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.

Acyl moiety and temperature affects thermo-oxidative degradation of steryl esters. Cytotoxicity of the degradation products.

Phytosterols and their esters are often used as functional ingredients in food products due to its lowering blood cholesterol properties. Products containing phytosterols and its esters are recommended for direct consumption, cooking, baking and frying, however during food preparation it is possible thermo-oxidative degradation is possible. Unsaturation of fatty acid present in steryl ester may further stimulates degradation. Free stigmasterol degraded faster than its esters, even with linoleic acid attached. The highest amount of degradation products was observed for free stigmasterol, followed by esters with linoleic and oleic acids. Polar dimers were fund in all heated samples, although for free stigmasterol heated at 60 °C were not detected. Whereas non-polar dimers were observed only in heated stigmasterol. Degradation of esterified stigmasterol generated degradation products with lower cytotoxicity.

New steroidal alkaloid and furostanol glycosides isolated from Solanum lyratum with cytotoxicity.

Two previously undescribed steroidal compounds, 16, 23-epoxy-22, 26-epimino-cholest-22(N), 23, 25(26)-trien-3ß-ol-3-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→4)-ß-D-galactopyranoside (1) and 26-O-ß-D-glucopyranosyl-(25R)-5α-furost-20(22)-en-3ß, 26-diol (2), together with 7 known ones including 26-O-ß-D-glucopyranosyl-(25R)-5, 20(22)-dien-furost-3ß, 26-diol (3), (25R)-5-en-spirost-3ß-ol-O-ß-D-glucopyranosyl-(1→4)-[α-L-rhmanopyranosyl-(1→2)]-ß-D-galactopyranoside (4), funkioside D (5), aspidistrin (6), tigogenin-3-O-ß-D-lucotrioside (7), desglucolanatigonin II (8), and degalactotigonin (9), were isolated from Solanum lyratum Thunb. Their cytotoxic activities were tested in two cancer cell lines by MTT method. One of the steroidal glycosides (6) showed significant cytotoxic activity against gastric cancer SGC7901 and liver cancer BEL-7402 cells.

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.

Safety assessment of the aqueous extract of the flowers of Nymphaea lotus Linn (Nymphaeaceae): Acute, neuro- and subchronic oral toxicity studies in albinos Wistar rats.

Background Nymphaea lotus Linn (N. lotus) is a medicinal plant widely used in Cameroon popular medicine, to treat neuropsychiatric conditions, male sexual disorders or as food supplement. However, scientific data on the pharmacotoxic profile of this plant are not available. The safety of N. lotus was assessed in acute, neuro- and subchronic toxicity studies by following the OECD guidelines. Effectively, no data have been published until now in regard to its safety on the nervous system. Methods Aqueous extract of N. lotus at doses of 200, 400 and 600 mg/kg body weight (BW) was evaluated for nitrites contents and orally administered to rats daily for 28 days (5 male, 5 female per group). The control group received distilled water (10 mL/kg) and a satellite group was used to observe reversal effects. Neurotoxicity of the plant was determined using open field test for motor coordination, ataxia and gait analysis. Clinical signs and state of livelihood were recorded during the 24 h, then for 28 days of treatments. At the end of 28-day period, animals were anesthetized and decapitated. The whole brain was homogenized for neurobiochemical analysis. Blood samples were collected with or without anticoagulant for hematological examinations and serum analysis. Specimens of liver, kidney, testis, ovaries, and brain were fixed in 10 % formalin and processed for histopathological examinations. Results Our findings indicate dose-dependent elevation of nitrites contents in the flowers aqueous extract of N. lotus. Acute toxicity study revealed no signs of toxicity neither at the dose 2,000 mg/kg nor at 5,000 mg/kg. Thus the LD50 value of aqueous extract of N. lotus flowers is superior to 5,000 mg/kg. The repeated administration of N. lotus during 28 days, induced no signs of neurobehavioral changes in male, but female rats exhibited dose-dependent response in the open field test, suggesting sex and dose-relative psychotropic effects of N. lotus. The evaluation of neurobiochemistry revealed consistent rise of brain cholesterol by 44.05 %; 158.10 % and 147.62 % respectively in male rats treated with the doses of 200, 400 and 600 mg/kg. In female rats, these levels were significantly increased (p<0.001) only at the dose of 600 mg/kg compared to control. This trend persisted after 14 days withdrawal. Brain potassium and calcium concentrations were increased in all rats compared to their respective control receiving distilled water, suggesting transmembrane current stabilizing properties of brain cells by our extract. Further, serum biochemical analysis demonstrated that 28-day administration of N. lotus flowers increased depending on the dose and sex, the levels of serum urea, proteins, creatinine and bilirubin and reduced γ-glutamyltransferase (GGT) and alkaline phosphatase (ALP) activities. These results suggest liver alterations that are endowed by lower liver relative weight and histology damages observed in female rats treated with the dose of 600 mg/kg of our extract. We also observed a rise in the low-density lipoprotein (LDL) fraction and AI of male rats undergoing N. lotus treatment. In female rats, the latter remains unaltered, confirming the dose- and sex-dependent response of our extract. The levels of white blood cells (WBC) and granulocytes were higher in male irrespective to their control, revealing stimulatory properties of the male hematopoietic system. Such variations (sex- and dose-dependent) are without biological relevance for the majority of the biochemical parameters evaluated, indicating a wide margin of safety for the traditional use of N. lotus. The alkaloids, nitrites and phytosterols contained in N. lotus flowers extract may probably account for its neuroprotective, anti-oxidant, and immunoboosting properties. Conclusions N. lotus do not possesses neurotoxicity but is able to induce behavioral changes in rats. Therefore, the application of this plant as either drug or supplementary food should be carefully considered.

Antiproliferative and toxicological properties of methanolic extract obtained from Solanum capsicoides All. seeds and carpesterol.

Natural products are considered important sources of potential chemotherapeutic agents. Here, we evaluated the antiproliferative activity and the toxicological effects of the methanolic extract and a pure compound obtained from Solanum capsicoides seeds. The phytochemical profile was analyzed by chromatographic and spectroscopy methods. The acute toxicity was assessed in mice orally treated with the extract (2000 mg/kg), in vitro hemolytic activity and micronucleus test. The mutagenicity, developmental toxicity, and lethal dose (LD50) of carpesterol were estimated by the Toxicity Estimation Software Tool (TEST) software. A sulforhodamine B assay was employed to evaluate the antiproliferative activity. The toxicological assays did not observe signs of toxicity, either during the behavioral observations or in the autopsies, as well as no mutagenicity and hemolytic activity. The carpesterol did not present mutagenic effect and hemolytic activity but presents developmental toxicology and LD50 of 410 mg/kg in toxicity estimations by the TEST software. The S. capsicoides extract exhibited antiproliferative activity mainly in leukemia (K562) cell lineage. However, carpesterol presented antiproliferative activity in glioma (U251), breast (MCF-7), kidney (786-0), ovary (OVCAR-03), and K562 cell lineages. The data obtained show that S. capsicoides extract presents antiproliferative and does not present toxicological effects. In addition, it was shown for the first time the antiproliferative and toxicological parameters of carpesterol.

Phytosterol oxidation products in enriched foods: Occurrence, exposure, and biological effects.

Hypercholesterolemia is an important risk factor for the development of cardiovascular diseases. Dietary intake of phytosterols/phytostanols and their fatty acid esters results in a reduction of the LDL and total plasma cholesterol levels. Therefore, these constituents are added to a broad spectrum of foods. As in the case of cholesterol, thermo-oxidative treatment of phytosterols may result in the formation of phytosterol oxidation products (POPs), i.e. keto-, hydroxy-, and epoxy-derivatives. This review summarizes and evaluates the current knowledge regarding POPs in the light of the potentially increasing dietary exposure to these constituents via the consumption of foods enriched with phytosterols/phytostanols and their esters. Data on the occurrence of POPs and approaches to assess the potential intake of POPs resulting from the consumption of enriched foods are described. The knowledge on the uptake of POPs and the presently available data on the impact of the consumption of enriched foods on the levels of POPs in humans are discussed. Biological effects of POPs, such as potential proatherogenic properties or the loss of the cholesterol-lowering effects compared to nonoxidized phytosterols, are discussed. Finally, knowledge gaps are outlined and recommendations for further research needed for a safety assessment of POPs are presented.

Cytotoxicity potentials of eleven Bangladeshi medicinal plants.

Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50 = 2.93 µg/mL) and the lowest by Litsea glutinosa leaves (LC50 = 114.71 µg/mL) in comparison with standard vincristine sulphate (LC50 = 2.04 µg/mL). Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action.

The effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminosilicate (NSAS) on the expression and activity of P-glycoprotein within Caco-2 cells.

BACKGROUND: Many drugs are substrates for P-glycoprotein (P-gp) and interactions involving P-gp may be relevant to clinical practice. Co-administration with P-gp inhibitors or inducers changes the absorption profile as well as the risk for drug toxicity, therefore it is important to evaluate possible P-gp alterations. The purpose of this study was to investigate the effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminium silicate (NSAS), a protonated montmorillonite clay, on mdr-1 gene expression and its protein, P-glycoprotein (P-gp) within Caco-2 cells. METHODS: The effects of DAPP and NSAS on the regulation of mdr-1 gene, P-gp protein expression and activity within Caco-2 cells, were determined using cell viability and cytotoxicity tests, RT-PCR, Western Blot analysis and bi-directional transport studies. RESULTS: We observed a significant down-regulation of mdr-1 mRNA (e.g. 38.5 ± 17% decrease vs. control at 5 µM DAPP and 61.2 ± 25% versus control at 10 µM DAPP; n = 6, P* < 0.05) within Caco-2 cells. Western Blot analysis of P-gp expression showed that changes in mdr-1 gene expression lead to correlating changes in P-gp protein expression. This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control. In contrast, when Caco-2 cells were treated with NSAS, no changes in mdr-1 gene expression, P-gp protein expression nor P-gp activity were observed. CONCLUSIONS: DAPP but not NSAS decreases P-gp mediated drug efflux through decreased mdr-1 gene expression and consequently decreased P-gp protein expression. These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions. We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

Phytosterols promote liver injury and Kupffer cell activation in parenteral nutrition-associated liver disease.

Parenteral nutrition-associated liver disease (PNALD) is a serious complication of PN in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Yet, the mechanisms underlying PNALD are poorly understood. It has been suggested that a component of soy oil (SO) lipid emulsions in PN solutions, such as plant sterols (phytosterols), may be responsible for PNALD, and that use of fish oil (FO)-based lipid emulsions may be protective. We used a mouse model of PNALD combining PN infusion with intestinal injury to demonstrate that SO-based PN solution causes liver damage and hepatic macrophage activation and that PN solutions that are FO-based or devoid of all lipids prevent these processes. We have furthermore demonstrated that a factor in the SO lipid emulsions, stigmasterol, promotes cholestasis, liver injury, and liver macrophage activation in this model and that this effect may be mediated through suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Fxr and Lxr, and failure of up-regulation of the hepatic sterol exporters (Abcg5/g8/ABCG5/8). This study provides experimental evidence that plant sterols in lipid emulsions are a major factor responsible for PNALD and that the absence or reduction of plant sterols is one of the mechanisms for hepatic protection in infants receiving FO-based PN or lipid minimization PN treatment. Modification of lipid constituents in PN solutions is thus a promising strategy to reduce incidence and severity of PNALD.

Phytosterol feeding causes toxicity in ABCG5/G8 knockout mice.

Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels.

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells.

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and ß-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, ß-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7ß-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3ß,5α,6ß-triol and 2CMP:1DHB-5ß,6ß-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.

Genotoxicity of polar and apolar extracts obtained from Qualea multiflora and Qualea grandiflora.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Qualea grandiflora and Qualea multiflora, which belong to the Vochysiaceae family, are common in the Brazilian savannah (Cerrado biome), and the local inhabitants use these species to treat external ulcers and gastric diseases and as an anti-inflammatory agent. Studies have demonstrated that these plants contain compounds that exhibit pharmacological activities; however, the risks associated with their consumption are not known. MATERIAL AND METHODS: In the present study, the mutagenicity of polar and apolar extracts from Qualea grandiflora and Qualea multiflora were assessed by employing the Ames assay with and without metabolic activation. Additionally, phytochemical analyses (HPLC-ESI-IT-MS, HPLC-UV-PDA and GC-IT-MS) were performed to identify the chemical constituents present in these species, including the evaluation of physico-chemical properties, such as polarity or apolarity of the organic compounds, which are related to each fraction obtained. These studies provide important information regarding the biochemical behaviour of these compounds. RESULTS: All extracts exhibited mutagenicity, inducing frameshift mutations and base substitutions in DNA. Phytochemical analysis identified terpenes, ellagic acid derivatives and phytosteroids. CONCLUSIONS: The mutagenicity observed might be due to the presence of pentacyclic triterpenes and polyphenols, which are able to generate reactive oxygen species (ROS) and result in the potential to cause DNA damage. The genetic risk identified in this present work shows that special attention should be considered for the use of compounds obtained from these plant species in medicinal treatments. Further studies must be conducted to identify safe therapeutic doses.

Contents of phytosterols in vegetables and fruits commonly consumed in China.

OBJECTIVE: To quantify five specific dietary phytosterols and phytostanols in vegetables and fruits commonly consumed in China. METHODS: A total of 34 different kinds of vegetables and 33 kinds of fruits were chosen according to the consuming habit of Chinese people. All the samples were purchased from two shops in Beijing. The contents of phytosterols (beta-sitosterol, campesterol, stigmasterol, beta-sitostanol, and campestanol) were analyzed by GLC method which was established by our laboratory, and the total phytosterols were calculated. RESULTS: The total phytosterol content in vegetables ranged 1.1-53.7 mg/100 g edible portion. The highest concentration was found in pea, cauliflower, broccoli, and romaine lettuce. The phytosterol contents in fruits ranged 1.6-32.6 mg/100 g, the highest concentration was found in navel orange, tangerine, and mango. CONCLUSION: The phytosterol contents in vegetables and fruits are not as high as those in edible oils, but because of the large amount of consumption, they also play an important role in increasing the people's phytosterols intake, indicating that increased intake of vegetables and fruits with higher phytosterol contents helps increase the phytosterol intake in China.

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.

Multigenerational exposure to phytosterols in the mouse.

Due to beneficial health effects phytosterols (PS) are increasingly added to functional foods. The aim of the present study was to investigate the chronic effects of a dietary PS mixture (5mg/kg/day), containing mainly beta-sitosterol, on the reproduction of the mouse. General reproductive parameters, postnatal development, growth and survival of pups, weight of sex organs, the concentrations of plasma sex steroids and testicular testosterone were monitored across five generations (F(0)-F(4)). PS exposure increased the plasma levels of testosterone and decreased the relative uterine weights in the pups of F(2) and F(4) generations. Furthermore, PS exposure increased the concentrations of plasma estradiol in the female pups of F(3) generation. PS supplement also increased the testicular levels of testosterone in the male pups of F(2) generation. In spite of these transitory changes, PS exposure caused no permanent deleterious effects on the reproduction of the mouse.