Antiviral and Antibacterial Effect of Honey Enriched with Rubus spp. as a Functional Food with Enhanced Antioxidant Properties

Abstract

The aim of this study was to investigate the effect of blackberry and raspberry fruits (1 and 4%) and leaves (0.5 and 1%) on the biological activities of rape honey. Honey and plant material extracts were analyzed regarding total phenolic, flavonoid, anthocyanin contents, HPTLC and HPLC polyphenol profiles, as well as antioxidant activity. The antiviral potential was analyzed against bacteriophage phi 6-a coronavirus surrogate-whereas antimicrobial was tested against S. aureus and E. coli. Blackberry extracts were more abundant in antioxidants than raspberry extracts, with better properties found for leaves than fruits and for cultivated rather than commercial plants. The addition of both Rubus plant additives significantly increased the antioxidant potential of honey by four-fold (for 4% fruits additive) to five-fold (for 1% of leaves). Honey with the addition of fruits possessed higher antiviral potential compared with raw rape honey (the highest for 4% of raspberry fruit and 1% of blackberry leaf additive). Honey enriched with Rubus materials showed higher antibacterial potential against S. aureus than rape honey and effectively inhibited S. aureus biofilm formation. To summarize, honey enriched with Rubus fruit or leaves are characterized by increased pro-health value and can be recommended as a novel functional food.

Keywords: Rubus; antibacterial; antioxidants; antiviral; blackberry; fruit; honey; leaves; raspberry.

Figure 1

Changes in TPC and FRAP values of enriched honey after 90 days of storage under room conditions. *—means differ significantly (t test, p = 0.05).

Figure 2

HPTLC plate images after NP-reagent derivatization: in visible light (top) and in UV 366 nm (bottom). Anthocyanins standards mix (track 19): cyanidin-3- galactoside, cyanidin-3-glucoside, and cyanidin-3-arabinoside; flavonoids mix: rutin, hyperoside, (+)-catechin (in order of increasing Rf values).

Figure 3

HPLC-DAD chromatograms for control honey (A), honey enriched with R. idaeus fruits (B) and leaves (C).

Figure 4

Reduction in viral particles after 10 min. of incubation with blackberry (BH—harvested; BC—commercial) and raspberry (RH—harvested; RC—commercial) fruits and leaves extracts, dissolved in 5% DMSO at a concentration of 5 mg/mL. ^a,b,c—means sharing the same superscript letter in a column do not differ significantly (Tukey’s test, p = 0.05).

Figure 5

Reduction in viral particles after different times of incubation with 25% (w/v) water honey samples enriched with Rubus fruits. Rape—rape honey control, BH—honey with harvested blackberry, BC—honey with commercial blackberry, RH—honey with harvested raspberry, RC—honey with commercial raspberry; 1–4%—concentration of additive. *—means differ significantly (t test, p = 0.05).

Figure 6

Reduction in viral particles after 24 h of incubation with honey enriched with Rubus leaves (50% w/v water honey samples) (on the left). Plaques formed by phi6 in the double agar overlay method after 24 h of virus incubation with 50% (w/v) water honey samples—(A)—control phage phi6 in STM buffer, sample dilution 10⁻⁴, (B)—rape honey, sample dilution 10⁻², (C)—honey with 1% blackberry leaves extract (BH), sample dilution 10⁻² (D)—honey with 1% raspberry leaves extract (RH), sample dilution 10⁻² (on the right). ^a,b—means sharing the same superscript letter in a column do not differ significantly (Tukey’s test, p = 0.05).

Figure 7

Bacterial growth inhibition of blackberry and raspberry leaves and fruits at a concentration of 10 mg/mL. BH—harvested blackberry, BC—commercial blackberry, RH—harvested raspberry, RC—commercial raspberry. ^a,b,c,d,e—means sharing the same superscript letter in a column do not differ significantly (Tukey’s test, p = 0.05).

Figure 8

Bacterial growth inhibition at different honey concentrations (6.25%, 12.5%, and 25%) of the control (rape) and enriched honey. (A,B)—honey enriched with fruits, (C,D)—honey enriched with leaves. Rape—rape honey control, BH—honey with harvested blackberry, BC—honey with commercial blackberry, RH—honey with harvested raspberry, RC—honey with commercial raspberry. 0.5–4%—concentration of additive. *—means differ significantly (t test, p = 0.05).

Figure 9

Effect of different concentrations of Rubus-enriched honey on S. aureus biofilm formation (A) and elimination of pre-established biofilms (B)—1% of leaves addition, 4% of fruits addition. Rape—rape honey-control, BH—honey with harvested blackberry, BC—honey with commercial blackberry, RH—honey with harvested raspberry, RC—honey with commercial raspberry. *—means differ significantly (t test, p = 0.05).