Silymarin administration during pregnancy and breastfeeding: evaluation of initial development and adult behavior of mice.

Silymarin is a phytotherapeutic agent derived from the species Silybum marianum (Asteraceae), commonly is known as milk thistle, and traditionally used as a hepatoprotective; however, recent studies have proposed its use in order to promote lactogenesis, but there are few reports of its effects on the development of offspring. Thus, the objective of this study was to evaluate the effect of silymarin treatment during pregnancy and breastfeeding on the sensory-somatic-motor development and adult behavior of F1-generation Swiss mice. The pregnant females of the parental generation were distributed in four experimental groups and treated orally with doses of 100, 200 or 300 mg/kg of silymarin, with a control group receiving vehicle - vegetable oil (VEH), to obtain the F1-generation. At the end of lactation, the parental generation were submitted to euthanasia. Body mass evolution was determined in both generations. The sensory-motor development of the offspring (F1-generation) was evaluated, and one male pup from each litter was followed up for an analysis of adult behavior. In the F1 analysis, no differences between the groups were observed in initial development from the sensory-somatic-motor analysis performed during the 1st to 21st postnatal days. In the behavioral evaluation of adults from the F1 generation, all the groups from dams treated with silymarin in open field (OF) analysis showed a decrease in the time spent in the periphery and an increase in the time spent in the center, but the ambulation observed by the number of quadrant crossed showed no difference. In addition, during OF, the 100 and 200 mg/kg groups presented an increase in fecal bolus compared with the VEH group. There was a decrease in immobility time in the forced swimming test in the 300 mg/kg group compared to the VEH group. Regarding the memory and learning test, the groups did not differ in their recognition scores. The results of this study using an animal model indicate that treatment with silymarin during pregnancy and breastfeeding does not promote significant morpho-functional changes in the offspring in their initial development and adult behavior, indicating the safety of its use during gestation and lactation.

Cytotoxic, hepatoprotective and antioxidant activities of Silybum marianum variety albiflorum growing in Egypt.

Silymarin prepared from the fruits of Silybum marianum (L.) Gaertn. (Asteraceae) has long been used for the treatment of liver disorders. This study was carried out to evaluate the protective effect of the fruit extract of white-flowered S. marianum variety albiflorum Eig. (WSE) against paracetamol-induced liver toxicity in rats. Silyhermin, isosilandrins A/B were identified as the major flavonolignans in WSE. Cytotoxic activities of WSE and isolated flavonolignans compared to silymarin were carried out using sulforhodamine B assay. WSE, silyhermin and isosilandrins had no obvious harmful effect on normal human cell line compared to silymarin with IC50 values 78.95, 84.34, 72.14 and 16.83 µg/ml, respectively. The hepatoprotective activity of WSE at dose 50 mg/kg was comparable to silymarin (100 mg/kg). These data were supplemented with histopathological studies on liver sections. The hepatoprotective effects of WSE on oxidative stress induced by administration of paracetamol are probably associated with its antioxidant properties.

Nanoencapsulation Improves Scavenging Capacity and Decreases Cytotoxicity of Silibinin and Pomegranate Oil Association.

Silibinin (SB) and pomegranate oil (PO) present therapeutic potential due to antioxidant activity, but the biological performance of both bioactives is limited by their low aqueous solubility. To overcome this issue, the aim of the present investigation was to develop nanocapsule suspensions with PO as oil core for SB encapsulation, as well as assess their toxicity in vitro and radical scavenging activity. The nanocapsule suspensions were prepared by interfacial deposition of preformed polymer method. SB-loaded PO-based nanocapsules (SBNC) showed an average diameter of 157 ± 3 nm, homogenous size distribution, zeta potential of -14.1 ± 1.7 mV, pH of 5.6 ± 0.4 and SB content close to 100%. Similar results were obtained for the unloaded formulation (PONC). The nanocapsules controlled SB release at least 10 times as compared with free SB in methanolic solution. The SBNC scavenging capacity in vitro was statistically higher than free SB (p < 0.05). Cell viability in monocytes and lymphocytes was kept around 100% in the treatments with SBNC and PONC, while the SB and the PO caused a decrease around 30% at 50 µM (SB) and 724 µg/mL (PO). Protein carbonyls and DNA damage were minimized by SB and PO nanoencapsulation. Lipid peroxidation occurred in nanocapsule treatments regardless of the SB presence, which may be attributed to PO acting as substrate in reaction. The free compounds also caused lipid peroxidation. The results show that SBNC and PONC presented adequate physicochemical characteristics and low toxicity against human blood cells. Thereby, this novel nanocarrier may be a promising formulation for therapeutic applications.

The Potential of Flavonolignans in Prostate Cancer Management.

BACKGROUND: The generic name "flavonolignan" was created in 1968 for a relatively small class of naturally occurring hybrid molecules biogenetically originated from ubiquitous flavonoids and lignans (phenylpropanoids). The first group of flavonolignans was extracted from Silybum marianum that has long been used for hepatoprotection. Recently, the medicinal merit of flavonolignans has been extended to the prostate cancer management. METHODS: Systematic interpretation and summarization of the relevant literature. RESULTS: Over forty naturally occurring flavonolignans have so far been obtained from various plants. Certain flavonolignans have been demonstrated by in vitro cell-based and in vivo animal-based experiments, and human clinical studies i) to possess effective chemopreventive function against various tumor promoters; ii) to show the anti-angiogenic efficacy; iii) to have potential in treating prostate cancer; iv) to sensitize prostate tumors to chemotherapeutic agents through down-regulation of P-glycoprotein and other mechanisms; and v) to be used by prostate cancer patients to protect or treat the hepatotoxicity caused by several chemotherapies. Certain flavonolignans can synergize with well-established chemotherapeutic agents for prostate cancer. CONCLUSION: This review provides a systematic and in-depth overview of the promise and potential of flavonolignans in prostate cancer management, which covers their chemopreventive effect, chemotherapeutic treatment, mechanisms of actions, synthetic derivatives, structure-activity relationships, and the difference in inhibiting prostate cancer cell proliferation between certain flavonoligans and their respective flavonoid counterpart. This summarization aims to provide valuable insights into further and rational development of flavonolignans for prostate cancer management by interpreting the data reported in the literature.

Effects of benzo[a]pyrene as an environmental pollutant and two natural antioxidants on biomarkers of reproductive dysfunction in male rats.

Benzo[a]pyrene (B[a]P) is an environmental toxicant and endocrine disruptor. Therefore, the aim of the present study was to investigate the toxicity of B[a]P in testis of rats and also to study the role of silymarin and thymoquinone (TQ) as natural antioxidants in the alleviation of such toxicity. Data of the present study showed that levels of testosterone, estrogen and progesterone were significantly decreased after treatment of rats with B[a]P. In addition, B[a]P caused downregulation of the expressions of steroidogenic enzymes including CYP17A1 and CP19A1, and decreased the activity of 17-ß hydroxysteroid dehydrogenase (17ß-HSD). Moreover, B[a]P decreased the activities of antioxidant enzymes including catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD), and significantly increased free radicals levels in testis of male rats. However, pretreatment of rats with silymarin prior to administration of B[a]P was found to restore the level of free radicals, antioxidant status, and activities of steroidogenic enzymes to their normal levels in testicular tissues. Moreover, histopathological finding showed that silymarin recovered the abnormalities occurred in tubules caused by B[a] P in testis of rats. On the other hand, TQ showed pro-oxidant effects and did not ameliorate the toxic effects of B[a] P on the testicular tissue since it decreased antioxidant enzymes activities and inhibited the protein expression of CYP11A1 and CYP21A2 compared to control rats. Moreover, TQ decreased the levels of testosterone, estrogen, and progesterone either in the presence or absence of B[a]P. It is concluded that B[a]P decreased testosterone levels, inhibited antioxidant enzymes activities, caused downregulation of CYP isozymes involved in steroidogenesis, and increased free radical levels in testis. Moreover, silymarin was more effective than TQ in restoring organism health and alleviating the deleterious effects caused by B[a]P in the testis of rats. Due to its negative impact, it is highly recommended to limit the use of TQ as a dietary supplement since millions of people in the Middle East are using it to improve their health.

Milk thistle: a future potential anti-osteoporotic and fracture healing agent.

Osteoporosis is a progressive disease of the skeleton characterised by bone fragility due to a reduction in bone mass and possibly to alteration in bone architecture that lead to a propensity to fracture with minimum trauma. Most osteoporotic fractures occur at locations rich in trabecular or cancellous bone and usually related to post menopausal women. Recently, silymarin received attention due to its alternative beneficial effect on bone formation. It is a mixture of flavonoids with powerful antioxidant properties. This review focuses on the use of milk thistle or silymarin for the treatment of osteoporosis that may be related to fracture bone. Silymarin shows potent antioxidant herb that may modulate multiple genes in favour of helping to build bone and prevent bone loss. In the mouse fracture healing model, silymarin supplementation improved tibial healing with elevated BMD and serum levels of ALP and osteocalcin. Silymarin also demonstrated clear estrogenic antiosteoporotic effects in bone structure. Silymarin appears to play a crucial role to prevent bone loss and might regulate osteogenesis and may be beneficial for fracture healing. If silymarin is considered for the use of post menopausal women, it may be used for the treatment of osteoporosis. It would be of great benefit to postmenopausal women to develop an oestrogen antagonist that is as potent and efficacious as oestrogen in preventing bone loss without the major side effect associated with HRT.

Silibinin reverses drug resistance in human small-cell lung carcinoma cells.

Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Silibinin is a flavonolignan extracted from milk thistle (Silybum marianum), extracts of which are used in traditional medicine. We tested the effects of silibinin on drug-sensitive (H69) and multi-drug resistant (VPA17) SCLC cells. VPA17 cells did not show resistance to silibinin (IC50 = 60 µM for H69 and VPA17). Flow cytometry analysis after incubation in 30 µM silibinin showed no changes in cell cycle phases in VPA17 or H69 cells compared with untreated cells. Silibinin (30 µM) incubation was pro-apoptotic in VPA17 cells after > 3 days, as measured by ELISA of BUdR labeled DNA fragments. Apoptosis was also indicated by an increase in caspase-3 specific activity and decrease in survivin in VPA17 MDR cells. VPA17 cells had increased Pgp-mediated efflux of calcein acetoxymethyl ester (calcein AM); however, this was inhibited in cells pre-incubated in silibinin for 5 days. Pre-incubation of VPA17 cells in 30 µM silibinin for 5 days also reversed resistance to etoposide (IC50 = 5.50 to 0.65 µM) and doxorubicin (IC50 = 0.625 to 0.035 µM). The possible synergistic relationship between silibinin and chemotherapy drugs was determined by exposure of VPA17 cells to 1:1 ratios of their respective IC50 values, with serial dilutions at 0.25 to 2.0 × IC50 and calculation of the combination index (CI). Silibinin and etoposide showed synergism (CI = 0.46 at ED50), as did silibinin and doxorubicin (CI = 0.24 at ED50). These data indicate that in SCLC, silibinin is pro-apoptotic, reverses MDR and acts synergistically with chemotherapy drugs. Silibinin, a non-toxic natural product may be useful in the treatment of drug-resistant SCLC.

Hepatoprotective activity of punarnavashtak kwath, an Ayurvedic formulation, against CCl4-induced hepatotoxicity in rats and on the HepG2 cell line.

OBJECTIVE: Punarnavashtak kwath (PNK) is a classical Ayurvedic formulation, mentioned in Ayurvedic literature Bhaishajya Ratnavali, for hepatic disorders and asthma. This study investigated the hepatoprotective activity of PNK to validate the traditional use of this formulation. MATERIALS AND METHODS: PNK was prepared in the laboratory according to the method given in Ayurvedic literature. Phytochemical screening was performed to determine the presence of phytoconstituents. Hepatoprotective activity was evaluated against CCl(4)-induced hepatotoxicity in rats and by its effect on the HepG2 cell line. RESULTS: Preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins, and a bitter principle in PNK. Administration of PNK produced significant hepatoprotective effect as demonstrated by decreased levels of serum liver marker enzymes such as aspartate transaminase, serum alanine transaminase, serum alkaline phosphatase, and serum bilirubin and an increase in protein level. Thiopentone-induced sleeping time was also decreased in the PNK-treated animals compared with the CCl(4)-treated group. It also showed antioxidant activity by increase in activity of glutathione, superoxide dismutase, and catalase and by a decrease in thiobarbituric acid reactive substance level compared with the CCl(4)-treated group. Results of a histopathological study also support the hepatoprotective activity of PNK. Investigation carried out on the HepG2 cell line depicted significant increase in viability of cells exposed to PNK as compared with CCl(4)-treated cells. DISCUSSION AND CONCLUSION: It can be concluded that PNK protects hepatocytes from CCl(4)-induced liver damages due to its antioxidant effect on hepatocytes. An in vitro study on HepG2 cell lines also supports its protective effect.

Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities.

The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 µg/ml. The EC(50) value was calculated to be 72.0 µg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 µg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c(max) at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 µg/ml, which is close to c(max). The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 µg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 µg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 µg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 µg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K(i) values were determined. All K(i) values exceeded c(max) by at least a factor of 10-fold. According to FDA regulations 1>c(max)/K(i)>0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.

Evaluation of the genotoxic and anti-genotoxic activities of silybin in human hepatoma cells (HepG2).

Silybin (SB), a constituent of the medicinal plant Silybum marianum, is reported to be a potent hepatoprotective agent, but little is currently known regarding its genotoxicity, mutagenicity and potential chemopreventive properties. In this study, we evaluated the ability of SB to induce DNA migration and micronuclei (MN) formation in human hepatoma cells (HepG2). Also, possible preventive effects of SB on MN formation induced by three different mutagens, bleomycin (BLEO), benzo[a]pyrene (B[a]P) and aflatoxin B(1) (AFB(1)), were studied. To clarify the possible mechanism of SB antimutagenicity, three treatment protocols were applied: pretreatment, in which SB was added before the application of the mutagens; simultaneous treatment, in which SB was added during treatment and post-treatment, in which SB was added after the application of the mutagens. At concentrations up to 100 microM, SB was non-genotoxic, while at a concentration of 200 microM, SB induced DNA migration, generated oxidized DNA bases, reduced cell viability, decreased the replicative index of the cells and induced oxidative stress. It is noteworthy that SB was able to reduce the genotoxic effect induced by B[a]P, BLEO and AFB(1) in pretreatment and simultaneous treatments but had no significant effect on DNA damage induction in post-treatment. Taken together, our findings indicate that SB presents anti-genotoxic activity in vitro, which suggests potential use as a chemopreventive agent.

The effect of an iron (III) chelator, silybin, on the proliferation and cell cycle of Jurkat cells: a comparison with desferrioxamine.

Silybin is a flavonoid with antioxidant and free radical scavenging abilities. Silybin also acts as an iron chelator by binding Fe (III). The present study was undertaken to assess the biological effects of silybin on T leukemia cells in the presence or absence of iron and compare its effects with a well-known iron chelator; desferrioxamine. In these experiments, we studied the growth capacity of Jurkat while varying iron availability in the environment. Desferrioxamine significantly inhibited growth and proliferation of Jurkat cells, blocking treated cells in the G0/G1 phase and inducing apoptosis. In contrast, silybin showed a bimodal effect, inducing cell proliferation at lower concentrations whereas inhibition of DNA synthesis and significant cell death was observed at higher concentrations. Chelation of Fe totally abrogated antiproliferative, cytotoxic and apoptotic effects of desferrioxamine on Jurkat cells. Conversely, the silybin-Fe complex had no appreciable effect on its antiproliferative and cytotoxic activities. The cytotoxic effect of desferrioxamine was also prevented in iron-loaded Jurkat cells; however, the effect of silybin on the growth and viability of iron-loaded cells was similar to the effect of its iron complex on untreated Jurkat cells. Despite the Fe chelating activity of silybin that suggests its possible application in chelation therapy of chronic iron overload, the biological effects of silybin on Jurkat cells are different than those of desferrioxamine, probably due to antioxidant activity of silybin, which causes pro-oxidant effect via iron-catalyzed oxidation with the subsequent generation of reactive oxygen species.

Recent advances in plant hepatoprotectives: a chemical and biological profile of some important leads.

Medicinal plants have been traditionally used for treating liver diseases since centuries. Several leads from plant sources have been found as potential hepatoprotective agents with diverse chemical structures. Although, a big list of hepatoprotective phytomolecules was reported in the scientific literature, only a few were potent against various types of liver damages. Of which, silymarin, andrographolide, neoandrographolide, curcumin, picroside, kutkoside, phyllanthin, hypophyllanthin, and glycyrrhizin have largely attracted the scientific community. This review focuses discussion on the chemistry, biological activity, mode of action, toxicity, and future prospects of these leads.

Physiological responses of a natural antioxidant flavonoid mixture, silymarin, in BALB/c mice: III. Silymarin inhibits T-lymphocyte function at low doses but stimulates inflammatory processes at high doses.

Silymarin is a mixture of bioactive flavonoids isolated from Milk Thistle (Silybum marianum). Crude extracts from this plant have been used for centuries as a natural remedy and silymarin is now effectively used in the treatment of inflammatory liver toxicity and disease in humans. In vitro studies show that silymarin can inhibit the production and damage caused by tumor necrosis factor alpha (TNFalpha) and is a potent antioxidant both in vitro and in vivo. Such findings suggest silymarin may impact the immune system but little information exists following in vivo exposure. Therefore, we tested the hypothesis that exposure to silymarin will modulate the inflammatory immune response. Male BABL/c mice (6/group) were treated intraperitoneally once daily for five days with 0, 10, 50 or 250 mg/kg of silymarin. Silymarin exposure did not produce any signs of overt toxicity or any changes in relative organ weights. Flow cytometric examination of splenic lymphocyte populations showed that the absolute number of CD3+ T-lymphocytes was reduced in the 10 and 50 mg/kg groups although significance was evident only in the 10 mg/kg group. Concomitant decreases in CD4+ and CD8+ T-cell populations were observed but only the CD4+ population in mice treated with 10 mg/kg of silymarin was significantly different from control. Functional examination of secondary lymphoid cells revealed that phytohemagglutinin-induced T-lymphocyte proliferation was increased in the lowest dose group only. B-lymphocyte blastogenesis induced by lipopolysaccharide was increased following exposure to 10 and 50 mg/kg of silymarin. Similarly, expression of TNFalpha, inducible nitric oxide synthase, IL-1beta and IL-6 mRNA were increased dose-dependently. The expression of IL-2 and IL-4 were reduced in mice treated with 10 and 50 mg/kg of silymarin although only the 10 mg/kg group was significantly different from control. The results indicate that in vivo parenteral exposure to silymarin results in suppression of T-lymphocyte function at low doses and stimulation of inflammatory processes at higher doses. Further studies investigating the effects of silymarin on the immune system are warranted.

Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels.

We have reported recently the anticancer effect of flavonoid antioxidant silymarin, the major part of milk thistle extract, against advanced human prostate carcinoma DU145 cells (X. Zi et al., Cancer Res., 58: 1920-1929, 1998) and later identified that silibinin is the main active component in silymarin responsible for its effect in cell culture studies. On the basis of these observations, here we assessed in vivo growth inhibitory potential of silibinin against advanced human prostate cancer (PCA). Dietary feeding of silibinin at 0.05 and 0.1% doses (w/w) for 60 days, 24 h after s.c. DU145 tumor xenograft implantation in athymic male nude mice, significantly inhibited tumor volume by 35 and 58% (P < 0.05), and wet weight of tumor by 29 and 40% (P < 0.05), respectively. In a second experiment where mice were fed with these test diets for 3 weeks before tumor xenograft implantation and continued on these diets for a total of 63 days, tumor volume and wet weight of tumor were reduced by 53-64% (P < 0.001-0.05) and 31-52% (P < 0.05), respectively. In both studies, animals did not show weight loss or reduced food consumption. These in vivo anticancer effects of silibinin were associated with an increased accumulation (up to 5.8 fold; P < 0.05) of human insulin-like growth factor-binding protein-3 in mouse plasma. In additional studies assessing biological availability of silibinin in nude mice and its antiproliferative activity at such doses in DU145 cells in culture, silibinin levels in plasma and prostate were found to be in the range of 7-13 microg/ml and 3.7-4.6 microg/g, respectively. At these biologically achievable silibinin concentrations, increased IGFBP-3 level in DU145 cell culture medium and a strong DU145 cell growth inhibition were observed that were irreversible in the absence of silibinin in culture medium. These findings extend and translate our observations on in vitro anticancer effect of silibinin/silymarin to an in vivo preclinical PCA model, which may form the basis for a Phase I clinical trial in PCA patients.

Antihepatotoxic properties of picroliv: an active fraction from rhizomes of Picrorhiza kurrooa.

The hepatoprotective activity of picroliv, the irridoid glycoside mixture from Picrorhiza kurrooa, was determined in adult male albino rats. Pretreatment with picroliv prevented the hepatotoxic effects of paracetamol and galactosamine as evidenced by various biochemical and histopathological observations. Maximum hepatoprotective effect was observed with daily oral doses of 6 and 12 mg/kg for 7 or 8 days. The antihepatotoxic action of picroliv seems likely due to an alteration in the biotransformation of the toxic substances resulting in decreased formation of reactive metabolites.

The effects of silymarin on experimental phalloidine poisoning.

The hepatoprotective action of silymarin, the active principle extracted from the fruit of Silybum marianum (L.) Gaertn., in animals (dogs, rabbits, rats, mice) intoxicated with phalloidine is evident, both after protective and curative treatment. A dose of 15 mg/kg of silymarin protects every animal when given 60 min before the toxin. When injected 10 mim after phalloidine, a dose of 100 mg/kg of silymarin again provides total protection. However, as the time span between administration of the toxic substance and start of treatment increases, so the efficacy of silymarin decreases; after 30 min its curative effect is negligible. The histochemical and histoenzymological studies show that during intoxication of the mice by phalloidine, silymarin inhibits the effect of the toxic substance and regulates the functions of the hepatocyte, when given either 60 min before or 10 min after phalloidine.