Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

New C-23 modified of silybin and 2,3-dehydrosilybin: synthesis and preliminary evaluation of antioxidant properties

Silybin is the major flavonolignan of silymarin and it displays a plethora of biological effects, generally ascribed to its antioxidant properties. Herein we shall describe an efficient synthetic strategy to obtain a variety of new and more water-soluble silybin and 2,3-dehydrosilybin (DHS) derivatives in which the 23-hydroxyl group was converted to a sulfate, phosphodiester, or amine group, using a solution-phase approach. Furthermore a new and efficient method for the preparation of DHS from silybin was developed and optimised.The antioxidant properties of the new compounds were evaluated in a cellular model in vivo and they displayed an antioxidant activity comparable to or higher than silybin and DHS, being able to prevent H₂O₂-induced generation of intracellular reactive oxygen species (ROS). Most of the derivatives also displayed a better hydrophilicity while retaining the biological activities of silybin and they might broaden the in vivo applications of this class of natural compounds.     

A new approach to enhance oral bioavailability of Silybum Marianum dry extract: Association of mechanochemical activation and spray congealing

The aim of the work was to produce a delivery system for Silybum Marianum dry extract with enhanced oral bioavailability by combining two technologies (mechanochemical activation and spray congealing). Initially, the active was coground with sodium croscarmellose in a planetary mill in order to reach an activated state more prone to dissolution. DSC, XRD, FT-IR and LD analyses showed the formation of nanosized particles of dry extract, with reduced degree of crystallinity of the main crystalline flavolignans (silybine A and B). Then, microparticles containing the activated coground and, as comparison, the corresponding physical mixture of extract and polymer and the dry extract alone were produced by spray congealing technology using Gelucire^® 50/13 as a hydrophilic low m.p. carrier. Microparticles containing the activated coground were produced spherical in shape, achieved satisfactory yield and high encapsulation efficiency. These microparticles, in addition to a favourable in vitro solubilisation kinetic, in a preliminary in vivo study in five rats demonstrated their ability to improve very significantly the oral bioavailability of the main flavolignans of Silybum Marianum dry extract (silybin A and B). These results suggested that the association of mechanochemical activation and spray congealing could be considered an innovative and very useful approach to the oral delivery of Silybum Marianum. Furthermore, for the first time the possibility of successfully applying the spray congealing technology for the preparation of a herbal drug delivery system was shown.     

Silibinin phosphodiester glyco-conjugates: Synthesis,redox behaviour and biological investigations

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 (IC₅₀ = 73 μM) than silibinin.     

Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

Optimisation of enzyme assisted extraction of silybin from the seeds of Silybum marianum by Box–Behnken experimental design

Introduction – Silybin, a standardised extract of flavanolignans from the seeds of Silybum marianum, has been used for centuries as a natural remedy in the treatment of hepatitis and cirrhosis. The higher yield of silybin by using more efficient extraction technique is of particular interest in the herbal products manufacture. Objective – To systematically investigate the important factors of enzyme‐assisted extraction of flavanolignans from the seeds of Silybum marianum to enhance the extraction yield of silybin. Methodology – The important factors of enzyme‐assisted extraction were optimised by employing Box–Behnken design with the aid of the orthogonal array design (OAD) OA₈ (2⁷). The effects of enzyme incubation temperature (EIT), the pH of enzyme solution (PES) and the size of seeds (SS) on the yield of silybin were visualised as three‐dimensional response surface and contour plots. Results – The predictive yield was 24.6 mg/g defatted seeds under the optimum enzymolysis conditions (EIT 40°C, PES 4.5 and SS 7003 μm). The coefficient of the model was r² > 0.97 (n = 15). The actual yield of silybin was 24.81 ± 1.93 mg/g defatted seeds, higher by 138 and 123.6% than that from ethanol extraction in this study and in the previous literature, respectively. IR spectra and HPLC of the extracts by EAE were in agreement with those from ethanol extraction. SEM and TEM pictures of defatted seeds by variant extractions demonstrate that the extraction of silybin depends on the destruction of cell walls. Conclusion – The results suggest that EAE is a promising alternative for the extraction of silybin by the use of traditional ethanol extraction. Copyright © 2009 John Wiley & Sons, Ltd.     

Iron and iron chelating agents modulate Mycobacterium tuberculosis growth and monocyte-macrophage viability and effector functions

Excess of iron promotes Mycobacterium tuberculosis infection, its replication and progression to clinical disease and death from tuberculosis. Chelation of iron may reduce M. tuberculosis replication, restore host defence mechanisms and it could constitute an application in the prevention and treatment strategies where both iron overload and tuberculosis are prevalent. We investigated the effect of iron and iron chelating agents, like desferrioxamine and silybin, individually and in combination with iron on mycobacterial number, viability in culture and after recovery from monocyte-macrophages, together with monocyte-macrophages viability and oxidative defence. Mycobacterial number and viability in culture were assessed using real-time quantitative PCR of H37Rv IS6110 DNA, 16S rRNA and 85B mRNA, whereas the microplate AlamarBlue(TM) assay was used to detect viability in culture post-infection. Mitochondrial membrane potential and phosphatidyl serine exposure of monocyte-macrophages, detected using Mitotracker Red fluorescence and Annexin V binding, respectively, served as indicators of host cell viability. Superoxide generation served as marker of monocyte-macrophage effector functions. Extracellular H37Rv showed a significant increase in number and viability in presence of excess iron and, by large, a significant decrease in number and viability in presence of the iron chelating agents, silybin and desferrioxamine, compared to cultivation without supplementation. Intracellularly, excess iron increased H37Rv viability significantly but reduced monocyte-macrophages mitochondrial membrane potential and compromised superoxide production. Desferrioxamine had little influence on intracellular parameters, but consistently prevented effects of excess iron, while silybin significantly altered most intracellular parameters and mostly failed to prevent effects of excess iron. These findings suggest that chelation therapy should be considered in conditions of iron overload and that effective chelating agents like desferrioxamine, with limited intracellular access might need to be used in combination with lypophilic chelating agents.     

Silybin and its bioavailable phospholipid complex (IdB 1016) potentiate in vitro and in vivo the activity of cisplatin

In this study we investigated whether the flavonoid silybin and its bioavailable derivative IdB 1016 (silipide) could enhance the antitumour activity of cisplatin (CDDP), the most commonly used drug in the treatment of gynaecological malignancies. Silybin alone up to 10 (M was unable to produce a relevant in vitro growth inhibition of A2780 cells, whereas CDDP was effective, giving an IC50 value of 0.5+/-0.14 microM. When silybin was combined with CDDP, a dose-dependent and statistically significant (p<0.05) increase of the CDDP activity was noticed, yielding IC50 values of 0.35+/-0.07 and 0.263+/-0.004 microM at silybin concentrations of 1 and 10 microM, respectively. The same trend was observed for in vivo experiments. IdB 1016 alone (1350 mg/kg) did not significantly affect tumour growth, whereas CDDP at the Maximum Tolerated Dose (12 mg/kg) produced a tumour weight inhibition (TWI%) of 80% and a log10 cell kill (LCK) of 0.7. Administration of both drugs resulted in a potentiation of the antitumour activity and TWI% and LCK increased to 90% and 1, respectively. Interestingly, mice receiving the combination recovered earlier in terms of body weight loss as compared to CDDP-treated mice. CDDP at 6 mg/kg yielded TWI of 44% and LCK of 0. The concomitant administration of IdB 1016 (1800 mg/kg) enhanced CDDP anti-tumour activity, with 68% TWI and 0.6 LCK. Finally, an antiangiogenic effect of IdB 1016 in an in vivo experimental model was demonstrated. Median haemoglobin value for the Matrigel from the vehicle-treated controls was 2.43 versus a value of 0.321 for the IdB 1016-treated animals.