anthocyanins , as ubiquitous constituents of berries and coloured vegetables , are widespread 
 in the plant dominion .
much attention has been paid in recent 
 years to their radicals scavenging activity in vitro and in vivo 
 and possible health benefits .
the main anthocyanins being 
 analysed in elderberries are various glucosylated cyanidins 
 ( figure 1 ) . besides glucose , sambubiose is the 
 typical sugar conjugate in elderberries [ 1 , 
 2 ] .
rate and 
 extent of absorption , metabolisation , and excretion of elderberry 
 anthocyanins are to date not fully understood .
first of all , 
 controversially discussed hitherto is whether also conjugated 
 glucuronides of cyanidin are formed and excreted in vivo .
time - course plots ( mean  sd ) of total anthocyanins 
 ( glucosides + glucuronides ) and anthocyanin glucuronides in human 
 urine following ingestion of 150  ml of elderberry juice .
individual amounts excreted   of total anthocyanins ( glucosides + glucuronides ) and 
 anthocyanin glucuronides within 5 hours following ingestion of 150  ml of elderberry juice .
urinary excretion of anthocyanins as unchanged glucosides 
 and glucuronides after elderberry juice consumption ( means  sd ) .
calculated as the ratio of amounts excreted ( within 5  h ) to 
 amounts / doses ingested .
the study protocol was approved by the ethics 
 committee of the giessen university , germany .
after an overnight 
 fasting , seven healthy nonsmoking volunteers ( 6 female and 1 male 
 with a mean bmi of 21.5 ) consumed a bolus quantity of 
 150  ml of concentrated elderberry juice ( containing 
 3.57  g of total anthocyanins ) together with rolls and 
 cheese
. the elderberry juice concentrate was obtained from wild 
 ( heidelberg , germany ) .
urine samples were collected initially and 
 in hourly intervals over a period of 5 hours .
hplc - dad analyses of urinary samples ( 05 hours after ingestion ) 
 were performed before and after hydrolysis of glucuronide 
 conjugates with -glucuronidase [ 3 , 
 4 , 5 , 
 6 ] .
the 
 anthocyanins were extracted by using a solid phase extraction 
 cartridge ( sep - pakvac 12 , waters , milford , mass ) .
the cartridge 
 was first washed with 5  ml methanol and 
 equilibrated with the same volume of 5% aqueous formic acid . 

seven  ml urine samples diluted with 2  ml formic acid 
 were applied to the equilibrated cartridge . after washing with 
 5  ml of 5% formic acid , the anthocyanins were eluted with 5% 
 formic acid in 5  ml methanol .
the eluate was evaporated to 
 dryness in a vacuum rotary evaporator at 30c and the 
 extract redissolved with 200  l mobile phase before hplc 
 analysis .
the chromatographic conditions adopted from netzel et 
 al   comprised separation on a prontosil eurobond rp-18 
 ( 5  m , 250  4  mm i d , bischoff , 
 leonberg , germany ) , protected by a guard column ( lichrospher 100 
 rp-18 , 4  4  mm , merck , germany ) , and 
 isocratic elution with water / formic acid / acetonitril 
 ( v / v / v ) = 81/10/9 ( flow rate 
 0.5  ml / min ) by using a high - precision pump ( model l-6200 , 
 merck - hitachi , darmstadt , germany ) .
the cyanidin glucosides were 
 detected at 520  nm with the aid of a uv - vis detector ( l-4200 , 
 merck - hitachi ) .
cyanidin glucosides excreted were identified by spiking blank 
 urine samples with authentic compounds and comparing the 
 retention time in the hplc analysis and uv - visible spectra . 

standard curves were prepared for quantification prior to the 
 preparation procedure . the detection limit ( s / n  10 ) was between 
 0.13  ng and 0.60  ng / injection volume ( 100  l ) . 

aliquots of each urine sample were acidified with formic acid 
 ( 2  ml/0.2  ml ) and stored frozen at 80c until 
 analysis .
the urinary excretion of total 
 anthocyanins ( unchanged cyanidin glucosides and their glucuronide 
 conjugates ) within 5 hours was 0.053  0.030% of the 
 administered dose .
only 6.2 2.2% of the excreted amount 
 consisted of glucuronides . based on this recovery ,
the percentage 
 of glucuronides in urinary excretion was 0.003  0.001% 
 ( calculated as the ratio of anthocyanin glucuronides excreted to 
 anthocyanin glucosides ingested ) .
the excretion pattern of total 
 anthocyanins in figure 2 demonstrates that a maximal 
 excretion rate is reached 1 hour after intake , followed by a 
 rapid drop to initial values about 5 hours after intake , 
 resembling a first - order excretion kinetics .
the metabolic conversion of anthocyanins within the organism 
 remains to be elucidated . in principle , glucuronidation or 
 conjugation with sulphuric acid is a common final metabolic step 
 to facilitate urinary excretion .
tsuda et 
 al   found no cyanidin glucuronides in livers and kidneys of 
 rats after oral administration of cyanidin 3-glycoside , but 
 cyanidin was converted to peonidin and protocatechuic acid .
a different situation seems to exist to date in man . in the 
 urinary excretion of elderly women ,
minor amounts of glucuronides 
 of peonidin and cyanidin 3-glucoside could be deteced in half of 
 the volunteers besides unchanged glucosides after elderberry 
 consumption , which does correspond to our results [ 2 , 
 5 ] . 

only glucosides of elderberry anthocyanins , however , could 
 be found in plasma and urine of 
 volunteers by   other authors [ 8 , 
 9 ] .
to entirely estimate the extent of glucuronidation of 
 anthocyanins as metabolic fate , it has to be considered that 
 besides urinary excretion , biliary secretion may also serve as a 
 possible way of elimination , particularly known for glucuronides . 

newer studies , however , revealed that after intake of elderberry 
 extract , the identical pattern of glucosylated cyanidins could be 
 detected in plasma as in urine .
thus , the results of the 
 quantification of excretion products in the present study 
 demonstrate that , at least in the given dose range , glucuronidation 
 of cyanidin obviously represents a negligible conversion step in 
 the metabolism of cyanidin ingested from elderberries .
the 
 proportion of glucuronide conjugates seems to represent a rather 
 constant but very small proportion despite interindividual 
 differences of total anthocyanin excretion ( figure 3 )
. 
 this may be in contrast to other fruits such as strawberry 
 anthocyanins , being predominantly excreted in urine as glucuronides 
 besides small amounts of sulfoconjugates .
it remains to show the extent to which the administered dose level does 
 determine the site of metabolism .
there exists some body of 
 evidence that large doses of polyphenols are primarily 
 metabolised in the liver , whereas small doses may be metabolised 
 by the intestinal mucosa , with the liver playing a secondary role 
 to further modify the polyphenol conjugates .
 in a pilot study with 6 females and 1 male , the 
 metabolism of various cyanidin glucosides after oral 
 administration of elderberry juice was investigated . 
 the 
 anthocyanin metabolites were detected in urinary excretion . 
 after 
 ingestion of a bolus quantity of 3.57  g total anthocyanins 
 in a 150  ml elderberry juice concentrate , 0.053% of the 
 administered dose was excreted in urine as glucosidically bound 
 cyanidins within the first 5 hours . 
 only 0.003% of 
 the ingested anthocyanin glucosides was excreted as cyanidin 
 glucuronide , suggesting that this conversion step might be of 
 minor importance in urinary excretion . 
INTRODUCTION
STUDY DESIGN
SAMPLE EXTRACTION AND HPLC ANALYSIS
RESULTS AND DISCUSSION