European Food Research and Technology

, Volume 234, Issue 1, pp 173–186 | Cite as

Comparative study of cytotoxic and cytoprotective activities of cocoa products affected by their cocoa solids content and bioactive composition

  • Ana Belščak-CvitanovićEmail author
  • Ksenija Durgo
  • Tea Gačina
  • Dunja Horžić
  • Jasna Franekić
  • Draženka Komes
Original Paper


Polyphenolic profile and antioxidant properties of water extracts of milk, semisweet and dark chocolates, as well as cocoa liquor, were determined and examined for potential biological activity. Non-fat cocoa solids (NFCS), phenolic compounds content and the antioxidant capacity of cocoa product extracts were determined using UV/Vis spectrophotometric methods and HPLC analysis. The increase in NFCS was consistent with the increase in polyphenol content and antioxidant properties. Methylxanthines, theobromine and caffeine constituted the most abundant bioactive compounds, followed by flavan-3-ols epicatechin and procyanidin B2. Cytotoxic and antioxidative/prooxidative effects of cocoa product extracts were determined on human laryngeal carcinoma cell line (HEp2). Cocoa liquor containing the highest NFCS exhibited the lowest HEp2 cell viability, while milk chocolate characterized by the lowest NFCS exhibited no cytotoxic effect. Experiments revealed a strong relationship between the type of product/concentration/time of exposure and antioxidant/prooxidant character of cocoa products. Lower concentrations of semisweet, dark chocolate and cocoa liquor induced an increase in ROS formation, while the higher concentrations resulted in a decrease in ROS formation when compared with control (growth medium). Principal component analysis of the obtained results revealed specific grouping of samples (milk chocolate and cocoa liquor), while the observed dispersions indicated that the outcome of cytotoxic and cytoprotective activities of cocoa products are greatly affected by their concentration.


Antioxidant activity Cocoa Cytotoxicity Non-fat cocoa solids Polyphenols Principal component analysis 



Non-fat cocoa solids


Human laryngeal carcinoma cell line


Reactive oxygen species


Total phenol content


Total flavonoid content




2,2′-Azino- bis(3-ethylbenzthiazoline-6-sulphonic acid)diammonium salt



proc B2

Procyanidin B2


Gallic acid


Caffeic acid


Gallic acid equivalents


Catechin equivalents


Cyanidin chloride equivalents


Principal component analysis


Limit of detection


Limit of quantification



This work was supported by the Ministry of Science, Education and Sports, Republic of Croatia projects Nr. 058-0000000-3470 and 058-0582261-2246. The authors are grateful to Croatian food industry Zvečevo d.d. for supplying the cocoa product samples used in the study and to the Rudjer Boskovic Institute for providing the human laryngeal carcinoma (HEp2) cells.

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Ana Belščak-Cvitanović
    • 1
    Email author
  • Ksenija Durgo
    • 1
  • Tea Gačina
    • 1
  • Dunja Horžić
    • 1
  • Jasna Franekić
    • 1
  • Draženka Komes
    • 1
  1. 1.Department of Food Engineering, Faculty of Food Technology and BiotechnologyUniversity of ZagrebZagrebCroatia

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