LC-MS and GC-MS analyses reveal that OxiCyan® is characterized by the presence of the typical chemical constituents of spirulina and bilberry. A direct comparison of OxiCyan® with the plant extracts of spirulina and bilberry shows that spirulina contributes mainly to the phycobiliproteins and fatty acid content, whereas bilberry provides the anthocyanin moiety (Table 1)

Table 1. Chemical composition of OxiCyan®, Spirulina platensis (Spirulina) and Vaccinium myrtillus (Bilberry) extracts. Values are expressed as mg g-1 d. wt. (± standard deviation). n.d. = not detectable (below the detection limit). In the same row, different letters indicate significant (P < 0.05) differences.

Phycocyanin49.70a ± 1.97256.12b ± 9.26n.d.
Allophycocyanin32.12a ± 1.7444.12b ± 1.80n.d.
Phycoerithrin29.36a ± 1.2721.79b ± 1.13n.d.
Total Phycobiliproteins111.18a ± 1.58322.03b ± 3.85n.d.
Total Anthocyanins10.24a ± 0.85n.d.261.25b ± 10.94
Total Unsaturated Fatty Acids14.39a ± 1.9912.18a ± 0.282.49b ± 0.34
Total Saturated Fatty Acids18.35a ± 0.5820.00a ± 0.030.82b ± 0.04

With regards anthocyanins, OxiCyan® is characterized by the presence of delphinidin, cyanidin, petunidin and peonidin glycosides (Figure 1).


Figure 1. HPLC chromatogram of OxiCyan® anthocyanins. A, Delphinidin-3-O-galactoside; B, Delphinidin-3-O-glucoside; C, Delphinidin-3-O-arabinoside; D, Cyanidin-3-O-galactoside; E, Cyanidin-3-O-glucoside; F, Cyanidin-3-O-arabinoside; G, Petunidin-3-O-galactoside; H, Petunidin-3-O-glucoside; I, Petunidin-3-O-arabinoside; L, Peonidin-3-O-galactoside; M, Peonidin-3-O-glucoside; N, Peonidin-3-O-arabinoside.

The lipid composition of OxiCyan® is characterized by the presence of both saturated and unsaturated fatty acids. The unsaturated fatty acid contribution to OxiCyan® is given by spirulina, in which these compounds are the most abundant fatty acids (Table 2).

Table 2. Fatty acid chemical composition of OxiCyan®, Spirulina platensis (Spirulina) and Vaccinium myrtillus (Bilberry) extracts. Values are expressed as mg g-1 d. wt. (± standard deviation), Ki = Kovats Index. n.d. = not detectable (below the detection limit). In the same row, different letters indicate significant (P < 0.05) differences.

Palmitoleic acid18782.47 a ± 0.172.03 a ± 0.03n.d.
Palmitic Acid188617.89 a ± 0.5919.49 a ± 0.020.72 b ± 0.08
Linoleic acid20826.31 a ± 0.904.89 b ± 0.161.22 c ± 0.17
Oleic acid20850.88 a ± 0.050.91 a ± 0.010.14 b ± 0.01
Elaidic acid20930.34 a ± 0.020.12 b ± 0.10n.d.
Stearic Acid21330.47 a ± 0.010.51 a ± 0.010.11 b ± 0.03
γ-Linolenic acid22204.37 a ± 0.854.23 a ± 0.181.12 b ± 0.19


The oxidative stress is caused by the unbalance in the prooxidant-antioxidant activity of living cells, with prooxidant activities leading to the production of Reactive Oxygen Species (ROS), which cause cell damage. Uptake of antioxidant can be supported by the diet through intake of fruits, vegetables and dietary supplements. However, the evaluation of the antioxidant capacity of foods and food supplements is often biased by analytical methods. When the chemical antioxidant power of molecules and extracts tested with chemical methods (ABTS, ORAC, DPPH, etc.) is compared with a cellular-based activity, different and often contrasting results are obtained. This is because these traditional chemical tests do not inform about the physiological functions of antioxidants.

Human HepG2 cells are a suitable in vitro model system for different studies. Because of their high degree of morphological and functional differentiation in vitro, HepG2 cells are a suitable model to study the intracellular trafficking and dynamics of membrane proteins and lipids in human hepatocytes in vitro. HepG2 cells and their derivatives are also used as a model system for studies of liver metabolism and toxicity of xenobiotics, the detection of environmental and dietary cytotoxic and genotoxic (and thus cytoprotective, anti-genotoxic, and cogenotoxic) agents, and for drug targeting studies (Moscato et al., 2015, J. Funct. Biomat. 6: 16–32; Mersch-Sundermann et al., 2004, Toxicol. 198 : 329–340).

We evaluated the cellular-based antioxidant activity of OxiCyan® by using HepG2 human hepatocyte carcinoma 85011430 cell lines and the technology based on Light-Up Cell System (LUCS, see Oxi-P facts for more details). Besides evaluating the direct antioxidant activity, we also performed the ARE/Nrf2 live cell assay, which is a reporter gene approach that measures the ability of OxiCyan® to activate ARE (Antioxidant Response Element) DNA promoter sequence following the nuclear release of Nrf2 transcription factor from the Keap1/Nrf2 cytosolic complex. This genomic pathway (also called “natural antioxidant cell defense”) is well-known to increase cell capacity to adapt to oxidant stress or aggression (Ma, 2013, Annu. Rev. Pharmacol. Toxicol. 53, 401-426) and the Nrf2-dependent luciferase reporter gene assay has been successfully used to revealed the activation of the expression of Nrf2-regulated gene by natural products (Wu et al., 2018, Chin. J. Nat. Med. 16, 219-224).

The ability of OxiCyan® to directly quench HepG2 ROS is shown in Table 3.

Table 3. Antioxidant power of OxiCyan® in HepG2 cells by using the LUCS approach.

SpecificationOxiCyan® (mg ml-1)
Highest antioxidant index10.00

OxiCyan® shows a strong effect on natural antioxidant cell defence with a maximum at 20 mg ml-1 (2.77 times the gene expression obtained in control conditions). Induction of ARE/Nrf2 pathway is significant in the range between 0.31 to 20 mg ml-1. Comparison of OxiCyan® vs sulforaphane (a classical positive control of ARE/Nrf2 pathway – Soane et al., 2010, J. Neurosci. Res. 88, 1355-1363) reveals that OxiCyan® exerts a cell effect equivalent to the EC23 (1.1 μM) of sulforaphane (Figure 2).


Figure 2. Effect of OxiCyan® on HepG2 human cell natural antioxidant defense. In the left panel, luminescence values reveal luciferase gene expression following ARE promotion. Results are presented as gene expression fold increase with respect to control values. The right panel shows a comparison between OxiCyan® and sulforaphane activity. OxiCyan® exerts an antioxidant promotion equivalent to the EC23 (1.1 μM) of sulforaphane, with a gene expression fold increase (FI) 2.77 times with respect to control. Bars indicate standard deviation.

OxiCyan®, a phytocomplex composed of bilberry and spirulina, shows a consistent antioxidant power when tested with cellular-based assays. OxiCyan® acts on HepG2 cells in a dual mode by both exerting a direct effect on ROS scavenging and as a cytoprotective agent via induction of ARE/Nrf2 pathway. These results may be used as cell-based reference to improve preparation processes of products based on OxiCyan® and provide evidence of its antioxidant power at the cellular level.

For more information, refer to the paper:

Vigliante I., Mannino G. and Massimo M.E. (2019)
OxiCyan®, a phytocomplex of Bilberry (Vaccinium myrtillus) and Spirulina (Spirulina platensis), exerts both direct antioxidant activity and modulation of ARE/Nrf2 pathway in HepG2 cells.
Journal of Functional Foods, 2019, 61: 103508(read online) – (read pdf)

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