Anti-glycation activities of phenolic constituents from Silybum marianum (Milk Thistle) flower in vitro and on human explants

Abstract

Glycation is an ageing reaction of naturally occurring sugars with dermal proteins, with clinical signs appearing in vivo around age 30, and increasing steadily/regularly with age. The suppleness of the dermis is affected by the formation of bridges between proteins and sugars (Maillard's reaction). The accumulation of advanced glycation end products (AGEs) in skin plays a very important role in skin ageing. Therefore, natural compounds or extracts that possess antiglycation activities may have great anti-ageing potential. In the present study, Silybum marianum flower extract (SMFE) was demonstrated to possess antiglycation activity. We found that SMFE inhibits glycation reaction between BSA and glucose. In addition, antiglycation activity of SMFE was confirmed in a human skin explants model. SMFE reduced Nε-(carboxymethyl) lysine (CML) expression, whereas SMFE stimulated fibrillin-1 expression compared to treatment with methyglyoxal. An active ingredient contributing to the observed activities was identified as silibinin. The antiglycation activity of silibinin was dose-dependent. The beneficial effects of silibinin may be applied to prevention or management of AGE-mediated pathologies, targeting in a pleiotropic and complementary way the biochemical and cellular bases of skin aging.

Figure 1

The effects of SMFE on the formation of fluorescent advanced glycation end products (AGEs) in BSA incubated with glucose. Data are mean ± standard deviation of thirty independent experiments. ^§ p < 0.01 compared with the vehicle-treated group, ^## p < 0.01 compared with the BSA/glucose treated group.

Figure 2

Immunostaining of fibrillin-1 in untreated batch (A), treated with AG (B), treated with SMFE (C), treated with MG (D), treated with MG + AG (E) and treated with MG + SMFE (F). (G) Image analysis of the surface percentage occupied by fibrillin-1 under the dermal-epidermal junction, as a function of the product applied. The average fluorescence intensity values were calculated using Image J software. Data are mean ± standard deviation. ^§ p < 0.01 compared with the vehicle-treated group, ^# p < 0.05 compared with the MG treated group (n = 3).

Figure 2

Immunostaining of fibrillin-1 in untreated batch (A), treated with AG (B), treated with SMFE (C), treated with MG (D), treated with MG + AG (E) and treated with MG + SMFE (F). (G) Image analysis of the surface percentage occupied by fibrillin-1 under the dermal-epidermal junction, as a function of the product applied. The average fluorescence intensity values were calculated using Image J software. Data are mean ± standard deviation. ^§ p < 0.01 compared with the vehicle-treated group, ^# p < 0.05 compared with the MG treated group (n = 3).

Figure 3

Activity of silibinin against methylglyoxal-induced glycation, revealed by the immunostaining of CML. Immunostaining of CML in untreated batch (A), treated with AG (B), treated with SMFE (C), treated with MG (D), treated with MG + AG (E) and treated with MG + SMFE (F). (G) Staining intensity on batches treated with active ingredients on CML immunostaining. The average staining intensity values were calculated using Image J software. Data are mean ± standard deviation. ^§ p < 0.01 compared with the vehicle-treated group, ^# p < 0.05 compared with the MG treated group, ^## p < 0.01 compared with the MG treated group (n = 3).

Figure 3

Activity of silibinin against methylglyoxal-induced glycation, revealed by the immunostaining of CML. Immunostaining of CML in untreated batch (A), treated with AG (B), treated with SMFE (C), treated with MG (D), treated with MG + AG (E) and treated with MG + SMFE (F). (G) Staining intensity on batches treated with active ingredients on CML immunostaining. The average staining intensity values were calculated using Image J software. Data are mean ± standard deviation. ^§ p < 0.01 compared with the vehicle-treated group, ^# p < 0.05 compared with the MG treated group, ^## p < 0.01 compared with the MG treated group (n = 3).

Figure 4

HPLC chromatogram of Silybum marianum flower extract at 290 nm.

Figure 5

The effects of silibinin on the formation of fluorescent advanced glycation end products (AGEs) in BSA incubated with glucose. Data are mean ± standard deviation. ^§ p < 0.01 compared with the vehicle-treated group, ^## p < 0.01 compared with the BSA/glucose treated group. (n = 3).

Figure 6

The effects of silibinin on the level of N^ϵ-(carboxymethyl) lysine (CML) in BSA incubated with glucose after 3 weeks of incubation. Each value represents the mean ± SEM. ^# p < 0.05 compared to BSA/ glucose; ^## p < 0.01 when compared to BSA/glucose at week 3 (n = 3).