1. Safety assessment and antioxidant activity of sodium copper chlorophyllin
Goran Gajski
Institute for Medical Research and Occupational Health
Mutagenesis Unit
Zagreb, Croatia
Zagreb, 2013

2. ••• Background
Chlorophyllins are derivatives of natural green pigment chlorophyll in which the central magnesium atom is replaced by other metals, such as cobalt, copper or iron.
Sodium copper chlorophyllin (CHL) is a semi-synthetic mixture of water-soluble sodium copper salts derived from chlorophyll.
CHL is used as a colour additive in foods, drugs, cosmetics and as a dietary supplement.

3. Fig 1. Chemical structures of some chlorin-based compounds that may be found in commercial preparations of sodium copper chlorophyllin.

4. ••• Background
Although both chlorophyll and copper chlorophyllin are allowed as food colorants in Europe (EC, 1994) and Brazil (ANVS, 2008); the latter is permitted only in one type of citrus drink in the United States (FDA, 2002).

5. ••• Aim Impact of CHL towards human cells
human peripheral blood lymphocytes
Impact of CHL on DNA molecule
Free radical scavenging capacity of CHL

6. Formula:
C34H31CuN4Na3O6
Molecular Weight:
724.15
Purity:
≥99%
Fig 2. Structure of chlorophyllin sodium copper salt.

7. ••• Methods Cytotoxicity assay Comet assay Micronucleus assay
staining with AO and EtBr
Comet assay
DNA damage
Micronucleus assay
micronuclei, nucleoplasmic bridges, nuclear buds
DPPH assay
free radical scavenging ability

8. ••• Results A B B
Fig 3. The effects of CHL on the viability and DNA damage in HPBLs (A) The cell viability was determined by differential staining with AO and EtBr after the exposure to different concentrations of CHL (0.1 to 100 µg/ml) for 4 and 24 h. (B) DNA damage was assessed with the alkaline comet assay and is expressed as percentage of tail DNA. Note: there were no statistically significant differences between treated samples compared to corresponding control (P<0.05).

9. ••• Results A B C D
Fig 4. Parameters of cytokinesis-block micronucleus (CBMN) assay. Number of (A) micronuclei, MNi; (B) micronucleated cells, MNed; (C) nucleoplasmic bridges, NPBs; and (D) nuclear buds, NBUDs in binucleated HPBLs exposed to CHL (0.1 to 100 µg/ml) for 4 and 24 h. Incidence of MNi, MNed, NPBs and NBUDs was evaluated by analyzing 1000 binucleated cells. Note: there were no statistically significant differences between treated samples compared to corresponding control (P<0.05).

10. ••• Results Antioxidant EC50 (μM) Ascorbic acid 11.06 Trolox 13.40 CHL
21.06
BHT
91.23 B A B
Fig 5. The antioxidant activity of CHL assessed with DPPH assay (A) The antioxidant activity/DPPH free radical scavenging activity of CHL in concentration range (1.0 to 30 μM) was concentration-dependent (B) Values of EC50 index of different antioxidants (L-ascorbic acid (vitamin C), Trolox (water-soluble derivative of vitamin E) and buthylated hydroxytoluene (BHT) - as reference compounds ) assessed with DPPH assay.

11. ••• Conclusions
CHL in concentration range tested (0.1 to 100 μg/ml) was not cyto/genotoxic to HPBLs.
DPPH assay confirmed that CHL has a marked capacity to scavenge DPPH· free radical indicating that CHL acts as a free radical scavenger probably due to its facile electron-donating ability.
CHL could be a perfect candidate for testing as a novel antioxidant.

12. ••• Future prospects
Since there is wide variation in the health-related biological activities of CHL that may arise from differences in the degree of purity and composition of the commercial grade CHL preparations there is a need for the standardisation of CHL.
Further investigations are needed to confirm and to determine the most effective CHL dose ranges as well as the existence of possible adverse effects.
There is also an urge for the development of a simplified method for the characterization of CHL preparations for lab and field use.

13. ••• Acknowledgement Ivana Novak, PhD Ana-Marija Domijan, PhD
Institute for Medical Research and Occupational Health
Toxicology Unit
Zagreb, Croatia
Ana-Marija Domijan, PhD
University of Zagreb
Faculty of Pharmacy and Biochemistry
Department of Pharmaceutical Botany
Marko Gerić and Vera Garaj-Vrhovac, PhD
Mutagenesis Unit

14. Thank you for your attention!!!