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Numerous in vitro assays are used to characterize the antioxidant properties of natural-based matrices. However, many of them generate contradictory and non-compliant results. In our study, we focused on the characterization of traditionally used biochemical (2,2′-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS), Oxygen Radical Absorption Capacity (ORAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and cellular (CAA) antioxidant tests on a broad set of milk thistle dietary supplements containing silymarin. In addition to 26 commercially available preparations, also the natural silymarin extract available from Sigma Aldrich, St. Louis, MI, USA, and a model mixture of pure flavonoid/flavonolignans mimicking the silymarin composition were investigated as control samples. Significant differences in the antioxidant capacity of the supplements were observed. Unlike the DPPH, the results of the ABTS and ORAC methods correlated with the silymarin components determined by U-HPLC-HRMS/MS. The responses in CAA were considerably lower than in other assays. Silymarin exhibited a significantly higher antioxidant capacity than the artificially prepared flavonoid/flavonolignans mixture in all tests, indicating possible presence of other antioxidants of natural origin. The follow-up U-HPLC-HRMS/MS screening revealed the presence of tens of non-silymarin compounds with reported antioxidant activity (not only in the silymarin extract, but also in the milk thistle preparations). The sum of the total phenolics and the sum of the simple phenolics correlated with CAA results more than silymarin.

2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.

2,3-Dehydrosilybin (DHS) was previously shown to chelate and reduce both copper and iron ions. In this study, similar experiments with 2,3-dehydrosilychristin (DHSCH) showed that this congener of DHS also chelates and reduces both metals. Statistical analysis pointed to some differences between both compounds: in general, DHS appeared to be a more potent iron and copper chelator, and a copper reducing agent under acidic conditions, while DHSCH was a more potent copper reducing agent under neutral conditions. In the next step, both DHS and DHSCH were tested for metal-based Fenton chemistry in vitro using HPLC with coulometric detection. Neither of these compounds were able to block the iron-based Fenton reaction and, in addition, they mostly intensified hydroxyl radical production. In the copper-based Fenton reaction, the effect of DHSCH was again prooxidant or neutral, while the effect of DHS was profoundly condition-dependent. DHS was even able to attenuate the reaction under some conditions. Interestingly, both compounds were strongly protective against the copper-triggered lysis of red blood cells, with DHSCH being more potent. The results from this study indicated that, notwithstanding the prooxidative effects of both dehydroflavonolignans, their in vivo effect could be protective.

Silybin is considered to be the main biologically active component of silymarin. Its oxidized derivative 2,3-dehydrosilybin typically occurs in silymarin in small, but non-negligible amounts (up to 3%). Here, we investigated in detail complex biological activities of silybin and 2,3-dehydrosilybin optical isomers. Antioxidant activities of pure stereomers A and B of silybin and 2,3-dehydrosilybin, as well as their racemic mixtures, were investigated by using oxygen radical absorption capacity (ORAC) and cellular antioxidant activity (CAA) assay. All substances efficiently reduced nitric oxide production and cytokines (TNF-α, IL-6) release in a dose-dependent manner. Multidrug resistance (MDR) modulating potential was evaluated as inhibition of P-glycoprotein (P-gp) ATPase activity and regulation of ATP-binding cassette (ABC) protein expression. All the tested compounds showed strong dose-dependent inhibition of P-gp pump. Moreover, 2,3-dehydrosilybin A (30 µM) displayed the strongest sensitization of doxorubicin-resistant ovarian carcinoma. Despite these significant effects, silybin B was the only compound acting directly upon P-gp in vitro and also downregulating the expression of respective MDR genes. This compound altered the expression of P-glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2). 2,3-Dehydrosilybin AB exhibited the most effective inhibition of acetylcholinesterase activity. We can clearly postulate that silybin derivatives could serve well as modulators of a cancer drug-resistant phenotype.

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

Silychristin A is the second most abundant compound of silymarin. Silymarin complex was previously described as an antioxidant with multidrug resistance modulation activity. Here, the results of a classical biochemical antioxidant assay (ORAC) were compared with a cellular assay evaluating the antioxidant capacity of pure silychristin A and its derivatives (anhydrosilychristin, isosilychristin and 2,3-dehydrosilychristin A). All the tested compounds acted as antioxidants within the cells, but 2,3-dehydro- and anhydro derivatives were almost twice as potent as the other tested compounds. Similar results were obtained in LPS-stimulated macrophages, where 2,3-dehydro- and anhydrosilychristin inhibited NO production nearly twice as efficiently as silychristin A. The inhibition of P-glycoprotein (P-gp) was determined in vitro, and the respective sensitization of doxorubicin-resistant ovarian carcinoma overproducing P-gp was detected. Despite the fact that the inhibition of P-gp was demonstrated in a concentration-dependent manner for each tested compound, the sensitization of the resistant cell line was observed predominantly for silychristin A and 2,3-dehydrosilychristin A. However, anhydrosilychristin and isosilychristin affected the expression of both the P-gp (ABCB1) and ABCG2 genes. This is the first report showing that silychristin A and its 2,3-dehydro-derivative modulate multidrug resistance by the direct inhibition of P-gp, in contrast to anhydrosilychristin and isosilychristin modulating multidrug resistance by downregulating the expression of the dominant transmembrane efflux pumps.

Antioxidants protect the structural and functional components in organisms against oxidative stress. Most antioxidants are of plant origin as the plants are permanently exposed to oxidative stress (UV radiation, photosynthetic reactions). Both carotenoids and flavonoids are prominent antioxidant and anti-radical agents often occurring together in the plant tissues and acting in lipophilic and hydrophilic milieu, respectively. They are complementary in their anti-radical activity. This study describes the synthesis of a series of hybrid ester conjugates of retinoic acid with various flavonolignans, such as silybin, 2,3-dehydrosilybin and isosilybin. Antioxidant/anti-radical activities and bio-physical properties of novel covalent carotenoid-flavonoid hybrids, as well as various mixtures of the respective parent components, were investigated. Retinoyl conjugates with silybin—which is the most important flavonolignan in silymarin complex—(and its pure diastereomers) displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than both the parent compounds and their equimolar mixtures.