Characterization of oxyphytosterols generated by ß-sitosterol ozonization.

ß-Sitosterol (ßSito) is the most abundant phytosterol found in elevated concentrations in vegetable oils, nuts, seeds, cereals, fruits, and in many phytosterol-enriched foods. Although the benefits, there is a concern in terms of food quality and health due to the increasing consumption of phytosterols and the possible adverse side effects of their oxidation products, oxyphytosterols. ßSito has a similar structure to cholesterol, with an unsaturated double bond at C5-C6, which is susceptible to oxidation by reactive oxygen species like ozone, generating oxyphytosterols. In this work we propose a mechanism of formation of three oxyphytosterols 2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7- octahydroinden-4-yl] acetaldehyde (ßSec), (2-[(7aR)-5-[(2R,5S)-5-hydroxy-2-methyl-7-oxo-oxepan- 2-yl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4- yl] acetaldehyde (ßLac) and 2-((7aR)-5-((1R,4S)-4-hydroxy-1-methyl-2- oxocyclohexyl)-1,7a-dimethyloctahydro-1Hinden-4-yl) acetic acid (ßCOOH) generated by ozonization of ßSito, through their synthesis and molecular characterization. The cytotoxic effect of ßSito and its main oxyphytosterol ßSec was evaluated and both reduced the HepG2 cell viability.

Spinasterol, 22,23-Dihydrospinasterol and Fernenol from Citrullus Colocynthis L. with Aphicidal Activity against Cabbage Aphid Brevicoryne Brassicae L.

Brevicoryne brassicae is a problematic pest in cabbage and other field crops. Synthetic pesticides are used to control this pest, but they are injurious for human health and the environment. The present study aimed to purify and identify the active compounds from Citrullus colocynthis leaves with an appraisal of their efficacy against B. brassicae. Separation and purification were performed via different chromatographic techniques. Molecular analysis and chemical structures were recognized by mass spectrum (MS) and nuclear magnetic resonance (NMR), respectively. Moreover, in vitro and in vivo aphicidal activity was assessed using various concentrations, i.e., 6.25, 12.5, 25 and 50 µg/mL at 12, 24, 48 and 72 h exposure. The outcome shows that mass spectrum analyses of the purified compounds suggested the molecular formulae are C30H50O and C29H50O, C29H48O. The compounds were characterized as fernenol and a mixture of spinasterol, 22,23-dihydrospinasterol by 1H-NMR and 13C-NMR spectrum analysis. The toxicity results showed that the mixture of spinasterol and 22,23-dihydrospinasterol showed LC50 values of 32.36, 44.49 and 37.50 µg/mL by contact, residual and greenhouse assay at 72 h exposure, respectively. In contrast, fernenol recorded LC50 values as 47.99, 57.46 and 58.67 µg/mL, respectively. On the other hand, spinasterol, 22,23-dihydrospinasterol showed the highest mortality, i.e., 66.67%, 53.33% and 60% while, 30%, 23.33% and 25% mortality was recorded by fernenol after 72 h at 50 µg/mL by contact, residual and greenhouse assay, respectively. This study suggests that spinasterol, 22,23-dihydrospinasterol are more effective against B. brassicae which may be introduced as an effective and suitable substitute of synthetic chemical pesticides.

Unilateral intranigral administration of ß-sitosterol ß-D-glucoside triggers pathological α-synuclein spreading and bilateral nigrostriatal dopaminergic neurodegeneration in the rat.

The spreading and accumulation of α-synuclein and dopaminergic neurodegeneration, two hallmarks of Parkinson's disease (PD), have been faithfully reproduced in rodent brains by chronic, oral administration of ß-sitosterol ß-D-glucoside (BSSG). We investigated whether a single injection of BSSG (6 µg BSSG/µL DMSO) in the left substantia nigra of Wistar rats causes the same effects. Mock DMSO injections and untreated rats formed control groups. We performed immunostainings against the pathological α-synuclein, the dopaminergic marker tyrosine hydroxylase (TH), the neuroskeleton marker ß-III tubulin, the neurotensin receptor type 1 (NTSR1) as non-dopaminergic phenotype marker and Fluro-Jade C (F-J C) label for neurodegeneration. Using ß-galactosidase (ß-Gal) assay and active caspase-3 immunostaining, we assessed cell death mechanisms. Golgi-Cox staining was used to measure the density and types of dendritic spines of striatal medium spiny neurons. Motor and non-motor alterations were also evaluated. The study period comprised 15 to 120 days after the lesion. In the injured substantia nigra, BSSG caused a progressive α-synuclein aggregation and dopaminergic neurodegeneration caused by senescence and apoptosis. The α-synuclein immunoreactivity was also present within microglia cells. Decreased density of dopaminergic fibers and dendritic spines also occurred in the striatum. Remarkably, all the histopathological changes also appeared on the contralateral nigrostriatal system, and α-synuclein aggregates were present in other brain regions. Motor and non-motor behavioral alterations were progressive. Our data show that the stereotaxic BSSG administration reproduces PD α-synucleinopathy phenotype in the rat. This approach will aid in identifying the spread mechanism of α-synuclein pathology and validate anti-synucleinopathy therapies.

Anti-Trypanosoma cruzi Activity in vitro of Phases and Isolated Compounds from Excoecaria lucida Leaves.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. This illness is found mainly in 21 Latin American countries and an estimated 8 million people are infected worldwide. The unsatisfactory chemotherapy provokes severe toxicity and resistant strains. Medicinal plants constitute a promising source of new drugs and remedies against all kinds of disorders, mainly infectious diseases arousing interest worldwide. OBJECTIVE: The aim of this study is the isolation, structural identification and evaluation of the trypanocidal activity of samples present in the Excoecaria lucida Sw. leaves. METHODS: Total extract (TE) of E. lucida Sw. leaves was obtained by ethanol extract therefore fractionated sequentially with hexane, ethyl acetate and n-butanol, to obtain three phases: Hex, EA and But, respectively. Ellagic acid (EL1) was purified from both EA and But phases, while EL2; a 1:1 stigmasterol-3-O-ß-D-glucopyranoside plus sitosterol-3-O-ß-D-glucopyranoside mixture was obtained from the Hex phase. Activity assays were performed using bloodstream and intracellular forms of T. cruzi and cytotoxicity assays using L929 fibroblasts. RESULTS: The EL1 and EL2 samples were more active against bloodstream trypomastigote forms with EC50 of 53.0±3.6 and 58.2±29.0 µg/mL, respectively; at 100 µg/mL. These samples also showed 70% of inhibition of L929 cells infection. Toxicity assays demonstrated that after 96 h of treatment only the fractions Hex and EA presented detectable cytotoxicity. CONCLUSION: Ellagic acid, stigmasterol-3-O-ß-D-glucopyranoside and sitosterol-3-O-ß-Dglucopyranoside are reported for the first time in E. lucida Sw. leaves as well as their biological activity studies supporting further investigations for Chagas disease treatment.

Autoinhibitory sterol sulfates mediate programmed cell death in a bloom-forming marine diatom.

Cell mortality is a key mechanism that shapes phytoplankton blooms and species dynamics in aquatic environments. Here we show that sterol sulfates (StS) are regulatory molecules of a cell death program in Skeletonema marinoi, a marine diatom-blooming species in temperate coastal waters. The molecules trigger an oxidative burst and production of nitric oxide in a dose-dependent manner. The intracellular level of StS increases with cell ageing and ultimately leads to a mechanism of apoptosis-like death. Disrupting StS biosynthesis by inhibition of the sulfonation step significantly delays the onset of this fatal process and maintains steady growth in algal cells for several days. The autoinhibitory activity of StS demonstrates the functional significance of small metabolites in diatoms. The StS pathway provides another view on cell regulation during bloom dynamics in marine habitats and opens new opportunities for the biochemical control of mass-cultivation of microalgae.

ß-Sitosterol Bioconversion to Androstenedione in Microtiter Plates.

Microtiter plates are routinely used as low-cost miniaturized bioreactors due to the large number of experiments that can be conducted simultaneously under similar conditions and replicate all functions of bench-scale reactors at dramatically smaller volumes. These plates, due to the standard footprint, can be integrated with liquid-handling systems and associated equipment expanding considerably their application and use. However, care has to be taken to operate the microtiter plates in optimized mixing and oxygen transfer conditions, preventing medium evaporation in prolonged experiment runs. Here, we describe the production of 4-androstene-3,17-dione (androstenedione; AD), a key pharmaceutical steroid intermediate, by Mycobacterium sp. NRRL B-3805 via the selective cleavage of the side-chain of ß-sitosterol using 24-well microtiter plates.

Identification of beta-sitosterol and stigmasterol in Bambusa bambos (L.) Voss leaf extract using HPLC and its estrogenic effect in vitro.

Focus of the study is to identify a safe alternate to Hormone Replacement Therapy by identifying the presence of ß-sitosterol and stigmasterol in the hydroalcoholic extract of Bambusa bambos using HPTLC and RP-HPLC-PDA; by evaluating the estrogenic effects of extract containing ß-sitosterol and stigmasterol on the growth of MCF-7 cells in vitro. Plant material was identified by DNA sequencing analysis. Presence of ß-sitosterol and stigmasterol was confirmed by HPTLC and direct RP-HPLC-PDA. Peaks with retention time about 19.13 and 21.16min were found to be stigmasterol and ß-sitosterol in extract. Extract was not cytotoxic to MCF-7 cells in any of the dilutions. It induced cell proliferation and all the dilutions except <500µg/ml have significantly increased cell multiplication. 15.6, 31.2, 62.5 and 125µg/ml of HEBB have shown influence on the proliferation rates similar to the standard 17ß-estradiol. The results suggest that HEBB might be used as a safe alternative to estrogen replacement therapies.

Fathead minnow response to broad-range exposure of ß-sitosterol concentrations during life-cycle testing.

The ß-sitosterol concentration in pulp and paper mill effluents is typically greater than that of other phytosterols and has been shown to cause a variety of effects in fish. The authors exposed fathead minnow (Pimephales promelas) to low (22 ± 0.93 µg/L), medium-low (70 ± 2.1 µg/L), medium-high (237 ± 5.5 µg/L), and high (745 ± 16.2 µg/L) concentrations of ß-sitosterol as well as negative (water), positive (ethynyl estradiol, 16 ± 0.58 ng/L), and carrier (0.6 mL/L acetone) controls. Fish were monitored over a full life cycle for population-level endpoints including growth and survival, reproductive endpoints (e.g. fecundity, sex steroids and vitellogenin, gonado-/hepatosomatic indices, and gonad histology). No significant differences were seen in fish growth, mortality, or reproduction with ß-sitosterol exposure, although a trend for lower egg production in ß-sitosterol exposures relative to the water control may be related to the acetone carrier. All ethynyl estradiol-exposed fish were smaller, showed female characteristics, and did not spawn. Sex steroid and vitellogenin were highly variable with no detectable treatment-related differences. Gonadal tissue showed no ß-sitosterol-related differences in reproductive development and spawning capability, although most ethynyl estradiol-exposed males had ovarian tissue and were not spawning-capable. The results indicate that ß-sitosterol exposure had little apparent impact on fathead minnow survival, growth, and reproduction even at concentrations >10 times that of typical effluents, although small sample size and variability precluded fully evaluating treatment responses on sex steroids and vitellogenin.

Chronic effects of Pinus radiata and Eucalyptus globulus kraft mill effluents and phytosterols on Daphnia magna.

Two kraft pulp mill effluents were compared in terms of their chronic toxicity to Daphnia magna. One resulted from pulping Pinus radiata and the other came from a parallel processing of Pinus radiata and Eucalyptus globulus (mixed kraft pulp mill effluent). The concentration of phytosterols found in the mixed kraft pulp mill effluent was higher than in the effluent from Pinus radiata, with values of 0.1082 and 0.02 µg/L, respectively. The phytosterols per se are responsible for 12.9% and 8.1% of the deviation from the natural shape, while the kraft pulp mill effluents account for 25.6%-27.8% of shape deviation. The role of ß-sitosterol and stigmasterol is discussed in relation to endocrine disruption.

Estrogenic and anti-estrogenic effects of wood extractives present in pulp and paper mill effluents on rainbow trout.

Wood extractives present in pulp and paper mill effluents may cause reproductive disturbances in fish. A chronic-exposure toxicity experiment using immature rainbow trout (Oncorhynchus mykiss) was conducted in order to assess the endocrine disrupting effects of two Chilean pulp and paper mill specific extracts (solid phase extraction, SPE) obtained from primary and secondary treated effluents. The (anti)estrogenic potencies and toxicity of the wood extractives regularly present in pulp mill effluent such as dehydroabietic acid (DHAA), beta-sitosterol (BS), and model estrogen 17beta-estradiol (E2) were evaluated by analysis of plasma vitellogenin (VTG) levels, gonadal somatic index (GSI) and liver ethoxyresorufin-O-deethylase (EROD) activity, respectively. The protocol involved the use of multiple intra-peritoneal injections (1 injection every 7 days for a total exposure period of 28 days). Analysis of variance/covariance, demonstrated no differences associated with fish gender other than GSI. The phytosterol BS, E2 and both pulp mill effluent extracts showed significant inductions of EROD and increased VTG levels after 4, 7, 14, 21 and 28 of exposure. While fish injected with secondary treated effluent extract showed a delayed induction in VTG levels compared to primary effluent injected fish, no effects on VTG and EROD levels were observed in DHAA injected fish. Moreover simultaneous injection of DHAA+E2 reduced the VTG levels found in E2 injected fish, indicating a potential indirect anti-estrogenic effect of this resin acid. The results of this study indicate that Chilean pulp and paper mill effluent extracts are estrogenic in rainbow trout males and females.

Ethnobotanical survey and cytotoxicity testing of plants of South-western Nigeria used to treat cancer, with isolation of cytotoxic constituents from Cajanus cajan Millsp. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: There is only scant literature on the anticancer components of medicinal plants from Nigeria, yet traditional healers in the area under study claim to have been managing the disease in their patients with some success using the species studied. AIM OF STUDY: To document plants commonly used to treat cancer in South-western Nigeria and to test the scientific basis of the claims using in vitro cytotoxicity tests. METHODS: Structured questionnaires were used to explore the ethnobotanical practices amongst the traditional healers. Methanol extracts of the most common species cited were screened for cytotoxicity using the sulforhodamine B (SRB) assay in both exposure and recovery experiments. Three cancer cell lines (human breast adenocarcinoma cell line MCF-7, human large cell lung carcinoma cell line COR-L23 and human amelanotic melanoma C32) and one normal cell line (normal human keratinocytes SVK-14) were used for the screening of the extracts and the fractions obtained. The extract of Cajanus cajan showed considerable activity and was further partitioned and the dichloromethane fraction was subjected to preparative chomatography to yield six compounds: hexadecanoic acid methyl ester, alpha-amyrin, beta-sitosterol, pinostrobin, longistylin A and longistylin C. Pinostrobin and longistylins A and C were tested for cytotoxicity on the cancer cell lines. In addition, an adriamycin-sensitive acute T-lymphoblastic leukaemia cell line (CCRF-CEM) and its multidrug-resistant sub-line (CEM/ADR5000) were used in an XTT assay to evaluate the activity of the pure compounds obtained. RESULTS: A total of 30 healers from S W Nigeria were involved in the study. 45 species were recorded with their local names with parts used in the traditional therapeutic preparations. Cytotoxicity (IC(50) values less than 50 microg/mL) was observed in 5 species (Acanthospermum hispidum, Cajanus cajan, Morinda lucida, Nymphaea lotus and Pycnanthus angolensis). Acanthospermum hispidum and Cajanus cajan were the most active. The dichloromethane fraction of Cajanus cajan had IC(50) value 5-10 microg/mL, with the two constituent stilbenes, longistylins A and C, being primarily responsible, with IC(50) values of 0.7-14.7 microM against the range of cancer cell lines. CONCLUSIONS: Most of the species tested had some cytotoxic effect on the cancer cell lines, which to some extent supports their traditional inclusion in herbal preparations for treatment of cancer. However, little selectivity for cancer cells was observed, which raises concerns over their safety and efficacy in traditional treatment. The longistylins A and C appear to be responsible for much of the activity of Cajanus cajan extract.

The regulation of inflammatory cytokine secretion in macrophage cell line by the chemical constituents of Rhus sylvestris.

In our preliminary screening study on the anti-inflammatory activity, eight triterpenes, one sterol, and one chalcone were isolated from the CH(2)Cl(2)-soluble extract of the stems and leaves of Rhus sylvestris Siebold and Zucc (Anacardiaceae). On the basis of their spectroscopic data, these compounds were identified as 10alpha-cucurbitadienol (1), glut-5-en-3-ol (2), beta-amyrin acetate (3), beta-amyrin (4) and lupeol (5), cycloart-24-en-3-one (6), cycloart-25-en-3,24-dione (7), 24-hydroxycycloart-25-en-3-one (8), beta-sitosterol (9), and 2'-hydroxy-4,4'-dimethoxychalcone (10). All of them were isolated from this plant for the first. Furthermore, the compounds in non-cytotoxic concentrations (0-1.0microM) were tested for their ability to block inflammatory cytokine secretion in the presence of LPS in the murine RAW264.7 macrophage cell line. Among the compounds that were tested, compounds 8 and 9 reduced the LPS-induced secretion of IL-6, as well as TNF-alpha, in a mouse RAW264.7 macrophage cell line. Moreover, compounds 2, 3, 7, and 10 specifically diminished only the secretion of TNF-alpha even in 0.01microM concentrations. It is thus suggested that they are potential therapeutics of TNF-alpha-related diseases and conditions, such as transplant rejection, type II diabetes, and atherosclerosis.

Cytotoxic and apoptotic effects of single and mixed oxides of beta-sitosterol on HepG2-cells.

While health implications caused by cholesterol oxidation products (COPs) seem to be generally accepted, research on phytosterol oxidation products (POPs) is still limited. Since POPs are commercially not available knowledge on their toxic activities is mainly derived from blends instead of pure compounds. Therefore the aim of the present study was to examine the cytotoxicity of three individual oxidation products of beta-sitosterol, 7-ketositosterol, 7beta-OH-sitosterol, 7alpha-OH-sitosterol, a mixture of 6beta-OH-3-keto-sitosterol/6alpha-OH-3-keto-sitosterol (ratio 4:3) and a mixture of polar oxides towards HepG2-cells. All tested compounds were found to reduce cell viability in a significant and concentration dependent way, particularly 7-keto- and 7alpha-OH-sitosterol showed to be highly active. Only for 7-ketositosterol an increase in early apoptotic cells was observed. Enhancement of O(2)(-) production was assessed for all oxides, whereas malondialdehyd (MDA) levels were increased by 7-keto- and 7alpha-OH-sitosterol only. However, cell death did not appear to be necessarily dependent on the generation of oxidative stress. Further no DNA strand breaks were observed with the COMET assay. By assessing the accumulation of single oxidation products in the cells a link between higher proportions of oxides inside the cells and their cytotoxic potential could be found.

Liver X receptor beta (LXRbeta): a link between beta-sitosterol and amyotrophic lateral sclerosis-Parkinson's dementia.

Administration of beta-sitosterol (42 mg/kg per day) for 3 weeks to 8-month-old male LXRbeta-/- mice resulted in the death of motor neurons in the lumbar region of the spinal cord and loss of tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra. In mice at 5 months of age, beta-sitosterol had no observed toxicity but at 16 months of age, it caused severe paralysis and symptoms typical of dopaminergic dysfunction in LXRbeta-/- mice. WT mice were not affected by these doses of beta-sitosterol. In 5-month-old mice, levels of the intestinal transporters, ABCG5/8 and Niemann-Pick C1 Like 1, were not affected by loss of liver X receptor (LXR) beta and/or treatment with beta-sitosterol nor were there changes in plasma levels of cholesterol or beta-sitosterol. In 8-month-old LXRbeta-/- mice there was activation of microglia in the substantia nigra pars reticulata and aggregates of ubiquitin and TDP-43 in the cytoplasm of large motor neurons in the lumbar spinal cord. Brain cholesterol concentrations were higher in LXRbeta-/- than in their WT counterparts, and treatment with beta-sitosterol reduced brain cholesterol in both WT and LXRbeta-/- mice. In LXRbeta-/- mice but not in WT mice levels of 24-hydrocholesterol were increased upon beta-sitosterol treatment. These data indicate that multiple mechanisms are involved in the sensitivity of LXRbeta-/- mice to beta-sitosterol. These include activation of microglia, accumulation of protein aggregates in the cytoplasm of large motor neurons, and depletion of brain cholesterol.

Evaluation of reproductive safety of beta-sitosterol on the American mink (Neovison vison).

beta-Sitosterol is a weakly estrogenic phytosterol used extensively in functional foods to lower elevated serum cholesterol concentrations due to its inhibitory action on intestinal cholesterol absorption. It caused previously decreased sex steroid concentrations in fish and lowered sperm counts in rats. In the American mink (Neovison vison), litter size increased slightly due to dietary beta-sitosterol supplement. The aim of the present experiment was to conduct a dose-response study on the effects of beta-sitosterol on the reproduction of the American mink. Juvenile male and female mink (n=480) were exposed to 0, 5, 10 or 50 mg of peroral beta-sitosterol kg(-1)d(-1) for 10 months. After 3 months of exposure in November, 15 males per group were sacrificed and general biochemical variables reflecting overall health were determined. The beta-sitosterol-treated male mink had increased absolute and relative masses of intraabdominal fat and higher blood hemoglobin and serum high-density lipoprotein cholesterol concentrations. In spring, the top-rated male mink were mated with multiple females within each study group and reproductive success was assessed. No differences in the reproductive performance of the males (10-11 per group) or females (47-50 per group) could be detected in the exposed groups and the kits of all groups developed in a similar manner. The results suggest that dietary beta-sitosterol presents no significant risk to mammalian fertility.

No evidence of genotoxic effect in vivo of the phytosterol oxidation products triols and epoxides.

Phytosterols (PS) are naturally occurring compounds present in food products of plant origin. Due to reported positive health effects, some food products are also enriched with PS. In the same way as cholesterol is oxidised, PS also oxidise to a variety of oxidation products (POPs). The biological effects and safety aspects of POPs are still unclear. This study investigated whether POPs are genotoxic in vivo, using a flow cytometer-based micronucleus assay in mice. The highest dose of oxidation products administered was 67mg/kg b.w. for epoxides and 9.4mg/kg b.w. for triols. Synthesised and separated triols and epoxides from a mixture of sitosterol and campesterol were investigated. The frequency of micronucleated polychromatic erythrocytes (fMNPCE) in POP-exposed mice did not significantly differ from the control values in either of two experiments performed. The flow cytometer-based method also allows for restriction of the analysis to micronuclei with a high DNA content, indicating an aneugenic potency. Even with this approach, there was no significant increase in the frequency of micronucleated erythrocytes among POP-treated mice compared with control mice. Furthermore, no significant deviation in cell proliferation rate (%PCE) was observed.

Steroidal constituents of rice (Rryza sativa) hulls with algicidal and herbicidal activity against blue-green algae and duckweed.

Two new compounds, 14-methyl stigmast-9(11)-en-3alpha-ol-3beta-D-glucopyranoside (1) and cholest-11-en-3beta, 6beta, 7alpha, 22beta-tetraol-24-one-3beta-palmitoleate (2), along with the known compound beta-sitosteryl-3beta-D-glucopyranosyl-6'-linoleiate (3), were isolated from the methanolic extract of rice (Oryza sativa) hulls. The structures of the two new compounds were elucidated using one- and two-dimensional NMR in combination with IR, EI/MS, FAB/MS, HR-EI/MS and HR-FAB/MS. In bioassays with blue-green algae, Microcystis aeruginosa UTEX 2388 and duckweed, Lemna paucicostata Hegelm 381, the efficacy of bioactivity of the two new compounds linearly increased as the concentration increased from 0.3 to 300 IgM. Compared with momilactone A, compounds 1 and 2 showed similar and higher inhibitory activities against the growth of M. aeruginosa at a concentration of 300 microM. However, compound 2 was similar to momilactone A in inhibiting L. paucicostata growth at a concentration of 300 microM. As a result, compound 2 appears to have a strong potential for the environmentally friendly control of weed and algae that are harmful to water-logged rice.

Clinical and molecular genetic analysis of a family with sitosterolemia and co-existing erythrocyte and platelet abnormalities.

We describe the clinical, biochemical and molecular genetic features of a Chinese family with sitosterolemia, mainly manifested by hematologic abnormalities. The clinical features of three patients were analyzed. Their plasma sterol levels were measured, and ABCG5 and ABCG8 genes sequenced to search for the causative mutation. The main clinical features of these patients were hemolysis and macrothrombocytopenia; they had increased plasma sitosterol but maintained normal cholesterol levels. Sequence analysis revealed a novel Gln22X nonsense mutation in exon 1 or ABCG5. Our results suggest that blood cells could be a target for the toxic effect of plasma phytosterols; the coexisting hematologic abnormalities might represent a specific subtype of sitosterolemia.

Antineoplastic activity of selected constituents of Duguetia glabriuscula.

The cytotoxic effects of seven constituents isolated from Duguetia glabriuscula were evaluated against Hep2 human larynx carcinoma cells. The cytotoxicity exhibited by beta-sitosterol was as strong as that of cis-platin. (+)-Alloaromadendran-10,14beta-diol caused inhibition of cellular growth with IC50 values lower than 25 microg/ml, a feature that was considered as revealing significant activity. Polycarpol showed borderline cytotoxicity, whereas the other compounds were inactive.

Toxicity of a phytosterol mixture to grayling (Thymallus thymallus) during early developmental stages.

The study concerns the toxicity of a phytosterol mixture, ultrasitosterol, consisting mainly of beta-sitosterol 75.7% and beta-sitostanol 13.0%, to grayling (Thymallus thymallus) embryos. Eyed eggs were exposed to three concentrations (1 microg/l, 10 microg/l, and 50 microg/l) of ultrasitosterol for 4 weeks. Embryos and later on hatched fry were taken for triiodothyronine (T3), thyroxine (T4), and histopathological analyses after 7, 14, 21, and 28 days of exposure. Most of the eggs (>95%) hatched during the first week of exposure, and ultrasitosterol treatment shortened hatching time significantly (p < or = 0.0001) at all exposure concentrations in comparison to the control. Ultrasitosterol did not have any significant effect on T3 or T4 levels in the embryo extracts. However, an interesting observation was that T3 levels increased in all treatments and in the control near the time of hatching. In conclusion, ultrasitosterol showed potential to affect the development of grayling embryos and fry, but further long-term exposure experiments are needed to verify these changes in more detail.