STABILITY OF ANTHOCYANINS FROM COMMERCIAL BLACK CURRANT JUICE UNDER SIMULATED GASTROINTESTINAL DIGESTION Introduction

Anthocyanins are eff ective antioxidants but they have also been proposed to have other biological activities independent of their antioxidant capacities that produce health benefi ts. Examples range from inhibition of cancer cell growth in vitro, induction of insulin production in isolated pancreatic cells, reduction of starch digestion through inhibition of a-glucosidase activity, suppression of infl ammatory responses as well as protection against age-related declines in cognitive behavior and neuronal dysfunction in the central nervous system. However, to achieve any biological eff ect in a specifi c tissue or organ, anthocyanins must be bioavailable; i.e. eff ectively absorbed from the gastrointestinal tract (GIT) into the circulation and delivered to the appropriate location within the body. In this study, we assess the stability of anthocyanins from commercial Black currant (Ribes nigrum L.) juice using an in vitro digestion procedure that mimics the physiochemical and biochemical conditions encountered in the gastrointestinal tract (GIT). Th e main objective of this work was the evaluation of stability of anthocyanins during in vitro digestion in gastric and intestinal fl uid regarding whether appropriate enzyme (pepsin or pancreatin) was added or not. Anthocyanins present in commercial black currant juice remain stable during in vitro digestion in gastric fl uid regardless whether pepsin was added into the medium or not. Also, they remain stable during in vitro digestion in simulated intestinal fl uid without pancreatin. Th e stability studies of anthocyanins in the intestinal fl uid containing pancreatin indicated reduced stability, which also mainly contribute to slight reduction of total anthocyanins content (-,) in commercial black currant juice.


Introduction
Anthocyanins belong to the flavonoid group of polyphenolic compounds and are responsible for the red, purple and blue hues present in plant organs such as fruits, fl owers, grains and leaves as well as in products made from those sources ().Pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin are the six common anthocyanidins found in nature.Th eir structures may be altered by glycosidic substitution (glucose, galactose, rhamnose, xylose, and arabinose) at the  and  positions in the A and C rings.Th ey are industrially important natural food colorings ().Additional variations occur by acylation of the sugar groups with acids.Acetic acid, p-coumaric acid, caff eic acid, malonic acid, sinapic acid, ferulic acid, oxalic acid, and succinic acid are some of the commonly found acylating groups (, ).
Anthocyanins are eff ective antioxidants (, ) but they have also been proposed to have other biological activities that are independent of their antioxidative capacities and produce health benefi ts.Examples range from inhibition of cancer cell growth in vitro (), induction of insulin production in isolated pancreatic cells (), reduction of starch digestion through inhibition of a-glucosidase activity (), suppression of infl ammatory responses () as well as protection against age-related declines in cognitive behavior and neuronal dysfunction in the central nervous system ().
However, to achieve any biological eff ect in a specifi c tissue or organ, anthocyanins must be bioavailable; i.e. effectively absorbed from the gastrointestinal tract (GIT) into the circulation and delivered to the appropriate location within the body.Oral intake of anthocyaninrich fruits, extracts or pure anthocyanins has proved to have benefi cial eff ects in preventing or suppressing ailments in vivo (, ).Studies of oral administration of anthocyanins have confi rmed increased antioxidant status of the serum (, , ) but this is usually accompanied by very low uptake of anthocyanins into the serum (< of dose) (, , )) and correspondingly low levels of urinary excretion as intact or conjugated forms.Th e apparent low bioavailability of anthocyanins seems to cast doubt on their ability to exert their proposed benefi cial eff ects throughout the body.
Assessment of true bioavailability of any class of phytochemicals requires data concerning their absorption, metabolism, tissue and organ distribution and excretion ().Such stud-ies carried out in animals or human subjects are complex, expensive and raise moral and ethical questions.The relative stability of the anthocyanins under GIT conditions is crucial for the bioavailability of these compounds, as it will determine the pool size for whatever active mechanisms are present in the stomach () or the small intestine ().
In this study, we assess the stability of anthocyanins from Black currant (Ribes nigrum L.) juice using an in vitro digestion procedure that mimics the physiochemical and biochemical conditions encountered in the gastrointestinal tract (GIT).The main objective of this work was the evaluation of stability of anthocyanins during in vitro digestion in gastric and intestinal fluid regarding whether appropriate enzyme (pepsin or pancreatin) was added or not.

Samples
Juice sample, that was not blend of different fruits, and that represented an array of anthocyanin containing products available at a local market was purchased, brought to the laboratory, aliquoted and stored at -oC until analyzed.

Chemicals and Reagents
Th e used reagents were all of analytical grade, unless otherwise stated.Glacial acetic acid (), orthophosphoric acid () and sodium chloride were provided by Carlo Erba; monobasic potassium phosphate, concentrated hydrochloric acid () and sodium hydroxide were provided by Merck.Acetonitrile was HPLC grade (Sigma-Aldrich), pepsin from hog stomach (Fluka), pancreatin from porcine pancreas (Sigma).

In vitro digestion procedure
The diluted black currant juice was subjected to successive in vitro gastric and pancreatic digestion, following the procedure: TEST : An aliquot of juice was diluted with hydrochloric acid solution, supplemented with sodium chloride and adjusted to pH , ( ml of juice +  ml hydrochloric acid solution at pH , + NaCl); chromatograms were analyzed before and after dissolution procedure ( hour  TEST : An aliquot of juice was diluted with phosphate buffer at pH , ( ml of juice +  ml of phosphate buffer at pH ,); chromatograms were analyzed before and after dissolution procedure ( hours at C, paddle method; stirring speed  rpm) TEST : An aliquot of juice was diluted with phosphate buffer at pH , and pancreatin ( ml of juice +  ml of phosphate buffer at pH , + pancreatin); chromatograms were analyzed before and after dissolution procedure ( hours at C, paddle method; stirring speed  rpm) Th e tests were performed using USP apparatus , Van Kel VK  dissolution tester (Van Kel, Cary).Th e dissolution apparatus was maintained at °C throughout the experiment.Test samples were fi ltered using a , μm nylon syringe fi lters for HPLC (Cronus).All tests were performed in triplicate.Prior to use, the dissolution media and juice samples were also equilibrated at °C.Samples were drawn at the following points: at the beginning (immediately thereafter stirring was started) and at the end of test procedure (after one hour for testing in gastric conditions with/without pepsin added and after two hours for testing in intestinal conditions with/without pancreatin added).

HPLC analysis
Chromatographic analysis (gradient HPLC analysis) of anthocyanins was done according to the method of Lee at all.() using a Zorbax StableBond-C column (mm×, mm× μm).Th e analysis was performed on an HPLC system composed of a pump, injection valve, autosampler and variable (DAD) wavelength detector (Shimadzu).Mobile phase A was  acetonitrile, while mobile phase B consisted of  phosphoric acid and  acetic acid (glacial) (v/v) in deionized water.Th e material used as a reference sample was native juice prepared from fresh black currant fruits using pressure procedure.Th e program was as follows: - min linear gradient from  to  B;  - min linear gradient from  to  B;  - min linear gradient from  to  B and - min linear gradient  B. Th e fl ow rate was , ml/min, the injection volume  μl, the column temperature  o C and the detection wavelength  nm.
Wavelength selection was in accordance with maximum absorption for anthocyanins.Media was selected with the intention to determine possible influence of gastric and intestinal fluids on stability of anthocyanins in acidic/basic conditions in presence/ absence of digestive enzymes (pepsin and pancreatin).
ALIJA UZUNOVIĆ, EDINA VRANIĆ: STABILITY OF ANTHOCYANINS FROM COMMERCIAL BLACK CURRANT JUICE UNDER SIMULATED GASTROINTESTINAL DIGESTION

Results and Discussion
Stability of anthocyanins in commercial black currant juice was analyzed on the basis of four characteristic peaks which were observed in native black currant juice chromatogram (Figure .).The native juice was prepared from fresh black currants using pressure procedure.This chromatogram was used as fingerprint chromatogram of black currant juice.The same peaks were observed in chromatograms of the analyzed commercial juice, which conveniently served as identity confirmation as well.
Total anthocyanins content and percentage changes during in vitro digestion of black currant juice are presented in Table .
Th e analysis of content of four basic anthocyanin components in commercial black currant juice in simulated gas-tric fl uid without pepsin indicated slight increase which is in accordance with previously published data ().Decreased content was noted for anthocyanin components  and  (-, and -,) after in vitro digestion in intestinal fluid containing pancreatin.
When the intestinal fluid without pancreatin was used, increased content of anthocyanin components  and  (, and ,) could be determined.The effective content reduction of anthocyanin components  and  is -, and -,, respectively, and does not represent significant reduction as it was perceived in some previously published studies ().
Negative level of anthocyanin components content (T -T) was perceived for components  and  (-, and -,) in intestinal fluid containing pancreatin, regardless of the fact that if we consider them individually, in media at pH , with/ without enzyme addition, the content of these components increased.

Conclusion
◊ Anthocyanins present in commercial black currant juice remain stable during in vitro digestion in gastric fl uid regardless whether pepsin was added into medium or not; ◊ Anthocyanins present in commercial black currant juice remain stable during in vitro digestion in simulated intestinal fl uid without pancreatin; ◊ Th e stability studies of anthocyanins in the intestinal fl uid containing pancreatin indicated reduced stability, primarily of anthocyanin components  and ; which also mainly contribute to slight reduction in total anthocyanins content (-,) in commercial black currant juice.
at C, paddle method; stirring speed  rpm) ALIJA UZUNOVIĆ, EDINA VRANIĆ: STABILITY OF ANTHOCYANINS FROM COMMERCIAL BLACK CURRANT JUICE UNDER SIMULATED GASTROINTESTINAL DIGESTION EST : An aliquot of juice was diluted with hydrochloric acid solution, supplemented with sodium chloride and pepsin and adjusted to pH , ( ml of juice +  ml of hydrochloric acid solution at pH , + NaCl+ pepsin); chromatograms were analyzed before and after dissolution procedure ( hour at C, paddle method; stirring speed  rpm)

TABLE 1 .
Total anthocyanins content and percentage changes during in vitro digestion of black currant juice UZUNOVIĆ, EDINA VRANIĆ: STABILITY OF ANTHOCYANINS FROM COMMERCIAL BLACK CURRANT JUICE UNDER SIMULATED GASTROINTESTINAL DIGESTION ALIJA