The Combined Effect of Green Tea, Saffron, Resveratrol, and Citicoline against Neurodegeneration Induced by Oxidative Stress in an In Vitro Model of Cognitive Decline – PubMed Black Hawk Supplements

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During ageing, the brain is vulnerable to a growing imbalance of the antioxidant defence system, resulting in increased oxidative stress. This condition may be mainly responsible for cognitive decline, resulting in synaptic transmission disruptions and the onset of neuronal dysfunction. In this context, developing efficient preventive and therapeutic strategies against increased oxidative stress and decreased antioxidant defence mechanisms should be considered a public health priority to promote…
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The Combined Effect of Green Tea, Saffron, Resveratrol, and Citicoline against Neurodegeneration Induced by Oxidative Stress in an In Vitro Model of Cognitive Decline - PubMed

The Combined Effect of Green Tea, Saffron, Resveratrol, and Citicoline against Neurodegeneration Induced by Oxidative Stress in an In Vitro Model of Cognitive Decline

Simone Mulè et al. Oxid Med Cell Longev. .

Abstract

During ageing, the brain is vulnerable to a growing imbalance of the antioxidant defence system, resulting in increased oxidative stress. This condition may be mainly responsible for cognitive decline, resulting in synaptic transmission disruptions and the onset of neuronal dysfunction. In this context, developing efficient preventive and therapeutic strategies against increased oxidative stress and decreased antioxidant defence mechanisms should be considered a public health priority to promote healthy ageing. Therefore, the current study explored the benefits of a novel combination of green tea, saffron, trans-Reveratrol, and citicoline, called MIX, on improving intracellular processes to ameliorate the mechanisms linked to cognitive decline under oxidative stress conditions. First, the ability of MIX to cross the blood-brain barrier (BBB) was evaluated in an in vitro model, analysing TEER value and the specific tight junctions; second, the CCF-STTG1 cell line was pretreated with 200 µM H2O2 for 30 min to explore the effects of the single active compounds and their combination under oxidative stress conditions. Our results demonstrated for the first time the synergistic effects of the new combination to improve the absorption rate of individual agents through the BBB and maintain its integrity. Subsequently, further research was done to assess the positive role of the combination to counteract oxidative damage; as expected, MIX restored the neurodegenerative state activated by 200 µM H2O2, reducing mitochondrial damage, and improving survival pathways. Additionally, MIX acted as a regulator of both cellular energy metabolism and apoptosis, reducing the inflammatory state activated by oxidative stress. Finally, MIX can balance neurotrophin production to prevent mitochondrial disruption. In conclusion, MIX counteracted the adverse effects of brain oxidative stress, suggesting that this new proposed formulation prevents the molecular mechanisms underlying the onset of cognitive decline, even in support of conventional therapy.

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Conflict of interest statement

The authors declare that they have no competing interests. Francesca Uberti is a cofounder of noiVita Srls. Dr. Stefania Battaglia and Dr. Valeria Curti are employees of Kolinpharma S.p.A., a nutraceutical company that produces and commercialises food supplements. These authors report no involvement of the company in the research that could have influenced the outcome of this work.

Figures

Figure 1
Figure 1

Dose-response study on cell viability in CCF-STTG1 cells tested by MTT test. (a) Dose-response study after green tea administration titrated 40% EGCG (200−25 µM). The results are expressed as mean ± SD (%) of five independent experiments normalised to the control (0 lines corresponding to 100% cell viability), each performed in triplicate and expressed as the percentage increased. a, b, c and d p  < 0.05 vs. control; a is p  < 0.05 vs. c and d; b is p  < 0.05 vs. a, c and d; c is p  < 0.05 vs. d. (b) Dose-response study after trans-R administration (10−1 µM). The results are expressed as mean ± SD (%) of five independent experiments normalised to the control (0 line corresponding to 100% cell viability), each performed in triplicate and expressed as the percentage increased. a, b, c and d p  < 0.05 vs. control; b is p  < 0.05vs a; c is p  < 0.05 vs. a and b; d is p  < 0.05 vs. a, b and c. (c) Dose-response study after saffron administration titrated 0.3% crocin (200−25 µM). The results are expressed as mean ± SD (%) of five independent experiments normalised to the control (0 line corresponding to 100% cell viability), each performed in triplicate and expressed as the percentage increased. a, b, c and d p  < 0.05 vs. control; b and c are p  < 0.05 vs. a; d is p  < 0.05 vs. a, b and c.

Figure 2
Figure 2

Cell viability on CCF-STTG1 cells measured by MTT test. Green tea titrated 40% EGCG = 100 μM; saffron titrated 0.3% crocin = 25 μM; citicoline = 100 µM; trans-R = 1 µM; MIX = green tea 100 μM + saffron 25 μM +citicoline 100 µM + trans-R 1 µM. The results are expressed as mean ± SD (%) of five independent experiments normalised to the control (0 line corresponding to 100% cell viability), each performed in triplicate and expressed as the percentage increased.  ∗p < 0.05 vs. control; #p < 0.05 vs. green tea, saffron, citicoline, trans-R.

Figure 3
Figure 3

Permeability of the agents selected on BBB in vitro model. Absorption through BBB evaluated by fluorescent tracer. The abbreviations are the same as those used in Figure 2. Data are expressed as means ± SD (%) of five independent experiments normalised to control values (0% line), each performed in triplicate. All agents are p  < 0.05 vs. control;  ∗p  < 0.05 vs. green tea; γp <0.05 vs. saffron; φp  < 0.05 vs. citicoline; ψp  < 0.05 vs. trans-R.

Figure 4
Figure 4

The integrity of BBB in vitro model. (a) the TEER values were measured using EVOM3. The breakpoint between the axes corresponds to the threshold value below which the experimental model does not mature. The abbreviations are the same as in Figure 2. The data are expressed as means ± SD (%) of five independent experiments performed in triplicates. All agents are p < 0.05 vs. control;  ∗p < 0.05 vs. green tea; γp < 0.05 vs. saffron; φp < 0.05 vs. citicoline; ψp < 0.05 vs. trans-R. (b) marveld and (c) claudin 5 levels were measured by the ELISA Kit under the same conditions as the TEER analysis. The abbreviations are the same as in Figure 2. The data are expressed as means ± SD (%) of five independent experiments normalised to control values (0% line), each performed in triplicates.  ∗p < 0.05 vs. control; #p < 0.05 vs. green tea, saffron, citicoline, and trans-R.

Figure 5
Figure 5

Analysis of the stimulation under oxidative stress conditions on CCF-STTG1 cells. (a) cell viability (percentage increased), (b) ROS production, and (c) NO production measured on CCF-STTG1 cells pretreated with H2O2 200 μM and then treated for 24 hr with the agents alone and MIX are illustrated. H2O2 = 200 μM H2O2; H2O2 + green tea = 200 μM H2O2 + green tea titrated 40% EGCG = 100 μM; H2O2; H2O2 + saffron = 200 μM H2O2 + saffron titrated 0.3% crocin = 25 μM; H2O2; H2O2 + citicoline = 200 μM H2O2 + 100 µM citicoline; H2O2 + trans-R = 200 μM H2O2 + 1 µM trans-R; H2O2 + MIX = 200 μM H2O2 + green tea 100 μM + saffron 25 μM + citicoline 100 µM + trans-R 1 µM. The results are expressed as mean ± SD (%) of 5 independent experiments performed in triplicates normalised to control (0% line).  ∗p < 0.05 vs. control; #p  < 0.05 vs. H2O2; φp  < 0.05 vs. green tea, saffron, citicoline, and trans-R.

Figure 6
Figure 6

Analysis of the inflammatory panel on CCF-STTG1 cells under oxidative stress conditions. (a) IL-2 and (b) TNFα productions were measured on CCF-STTG1 pretreated with H2O2 200 μM and then treated for 24 hr with single agents alone and MIX. The results are expressed as mean ± SD (pg/mL) of 5 independent experiments performed in triplicate. The abbreviations are the same as those used in Figure 5.  ∗p < 0.05 vs. control; #p  < 0.05 vs. H2O2; φp  < 0.05 vs. green tea, saffron, citicoline, and trans-R.

Figure 7
Figure 7

BDNF production under oxidative stress conditions. BDNF = 10 ng/mL. The abbreviations are the same as reported in Figure 5. The results are expressed as the mean ± SD (pg/mL) of five independent experiments performed in triplicate normalised to the control (0 line).  ∗p < 0.05 vs. control; #p  < 0.05 vs. H2O2; φp  < 0.05 vs. green tea, saffron, citicoline, and trans-R.

Figure 8
Figure 8

Analysis of biomarkers activity in CCF-STTG1 cells under oxidative stress condition. (a) p53, (b) cytochrome C (c), SIRT-1, (d) Nrf2, (e) Aβ 1-40, and (f) pTAU protein were reported. The tests were performed on CCF-STTG1 cells pretreated with H2O2 200 μM and then treated for 24 hr with single agents alone and MIX. The abbreviations are the same as those used in Figure 5. The results are expressed as mean ± SD (%) of five independent experiments normalised to control, each performed in triplicate.  ∗p  < 0.05 vs. control;  #p  < 0.05 vs. H2O2; φp  < 0.05 vs. green tea, saffron, citicoline, and trans-R.

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The Combined Effect of Green Tea, Saffron, Resveratrol, and Citicoline against Neurodegeneration Induced by Oxidative Stress in an In Vitro Model of Cognitive Decline – PubMed