are   reported   to   be   due   to   not   only   to  
AMPK  phosphorylation  but  also  Akt  and  Sirt1  signaling  pathway  (Zang,  M.,  et  al.,  2006,  
Ahn,   J.,   et   al.,   2008,   Floyd,   Z.   E.,   et   al.,   2008).   All   these   studies,   including   our   own,  
suggest  that  grape  derived  compound  resveratrol  contributes  to  prevention  of  obesity  
through   multiple   pathways.   However,   thorough   clinical   trials   on   healthy   and   obese  
subjects  are  needed  to  support  data  obtained  in  the  cell  cultures  and  animals.  
Another   compound   exhibiting   anti-­‐   obesity   properties   is   berberine   isolated   from  
Cortidis  rhizoma.  Cortidis  rhizoma  extract  is  known  in  traditional  Chinese  medicine  as  an  
anti-­‐bacterial  drug  and  in  the  recent  studies  of  rat  islets  pretreated  with  this  extract,  a  
retained  insulin-­‐  secretion  capacity  was  found.  Berberine  was  shown  to  inhibit  adipocyte  
proliferation  and  differentiation  and  decrease  lipid  accumulation  in  3T3-­‐L1  cells.  In  high  
fat  diet  fed  animals,  berberine  was  shown  to  reduce  serum  cholesterol,  triglycerides  and  
LDL-­‐cholesterol.   Reduction   of   LDL-­‐cholesterol   was   also   observed   in   hyper-­‐
cholesterolemic   patients   and   in   diabetic   animal   models   hypoglycemic   action   of   insulin  
was  enhanced  (Kwon,  K.  B.,  et  al.,  2005).    
  Berberine,   resveratrol   and   EGCG   were   reported   to   activate   multiple   pathways,  
including  PPARγ,  AMPK,  Akt,  Sirt1  and  ERK1/2  (Huang.  C.,  et  al.,  2006,  Kong,  W.,  et  al.,  
2004,  Brusq,  J.  M.,  et  al.,  2006,  Ko,  B.  S.,  et  al.,  2005).  It  has  been  suggested  that  natural  
compounds,  in  contrast  to  synthetic  ones,  may  work  through  many  pathways,  activating  
them   simultaneously.   Resveratrol,   berberine   and   EGCG   but   also:   curcumin   (isolated  
from  turmeric,  activating  AMPK  and  PPARγ  pathway),  kaempferol  and  quercetin  (main  
compounds   in   Euonymus   alatus,   activating   PPARγ   and   IκB),   artepillin   C   (isolated   from  
Brazilian   propolis,   working   via   PPARγ   and   PKC   pathways)   and   piperine   (component   of  
black   pepper,   activating   PPARγ   and   LXRα)   (Table   1)   are   the   examples   of   plant   derived  
compounds  supporting  multiple  pathway  hypothesis  (Ejaz,  A.  et  al.,  2009,  Wu,  Z.,  1995,  
Fang,  X.-­‐  K.,  et  al.,  2007,  Choi,  S.-­‐S.  et  al.,  2011,  Park,  U.-­‐H.,  et  al.,  2012).  
 
 
 
 
 
Compound   Plant  of  origin   Activated  pathways   Reference  

Curcumin   turmeric   AMPK,  PPARγ   Ejaz,  A.  et  al.,  2009  

Kaempferol   Euonymus  alatus   PPARγ,  IκB   Wu,   Z.,   1995,   Fang,   X.-­‐
K.,  et  al.,  2007  
Quercetin   Euonymus  alatus   PPARγ,  IκB   Wu,   Z.,   1995,   Fang,   X.-­‐
K.,  et  al.,  2007  
Artepillin  C   Brazilian  propolis   PPARγ,  PKC   Choi,  S.-­‐S.  et  al.,  2011  

Piperline   Black  pepper   PPARγ,  LXRα   Park,  U.-­‐H.,  et  al.,  2012  

Berberine   Cortidis  rhizoma     PPARγ,  AMPK   Kwon,  K.  B.,  et  al.,  2005    

Resveratrol   Grapes,   AMPK,  Akt,  Sirt1   Zang,   M.,   et   al.,   2006,  

pistachios,  berries   Ahn,   J.,   et   al.,   2008,  
Floyd,  Z.  E.,  et  al.,  2008    
EGCG   Camellia  sinensis   AMPK,  ERK1/2   Murase,  T.,  2009    

Table  1.  Examples  of  plant  derived  compounds  activating  multiple  signaling  pathways.  
 
 
  Isolation  and  identification  of  new,  bioactive  compounds  from  plant  extracts  can  
be  performed  in  two  ways:  1)  plant  extracts  showing  promising  properties  are  scanned  
and   one   or   two   major   compounds   are   isolated   and   tested   further,   or   2)   extracts   are  
fractionated   and   fractions   are   next   submitted   to   bioassay   guided   fractionations.  
Scanning   for   major   bioactive   compounds   is   more   commonly   used,   as   it   is   faster   and  
cheaper   to   perform.   However,   major   phytochemicals   are   not   always   the   ones  
responsible   for   beneficial   effects   of   the   plant.   Moreover,   synergistic   effects   of   two   or  
more  compounds  may  occur  and  will  be  missed  if  bioassay  guided  fractionation  is  not  
performed.      
   Bioassay   guided   fractionation   was   used   by   Shang,   N.,   et   al.   in   search   for  
adipogenic   constituents   from   bark   of   Larix   laricina   du   Roi   for   the   treatment   of   type   2  
diabetes   symptoms.   Starting   from   ethanol   extract   of   Larix   laricina   sixteen   primary  
fractions   was   obtained   from   silica   gel   column   chromatography.   These   fractions   were  
tested   alongside   with   crude   extract   for   ability   to   increase   triglyceride   accumulation   in  
3T3-­‐L1   adipocytes.   Six   of   the   tested   fractions   were   found   to   significantly   increase  
triglyceride   content   and   pure   compounds   were   isolated   from   these   fractions,   which  
resulted  in  discovery  of  new  cycloartane  triterpentoid  (23-­‐oxo-­‐3alpha-­‐hydroxycycloart-­‐
24-­‐en-­‐26-­‐oid   acid)   among   the   other   9   compounds.   Interestingly,   23-­‐oxo-­‐3alpha-­‐
hydroxycycloart-­‐24-­‐en-­‐26-­‐oid  acid  was  not  the  main  component  of  the  extract  and  still  
seemed   to   be   responsible   for   adipogenic   activity   of   the   fraction   it   was   isolated   from  
(Shang,  N.,  et  al.,  2012).
  Similar   work   was   performed   while   searching   for   anti-­‐adipogenic   activities   of   two  
extracts  isolated  from  Alnus  incana  and  Populus  balsamifera  barks  respectively  (Fig.1a,  
b).   However   no   new   compounds   were   discovered   during   this   study,   the   isolated  
phytochemicals  responsible  for  anti-­‐adipogenic  activity  (oregonin  from  Alnus  incana  and  
salicortin  from  Populus  balsamifera)  were  not  major  components  of  the  crude  extracts,  
similar  to  the  previously  described  studies  (Martineau,  L.  C.,  et  al.,  2010).  
 
 Fig. 1 a Bioassay-g
of Alnus incana (a) a
performed accordin
sulted in the isolatio
and salicortin as the
each level of fractio
were tested simulta
the crude extract te
performed at the sa
crude extract (50 µg
ulus). Fractionation
pogenesis. For the P
confirmed by simult
of proliferation. Mea
wells of a single cell
brackets under each
g or mg beside each

 
Fig.  1a.  From  Martineau,  L.  C.,  et  al.,  2010;  Outline  for  bioassay  guided  fractionation  of  
methanol   extract   of   Alnus   incana   and   chemical   structure   of   isolated   compound:  
oregonin.   Fig. 1 b Bioassay-g
of Populus balsamife
" Fig. 1 a).
l
 Fig. 1 b Bioassay-g
of Populus balsamife
" Fig. 1 a).
l

 
Fig.1b.  From  Martineau,  L.  C.,  et  al.,  2010;  Outline  for  bioassay  guided  fractionation  of  
methanol  extract  of  Populus  balsamifera  and  chemical  structure  of  isolated  compound:  
salicortin.   Martineau LC et al. Anti-adipogenic Activities of …
 
 
 
 
 
 
 
 
   In  2009  Christensen  K.  B.  et  al.,  published  a  bioassay  guided  screening  platform  
(Fig.2)   for   identification   of   plant   extracts   with   potential   antidiabetic   properties  
(Christensen   K.   B.,   et   al.,   2009).   The   screening   platform   used   in   this   study   served   as   a  
base  for  our  own  studies,  in  which  we  performed  bioassays  guided  fractionations  of  two  
dichloromethane  extracts  from  roots  of  Daucus  carota  (carrot)  and  Echinacea  purpurea  
(Fig.3).    
 
 

 Plant  extract  

No  
 PPARγ  transactivation  

Yes   S  
T
Yes  
0
 Adipocyte  differentiation    
P  

No  
No  
Insulin-­‐stimulated  
  glucose  uptake  

Yes

 PPARα+γ  transactivation  

 Co-­‐factor  recruitment  

 Pre-­‐clinical  trials
 
 
Fig.2.  Christensen  K.  B.  et  al.,  2008,  Outline  for  the  analytical  screening  platform  applied  
in  the  paper.  
  

 Plant  extract

Insulin-­‐stimulated  
  glucose  uptake
No
Yes S
T
  PPARγ  transactivation  Adipocyte  differentiation   0
P

 Fractionation  of  extract

Insulin-­‐stimulated  glucose  uptake  

of  fractions,  Adipocyte  
 
differentiation,   PPARγ  
transactivation

Pure  compound  identification  

 and  isolation

Insulin  stimulated  glucose  

uptake,  adipocyte  
differentiation,  PPARγ  
 transactivation,  gene  
expression,  mechanism  of  
action
 
 
Fig.3.     Outline   for   bioassays   guided   fractionation   and   compound   purification   used   on  
dichloromethane  extracts  of  Daucus  carrota  and  Echinacea  purpurea.    
 
 
 
   Bioassay   guided   fractionation   of   carrot   extract   led   us   to   two   pure   compounds:  
falcarinol   and   falcarindiol,   which   chemically   belong   to   the   class   of   polyacetylenes   and  
are  one  of  the  most  important  compounds  found  in  carrot  (Killeen,  D.  P.,  et  al.,  2013).  
Therefore,   this   study   stays   in   contradiction   to   classical   bioassay   guided   research.  
However,  polyacetylenes  are  wildly  researched  group  of  natural  compounds,  mostly  in  
anti-­‐cancer,   anti-­‐inflammatory,   anti-­‐bacterial   and   immune   stimulation   studies   (Brandt,  
K.,  et  al.,  2004,  Hansen,  S.  L.,  et  al.,  2003,  Metzeger,  B.  T.,  et  al.,  2009),  so  there  came  no  
surprise,   the   same   compounds   were   able   to   stimulate   glucose   uptake,   induce   lipolysis  
and  not  promote  adipogenesis  in  3T3-­‐L1  adipocytes.  
  Our  second  study  concentrated  on  bioassays  guided  fractionations  of  extract  of  
well-­‐studied  plant:  Echinacea  purpurea.  Echinacea  extracts  from  three  different  species  
of   this   flower   (E.   pallida,   E.   augustifolia   and   E.   purpurea)   are   used   commonly   for  
treatment   and   prevention   of   upper   respiratory   tract   infections   and   preparations   are  
made   from   both   roots   and   aerial   parts.   Main   bioactive   metabolites   of   Echinacea   are  
polysaccharides,   caffeic   acid   derivatives   and   alkamides   (Spelman,   K.,   et   al.,   2009,  
Starvaggi  Cucuzza,  L.,  et  al.,  2008,  Christensen,  K.  B.,  et  al.,  2009,  Goey,  A.  K.  L.,  et  al.,  
2012).   In   our   study,   isolated   and   purified   compound   responsible   for   insulin   sensitizing  
effect   in   3T3-­‐L1   adipocytes   belongs   to   the   group   of   alkamides,   therefore   one   of   the  
major   bioactive   metabolites   in   Echinacea.   However,   this   compound   (dodeca-­‐
2E,4E,8Z,10E/Z  tetraenoic  acid-­‐2-­‐methylbutylamides),  according  to  our  knowledge,  was  
never   purified   before   from   Echinacea   and   it’s   structure   was   never   published   and  
therefore  it  is  a  novel  compound  working  as  insulin  sensitizer,  inductor  of  adipogenesis  
and  partial  agonist  of  PPARγ  (Fig.4)  
 

Falcarinol Falcarindiol

dodeca-­‐2E,4E,8Z,10E/Z  tetraenoic  acid-­‐  2-­‐  methylbutylamides

 
 
Fig.4.   Chemical   structure   of   falcarinol   and   falcarindiol   (polyacetylenes,   isolated   from  
carrot)   and   dodeca-­‐2E,4E,8Z,10E/Z   tetraenoic   acid-­‐2-­‐methylbutylamides   (alkamide,  
isolated  from  Echinacea)      
 
 
   Isolated   plant   compounds,   can   be   used   in   their   naïve   form   or   can   be   further  
modified   and   introduced   modifications   can   modulate   their   potency.   In   our   study,   we  
have   used   formononetin   (4'-­‐O-­‐methyldaidzein,   isoflavon   found   in   red   clover)   main  
structure   and   modified   it   by   adding   2-­‐bromophenyl   or   2-­‐heptyl   groups   to   the  
formononetin   scaffold.   2-­‐(2-­‐bromophenyl)-­‐formononetin   (2BrPhF)   and   2-­‐heptyl  
formononetin  (C7F)  decreased  lipid  accumulation  in  3T3-­‐L1  adipocytes  and  were  acting  
as  partial  agonists  of  PPARγ,  while  formonotein  in  its  native  form  increased  triglyceride  
accumulation   and   did   not   bind   to   PPARγ.   C7F,   but   not   2BrPhF   and   formononetin,   also  
increased   glycerol   release   and   the   upregulation   of   lipolytic   genes   was   observed   when  
cells   were   treated   with   this   compound,   therefore   suggesting,   that   small   modifications  
can  have  big  impact  on  compounds  potency  and  molecular  mechanisms  (Fig.5)  (PAPER  
III).    
 

Formononetin 2BrPhF C7F

 
Fig.5.   Chemical   structure   of   native   structure   of   formononetin   and   its   two   derivatives:   2-­‐
(2-­‐bromophenyl)-­‐  formononetin  (2BrPhF)  and  2-­‐heptyl  formononetin  (C7F)  
 
 

1.3  Adipocytes  and  3T3-­‐L1  model  

Obesity   is   one   of   the   most   challenging   and   common   health   dilemma   throughout   the  
world   and   its   complex   etiology   is   an   obstacle   for   development   of   the   new   treatment.  
The   obese   state   is   mainly   caused   by   an   imbalance   between   energy   intake   and  
expenditure,  which  results  in  accumulation  of  lipids  and  triglycerides  in  adipose  tissue.  
Adipocytes  are  no  longer  believed  to  be  “storage  organ”;  on  the  contrary,  they  play  very  
important   role   in   regulation   of   lipid   homeostasis,   glucose   uptake   and   endocrine  
communication.   Under   the   conditions   of   excess   energy,   adipose   tissue   increases   the  
volume   first   due   to   hypertrophy   and   later   due   to   hyperplasia.   These   fully   functional  
adipocytes,   together   with   osteoblasts,   chondrocytes   and   myoblasts   originate   from  
mesenchymal   stem   cells   (MSC).   Due   to   differentiation   process   they   become   first  
committed  preadipocytes,  then  growth  arrested  preadipocytes,  which  go  through  2  or  3  
cycles  of  mitotic  clonal  expansion  and  terminal  differentiation  to  become  lipid-­‐laden  and  
insulin-­‐responsive   mature   adipocytes.   The   whole   adipogenesis   process   is   well   organized  
and  involves  cascade  of  transcription  factors  such  as  peroxisome  proliferator-­‐activated  
receptor   gamma   (PPARγ),   CCAAT/enhancer-­‐binding   proteins   (C/EBPs)   and   sterol  
regulatory  element  binding  protein  (SREBP)  family  (Rosen,  E.  D.,  et  al.,  2000).  PPARγ  and  
C/EBPα   represent   early   markers   of   adipogenesis   and   are   essential   for   activation   of  
downstream,   adipocyte   specific   genes,   such   as:   fatty   acid   binding   protein   2   (aP2),  
glucose   transporter   type   4   (Glut4),   leptin,   insulin   receptor   (IR),   fatty   acid   synthase   (FAS)  
and   acetyl-­‐CoA   carboxylase   (ACC)   (Lehrke,   M.,   et   al.,   2005).   SREBP-­‐1c   enhances   the  
expression  of  genes  related  to  lipid  and  cholesterol  metabolism  (Hsu,  J.  M.,  et  al.,  2003).    
  Adipocytes,   together   with   muscle   and   liver   are   responsible   for   regulation   of  
glucose  homeostasis  and  insulin  sensitivity.  
  Insulin   is   the   most   potent   anabolic   hormone   known.   It   promotes   storage   of  
triglycerides   in   adipocytes   by   multiple   mechanisms,   including   increasing   the  
differentiation   of   preadipocytes   to   mature   adipocytes,   synthesis   of   triglycerides  
inhibiting   lipolysis   and   stimulating   glucose   uptake   (Taniguchi,   C.   M.,   et   al.,   2006).   In  
adipose  tissue,  two  glucose  transporters  were  identified:  Glut4  and  Glut1.  While  Glut4  is  
insulin-­‐depended  glucose  transporter  and  its  movement  from  cell  surface  to  within  cell  
is   stimulated   via   PI3K   pathway,   Glut1   is   responsible   for   basal   glucose   uptake   required  
for   maintaining   respiration   in   cells   and   its   expression   levels   in   cell   membrane   are  
increased   by   reduced   glucose   levels   (Karnieli,   E.,   et   al.,   2008).   Simplified   schematic   of  
insulin  signaling  pathways  is  presented  on  Fig.6.  
  Most  of  the  knowledge  about  signaling  pathways  and  molecular  mechanisms  is  
obtained  primarily  from  in  vitro  studies.  In  the  subject  of  obesity  and  insulin  sensitivity,  
most  of  the  in  vitro  assays  are  performed  in  cell  culture  in  3T3-­‐L1  cell  line,  which  we  also  
used   in   our   experiments.   3T3-­‐L1   cell   line   was   established   in   late   seventies   and,  
developed  through  clonal  expansion  and  immortalization,  contains  only  one  single  cell  
type   (Green,   H.,   et   al.,   1974,   Green,   H.,   et   al.,   1976,   Fernyhough,   M.   E.,   et   al.,   2005).  
However   very   widely   used   in   laboratories   all   over   the   world,   3T3-­‐L1   cells   are   not  
identical   to   primary   cells   or   in   vivo   adipocytes.   They   follow   different   developmental  
patters   and   their   gene   expression,   despite   a   lot   of   similarities,   is   not   identical,   therefore  
the  results  obtained  from  cell  cultures  experiments  must  be  confirmed  in  in  vivo  studies  
(Poulos,  S.  P.,  et  al.,  2010).  
 
 
 
  
 
Fig.  6.  Outline  for  selected  insulin  signaling  pathways  in  mature  adipocyte.  
 
 
 

1.4  New  player:  microbiota  

  Plant   derived   phytochemicals   are   reported   to   influence   the   development   of  
adipose  tissue  and  connected  with  it  glucose  intolerance,  obesity  and  type  2  diabetes.  
As  components  of  every-­‐day  diet  they  also  have  an  impact  on  gut  microbiota:  an  active  
organ  within  the  host  body.    
  The   gut   microbiota   has   been   considered   forgotten   organ   within   the   body   and  
until   recently,   its   role   in   nutrition   and   metabolism   was   neglected.   Accumulating  
evidence   indicates   however   that   the   gut   microbiota   is   associated   with   the   etiology   or  
development  of  obesity  and  diabetes  (Backhed,  F.,  et  al.,  2004,  Turnbaugh,  P.  J.,  et  al.,  
2006).   Diet   and   gut   microbiota   are   strictly   connected:   gut   bacteria   help   to   digest  
otherwise   indigestible   food   components   such   as   plant   polysaccharides   or   increase   the  
capacity   of   energy   harvest   from   a   diet.   Due   to   mutualistic   host-­‐   bacteria   relationship,  
harvested  energy  can  be  stored  in  adipocytes  through  pathway,  that  involves  microbial  
regulation   of   the   intestinal   epithelial   expression   of   fasting-­‐induced   adipocyte   protein  
(Fiaf).   Bacterial   suppression   of   Fiaf   results   in   reduced   levels   of   circulating   inhibitor   of  
lipoprotein   lipase   (LPL)   which   in   turn   increases   the   LPL   activity   in   adipocytes   and  
enhance   storage   of   liver-­‐delivered   triglycerides   in   fat   tissue   (Ley,   R.   E.,   et   al.,   2005),  
therefore  influencing  the  host  energy  balance.  
  Gut  bacteria  not  only  influence  food  processing,  they  are  also  influenced  by  the  
dietary   intake.   It   has   been   shown,   the   high   fat   diet   promotes   changes   in   bacterial  
ecology   within   the   gut,   mostly   by   changing   the   ratio   of   different   taxa.   Also   selective  
modulation   of   the   structure   or   activity   of   intestinal   microbiome   by   using   prebiotics   or  
probiotics   has   been   demonstrated   in   both   human   and   animal   research   to   confer  
beneficial   effects.   Therefore,   gut   microbiota   can   be   considered   potential   target   of  
therapeutic  drugs  or  nutritional  interventions  (Jia,  W.,  et  al.,  2008,  Zhao,  L.,  et  al.,  2010).  
  Plant   enrichment   of   diet   had   been   shown   to   have   beneficial   effect   in   case   of  
obesity  and  diabetes,  and  plant  compounds,  isolated  from  the  dietary-­‐plants  had  been  
shown   to   work   via   multiple   pathways.   On   top   of   that   findings,   gut   bacteria   may   also  
contribute  to  the  health-­‐  promoting  effects  of  phytochemicals  by  digesting  them  or  by  
structural  changes  in  the  ecology  of  gut,  promoted  by  the  certain  compound.  Schematic  
interconnection   between   gut   microbiota,   diet,   inflammation   markers   and   body   fat   is  
shown  on  Fig.7.  
 
 
Mucin   Gut  microbiota
production

Adipokines Inflammation Fiaf Diet  composition

Body  fat
 
 
Fig.7.  From  Ravussin,  Y.,  et  al.,  2012;  modified.  Schematic  depicting  possible  interactions  
between   diet   composition,   body   fat,   gut   microbiota,   circulating   adipokines,   mucin  
production,  Fiaf  and  inflammatory  markers.  
   
 
  One   of   the   examples   of   combined   action   of   gut   bacteria   and   plant   derived  
compound   is   berberine.   As   mentioned   before,   berberine   was   reported   to   reduce   serum  
cholesterol,   triglycerides   and   LDL-­‐cholesterol   levels   in   high   fat   diet   fed   animals   and  
inhibit   differentiation   of   3T3-­‐L1   adipocytes.   Further   studies   suggest,   berberine   also  
modulates   gut   microbiota   by   inhibiting   wide   range   of   intestinal   bacteria,   which  
alleviates   systemic   inflammation   and   enriching   short   chain   fatty   acid   (SCFA)   producers  
and   therefore   increasing   SCFA   levels   in   the   intestine,   which   in   turn   contributes   to   the  
beneficial   effects   of   berberine   against   insulin   resistance   and   obesity   (Zhang,   X.   et   al.,  
2012).  
  Also  resveratrol  was  shown  to  interfere  with  bacterial  ecology  in  mice  fed  with  
high  fat  diet.  As  described  previously:  rodents  fed  with  resveratrol  supplementation  in  
the  high  fat  diet  had  significantly  reduced  body  fat  depots,  what  was  confirmed  in  our  
own   study,   too.   Gut   bacteria   seem   to   contribute   in   heath   promoting   effect   of  
resveratrol,   by   converting   it   into   dihydroresveratrol,   3,4’-­‐dihydroxy-­‐trans-­‐stilbene   and  
lunarin,  which  are  absorbed  from  the  gut  (Bode,  L.  M.,  et  al.,  2013).  From  our  study  we  
can   conclude,   resveratrol   supplementation   shifts   the   physiology   of   high   fat   diet   fed  
animals   towards   mice   on   standard   chow   diet   and   this   study   stays   in   agreement   with  
Baur,  J.  A.,  et  al.,  research  (Baur  J.  A.,  et  al.,  2006).  A  the  same  time,  we  were  not  able  to  
observe   any   significant   changes   in   bacterial   composition   in   the   gut   of   mice   provided  
with   high   fat   diet   and   resveratrol,   which   may   indicate,   that   resveratrol   does   not  
interfere  with  ecology  of  the  intestine  (PAPER  IV).      
 

2.  Discussion  of  the  papers  

  Maintaining   a   proper   balance   between   energy   intake   and   energy   expenditure  
and   therefore   between   storage   of   lipids   and   their   mobilization   is   of   great   importance.  
When   this   balance   is   disturbed   and   energy   intake   exceed   energy   expenditure,   obesity  
and  related  with  obesity  disorders  are  occurring.  Change  of  diet  and  lifestyle  is  the  first  
line   of   prevention   and   treatment   but   not   always   successful,   therefore   drugs   and   diet  
supplements   must   be   introduced.   However,   there   is   still   no   ideal   anti-­‐obesity   drug  
available  and  search  for  novel,  bioactive  compounds  is  continued.    
  Also,   for   obesity   related   disorders,   such   as   T2D,   no   perfect   drug   exists   and   much  
research   is   concentrating   on   finding   new   compounds,   which   can   work   as   insulin  
sensitizers  and  not  cause  the  patients  gaining  severe  weight.    
  Plant   extracts   seem   to   be   ideal   for   searching   for   novel   anti-­‐obesity   and   anti-­‐
diabetic  compounds.  Plants  have  been  used  for  this  purposes  for  hundreds  of  years  in  
folk   medicine,   therefore   there   is   a   strong   argument   that   plant   kingdom   may   hold   a   cure  
for  these  two  disorders.  However,  careful  and  thorough  research  is  needed,  before  any  
conclusions  can  be  draw.    
  The  work  presented  here  revolved  around  the  topic  of  plants  being  a  source  of  
bioactive   compounds   exploiting   promising   features   for   activation   of   nuclear   receptor  
PPARγ,   increasing   glucose   uptake   and   insulin   sensitivity,   promotion   of   adipogenesis   and  
lipolysis   in   in   vitro   assays   and   changing   gut   bacterial   composition   in   mice.     First   paper  
(PAPER   I)   presented   here   concentrated   on   PPARγ   activation   by   metabolites   of   sage,  
native   to   the   Mediterranean   region   herb,   while   PAPER   V   concentrates   on   novel  
compound  isolated  from  Echinacea  purpurea  and  it’s  insulin  sensitizing  effect  in  3T3-­‐L1  
adipocytes.  Second  paper  (PAPER  II)  takes  the  advantage  of  the  whole  plant  extracts  and  
concentrates   not   only   on   PPARγ   activation   but   also   modulation   of   glucose   uptake   and  
lipid  accumulation  in  adipocytes,  myotubes  and  C.  elegans.    
  Isolated   plant   compounds,   can   be   used   in   their   naïve   form   or   can   be   further  
modified  and  introduced  modifications  can  modulate  their  potency,  and  that  was  a  main  
focus   of   third   paper   (PAPER   III).   Using   formononetin   (4'-­‐O-­‐methyldaidzein,   isoflavon  
found   in   red   clover)   as   a   scaffold,   2-­‐(2-­‐bromophenyl)-­‐   formononetin   (2BRPHF)   and   2-­‐
heptyl-­‐  formononetin  (C7F)  analogues  were  synthetized  and  tested  together  with  native  
formononetin   in   3T3-­‐L1   cells.   Finally,   in   fourth   paper   (PAPER   IV)   we   investigate   plant  
derived  compound  resveratrol  and  its  influence  on  mouse  physiology  and  ecology  of  gut  
microbiota  in  mice.  
 
 

PAPER  I  
K.   B.   Christensen,   M.   Jorgensen,   D.   Kotowska,   R.   K.   Petersen,   K.   Kristiansen,   L.   P.  
Christensen,   2010,   Activation   of   the   nuclear   receptor   PPARγ   by   metabolites   isolated  
from  sage  (Salvia  officinalis  L.),  J.  Ethnophamacology,  132(1):127-­‐33  
 
  Nuclear  receptors  are  a  class  of  proteins  with  the  ability  to  bind  directly  to  DNA  
and   regulate   expression   of   specific   genes,   when   specific   ligand   is   present   and   therefore,  
they  constitute  potential  therapeutic  targets  for  many  disorders.  
  In  this  paper  we  focused  on  PPARγ  (peroxisome  proliferator-­‐  activated  receptor  
gamma),   a   nuclear   receptor   belonging   to   the   superfamily   of   ligand-­‐   dependent  
transcription   factors.   PPARγ   is   predominantly   present   in   adipose   tissue   and   has   been  
shown   to   be   a   master   regulator   of   adipocyte   differentiation   as   well   as   glucose  
homeostasis.  PPARγ,  when  activated  by  ligand,  binds  to  DNA  and  promotes  transcription  
of   genes   involved   in   lipid   and   glucose   metabolism.   Ligands   for   PPARγ   include   fatty   acids  
and   eicosanoids   and   despite   much   research,   PPARγ   endogenous   ligand(s)   is/are   still  
unknown.   Therefore,   it   is   speculated   that   PPARγ   regulates   gene   expression   based   on  
total  concentration  of  fatty  acids.    
  TZDs  are  synthetic  full  PPARγ  agonists,  binding  to  the  receptor  pocket  with  very  
high   affinity.   Glitazones   were   introduced   as   potent   insulin-­‐   sensitizing   drugs   for   the  
treatment  of  type  2  diabetes.  However,  TZDs  may  cause  unwanted  side  effects,  such  as:  
weight   gain,   edema,   heart   enlargement   and   hepatotoxicity.   Therefore,   it   was  
hypothesized,   that   PPARγ   ligand,   acting   as   partial   (instead   of   full)   agonist   might   not  
cause  unwanted  side  effects  while  maintaining  insulin-­‐  sensitizing  activity.    
  In   this   paper   we   investigate   the   known   traditional   medicinal   plant:   Salvia  
officinalis  L.  (Lamiaceae).  Sage  is  a  widely  used  culinary  and  medicinal  herb,  especially  in  
the   Mediterranean   region.   Leaves   are   used   as   spices   and   preparations   of   sage   leaves  
have   been   used   traditionally   as   a   remedy   towards   diabetes   in   Iran   and   Morocco.  
However,   compounds   responsible   for   anti-­‐diabetic   features   of   sage   extracts   are   still  
unknown,  despite  several  studies  reporting  that  aqueous  and  ethanol  extracts  of  Salvia  
officinalis  are  decreasing  blood  glucose  in  laboratory  animals  (Alarcon-­‐  Aguilar,  F.  J.,  et  
al.,   2002,   Eidi,   M.,   et   al.,   2005).   As   PPARγ   plays   significant   role   in   regulation   of   insulin  
sensitivity,   phytochemicals   isolated   from   sage   extract   and   activating   PPARγ   might,   at  
least  partially,  explain  the  glucose  lowering  effect  of  Salvia  officinalis.  
  In  contrast  to  previous  research,  we  prepared  dichloromethane  (DCM)  extracts  
in   place   of   aqueous   or   ethanol   extracts.   Solvent   and   extraction   method   are   crucial  
parameters   for   chemical   behavior   of   lipophilic   and   hydrophilic   compounds.   Choosing  
DCM  as  extraction  solvent  over  ethanol  or  water  and  dissolving  isolated  compounds  in  
DMSO   we   could   shift   our   spectra   of   compounds   towards   lipophilic   rather   than  
hydrophilic   phytochemicals.   As   a   result   of   this   choice,   we   were   able   to   isolate   and  
identify   compounds   different   from   those   reported   in   previous   studies   (for   example,  
viridiflorol,   α-­‐linoleic   acid,   oleanolic   acid   and   manool).   Also,   20-­‐hydroxyferruginol   was  
first   time   isolated   from   Salvia   officinalis   in   this   study   and   epirosmanol   ester   of   12-­‐O-­‐
methyl   carnosic   acid   was   new.   However   whereas   none   of   these   new   compounds  
activated  PPARγ,  we  found  two  known  metabolites  able  to  significantly  activate  PPARγ  
in   transfected   cell   cultures:   α-­‐linoleic   acid   and   12-­‐O-­‐methyl   carnosic   acid,   which   may  
suggest  that  the  anti-­‐diabetic  activity  of  sage  extract  may,  to  some  extend,  be  related  to  
PPARγ  activation  pathway.    
 
 

PAPER  II  
R.  B.  El-­‐Houri,  D.   Kotowska,  L.  C.  B.  Olsen,  S.  Bhattacharya,  L.  P.  Christensen,  K.  Grevsen,  
N.   Oksbjerg,   N.   Færgeman,   K.   Kristiansen,   K.   B.   Christensen,   Screening   for   Bioactive  
Metabolites   in   Plant   Extracts   Modulating   Glucose   Uptake   and   Fat   Accumulation,  
manuscript  
 
  Adipose   tissue,   systemic   insulin   resistance   and   skeletal   muscle   are   closely  
related.  Impaired  insulin  action  and  insulin  resistance  occur  first  in  adipose  tissue  before  
the   systemic   glucose   intolerance   develops,   therefore   adipose   tissue   is   recognized   as   the  
primary   site   of   the   type   two   diabetes   origin   (Hotamisligil,   G.   S.,   2000).   However,   also  
skeletal   muscle   from   obese   patients   exhibits   insulin   resistance   in   vivo   and   insulin  
resistance   effect   is   not   present   when   muscle   cells   obtained   from   obese   subjects   are  
cultured  in  vitro.  Therefore,  it  was  hypothesized,  that  negative  crosstalk  between  excess  
of  adipose  tissue  and  skeletal  muscle  results  in  first,  altered  insulin  signaling  and  later  in  
insulin   resistance   in   skeletal   muscle.   The   communication   between   adipose   tissue   and  
muscle   occurs   through   metabolic   mediators,   including   free   fatty   acids   and   adipokines,  
such   as   TNFα,   IL-­‐6,   leptin,   TIMP-­‐1   and   adiponectin.   Thus,   insulin   resistance   in   skeletal  
muscle   is   likely   to   result   from   a   combination   of   the   endocrine   effect   of   adipokines   on  
muscle  cells  and  the  metabolic  effect  of  free  fatty  acids  on  both  tissues  (Sell,  H.,  et  al.,  
2006).    
  In  the  light  of  this  research,  the  screening  for  novel  insulin  sensitizers  could  be  
developed   based   on,   not   only   adipocyte   cells,   but   also   myocytes   and   therefore   gives  
broader  idea  about  their  mechanisms  of  action.    
   In   vitro   studies   are   very   useful   but   they   never   mimic   the   whole   organism  
environment  and  therefore  the  results  obtained  from  cell  cultures  must  be  confirm  by  in  
vivo  studies.  However,  screening  in  search  for  novel  insulin  sensitizers  requires  fast  and  
efficient  way  of  testing  new  compounds  in  vivo.  Caenorhabditis  elegans  is  perfect  model  
for  this  purpose:  it  is  a  multicellular  organism,  with  simple  but  well  organized  anatomy  
and   large   (up   to   78%)   degree   of   homology   to   human   genes   and   cell   signaling   pathways.  
Many   studies,   including   investigation   of   therapeutic   drugs   and   their   possible   side  
effects,   development   of   neurons   and   lipid   accumulation   were   performed   in   worms   as  
confirmation  of  cell  culture  studies  or  novel,  exploratory  research.    
  In   this   study   we   concentrated   on   sixteen   extracts   from   seven   plants   (spices   or  
sources   of   food)   exhibiting   promising   results   in   treatment   of   obesity   and   insulin  
insensitivity.   Plant   extracts   in   this   study   were   tested   in   three   different   setups:   in  
adipocytes,  porcine  muscle  cells  and  C.  elegans.  This  combined  approach  led  us  to  two  
DCM  extracts  (golden  root  and  thyme  DCM  extract),  which  weakly  activated  PPARγ  and  
did   not   induce   adipocyte   differentiation   in   3T3-­‐L1   cells.   Thyme   significantly   stimulated  
insulin   dependent   glucose   uptake   in   both   adipocyte   cells   and   myotubes,   while   golden  
root   significantly   increased   glucose   uptake   in   myotubes   but   not   adipocytes.   However,  
both  extracts  did  not  increase  fat  accumulation  in  C.  elegans.  Therefore  we  speculated,  
that   golden   root   and   thyme   DCM   extracts   might   contain   bioactive   compounds,  
exhibiting  anti-­‐obesity  and  insulin-­‐sensitizing  activity.  However,  more  detailed  study  will  
be  needed  to  confirm  our  findings.  
 
 

PAPER  III  
C.  Andersen*,  D.   Kotowska*,  C.  G.  Tortzen,  K.  Kristiansen,  J.  Nielsen,  R.  K.  Petersen,  2-­‐
(2-­‐bromophenyl)-­‐formononetin   and   2-­‐heptyl-­‐formononetin   are   PPARγ   partial   agonists  
and  reduce  lipid  accumulation  in  3T3-­‐L1  adipocytes,  manuscript  
*Shared  first  author  
 
  Isoflavones   are   bioactive   compounds   occurring   naturally,   that   have   been  
reported   to   have   beneficial   health   promoting   effects,   including   anti-­‐obesity   and   anti-­‐
diabetic  effects.    
  Isoflavones  are  reported  to  be  PPARγ  agonists  (Shen,  P.,  et  al.,  2006,  Chacko,  B.  
K.,  et  al.,  2007,  Mueller,  M.  M.,  et  al.,  2008)  and  several  of  them  was  shown  to  act  as  
partial   agonists   and   compete   with   full   agonist   (if   present)   for   receptor   occupancy   and  
therefore   produce   a   net   decrease   in   PPARγ   activation.   Moreover,   despite   partial  
efficacy,   they   still   retain   anti-­‐diabetic   action.   Isoflavones   have   been   reported   to   also  
have  other  potencies  like:  osteogenic  and  antioxidant  activity,  modulation  of  enzymes  in  
steroid  biosynthesis  (Barnes,  S.,  2010),  and  modulation  of  lipid  metabolism.    
  Bioactive   phytochemicals,   like   isoflavones,   can   serve   as   starting   point   for  
creation  of  new  drugs  or  diet  supplements.  Small  modifications  introduced  to,  derived  
from  natural  source,  scaffold  can  alter  the  biological  effects  of  the  naïve  compound.  One  
of   the   examples   is   an   acetylation   of   salicylic   acid   derived   from   willow   extracts.   This  
modification  increased  pain-­‐killing  ability  of  the  native  compound  and  thus  a  new  drug  
was  created  (today  commonly  known  as  aspirin)  (Mahdi,  J.  G.,  et  al.,  2006).    
  Apigenin  (4’,5,7-­‐trihydroxyflavone)   belongs   to   flavone   class   is   present   in   many  
plants,   like   chamomile,   apples,   celery,   oregano,   basil   and   parsley.   Apigenin   has   been  
reported  to  exhibit  anti-­‐inflammatory,  anti-­‐tumor  and  anti-­‐bacterial  activity  (Kim,  H.P.,  
et   al.,   1998,   Basile,   A.,   et   al.,   1999).   However,   compared   to   already   existing   drugs,  
apigenin   does   not   exhibit   strong   enough   activities.   Introduced   small   modifications  
(aminomethyl   grups   substitutions   on   the   C-­‐8   position)   significantly   improved  
antiproliferative,   antibacterial,   and   antioxidant   activities   compared   with   the   parent  
apigenin  (Liu  R.,  et  al.,  2012).  

Fig.   8.   Chemical   structure   of   native   apigenin   (on   the   left)   and   formononetin   (on   the  
right)  
 
  Apigenin   and   formononetin   share   similar   core   structure.   Moreover,   small  
modifications   of   formononetin   scaffold   (made   by   substitutions   of   bromophenyl   and  
heptyl   groups   at   the   2-­‐position),   similarly   to   apigenin,   significantly   improved   parent  
compound   bioactive   abilities.   In   our   study,   2-­‐(2-­‐bromophenyl)-­‐formononetin   (2BrPhF)  
and  2-­‐heptyl-­‐formononetin  (C7F)  decreased  lipid  accumulation  in  3T3-­‐L1  adipocytes  and  
were  acting  as  PPARΥ  partial  agonists,  while  parent  formononetin  did  not  bind  to  PPARγ  
and   increased   triglyceride   accumulation.   Analogue   of   formononetin   with   2-­‐heptyl  
substitution  (C7F)  significantly  increased  glycerol  release  and  upregulated  lipolytic  genes  
in   3T3-­‐L1   cells   while   2BrPhF   and   parent   formononetin   did   not   exploit   these   features,  
therefore   suggesting   that   substitution   of   different   type   of   groups   can   have   different  
impact  on  bioactive  features.    
 

PAPER  IV  
D.   Kotowska*,  J.  Olesen*,  M.  Hansen,  W.  A.  Al-­‐Soud,  R.  S.  Bienso,  C.  M.  Kristensen,  N.  
Brandt,  S.  A.  B.  Larsson,  L.  Hansen,  K.  Kristiansen,  H.  Pilegaard,  Resveratrol,  exercise  and  
gut  microbiota,  manuscript  
*Shared  first  author  
 
  Microbial   research   was   dependent   on   culture-­‐   based   methods   for   decades,  
which,   however   useful,   were   limited   to   cultivable   microbes.   Recent   development   of  
molecular   techniques   independent   of   culturing   provided   novel   insight   into   complex  
microbial   ecology.   Especially   high-­‐   throughput   sequencing   technologies,   like  
pyrosequencing   allowed   simultaneous   assessments   and   in-­‐depth   analysis   of   microbial  
communities  and  emerging  biological  patterns  within  these  communities.  
  Gut   bacteria   and   their   hosts   co-­‐evolved   together   and   influence   each   other  
throughout   the   life   of   the   host.   Human   intestine   is   sterile   at   birth   and   colonized  
immediately  after,  first  with  bacteria  from  the  mother  and  later  with  bacteria  present  in  
the  environment.  Early  colonization  is  very  quick  and  seems  to  be  chaotic  but  there  are  
few  patterns  emerging  in  the  recent  research.  For  example:  children  born  by  caesarean  
section  had  been  found  to  have  higher  risk  of  developing  obesity  compared  to  children  
born  through  vaginal  canal  and  this  tendency  was  associated  with  early  colonization  of  
gut  by  different  groups  of  bacteria  (Isolauri,  E.,  2012,  Backhed,  F.,  2011).  Throughout  the  
life   of   the   host,   composition   and   complexity   of   gut   bacteria   increases   until   adulthood  
and  can  be  modified  by  different  dietary  products.    
  Gut   bacteria   influence   their   host   in   many   different   ways.   They   have   profound  
effects  on  host  gene  expression  in  the  enterohepatic  system,  including  genes  involved  in  
immunity,   metabolism   and   inflammation.   Gut   bacteria,   thanks   to   their   unique   sets   of  
enzymes,   can   also   digest   components   of   the   diet   such   as   plant   polysaccharides  
otherwise   unavailable   for   the   host   organism.   Intestinal   microbiota   ecology   can   be  
influenced   by   food   components   as   well,   for   example:   mice   fed   high   fat   diet   have  
completely   changed   bacterial   composition   compared   to   chow   fed   animals.   Moreover,  
this  change  is  reversible  by  modulations  of  the  diet.  (Turnbaugh,  P.  J.,  et  al.,  2008).  
  In  this  paper  we  focused  on  changes  in  gut  bacteria  ecology  in  mice  fed  HFD  with  
addition   of   resveratrol   (polyphenolic   compound   found   in   wine,   pistachios,   grapes   and  
berries)   and   trained-­‐   exercising.   Resveratrol   is   well-­‐studied   compound   and   its   anti-­‐
obesity  properties  have  been  described  (Baur,  J.  A.,  et  al.,  2006).  Also,  the  contribution  
of   bacteria   in   digestion   of   resveratrol   was   reported   by   Bode,   L.   M.   in   2013.   However,  
little  is  known  about  the  impact  of  resveratrol  on  the  gut  microbiota  and  therefore  it  is  
difficult  to  correlate  observed  physiological  shifts  of  mice  fed  HFD  with  supplementation  
of   resveratrol   towards   physiology   of   lean   mice   fed   standard   chow   diet.   However,   in  
agreement  with  Baur,  J.  A.  (Baur,  J.  A.,  et  al.,  2006),  we  did  not  observe  any  significant  
changes   in   intestinal   microbiota   composition   and   therefore   we   speculated,   that  
resveratrol  does  not  interfere  with  bacterial  ecology  of  the  intestine  in  mice.  
  Not   only   diet,   but   also   physical   activity   is  a   key   environmental   contributor   that  
should   be   taken   under   consideration   when   trying   to   understand   the   link   between   gut  
microbiota  and   obesity  (Kallus,  S.   J.,  et   al.,   2012).  Exercise  training  has   been   shown   to  
promote  weight  loss  even  when  animals  were  fed  HFD  (Bradley,  R.  L.,  et  al.,  2007;  Yan,  
L.,   et   al.,   2012).   However,   role   of   gut   microbiota   in   interconnecting   health   beneficial  
effect  of  exercise  and  host  physiology  is  not  well  elucidated.    
  In  our  study,  mice  fed  HFD  and  trained-­‐  exercise  did  not  lose  weight  compared  to  
sedentary,   HFD   fed   animals.   Therefore,   our   study   stays   in   contradiction   to   Bradley,   R.   L.  
(Bradley,  R.  L.,  et  al.,  2007)  research.  However,  to  our  surprise,  there  was  a  visible  and  
clear   shift   in   ecology   of   trained-­‐   exercised   animals’   gut   bacteria   and   this   shift   was  
towards   bacteria   of   chow   fed   mice.   Therefore   we   concluded,   that   diet   and   physical  
activity   determined   the   composition   of   murine   microbiome   independently   of   obese  
state   and   the   changes   may   be   observed   on   the   level   of   genus   and   class.   However,   exact  
link   between   changes   in   bacterial   flora   and   exercise   training   cannot   be   established  
based  on  the  present  findings  and  future  work  is  required  to  elucidate  this.  
 
   

PAPER  V  
 D.  Kotowska*,  R.  B.  El-­‐Houri*,  K.  B.  Christensen,  X.  Frette,  K.  Grevsen,  L.  P.  Christensen,  
K.   Kristiansen,   Novel   PPARγ   partial   agonist   isolated   from   Echinacea   purpurea   exploits  
insulin  sensitizing  effect  in  3T3-­‐L1  adipocytes,  manuscript  
*Shared  first  autor  
 
  In  search  for  novel,  bioactive  phytochemicals,  extracts  of  plants  commonly  used  
in  folk  medicine  are  examined.  However,  whereas  screening  for  major  phytochemicals  is  
useful  and  fast,  the  major  phytochemicals  are  not  always  the  ones  responsible  for  the  
beneficial  effects  of  extract.  Bioassay  guided  fractionations-­‐  based  screening  platforms  
have   an   advantage   of   focusing   on   active   fractions   and   compounds,   which   are   not  
necessarily   overrepresented   in   crude   extracts.   Moreover,   synergistic   effects   of   two   or  
more   compounds   may   occur   and   will   be   missed   if   simple   screening   for   major  
compounds  is  performed.      
  A   bioassay   guided   fractionation-­‐based   platform   was   developed   for   a   study,  
researching   adipogenic   constituents   from   the   bark   of   Larix   laricina   du   Roi   and   in   the  
study   researching   the   anti-­‐adipogenic   activities   of   two   extracts   isolated   from   Alnus  
incana   and   Populus   balsamifera   barks   (Shang,   N.,   et   al.,   2012,   Martineau,   L.   C.,   et   al.,  
2010).   Use   of   this   platform,   when   crude   extract,   its   fractions   and   purified   from   them  
compounds   were   tested   alongside   in   different   assays,   led   to   discovery   of   new  
cycloartane  triterpentoid  (23-­‐oxo-­‐3alpha-­‐hydroxycycloart-­‐24-­‐en-­‐26-­‐oid  acid)  among  the  
other  9  compounds.  Interestingly,  23-­‐oxo-­‐3alpha-­‐hydroxycycloart-­‐24-­‐en-­‐26-­‐oid  acid  was  
not   the   main   component   of   the   extract   and   still   seemed   to   be   responsible   for  
adipogenic  activity  of  the  fraction  it  was  isolated  from  (Shang,  N.,  et  al.,  2012).  
    Bioassay  screening  platform,  which  served  as  a  base,  for  the  platform  used  in  our  
study,  was  used  by  Christensen,  K.  B.,  et  al.,  (Christensen,  K.  B.,  et  al.,  2009)  to  screen  
plant   extracts   for   their   anti-­‐diabetic   and   anti-­‐obesity   properties.   Among   others,  
Echinacea   purpurea   DCM   extract   was   found   to   activate   PPARγ   and   increase   insulin  
dependent   glucose   uptake   while   not   promoting   adipogenesis   in   Christensen’s   study,  
which  stays  in  agreement  with  the  study  presented  in  this  paper.    
  Echinacea   extracts   from   three   different   species   of   this   plant   (E.   pallida,   E.  
augustifolia  and  E.  purpurea)  are  used  commonly  for  treatment  and  prevention  of  upper  
respiratory  track  infections  and  preparations  are  made  from  both  roots  and  aerial  parts.  
Main   bioactive   metabolites   of   Echinacea   are   polysaccharides,   caffeic   acid   derivatives  
and   alkamides   (Spelman,   K.,   et   al.,   2009,   Starvaggi   Cucuzza,   L.,   et   al.,   2008,   Christensen,  
K.  B.,  et  al.,  2009,  Goey,  A.  K.  L.,  et  al.,  2012).  However,  little  is  known  about  Echinacea  
and  Echinacea  derived  compounds  in  treatment  of  obesity  and  diabetes.  In  this  study,  
using   bioassay   guided   fractionations,   we   decided   to   investigate   an   effect   of   Echinacea  
purpurea   DCM   extract,   fractions   and   isolated   compound   on   adipocyte   differentiation  
and   glucose   uptake   in   3T3-­‐L1   adipocytes   and   try   to   reveal   mechanism   of   action   of  
purified  compound.  Our  approach  led  us  to  the  discovery  of  new  compound,  belonging  
to  the  group  of  alkamides:  dodeca-­‐2E,4E,8Z,10E/Z  tetraenoic  acid-­‐2-­‐methylbutylamides  
(Compound   X).   Compound   X   significantly   increased   basal   and   insulin   dependent   glucose  
uptake   in   3T3-­‐L1   adipocytes   in   dose   dependent   manner   and   promoted   adipocyte  
differentiation   in   maturing   adipocytes,   acting   as   partial   PPARγ   agonist.   Therefore,   we  
speculated,  that  Compound  X  exploits  anti-­‐diabetic  and  insulin  sensitizing  effect  in  vitro  
and  future  study  should  follow  into  testing  in  vivo  in  laboratory  animals.  
 
 

OTHER  PAPERS  
E.  Sokol,  R.  Almeida,  H.  K.  Hannibal-­‐Bach,  D.  Kotowska,  J.  Vogt,  J.  Baumgart,,  R.  Nitsch,  
O.  N.  Jensen,  J.  Knudsen,  C.  S.  Ejsing1,  Quantitative  profiling  of  lipid  species  by  normal-­‐
phase  liquid  chromatography,  chip-­‐based  nanoelectrospray  ionization  and  hybrid  ion  
trap-­‐orbitrap  mass  spectrometry,  manuscript  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.1  Concluding  remarks  
  The   treatment   of   obesity   and   related   disorders   usually   begins   with   dietary   and  
behavioral   modification   with   the   aim   of   reducing   caloric   intake   and   increase   energy  
expenditure.  However,  in  the  light  of  research  presented  in  this  thesis,  diet  (especially  
Mediterranean   type,   rich   in   fruits   and   vegetables)   can   not   only   be   used   as   a   way   to  
reduce   energy   intake,   but   also   as   a   source   of   health   promoting   compounds.   Bioactive  
compounds   from   dietary   products   may   influence   adipocytes   through   several  
mechanisms,   including:   suppression   of   adipocyte   differentiation   and   proliferation   or  
induction   of   apoptosis   in   mature   adipocytes,   inhibition/induction   of   fat  
absorption/release   of   triglycerides   or   induction/suppression   of   glucose   uptake   and  
therefore  facilitate  weight  loss  or  gain.    
  However  well  the  idea  of  therapeutic  food  sounds,  it  is  not  easy  to  achieve.  Food  
derived   compounds   are   first   digested   by   host   organism,   before   being   processed   by  
bacteria   living   in   the   guts.   Microbiota,   with   their   unique   sets   of   enzymes,   are   able   to  
cleavage  the  chemical  bonds,  animals  cannot  cut  and  make  available  for  host  organism  
complex   phytochemicals,   which   otherwise   cannot   be   absorbed   to   bloodstream.  
Bacterial  actions  also  introduce  changes  to  plant  derived  compounds  structures,  which  
can  increase  health-­‐  promoting  features  of  compounds  if  only  by  making  them  available  
for  the  host.  
  Dietary  intake  and  bacteria  are  interacting  with  each  other:  bacteria  are  able  to  
modify   delivered   compounds   and   compounds   are   able   to   influence   microbial   ecology,  
mostly   by   promoting   growth   of   selected   taxa   over   the   others   and   therefore   modulate  
the  quality  (and  amounts)  of  energy  intake.  
  The  net  of  processes  and  interplay  between  phytochemicals,  bacteria  and  host  is  
very  complex  and  still  not  fully  understood.  According  to  today’s  knowledge  it  is  clear,  
that  compounds  mechanism  of  action  and  their  fate  within  the  body  are  as  important  as  
identification   of   novel   phytochemicals.   The   work   presented   in   this   thesis   focuses   on  
initial   steps   of   compound   identification   and   elucidate   probable   molecular   mechanisms  
underlying   the   beneficial   effects   of   investigated   plants   and   therefore   tries   to   give   a   new  
meaning  to  common  adage:  “You  are  what  you  eat”.  
 
 
 
 
 
 
 
 
 
 
 
 
 
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Annex