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Supercritical fluid extraction with carbon dioxide is nowadays one of the best approaches on an economic and ecological point of view for the extraction of natural compounds from plants. Besides, many seeds or fruits contain both lipids, as triglycerides, which can be used for food or cosmetic industries, and polyphenols which do have many bioprotective effects for the human health. By using successively pure carbon dioxide and carbon dioxide added with a polar modifier, either pure ethanol or a ethanol/water mixture, we achieved first the extraction of triglycerides of the milk thistle seeds, followed by the extraction of the flavonolignans, which are bioactive compounds. Effects of pressure (15 and 25 MPa) and temperature (40 and 60 °C) were studied on the lipids extraction efficiency. For flavonolignans, the polar compounds, the impact of modifier amount (pure ethanol from 10% to 40%) and water percentage in ethanol (5–15%) were compared. The duration of the two sequential steps to achieve the complete seed defatting and to increase the flavonolignan recovery were also studied. The complete lipids extraction was achieved with pure CO2 at 40 °C, 25 MPa for 30 min whereas the extraction of flavonolignans was achieved with 20% of modifier (wate/ethanol 15/85) during 90 min. The analysis of the triglyceride composition of milk thistle seed were studied by using supercritical fluid chromatography (SFC), whereas the analysis of flavonolignans was carried out by an improved Ultra-high-pressure liquid chromatography (UHPLC) method.

This study examined the metabolism of flavonolignans and taxifolin in 33 healthy adult male volunteers, who received 200 mg of silymarin orally twice daily for three months. First-morning urine and feces collected on days 10 and 90 were analyzed by UHPLC-MS and bacterial composition of feces was investigated by Next Generation Sequencing. The analyses revealed 5/4 types of flavonolignan/taxifolin metabolites in urine and 13/10 types of metabolites in feces, with large differences in total metabolite levels in both urine and feces observed among individual participants. In urine, 21 and 23 participants showed some increase in the total amount of flavonolignan and taxifolin metabolites, respectively, on day 90 compared with day 10. In feces, some correlation was found between the amount of metabolites and the composition of the intestinal microbiota. In conclusion, silymarin flavonolignans and taxifolin undergo multiple biotransformation reactions, and the metabolic profiles of the constituents considerably vary among individuals.

Silybum marianum (L.) Gaertn. (Asteraceae) was hydroponically cultured using a nutrient film technique system. Silibinin, isosilibinin and silychristin were detected in the fruits of the cultured plants. The effect of salicylic acid on the improvement of flavonolignans production by the fruits of the hydroponically cultured S. marianum was investigated. Salicylic acid was added to the nutrient solution at different concentrations (100, 200 and 400 µM) and the mature fruits of the plant were collected five days after elicitor addition. The fruits were then analyzed for their total flavonolignans contents and individual components using quantitative proton nuclear magnetic resonance spectroscopy (qHNMR) and high-performance liquid chromatography (HPLC). The results showed that elicitation with salicylic acid at 200 µM for five days increased production of total flavonolignans (1.7-fold by qHNMR and 1.6-fold by HPLC) higher than the control cultures and (1.4-fold by qHNMR and 1.1-fold by HPLC) higher than the cultivated plants. Silychristin was the major flavonolignan produced by the cultured plant. Elicitation by 200 µM salicylic acid increased silychristin production (1.6-fold by qHNMR and HPLC) higher than the control cultures and (1.3-fold by qHNMR and 1.0-fold by HPLC) higher than the cultivated plants. The present study provides a chance to improve secondary metabolite yield, serves as a useful tool for studying the biosynthesis of these medicinally valuable compounds and its regulation in plant and spots more light on hydroponic system as an important agricultural technique.

Plant seeds, as those from milk thistle (Silybum marianum), are a valuable source of nonpolar and polar compounds with potentially interesting biological activity. The main nonpolar compounds are triglycerides, which are also the main components of all vegetable oils. In addition, specific polar compounds - flavonolignans, called silymarin, have been found in large amounts in milk thistle seeds extract. These flavonoids derivatives have different biological activity, for instance hepatoprotective effects. In order to extract and analyze both nonpolar (triglycerides) and polar compounds (flavonolignans) from milk thistle seeds through a sequential methodology, an on-line supercritical fluid extraction - supercritical fluid chromatography (SFE-SFC) method was developed. Different ways of transferring the extracts from SFE to SFC (i.e. direct on-column transfer and loop transfer) were compared, and particularly for their effect on chromatographic quality. In this respect, nonpolar and polar compounds caused different issues, especially as polar compounds required a significant portion of co-solvent in the extraction step, favoring early elution in the chromatographic column. First, on-line SFE-SFC was used for triglycerides analysis and allowed the comparison of transfer modes. Then, on-line kinetics were performed to measure defatting time before polar molecules extraction. Finally, the eventual benefit of loop transfer was also investigated for the analysis of flavonolignans, polar molecules whose analysis can be difficult by on-line SFE-SFC. The aim of this paper is to discuss the versatility of on-line SFE-SFC and how challenging the coupling can be, especially when both non-polar and polar molecules must be analyzed independently in a single sample., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Quentin Gros reports financial support was provided by French National Association for Research and Technology (ANRT). Quentin Gros, Johanna Duval, Shinnosuke Horie, Yasuhiro Funada and Yoshihiro Hayakawa report financial support provided by Shimadzu Corporation. Caroline West and Eric Lesellier report a relationship with Shimadzu Corporation that includes non-financial support (lending equipment)., (Copyright © 2023. Published by Elsevier B.V.)

Two pairs of new enantiomeric flavonolignans, ±stachyols A and B (±1 and ± 2), along with two novel isoflavanelignans, stachyols C and D (3 and 4) were isolated from the roots of Indigofera stachyodes. Their chemical structures and absolute configurations were determined using nuclear magnetic resonance and comparison of experimental and theoretical electronic circular dichroism (ECD) spectra, as well as quantum chemical calculations. Of those compounds, 1 and 2 represented the first examples of flavonolignans with 5-deoxyflavonoids adduct phenylpropanoids. Moreover, 3 and 4 possess an unprecedented skeleton with isoflavanes adduct phenylpropanoids. The antioxidant activity was evaluated for all compounds in terms of ABTS + and DPPH bioassays. Compounds 3 and 4 exhibited significant radical-scavenging activity in the ABTS + assay, with IC 50 values of 15.15 and 5.83 μM, respectively., (Copyright © 2022. Published by Elsevier B.V.)

Silybum marianum (L.) Gaertn. is an important medicinal plant belonging to Mediterranean flora. The medicinal properties of the species are mainly due to silymarin, a combination of different flavonolignans contained in the fruit. As for silymarin, so far a wide variability of possible S. marianum chemotypes has been described. In the present study the flavonolignan profile of 40 different S. marianum wild accessions was analysed at both population and single plant level, further extending the analysis to progenies derived from crosses between parental lines with different chemotypes. The results of this work indicate that S. marianum wild populations can be composed either of individuals with the same chemotype, or heterogeneous mixtures of individuals characterized by different chemotypes. Only three chemotypes (A, B and C) have been identified among Italian wild populations. Based on data collected we furthermore propose that chemotype C is the result of the hybridization between A and B chemotypes. If assessed at single plant level, chemotypes are extremely stable therefore evidencing a strong genetic control of silymarin biosynthetic pathway. Chemotypes A and B are present in all the analysed regions and no clear correlation between chemotypes and geographic features has been found. In conclusion, this work provides a general procedure for the characterization of different and stable chemotypes, for a deeper understanding of silymarin biosynthetic pathway, and in order to implement S. marianum breeding programmes aiming to improve silymarin quality., (Copyright © 2020 Elsevier B.V. All rights reserved.)

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS + and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC 50  = 73 μM) than silibinin., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The primary biological function of platelets is to form hemostatic thrombi that prevent blood loss and maintain vascular integrity. These multi-responding cells are activated by different endogenous, physiological agonists due to the vast number of receptors present on the surface of the platelets. Collagen represents up to 40% of the total protein presented in the vessel wall and is the major activator of the platelets' response after tissue injury, and is the only matrix protein which supports both platelet adhesion and complete activation. The aim of our study was to determine the effects of three major flavonolignans (silybin, silychristin and silydianin) on collagen-induced blood platelets' activation, adhesion, aggregation and secretion of PF-4. We observed that depending on the dose, silychristin and silybin have anti-platelet properties observed as inhibition of collagen-induced activation (formation of blood platelet aggregates and microparticles, as well as decreased expression of P-selectin and activation of integrin α IIb β 3 ), aggregation, adhesion and secretion of PF-4. These effects highlight the potential of silybin and silychristin as supplementation to prevent primary and secondary thrombotic events wherein excessive blood platelet response to a physiological agonist is observed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Silymarin is the phytochemical with medicinal properties extracted from Silybum marianum (L.) Gaertn. fruits. Yet, little information is available about silymarin biosynthesis. Moreover, the generally accepted pathway, formulated thus far, is not in agreement with actual experimental measurements on flavonolignan contents. The present work analyses flavonolignan and taxifolin content in 201 S. marianum samples taking into consideration a wide phenotypic variability. Two stable chemotypes were identified: one characterized by both high silychristin and silybin content (chemotype A) and another by a high silydianin content (chemotype B). Through the correlation analysis of samples divided according to chemotype, it was possible to construct a simplified silymarin biosynthetic pathway that is sufficiently versatile in explaining experimental results responding to the actually unresolved questions about this process. The proposed pathway highlights that three separate and equally sized metabolite pools exist, namely: diastereoisomers A (silybin A plus isosilybin A), diastereoisomers B (silybin B plus isosilybin B) and silychristin. In both A and B diastereoisomers pools, isosilybin A and isosilybin B always represent a given amount of the metabolite flux through the specific metabolite pool suggesting the possible involvement of dirigent protein-like enzymes. We suggest that chemotype B possesses a complete silymarin biosynthetic pathway in which silydianin biosynthesis is enzymatically controlled. On the contrary, chemotype A is probably a natural mutant unable to biosynthesize silydianin. The present simplified pathway for silymarin biosynthesis will constitute an important tool for the further understanding of the reactions that drive flavonolignan biosynthesis in S. marianum., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Silybum marianum (milk thistle) is a medicinal plant used for the treatment of various liver disorders. This study examined whether the main flavonolignans from S. marianum (i.e. silybin, silychristin, silydianin) and their 2,3-dehydro derivatives (i.e. 2,3-dehydrosilybin, 2,3-dehydrosilychristin, 2,3-dehydrosilydianin) activate the Nrf2 pathway, which regulates the expression of genes encoding many cytoprotective enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1). After 48h of exposure, 2,3-dehydrosilydianin at concentrations of 25μM and higher significantly elevated the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. In contrast, other tested compounds at non-cytotoxic concentrations had a mild or negligible effect on the NQO1 activity. Using a luciferase reporter assay, 2,3-dehydrosilydianin was found to significantly activate transcription via the antioxidant response element in stably transfected human AREc32 reporter cells. Moreover, 2,3-dehydrosilydianin caused the accumulation of Nrf2 and significantly induced the expression of the Nqo1 gene at both the mRNA and protein levels in Hepa1c1c7 cells. We found that 2,3-dehydrosilydianin also increased to some extent the expression of other Nrf2 target genes, namely of the heme oxygenase-1 gene (Hmox1) and the glutamate-cysteine ligase modifier subunit gene (Gclm). We conclude that 2,3-dehydrosilydianin activates Nrf2 and induces Nrf2-mediated gene expression in Hepa1c1c7 cells., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Flavonolignans constitute an important class of plant secondary metabolites formed by oxidative coupling of one flavonoid and one phenylpropanoid moiety. The standardized flavonolignan-rich extract prepared from the fruits of Silybum marianum is known as silymarin and has long been used medicinally, prominently as an antihepatotoxic and as a chemopreventive agent. Principal component analysis of the variation in flavonolignan content in S. marianum samples collected from different locations in Egypt revealed biosynthetic relationships between the flavonolignans. Silybin A, silybin B, and silychristin are positively correlated as are silydianin, isosilychristin, and isosilybin B. The detection of silyamandin in the extracts of S. marianum correlates with isosilychristin and silydianin content. The positive correlation between silydianin, isosilychristin, and silyamandin was demonstrated using quantitative 1 H nuclear magnetic resonance spectroscopy (qHNMR). These correlations can be interpreted as evidence for the involvement of a flavonoid radical in the biosynthesis of the flavonolignans in S. marianum. The predominance of silybins A & B over isosilybin A & B in the silybin-rich samples is discussed in light of the relative stabilities of their respective radical flavonoid biosynthetic intermediates., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A significant hurdle for discovery of plant-derived products is the numerous trial-and-error experiments required to develop an effective purification strategy. To overcome the experimental burden, a quantum mechanics-based molecular modeling approach - known as the COnductor-like Screening Model for Real Solvents (COSMO-RS) - was used to predict a suitable two-phase solvent system to purify six silymarins from an aqueous mixture. Silymarins, a class of flavonolignans found in milk thistle (Silybum marianum L.), are well suited for assessing the use of a molecular modeling approach to predict partitioning in a countercurrent chromatography (CCC) separation because they are well characterized and previous studies report low purity fractionation in liquid-liquid solvent systems. They also present an opportunity to evaluate the use of COSMO-RS in predicting the partitioning of structurally similar isomeric compounds that are present together in an aqueous solution upon extraction from their native source. The COSMO-RS model results predicted the partition coefficients in: three traditional ARIZONA solvent systems (composed of heptane, ethyl acetate, methanol, and water), nine additional variations of this quaternary solvent system, and two chloroform, methanol, and water solvent systems. Predicted results were concise but not accurate when compared to experimental results determined by the shake flask method. The 1:4:3:5 n-heptane:ethyl acetate:methanol:water (v/v/v/v) system was identified to be an improvement on the 1:4:3:4 system previously reported. The present study verified the ability of COSMO-RS to hone in on one or two solvent systems that will yield the best fractionation using CCC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Flavonolignans are a group of active chemical compounds presented in the silymarin - a standardized extract obtained from fruits and seeds of Milk thistle (Silybum marianum L. Gaernt.). Since the 70s of the last century, flavonolignans have been regarded to the official medicine as a substances having hepatoprotective properties. However many researches performed in recent years have demonstrated that flavonolignans posses many other healthy properties including modulation of variety cell-signaling pathways. The aim of our study was to examine the effects of three major flavonolignans (silybin, silychristin and silydianin) on ADP-induced blood platelet activation using the flow cytometry analysis as well as determine the mechanism of this interaction by bioinformatic ligand docking method. We observed that all tested flavonolignans in dose-dependent manner inhibit formation of blood platelet aggregates and microparticles as well as decrease expression of P-selectin and activation of integrin α IIb β 3 . Our computer-generated models confirm the flow cytometry analysis. We observed that all tested flavonolignans have conformations which are able to bind to the extracellular domain of P2Y12 receptor and probably block interaction with ADP. Our studies may help in the development of a new potential anti-platelet agent, which might be an alternative to the current using drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mature fruits collected from different milk thistle (Silybum marianum (L.) Gaertn.) plants, grown in various habitats in Europe, were analysed for silymarin content and variation in component composition. Two different German and Polish cultivars each, as well as fruits from Hungary and Bulgaria have been compared with respect to their ratio of flavonolignan regioisomers. Besides differences in total silymarin content (0.8%-4.9%), three distinct chemotypical variations in fruit flavonolignan regioisomer composition in the cultivars have been observed. Although the differences in the diastereomer ratios of silybin A/B and isosilybin A/B were not significant, they never appeared in a 1:1 ratio. In vitro cultures have been established from seedlings of three typical chemotypes for further insights into flavonolignan content and composition in suspension cultures and the release of these specialized compounds to the extracellular space. The differences in the three Silybum marianum chemotypes were also observed in the composition of the intracellular silymarin of suspension-cultured cells. Silymarin components released to the cell culture medium, however, showed a highly differing composition with only low amounts of silychristin and silydianin. Assays with crude protein extracts prepared from suspension cells or habituated medium of these three chemotypes did not result in differences in silymarin content or composition. In in vitro assays the formation of the regioisomers silydianin and silychristin were strongly influenced by the taxifolin:coniferyl alcohol concentration ratio., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Besides the well-known chemoprotective effects of polyphenols, their prooxidant activities via interactions with biomacromolecules as DNA and proteins are of the utmost importance. Current research focuses not only on natural polyphenols but also on synthetically prepared analogs with promising biological activities. In the present study, the antioxidant and prooxidant properties of a semi-synthetic flavonolignan 7-O-galloylsilybin (7-GSB) are described. The presence of the galloyl moiety significantly enhances the antioxidant capacity of 7-GSB compared to that of silybin (SB). These findings were supported by electrochemistry, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity, total antioxidant capacity (CL-TAC) and DFT (density functional theory) calculations. A three-step oxidation mechanism of 7-GSB is proposed at pH 7.4, in which the galloyl moiety is first oxidized at Ep,1=+0.20V (vs. Ag/AgCl3M KCl) followed by oxidation of the 20-OH (Ep,2=+0.55V) and most probably 5-OH (Ep,3=+0.95V) group of SB moiety. The molecular orbital analysis and the calculation of O-H bond dissociation enthalpies (BDE) fully rationalize the electrooxidation processes. The metal (Cu(2+)) complexation of 7-GSB was studied, which appeared to involve both the galloyl moiety and the 5-OH group. The prooxidant effects of the metal-complexes were then studied according to their capacity to oxidatively induce DNA modification and cleavage. These results paved the way towards the conclusion that 7-O-galloyl substitution to SB concomitantly (i) enhances antioxidant (ROS scavenging) capacity and (ii) decreases prooxidant effect/DNA damage after Cu complexation. This multidisciplinary approach provides a comprehensive mechanistic picture of the antioxidant vs. metal-induced prooxidant effects of flavonolignans at the molecular level, under ex vivo conditions., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)