Tannin profile, antioxidant properties, and antimicrobial activity of extracts from two Mediterranean species of parasitic plant Cytinus

Abstract

Background: Cytinus is small genus of endophytic parasitic plants distributed in South Africa, Madagascar, and in the Mediterranean region. In the latter area, two species occur, Cytinus hypocistis and C. ruber, distinguished by both morphological characters and ecological traits. We characterized the ethanolic and aqueous extracts obtained from the inflorescences of C. hypocistis and C. ruber collected in Sardinia, Italy, and explored their tannin content, antioxidant properties and antimicrobial activities.

Methods: Total phenolic contents were determined by Folin-Ciocalteu spectrophotometric method. Tannin content was determined by HPLC. Antioxidant activity of the extracts was tested with both electron transfer-based (FRAP, TEAC, DPPH) and spectrophotometric HAT methods (ORAC-PYR). The antimicrobial activities of extracts/compounds were evaluated using the broth microdilution method. The bactericidal activity was evaluated using the time-kill method. Biofilm formation was evaluated by crystal violet (CV) staining assay.

Results: Characterization of the tannin profile of C. hypocistis and C. ruber revealed a significant amount of gallotannins, in particular 1-O-galloyl-β-D-glucose. In addition, pentagalloyl-O-β-D-glucose was present in all extracts, reaching the concentration of 0.117 g/kg in the ethanolic extract of C. hypocistis. C. hypocistis extracts displayed a strongest antioxidant activity than C. ruber extracts. Three Gram-positive bacterial species tested (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium) resulted sensitive to both Cytinus extracts, with MICs ranging from 125 to 500 μg/ml for aqueous extracts and from 31.25 to 250 μg/ml for ethanolic extracts; on the contrary, Gram-negative strains (Pseudomonas aeruginosa and Klebsiella pneumoniae) were not affected by Cytinus extracts. Intriguingly, we observed the suppressive activity of ethanolic extracts of C. hypocistis and C. ruber on biofilm formation of S. epidermidis. Experiments performed with synthetic compounds indicated that pentagalloyl-O-β-D-glucose is likely to be one of the active antimicrobial components of Cytinus extracts.

Conclusions: These findings show that Cytinus extracts have antimicrobial and antioxidant activities, suggesting a possible application of Cytinus as sources of natural antimicrobials and antioxidants.

Keywords: Anti-biofilm; Antimicrobial; Antioxidant; Cytinus; Gallotannins; Tyrosinase inhibitors.

Fig. 1

a Cytinus hypocistis inflorescences, emerging directly from a Cistus monspeliensis’ root, Sardinia; (b) A classic portrait of C. hypocistis (from Reference [52]; in the public domain: https://en.wikipedia.org/wiki/Cytinus#/media/File:Illustration_Cytinus_hypocistis0.jpg)

Fig. 2

a Cytinus hypocistis inflorescences, Sardinia; (b) Cytinus ruber inflorescences, Sardinia

Fig. 3

Killing kinetics of Cytinus hypocystis and Cytinus ruber extracts (at concentrations ranging from 62.5 to 250 μg/ml) and penta-O-galloyl-β-D-glucose (abbreviated as 5-O-galloyl in the graphs, 125 μg/ml) against S. aureus ATCC 33591 (a) and S. epidermidis ATCC 35984 (b). Bacteria were incubated in sodium-phosphate buffer 10 mM, pH 7.4 supplemented with 2% LB in the presence of the antimicrobial compounds for 3 and 24 h. Control (CTRL) represents untreated bacteria. Dashed line represent 3 Logs reduction in CFU count as compared to inoculums. The numbers in the figure legend are the concentrations used expressed in μg/ml. Data are expressed as mean ± standard error of at least three independent experiments

Fig. 4

Inhibitory effect of Cytinus hypocystis and Cytinus ruber extracts (at concentrations ranging from 125 to 250 μg/ml) and penta-O-galloyl-β-D-glucose (abbreviated as 5-O-gall in the graph; 31.25 μg/ml) on biofilm formation of S. epidermidis ATCC 35984. The inhibitory effect was assessed by measuring the total biofilm biomass by crystal violet staining after 24 h of incubation with the extracts. Control (CTRL) represents untreated bacteria. Concentrations are expressed in μg/mL. Dashed lines represent 50% reduction in biofilm biomass as compared to untreated controls. Data are reported as mean ± standard error of at least three independent experiments. ∗∗p < 0.01; ∗∗∗p < 0.001 (one-way ANOVA followed by Tukey-Kramer post hoc test)