Probiotic Lactobacillus paracasei A221 improves the functionality and bioavailability of kaempferol-glucoside in kale by its glucosidase activity

Abstract

The interplay between food components and gut microbiota has been considered an important factor affecting the functionality of health-promoting foods. In this study, the effects of the probiotic Lactobacillus paracasei A221 on the functionality and bioavailability of kaempferol-3-o-sophroside (KP3S), a kaempferol-glucoside contained in kale, were investigated in vitro and in vivo. Unlike the type strain NBRC15889, the A221 strain converted standard KP3S as well as the kaempferol-glucosides in kale extract into kaempferol (KP). Using an intestinal barrier model, treatment with A221 significantly improved the effects of kale extract on the barrier integrity in vitro. KP, but not KP3S, clearly induced similar effects, suggesting that KP contributes to the functional improvement of the kale extract by A221. Pharmacokinetics analyses revealed that the co-administration of A221 and KP3S significantly enhanced the amount of deconjugated KP in murine plasma samples at 3 h post-administration. Finally, the oral administration of KP to Sod1-deficinet mice, which is a good mouse model of age-related disease, clearly ameliorated various pathologies, including skin thinning, fatty liver and anemia. These findings suggest that Lactobacillus paracasei A221 is effective for enhancing the anti-aging properties of kaempferol-glucosides by modulating their functionality and bioavailability through the direct bioconversion.

Figure 1

Effect of the Lactobacillus paracasei A221 strain on the bioconversion of kaempferol-3-o-sophoroside (KP3S) and kale components. (a) Chemical structure of kaempferol-3-o-sophoroside and schematic illustration of the bioconversion into kaempferol (KP) by the A221 strain. (b) The A221 strain was incubated with KP3S for 72 h, and the direct effect of A221 on the metabolism of KP3S was assessed with HPLC. (c) The A221 strain was incubated in water extract of kale for 72 h and subjected to an HPLC analysis. Arrowhead indicates the peak of KP.

Figure 2

The LC-MS³ analysis of the peak at 8.2 min for kale water extract. (a) The compound at 8.2 min was purified and subjected to an HPLC analysis. (b) Full scan spectra (LC-MS) of the compound at 8.2 min as well as LC-MS² and LC-MS³ spectra are shown. Arrowhead indicates unique ions shown by LC-MS, LC-MS₂ and LC-MS₃ analyses of KP3S7G. (c) The deduced chemical structure and fragment pattern of the compound at 8.2 min.

Figure 3

The role of A221 in improving the barrier-enhancing property of kale water extract. (a) The intestinal barrier-enhancing property was assessed for each test sample as described in the Methods section using a Caco-2 in vitro model. The Caco-2 cell monolayer was differentiated and treated with test samples on the apical side, and TEER values were recorded at the indicated time intervals. (b) KP and KP3S were administered to Caco-2 cells, and the TEER values were recorded as in the previous experiment. *P < 0.05 vs. Vehicle. **P < 0.01 vs. Vehicle.

Figure 4

A221-treated kale water extract as well as KP promoted tight junction formation in a Caco-2 intestinal barrier model. (a) Differentiated Caco-2 cells were treated with each of the test samples for 6 h, followed by fixation and immunohistochemical staining using anti-claudin-4 and anti-occludin antibody as described in the Methods section. (b) In order to assess the important role of aglycone, either KP or KP3S was administered to Caco-2 cells and processed in the same manner as (a).

Figure 5

The effect of the A221 strain on the bioavailability of KP3S. (a) An in vitro study on the permeability of KP and KP3S using the Caco-2 intestinal barrier model was carried out. KP and KP3S were administered to Caco-2 cells on the apical side at 28.6 and 61 mg/ml, respectively, and the concentration of each chemical in the basolateral compartment was quantified by an HPLC analysis. **P < 0.01 vs. initial (0 h). (b) The effects of A221 administration on the bioavailability of KP3S were assessed using C57/B6 mice with the protocol described in the Methods section. Deconjugated KP in the plasma sample was quantified by an HPLC analysis at different time points. *P < 0.05 vs. KP3S. (c) Representative HPLC chromatograms of a plasma sample are shown for the group treated with only KP3S and the group treated together with A221. Arrowhead indicates the peak of deconjugated KP, and BA indicates the peak of Biochanin A. (d) The bioconversion of KP3S into KP was confirmed in microtubes by incubating A221 with KP3S in the same sample preparation for the in vivo administration experiment. Arrowhead indicates the peak of KP.

Figure 6

KP ameliorates the pathological phenotypes of Sod1^−/− mice. (a) KP was orally administered for eight weeks to male Sod1^−/− mice at 10 mg/kg, and skin sections were prepared. Hematoxylin and eosin staining was carried out, and representative images were shown for the different subjects in each groups. (b) The thickness of whole skin, epidermis, and dermis were quantified and compared among groups. (c) The effects of KP administration on the ratio of liver/body weight, the number of red blood cells, and the amount of 8-isoprostans were quantified and compared among groups. *P < 0.05 vs. KO Vehicle. **P < 0.01 vs. KO Vehicle.