The Seed-Head Weevil,Larinus Latus Herbst (Coleoptera: Curculionidae) as a New Record in Egypt on the Milk Thistle,Silybum Marianum (L.) (Asteraceae: Compositaea)

The seed-head weevil, Larinus latus Herbst (Coleoptera: Curculionidae) was recorded for the first time in Egypt on the milk thistle, Silybum marianum (L.) (Asteraceae: Compositaea), which is considered as a weed in certain winter field crops and as a herb with medical importance. The general morphology and biology of this insect pest as well as the host plant were described.     

Enhanced silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.     

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.     

Identification of flavonolignans from Silybum marianum seeds as allosteric protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is an attractive molecular target for anti-diabetes, anti-obesity, and anti-cancer drug development. From the seeds of Silybum marianum, nine flavonolignans, namely, silybins A, B (1, 2), isosilybins A, B (3, 4), silychristins A, B (5, 6), isosilychristin A (7), dehydrosilychristin A (8), and silydianin (11) were identified as a novel class of natural PTP1B inhibitors (IC₅₀ 1.3 7–23.87?µM). Analysis of structure–activity relationship suggested that the absolute configurations at C-7" and C-8" greatly affected the PTP1B inhibitory activity. Compounds 1–5 were demonstrated to be non-competitive inhibitors of PTP1B based on kinetic analyses. Molecular docking simulations resulted that 1–5 docked into the allosteric site, including α3, α6, and α7 helix of PTP1B. At a concentration inhibiting PTP1B completely, compounds 1–5 moderately inhibited VHR and SHP-2, and weakly inhibited TCPTP and SHP-1. These results suggested the potentiality of these PTP1B inhibitors as lead compounds for further drug developments.     

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.     

Oral bioavailability of silymarin phytocomplex formulated as self-emulsifying pellets

The objective of this study was to develop new solid self-emulsifying pellets to deliver milk thistle extract (silymarin). These pellets were prepared via extrusion/spheronisation procedure, using a self-emulsifying system or SES (Akoline MCM^®, Miglyol^®, Tween 80^®, soy lecithin and propylene glycol), microcrystalline cellulose and lactose monohydrate. To select the most suitable formulations for extrusion and spheronisation, an experimental design of experiences was adopted. The screening amongst formulations (13 different blends) was performed preparing pellets and evaluating extrusion profiles and quality of the spheronised extrudates. The pellets were characterised for size and shape, density, force required to crush them. Although more than one type of pellets demonstrated adequate morphological and technological characteristics, pellets prepared from formulation 7 revealed the best properties and were selected for further biopharmaceutical investigations, including in vitro dissolution and in vivo trials on rats to study serum and lymph levels after oral administration of the pellets. These preliminary technological and pharmacokinetic data demonstrated that extrusion/spheronisation is a viable technology to produce self-emulsifying pellets of good quality and able to improve in vivo oral bioavailability of main components of a phytotherapeutic extract of more than 100 times by enhancing the lymphatic route of absorption.     

Silymarin BIO-C, an extract from Silybum marianum fruits, induces hyperprolactinemia in intact female rats

Breastfeeding is widely acknowledged to have important health benefits for infants and mothers. Milk thistle (Silybum marianum fruits) has been recently proposed to be used by nursing mothers for stimulating milk production; however, the mode of action of this herbal drug is still unknown. In this paper, we have evaluated the effect of a micronized standardized extract of S. marianum (Silymarin BIO-C^®=Piùlatte^®) on the serum levels of prolactin in female rats. A 14-day treatment with Silymarin BIO-C^® (25–200 mg/kg, given orally) increased, in a dose dependent manner, the serum prolactin levels. Moreover, after a 66-day discontinuation of Silymarin BIO-C^® treatment, prolactin levels were still significantly elevated although we observed a trend to decrease that was counteracted by a further 7-day treatment with Silymarin BIO-C^®. Bromocriptine, a dopamine D₂ receptor agonist, (1–10 mg/kg, os) significantly and in a dose dependent manner, reduced the serum prolactin levels; bromocriptine, at the dose of 1 mg/kg, significantly reduced the high serum prolactin levels induced by Silymarin BIO-C^®. In conclusion, we have shown that an extract from S. marianum fruits significantly increases circulating prolactin levels in female rats; this effect seems to involve, at least in part, dopamine D₂ receptors.     

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t‐resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β‐cyclodextrins

The growing demand for t‐resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large‐scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β‐cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t‐resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t‐resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non‐bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.     

Batch solvent extraction of flavanolignans from milk thistle (Silybum marianum L. Gaertner)

Seeds of milk thistle (Silybum marianum L. Gaertner) contain silymarins and ca. 25% (w/w) of oil. A pre-treatment step involving refluxing with petroleum ether is usually performed before extraction of the silymarins using organic solvents. This paper compares the extraction of whole and defatted milk thistle seeds in various solvents as a function of temperature. The extraction of whole seeds of milk thistle with water at 50, 70 and 85 degrees C was also examined: the yield of silymarin increased with increasing water temperature. In most cases, ethanol at 60 degrees C recovered the largest quantities of silymarins. However, boiling water proved to be an efficient extraction solvent for the more polar silymarins such as taxifolin and silychristin, even when using whole seeds. Extractions of defatted seed meal with boiling ethanol returned maximum yields of 0.62, 3.89, 4.04, and 6.86 mg/g defatted seed of taxifolin, silychristin, silybinin A and silybinin B, respectively. When extracting defatted seed meal with ethanol, yields of taxifolin, silybinin A and silybinin B were, respectively, 6.8-, 0.95-, 1.7- and 1.6-fold higher than when extracting whole seeds. When extracting with boiling water, the yields of silychristin, silybinin A, and silybinin B were 380, 47 and 50% higher for whole seeds compared with defatted seeds.     

Callus Formation from Protoplasts and Plant Regeneration from Tissue Culture of Silybum marianum Gaertn

Leaf calli of Silybum marianum Gaertn. subcultured for one year were used for protoplast isolation and culture. First division was observed three days after culture on medium M12, and the highest division frequency was 35.4%. One to three months later, small ralli were seen with naked eyes, and grew up gradually. Upon transferring them onto D6 differentiation medium, the green bud apices were observed two months later. However, no shoot differentiation was obtained. Hypocotyl calli were induced on MS+NAA 0.8mg/1, 6-BA 0.5mg/1. Two months after transferring calli onto D6 medium, shoots were regenerated from the surface of the calli. The freqency of shoot differentiation was 75%. On a MS rooting medium containing NAA 0.5 mg/1, IBA 0.1 mg/1, whole plants with healthy roots were obtained.