Anti-Inflammatory, Antioxidant, and Genoprotective Effects of Callus Cultures Obtained from the Pulp of Malus pumila cv Miller (Annurca Campana Apple)

Abstract

Apples are rich in phytochemicals useful for human health. However, environmental factors can greatly affect the accumulation of these compounds. To face this problem, the callus culture technique was used to obtain large quantities of phytochemicals. Specifically, two callus cultures were obtained from ripe Annurca apple pulp (Malus pumila cv Miller) and cultivated under different light conditions: darkness and an 18-h photoperiod. The hydro-alcoholic extracts from the calli underwent analysis using GC-MS, GC-FID, and HPLC-DAD-ESI-MSⁿ to determine the qualitative and quantitative content of phenolic and triterpenic acids. The study revealed the predominant presence of triterpenic compounds in both calli. Furthermore, we investigated their radical scavenging and antioxidant activities through DPPH, ABTS, ORAC assays, and lipoxygenase inhibition activity. Genoprotection was evaluated via nicking assay, and the anti-inflammatory effect was investigated via Griess assay on LPS-injured murine macrophages. All the analyses performed were compared with peel and pulp hydroalcoholic extracts. The results showed that both calli primarily show anti-inflammatory activity and moderate antioxidant effect and can protect DNA against oxidative stimuli. This data encouraged further research aimed at utilizing callus as a bioreactor to produce secondary metabolites for use in preventive and therapeutic applications to combat acute or chronic age-associated diseases.

Keywords: Annurca apple; biological activities; callus production; functional foods; secondary metabolites; triterpenic acids.

Figure 1

Annurca apple pulp-derived calli. (A) Callus grown in the dark (‘dark callus’); (B) callus maintained in an 18-h photoperiod (‘light callus’).

Figure 2

DPPH (A), ABTS (B), ORAC (C) tests, and lipoxygenase inhibition activity (D). The data represent the percentage of inhibition induced by increasing concentrations of Annurca apple extracts. Each value is the mean ± SD of three independent measurements.

Figure 3

Protective effect of Annurca apple extracts against strand breaks using the DNA nicking assay. In (a,c,e,g), an example of agarose gel of the pGEM plasmid after incubation with the oxidant system in the presence of decreasing amounts of extract; in (b,d,f,h), the quantification of gels obtained by the assay, expressed as the percentage ratio between the volume of the band of the plasmid supercoiled form (CCC) after incubation with oxidant and the same band volume in the control; each value represents the mean ± SEM of three independent measurements. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; Dunnett’s post hoc test).

Figure 4

Evaluation of extracellular NO release and cell viability of RAW 264.7 cells after LPS, Annurca extracts, and DEXA treatment. (a) RAW 264.7 cells were co-treated overnight with LPS 1 µg mL⁻¹ either in the absence or presence of Annurca extracts at different concentrations or DEXA 0.0039 mg mL⁻¹. CTRL: untreated cells. The results are expressed as NO reduction compared to LPS-treated cells (*** p < 0.001, § p < 0.0001); ANOVA followed by Dunnett’s multiple comparison test was performed). (b) Cell viability assay on Raw 264.7 cells co-treated with LPS and Annurca extracts (MTT). The results are expressed as % of CTRL ± SD of three independent. ANOVA followed by Dunnett’s multiple comparison test was performed; No statistical differences were found.