Combined acid hydrolysis and fermentation improves bioactivity of citrus flavonoids in vitro and in vivo

Abstract

Many bioactive plant compounds, known as phytochemicals, have the potential to improve health. Unfortunately, the bioavailability and bioactivity of phytochemicals such as polyphenolic flavonoids are reduced due to conjugation with sugar moieties. Here, we combine acid hydrolysis and tailored fermentation by lactic acid bacteria (Lactiplantibacillus plantarum) to convert the biologically less active flavonoid glycosides hesperidin and naringin into the more active aglycones hesperetin and naringenin. Using a comprehensive approach, we identify the most effective hydrolysis and fermentation conditions to increase the concentration of the aglycones in citrus extracts. The higher cellular transport and bioactivity of the biotransformed citrus extract are also demonstrated in vitro and in vivo. Superior antioxidant, anti-inflammatory and cell migration activities in vitro, as well as intestinal barrier protecting and antioxidant activities in Drosophila melanogaster are identified. In conclusion, the presented biotransformation approach improves the bioactivity of flavonoids, clearly traced back to the increase in aglycone content.

Fig. 1. Degradation of flavanones by α-L-rhamnosidase and β-D-glucosidase.

a Narirutin (naringenin-7-O-rutinoside) or naringin (naringenin-7-O-neohesperidoside) are converted to naringenin-7-O-glucoside by α-L-rhamnosidase. Next, β-D-glucosidase catalyzes the conversion of naringenin-7-O-glucoside to naringenin. b Hesperidin (hesperetin-7-O-rutinoside) or neohesperidin (hesperetin-7-O-neohesperidoside) are hydrolyzed to hesperetin-7-O-glucoside by α-L-rhamnosidase. Hesperetin-7-O-glucoside is converted to hesperetin by β-D-glucosidase.

Fig. 2. Enzymatic activity of various LAB strains for the formation of aglycones from 7-O-glucosides present in citric acid hydrolyzed citrus extract.

a Representative images of L. plantarum, L. rhamnosus, L. brevis and L. paracasei showing gram-positive rods. Scale bar: 10 µm. b Concentrations of flavanones in citric acid hydrolyzed extract (CAE) after 24 h incubation with each strain at 37 °C. Control refers to the sample without bacteria. Data are mean ± SD of n = 6 samples/strain. Differences to control are analyzed by Kruskal-Wallis’s test with Dunn’s multiple comparisons test, with exception of data set for hesperetin which is analyzed by ordinary one-way ANOVA with Dunnett’s multiple comparison test.

Fig. 3. Citric acid-based hydrolysis and fermentation affect flavanone profiles in citrus extracts.

a Schematic diagram of extract preparation. For citric acid hydrolyzed extract (CAE), bioflavonoid complex was treated with citric acid solution at 90 °C for 4 h. Then, sodium citrate solution was added to reach a final concentration of 10% (w/w). Aqueous extract (AQE) was prepared with water instead of citric acid. For fermentation, AQE and CAE were incubated with L. plantarum at 37 °C for 24 h (referred to as FermAQE and FermCAE). Control extracts were diluted in modified MRS and incubated without bacteria (referred to as AQE and CAE). b Influence of hydrolysis with increasing citric acid (CA) concentrations (0.25 M CA, 0.50 M CA, or 1.00 M CA) on flavanone concentrations in citrus extracts before incubation with bacteria. Data are mean ± SD of n = 4 samples/treatment. Differences to 0 M CA are analyzed by ordinary one-way ANOVAs with Dunnett’s multiple comparison test, with exception of data set for hesperidin which is analyzed with Kruskal’s test with Dunn’s multiple comparisons test. c Increase in aglycones after 24 h-incubation with L. plantarum. For calculation of the aglycone increase, the initial aglycone concentration (at 0 h) was subtracted from the total concentration at 24 h. Data are mean ± SD of n = 5 samples/treatment. Differences to 0 M CA are analyzed by Kruskal’s test with Dunn’s multiple comparisons test. d Flavonoid concentrations in the final extracts prepared with water (AQE and FermAQE) or 0.25 M CA (CAE and FermCAE). Data are mean ± SD of n = 9 samples/extract. Differences between treatments are analyzed by ordinary one-way ANOVAs with Tukey’s multiple comparison test (narirutin, neohesperidin and hesperetin) or Kruskal’s test with Dunn’s multiple comparisons test (naringin, naringenin-7-O-glucoside, naringenin, hesperidin and hesperetin-7-O-glucoside).

Fig. 4. Transport and uptake of flavonoids from AQE or FermCAE in Caco-2 cells.

a Schematic overview of uptake test (1) or transport test (2) in differentiated Caco-2 cells. b Comparison of aglycone uptake in Caco-2 cells treated with AQE or FermCAE for 4 h, normalized to cell protein content. Uptake of detectable flavonoids from (c) aqueous citrus extract (AQE) and (d) fermented citric acid hydrolyzed citrus extract (FermCAE) in Caco-2 cells, normalized to cell protein content. e TEER values of Caco-2 monolayers before and during transport studies. f Comparison of absolute aglycone concentrations detected on the basolateral side after treatment with AQE or FermCAE on the apical side for 4 h. Transport rate of detectable flavonoids from AQE (g) and FermCAE (h) across a Caco-2 monolayer. Data are mean ± SD of n = 6 samples/treatment. Differences are analyzed by ordinary one-way ANOVAs with Šídák’s multiple comparison test.

Fig. 5. Impact of citrus extracts of different biotransformation level on ROS production and cell migration of intestinal epithelial cells under challenging conditions.

Percentage of intracellular reactive oxygen species (ROS) accumulation in human Caco-2 cells after treatment with pure flavanones (a), or citrus extracts (b) and subsequent stress induction by 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH), normalized to stressor treatment. AQE stands for aqueous citrus extract, CAE for citric acid hydrolyzed citrus extract, FermAQE for fermented aqueous citrus extract, FermCAE for fermented citric acid hydrolyzed citrus extract. Percentage of intracellular ROS accumulation in swine epithelial IPEC-J2 cells after treatment with pure flavanones (c) or citrus extracts (d) and subsequent stress induction by tert-butylhydroperoxide (tBHP), normalized to stressor treatment. Control refers to untreated cells; quercetin was used as antioxidant positive control. Data are mean ± SD of n = 9 samples/treatment. Differences are analyzed by ordinary one-way ANOVAs with Šídák’s multiple comparison test. e Cell front velocity [µm · h⁻¹] of IPEC-J2 cells pre-treated with citrus extracts for 6 h and stressed with tBHP after scratching. Data are mean ± SD of n = 6 samples/treatment. Differences are analyzed by ordinary one-way ANOVAs with Šídák’s multiple comparison test. f Representative brightfield images of the scratch-wounded area at start (15 min after scratching) and end point (420 min). The wounded area is highlighted in yellow ocher.

Fig. 6. Change in cytokine expression profiles in LPS-stimulated THP-1 macrophages treated with citrus extracts of different biotransformation level.

a Representative images of cytokine arrays incubated with supernatants of differentiated THP-1 macrophages treated with aqueous citrus extract (AQE) or fermented citric acid hydrolyzed citrus extract (FermCAE) under lipopolysaccharide (LPS) challenge. Cytokines expressed are presented as duplicate spots. Important cytokines are outlined and marked with numbers. The corresponding spot intensities (stated as arbitrary units, A.U.) of these analytes are presented in b. Data of technical duplicates are shown. c Heat map of 105 cytokines with mean spot intensities in a color range from white (low level) to blue (high level).

Fig. 7. Biotransformed citrus extracts reduce pro-inflammatory cytokine levels in LPS-stimulated THP-1 macrophages.

a Schematic graph of lipopolysaccharide (LPS) challenge of THP-1 cells (1) with subsequent multiplex bead-based cytokine immunoassay (2). AQE stands for aqueous citrus extract, CAE for citric acid hydrolyzed citrus extract, FermAQE for fermented aqueous citrus extract, FermCAE for fermented citric acid hydrolyzed citrus extract. Effect of pure flavonoids (b−f) and citrus extracts (g−k) on expression of selected pro-inflammatory cytokines by differentiated THP-1 macrophages. Data are mean ± SD of n = 4 samples/treatment, measured in technical duplicates. Differences between treatments are analyzed by ordinary one-way ANOVAs with Šídák’s multiple comparison test with exception of CXCL9 in pure flavonoid treatments (e), which is analyzed with Kruskal-Wallis’s test with Dunn’s multiple comparisons test due to not normally distributed data.

Fig. 8. Biotransformed citrus extracts improve intestinal barrier integrity and reduce oxidative stress in female w¹¹¹⁸D. melanogaster under challenge conditions.

a Schematic overview of in vivo challenges: (1) dextran sulphate sodium (DSS) challenge to reduce intestinal barrier integrity; (2) ferrous iron induced oxidative stress in female w¹¹¹⁸ D. melanogaster. AQE stands for aqueous citrus extract, FermCAE stands for fermented citric acid hydrolyzed citrus extract, DCF(DA) stands for 2’,7’-dichlorodihydrofluorescein (diacetate). b D. melanogaster fed with blue dye (1) with DSS-challenged intestinal barrier (Smurf phenotype) and (2) in control group. Scale bar: 500 µm. c, d Mortality and Smurf phenotype observed in D. melanogaster challenged by DSS and treated with citrus extracts for 7 d. Data are mean ± SD of n = 300 flies/treatment. e, f Reactive oxygen species (ROS) level and metabolic activity of D. melanogaster stressed with ferrous iron and treated with citrus extracts for 70 h, normalized to protein content. RFU stands for relative fluorescence units. Data are mean ± SD of n = 12 samples (25 flies each)/treatment. g, h Mortality and climbing performance of D. melanogaster stressed with ferrous iron and treated with citrus extracts for 7 d. Data are mean ± SD of n = 225 flies/treatment. Differences are analyzed by ordinary one-way ANOVAs with Šídák’s multiple comparison test.