David Biedermann

Flavonolignan 2,3-dehydroderivatives: Preparation, antiradical and cytoprotective activity

The protective constituents of silymarin, an extract from Silybum marianum fruits, have been extensively studied in terms of their antioxidant and hepatoprotective activities. Here, we explore the electron-donor properties of the major silymarin flavonolignans. Silybin (SB), silychristin (SCH), silydianin (SD) and their respective 2,3-dehydroderivatives (DHSB, DHSCH and DHSD) were oxidized electrochemically and their antiradical/antioxidant properties were investigated. Namely, Folin-Ciocalteau reduction, DPPH and ABTS(+) radical scavenging, inhibition of microsomal lipid peroxidation and cytoprotective effects against tert-butyl hydroperoxide-induced damage to a human hepatocellular carcinoma HepG2 cell line were evaluated. Due to the presence of the highly reactive C3-OH group and the C-2,3 double bond (ring C) allowing electron delocalization across the whole structure in the 2,3-dehydroderivatives, these compounds are much more easily oxidized than the corresponding flavonolignans SB, SCH and SD. This finding was unequivocally confirmed not only by experimental approaches, but also by density functional theory (DFT) calculations. The hierarchy in terms of ability to undergo electrochemical oxidation (DHSCH~DHSD>DHSB>>SCH/SD>SB) was consistent with their antiradical activities, mainly DPPH scavenging, as well as in vitro cytoprotection of HepG2 cells. The results are discussed in the context of the antioxidant vs. prooxidant activities of flavonolignans and molecular interactions in complex biological systems.

Anti-cancer efficacy of silybin derivatives -- a structure-activity relationship.

Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-O-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-O-palmitoylsilybin (7OP), and 23-O-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.

Enzymatic Kinetic Resolution of Silybin Diastereoisomers

In nature, the flavonolignan silybin (1) occurs as a mixture of two diastereomers, silybin A and silybin B, which in a number of biological assays exhibit different activities. A library of hydrolases (lipases, esterases, and proteases) was tested for separating the silybin A and B diastereomers by selective transesterification or by stereoselective alcoholysis of 23-O-acetylsilybin (2). Novozym 435 proved to be the most suitable enzyme for the preparative production of both optically pure silybins A and B by enzymatic discrimination. Gram amounts of the optically pure substances can be produced within one week, and the new method is robust and readily scalable to tens of grams.

Phototoxic potential of silymarin and its bioactive components

Silymarin, a standardized extract of the seeds of the milk thistle (Silybum marianum) and its major component, silybin, is now used as an active component in a broad spectrum of dietary supplements, cosmetics and dermatological preparations. However, despite its use in skin products, there are no published data to exclude its phototoxic potential. The primary purpose of this study was to examine the phototoxicity of silymarin and its flavonolignans, silybin, isosilybin, silychristin, silydianin and 2,3-dehydrosilybin by validated 3T3 NRU assay. Further, we compared the validated biological system Balc/c 3T3 cell line with other cell models, particularly normal human dermal fibroblasts (NHDF), normal human epidermal keratinocytes (NHEK) and the human keratinocyte cell line (HaCaT). The results showed that silymarin and the flavonolignans silybin, isosilybin, silychristin and silydianin had no phototoxicity towards any of the cells used. In contrast, 2,3-dehydrosilybin was identified as a compound with phototoxic potential. Further study is needed to evaluate the health risks associated with 2,3-dehydrosilybin use in skin preparations.

The silymarin composition… and why does it matter???

The extract from milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin, contains a variety of flavonolignans and displays antioxidant, anti-inflammatory, immunomodulatory and hepatoprotective properties. As silybin is the main component of silymarin, the literature mainly focuses on this compound, ignoring all other components. This leads to problems in reproducibility of scientific results, as the exact composition of silymarin is often unknown and can vary to a certain degree depending on the processing, chemo-variety of the plant used and climatic conditions during the plant growth. There are studies dealing with the analytical separation and quantification of silymarin components as well as studies focused on silymarin content in clinically used drugs, in various plant parts, seasons, geographic locations etc. However, no comparison of detail flavonolignan profiles in various silymarin preparations is available to date. Also, as a result of the focus on the flavonolignans; the oil fraction, which contains linoleic, oleic and palmitic acids, sterols, tocopherol (vitamin E) and phospholipids, has been neglected. Due to all these factors, the whole plant is used e.g. as animal feed, the leaves can be eaten in salads and seed oil, besides culinary uses, can be also utilized for biodiesel or polymer production. Various HPLC separation techniques for the determination of the content of the flavonolignans have been vastly summarized in the present review.

Silibinin and its 2,3-dehydro-derivative inhibit basal cell carcinoma growth via suppression of mitogenic signaling and transcription factors activation

Basal cell carcinoma (BCC) is the most common cancer worldwide, and its current treatment options are insufficient and toxic. Surprisingly, unlike several other malignancies, chemopreventive efforts against BCC are almost lacking. Silibinin, a natural agent from milk thistle seeds, has shown strong efficacy against several cancers including ultraviolet radiation‐induced skin (squamous) cancer; however, its potential activity against BCC is not yet examined. Herein, for the first time, we report the efficacy of silibinin and its oxidation product 2,3‐dehydrosilibinin (DHS) against BCC both in vitro and in vivo using ASZ (p53 mutated) and BSZ (p53 deleted) cell lines derived from murine BCC tumors. Both silibinin and DHS significantly inhibited cell growth and clonogenicity while inducing apoptosis in a dose‐ and time‐dependent manner, with DHS showing higher activity at lower concentrations. Both agents also inhibited the mitogenic signaling by reducing EGFR, ERK1/2, Akt, and STAT3 phosphorylation and suppressed the activation of transcription factors NF‐κB and AP‐1. More importantly, in an ectopic allograft model, oral administration of silibinin and DHS (200 mg/kg body weight) strongly inhibited the ASZ tumor growth by 44% and 71% (P < 0.05), respectively, and decreased the expression of proliferation biomarkers (PCNA and cyclin D1) as well as NF‐κB p50 and c‐Fos in the tumor tissues. Taken together, these results provide the first evidence for the efficacy and usefulness of silibinin and its derivative DHS against BCC, and suggest the need for additional studies with these agents in pre‐clinical and clinical BCC chemoprevention and therapy models.

Silychristin: Skeletal Alterations and Biological Activities

Silychristin is the second most abundant flavonolignan (after silybin) present in the fruits of Silybum marianum. A group of compounds containing silychristin (3) and its derivatives such as 2,3-dehydrosilychristin (4), 2,3-dehydroanhydrosilychristin (5), anhydrosilychristin (6), silyhermin (7), and isosilychristin (8) were studied. Physicochemical data of these compounds acquired at high resolution were compared. The absolute configuration of silyhermin (7) was proposed to be identical to silychristin A (3a) in ring D (10R,11S). The preparation of 2,3-dehydrosilychristin (4) was optimized. The Folin-Ciocalteau reduction and DPPH and ABTS radical scavenging assays revealed silychristin and its analogues to be powerful antioxidants, which were found to be more potent than silybin and 2,3-dehydrosilybin. Compounds 4-6 exhibited inhibition of microsomal lipoperoxidation (IC50 4-6 μM). Moreover, compounds 4-8 were found to be almost noncytotoxic for 10 human cell lines of different histogenetic origins. On the basis of these results, compounds 3-6 are likely responsible for most of the antioxidant properties of silymarin attributed traditionally to silybin (silibinin).

Prokaryotic and Eukaryotic Aryl Sulfotransferases: Sulfation of Quercetin and Its Derivatives

Two types of sulfotransferases, namely recombinant rat liver aryl sulfotransferase AstIV and bacterial aryl sulfotransferase from Desulfitobacterium hafniense, were used for the sulfation of quercetin, its glycosylated derivatives (isoquercitrin and rutin), and dihydroquercetin ((+)‐taxifolin). The rat liver enzyme was able to sulfate only quercetin and taxifolin, whereas the quercetin glycosides remained intact. The D. hafniense enzyme sulfated isoquercitrin and rutin selectively at the C‐4′ position of the catechol moiety with very good yields. Taxifolin was sulfated at the C‐4′ position and a minor amount of the C‐3′ isomer was formed. Sulfation of quercetin proceeded preferentially at the C‐3′ position, but a lower proportion of the C‐4′ isomer was formed as well. A detailed analysis of the kinetics of this reaction is provided and a full structural analysis of all products is presented.

cis–trans Isomerization of silybins A and B

Summary Methods were developed and optimized for the preparation of the 2,3-cis- and the 10,11-cis-isomers of silybin by the Lewis acid catalyzed (BF3∙OEt2) isomerization of silybins A (1a) and B (1b) (trans-isomers). The absolute configuration of all optically pure compounds was determined by using NMR and comparing their electronic circular dichroism data with model compounds of known absolute configurations. Mechanisms for cis–trans-isomerization of silybin are proposed and supported by quantum mechanical calculations.

2,3-Dehydrosilybin A/B as a pro-longevity and anti-aggregation compound.

ABSTRACT Aging is an unavoidable process characterized by gradual failure of homeostasis that constitutes a critical risk factor for several age‐related disorders. It has been unveiled that manipulation of various key pathways may decelerate the aging progression and the triggering of age‐related diseases. As a consequence, the identification of compounds, preferably natural‐occurring, administered through diet, with lifespan‐extending, anti‐aggregation and anti‐oxidation properties that in parallel exhibit negligible side‐effects is the main goal in the battle against aging. Here we analyze the role of 2,3‐dehydrosilybin A/B (DHS A/B), a minor component of silymarin used in a plethora of dietary supplements. This flavonolignan is well‐known for its anti‐oxidative and neuroprotective properties, among others. We demonstrate that DHS A/B confers oxidative stress resistance not only in human primary cells but also in the context of a multi‐cellular aging model, namely Caenorhabditis elegans (C. elegans) where it also promotes lifespan extension. We reveal that these DHS A/B outcomes are FGT‐1 and DAF‐16 dependent. We additionally demonstrate the anti‐aggregation properties of DHS A/B in human cells of nervous origin but also in nematode models of Alzheimers disease (AD), eventually leading to decelerated progression of AD phenotype. Our results identify DHS A/B as the active component of silymarin extract and propose DHS A/B as a candidate anti‐aging and anti‐aggregation compound. Graphical abstract Figure. No Caption Available. Highlights2,3‐dehydrosilybin A/B (DHS A/B) promotes lifespan extension in C. elegans.DHS A/B promotes resistance to stress in human cells and in C. elegans.DHS A/B exerts anti‐aggregation properties in human cells and nematodes.DHS A/B leads to decelerated progression of Alzheimers disease phenotype.DHS A/B outcomes in C. elegans are FGT‐1‐ and DAF‐16‐ dependent.