Antiglycation potential and antioxidant activity of genipap (Genipa americana L.) in oxidative stress mediated by hydrogen peroxide on cell culture

Abstract Genipa americana L. is a plant widely used by folk medicine for the prevention and as an aid in the treatment of various diseases. In this work, we evaluated the anti-glycant and antioxidant activities of genipap fruit juice, as well as the influence of different temperatures (−6 °C and −80 °C) on the preservation of phenolic compounds. Purified extract from G. americana showed anti-glycant activity reducing the formation of fructosamine by up to 53% and recovered viability of cells under oxidative stress induced by H2O2. HPLC/UV-Vis analysis identified cinnamic acid as a bioactive substance and possibly responsible for the biological activities described above. Taken together, these results indicate that G. americana is a rich source of cinnamic acid with appreciable antioxidant and anti-glycant potential. Graphical Abstract


Introduction
Advanced glycation end products (AGEs) are proinflammatory molecules that induce oxidative stress through interaction with cell membrane receptors (RAGE) that activate NF-jB, increasing the gene expression and the production of inflammatory cytokines (Massaccesi et al. 2017; Schr€ oter and H€ ohn 2018; Song et al. 2021). AGEs accumulate in the body gradually and are involved in the pathogenesis of various diseases, including cardiovascular diseases, cancer, diabetes complications, and ageing (Oliveira et al. 2009;Adisakwattana et al. 2012;Cardoso et al. 2014;Ou et al. 2015) Bioactive substances found in fruits and vegetables, e.g. polyphenols, are able to scavenge free radicals and prevent AGEs formation, ameliorating oxidative stress-related disorders (Bartosz and Bartosz 2015;Sri Harsha and Lavelli 2019).
Genipap (Genipa americana L.) is a fruit widely consumed in Brazil with high content of phenolic compounds (Costa et al. 2010;Omena et al. 2012;Dickson et al. 2018;Neri-Numa et al. 2020). We provide here, an extended view of the genipap benefits, which includes the identification of beneficial compounds present in the genipap aqueous extract, evaluation of anti-glycation activity in vitro, and cytoprotective effect in a cell model of oxidative stress mediated by hydrogen peroxide. Finally, we show how both the concentration of polyphenols and polyphenol oxidase activity decrease over time under different freezing temperatures.

HPLC-UV/vis analysis and anti-glycant activity
During the formation of advanced glycation products in vivo, there is an intermediate stage characterised by a small fraction of amadori products, one of these products is the fructosamine (Schalkwijk and Miyata 2012; Sri Harsha and Lavelli 2019). The presence of G. americana purified extract in two concentrations (70 and 150 lg/mL), significantly decreased (p < 0.05) the level of fructosamine in a BSA/fructose in vitro system to 42% and 53%, respectively (Supplementary Figure S1). The anti-glycation activity can be explained by the antioxidant activity of cinnamic acid identified and quantified (8.2 lg/mL) through HPLC-UV/Vis analysis (Supplementary Figure S2). Previous studies showed that cinnamic acid can also block carbonyl or dicarbonyl groups in reducing sugars (e.g. Schiff bases, Amadori products) and cleavage of crosslinks with proteins (Velkov et al. 2007;Adisakwattana et al. 2012).

Cell viability by MTT assay
Pre-treatment with G. americana purified extract (1, 10 and 50 mg/mL) recovered mouse fibroblasts cell viability after damage induction by H 2 O 2 to the same level of untreated control cells (p < 0.05), similar to the positive control (pre-treatment with cinnamic acid) (Supplementary Figure S3). This cytoprotective effect can also be attributed to the presence of cinnamic acid that scavenges free radicals generated by H 2 O 2 and decreases the oxidative stress in the cell, as it was demonstrated in previous studies (Patra et al. 2012;Taner et al., 2017). These results suggest that genipap juice can be an ally in combating oxidative stress generated through the interaction of AGE and its receptors.

Antioxidant activity and quantification of total polyphenols
In the DPPH test, the lyophilised showed the highest percentage of inhibition of the DPPH radical dropping from 33.2% to 13.4% at À6 C, and from 36.2% to 21.7% at À80 C during 30 days (p < 0.05) (Supplementary Table S1). The concentration of total polyphenols in the samples was higher when stored at À80 C (p < 0.05) compared to temperatures at À6 C; this difference can be attributed to the freezing time which takes longer at À6 C. Moreover, at this temperature, oxidising enzymes present in the fruit, such as polyphenol oxidases (PPO) and peroxidases, could be more active when compared to the extracts stored at À80 C (Mahn and Rubio 2017;Oliveira et al., 2017).

Polyphenols stability and activity of polyphenol oxidase
The high reducing power provided by the number of polyphenols detected in the aqueous extract can be beneficial inside the organism if they are stable enough to become bioavailable after storage periods. After 30 days, the aqueous extract has its polyphenol concentration reduced by 20% when stored at À80 C and reduced by 42% when stored at À6 C (p < 0.05) (Supplementary Figure S4). On the other hand, the lyophilised has its polyphenol concentration reduced by 19% when stored at À80 C, and reduced by 10%, when stored at À6 C (p < 0.05). Even with the degradation process, as the graphs show in Supplementary Figure S4, the concentration of the polyphenols in the lyophilised is still much higher than the concentration found in the aqueous extract (300 mg/mL) (p < 0.05), evidencing that the lyophilisation process is still an efficient method for the conservation of polyphenols (Cheng et al. 2017). PPO presented higher activity on the first day of storage when compared to the 10th day of storage (p < 0.05) for both storage temperatures (Supplementary Figure S5). This could be associated with the higher concentration of total polyphenol seen on the first day of analysis in the aqueous extract (Supplementary Table S2).

Experimental
See supplementary material.

Conclusions
In the present study, we showed that aqueous extract from genipap pulp exhibits antioxidant activity when assayed with DPPH radical. The G. americana purified extract used in the pre-treatment of mouse fibroblasts, showed a protective effect against H 2 O 2 -induced damage and against the glycation of albumin by fructose (inhibiting the formation of fructosamine in vitro). At the same time, cinnamic acid was identified in the aqueous extract of this fruit, suggesting that the protective effect observed could be due to this compound. Furthermore, our results support that the lyophilisation process is an efficient method for polyphenols conservation. The pieces of evidence from this study suggest that the juice from genipap is a source of beneficial polyphenols, including cinnamic acid. Moreover, future in vivo studies in animals and thereafter in humans are necessary to evaluate the antioxidative potential of the lyophilised extract from genipap juice in an organism under oxidative stress condition such us diabetes.

Disclosure statement
No potential conflict of interest was reported by the authors.