Nutritional Value, Phytochemical Composition and Biological Activities of Lycium barbarum L. fruits from Serbia.

Cultivation of goji berries (GB), fruits of Lycium barbarum L. (Solanaceae), is expanding worldwide, including in Europe. In this study, a comparative analysis of the nutritional value, chemical composition and in vitro biological activities of GB from different locations in Serbia was performed. Proximate compositions were evaluated according to standard methods. Minerals were assessed by inductively coupled plasma techniques, while fatty acids, sterols, and phenolic profiles were analyzed by gas- and liquid chromatography-based techniques coupled with flame-ionization, mass spectrometry, or diode array detection. The total content of phenolics, flavonoids, carotenoids, and polysaccharides was assessed using spectrophotometric methods. Methanol extracts from GB were examined for their antioxidant, enzyme inhibitory (α-amylase, α-glucosidase, acetylcholinesterase and tyrosinase) and antibacterial activities. Despite significant variations among samples from different locations, the results confirmed that GB are a valuable source of dietary fiber and protein and are characterized by favorable fatty acid profiles. Phytochemical analysis revealed that ß-sitosterol, Δ5-avenasterol, and 24-methyldesmosterol are the predominant sterols and caffeic acid, gallic acid, quercetin and rutin are the main phenols. All GB samples showed both antioxidant and mild antimicrobial activity. A dose-dependent anti-enzymatic activity (IC50 ranging 1.68-6.88 mg/mL) was demonstrated. The results support further promotion of GB cultivation in Serbia and further investigations on their potential applications in various industries.

Potential of Kalanchoe pinnata as a Cancer Treatment Adjuvant and an Epigenetic Regulator.

Cancer is a global public health problem that is related to different environmental and lifestyle factors. Although the combination of screening, prevention, and treatment of cancer has resulted in increased patient survival, conventional treatments sometimes have therapeutic limitations such as resistance to drugs or severe side effects. Oriental culture includes herbal medicine as a complementary therapy in combination with chemotherapy or radiotherapy. This study aimed to identify the bioactive ingredients in Kalanchoe pinnata, a succulent herb with ethnomedical applications for several diseases, including cancer, and reveal its anticancer mechanisms through a molecular approach. The herb contains gallic acid, caffeic acid, coumaric acid, quercetin, quercitrin, isorhamnetin, kaempferol, bersaldegenin, bryophyllin a, bryophyllin c, bryophynol, bryophyllol and bryophollone, stigmasterol, campesterol, and other elements. Its phytochemicals participate in the regulation of proliferation, apoptosis, cell migration, angiogenesis, metastasis, oxidative stress, and autophagy. They have the potential to act as epigenetic drugs by reverting the acquired epigenetic changes associated with tumor resistance to therapy-such as the promoter methylation of suppressor genes, inhibition of DNMT1 and DNMT3b activity, and HDAC regulation-through methylation, thereby regulating the expression of genes involved in the PI3K/Akt/mTOR, Nrf2/Keap1, MEK/ERK, and Wnt/ß-catenin pathways. All of the data support the use of K. pinnata as an adjuvant in cancer treatment.

Yield, Characterization, and Possible Exploitation of Cannabis Sativa L. Roots Grown under Aeroponics Cultivation.

Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C.sativa is being increasingly reappraised; however, C. sativa roots remain poorly studied, despite citations in the scientific literature. In this direction, we identified and quantified the presence of valuable bioactives (namely, ß-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth-particularly of the roots-and a significant increase (up to 20-fold in the case of ß-sitosterol) in the total content of the aforementioned roots' bioactive molecules were observed in AP and AEP. In conclusion, aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.

Phytosterolaemia associated with parenteral nutrition administration in adult patients.

Vegetable lipid emulsions (LE) contain non-declared phytosterols (PS). We aimed to determine PS content depending on the brand and LE batch, and in adult hospitalised patients treated with parenteral nutrition (PN), to establish the association between plasma and administered PS. Part I was the LE study: totals and fractions of PS in three to four non-consecutive batches from six LE were analysed. Part II was the patient study: patients with at least 7 previous days of PN with 0·8 g/kg per d of an olive/soyabean (O/S) LE were randomised (day 0) 1:1 to O/S or 100 % fish oil (FO) at a dose of 0·4 g/kg per d for 7 d (day 7). Plasma PS, its fractions, total cholesterol on days 0 and 7, their clearance and their association with PS administered by LE were studied. In part I, LE study: differences were found in the total PS, their fractions and cholesterol among different LE brands and batches. Exclusive soyabean LE had the highest content of PS (422·36 (sd 130·46) µg/ml). In part II, patient study: nineteen patients were included. In the O/S group, PS levels were maintained (1·11 (sd 6·98) µg/ml) from day 0 to 7, while in the FO group, significant decreases were seen in total PS (-6·21 (sd 4·73) µg/ml) and their fractions, except for campesterol and stigmasterol. Plasma PS on day 7 were significantly associated with PS administered (R2 0·443). PS content in different LE brands had great variability. PS administered during PN resulted in accumulation and could be prevented with the exclusive administration of FO LE.

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.

Buddleja thyrsoides Lam. crude extract presents antinociceptive effect on an arthritic pain model in mice.

Arthritis is a chronic inflammatory disease which reduces the life quality of affected individuals. Therapeutic tools used for treating inflammatory pain are associated with several undesirable effects. Buddleja thyrsoides Lam., known as 'Barbasco' or 'Cambara', is mostly used in several disorders and possesses antirheumatic, anti-inflammatory, and analgesic properties. Here, we investigated the antinociceptive and anti-inflammatory effects of the B. thyrsoides crude extract applied orally and topically in acute pain models and an arthritic pain model induced by complete Freund's adjuvant (CFA) paw injection in male mice (25-30 g). The high-performance liquid chromatography (HPLC) of the B. thyrsoides extract crude revealed the presence of the lupeol, stigmasterol, and ß-sitosterol. The stability study of the B. thyrsoides gel did not show relevant changes at low temperatures. The oral treatment with the B. thrysoides extract prevented the capsaicin-induced spontaneous nociception and the acetic acid-induced abdominal writhing, but did not alter the thermal threshold in the tail immersion test. The B. thyrsoides antinociceptive effect was not reversed by naloxone in the capsaicin test. The B. thyrsoides oral or topical treatment reversed the CFA-induced mechanical allodynia and thermal hyperalgesia with maximum inhibition (Imax) of 69 ± 6 and 68 ± 5% as well as 78 ± 15 and 87 ± 12%, respectively. Moreover, the topical but not oral treatment inhibited the CFA-induced cell infiltration, but did not reduce the paw edema significantly. The oral treatment with B. thyrsoides did not cause adverse effects. These findings suggest that the oral or topical treatment with B. thyrsoides presents antinociceptive actions in an arthritic pain model without causing adverse effects.

Sterols in infant formulas: validation of a gas chromatographic method.

Sterols are components present in the fat fraction of infant formulas (IFs). Their characterization is therefore of interest, though there are no official reference methods for their analysis in these matrices. AIM: To validate a gas chromatographic method with flame ionization detection for the determination of animal (cholesterol and desmosterol) and plant sterols (brassicasterol, campesterol, stigmasterol, ß-sitosterol and sitostanol) found in IFs. All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (<1 µg/100 mL) and quantification (<4 µg/100 mL) are suitable for sterols determination in IFs. The within-assay precision ranged from 1.6% to 8.8%, while the between-assay precision was <10% for most of sterols. Accuracy was satisfactory and was calculated by recovery assays (ranging 93-108%). The analytical parameters obtained showed the suitability of the proposed method for the determination of sterols in IFs.

Influence of cultivation sites on sterol, nitrate, total phenolic contents and antioxidant activity in endive and stem chicory edible products.

Chicories produce a wide range of vegetables with important nutritional value. We determined the variation of sterol, total polyphenol, nitrate contents and antioxidant capacity (SC, TPC, NC, AC) in endive leaves and stem-chicory novel vegetables, cultivated in two Italian regions. Within a given area, the SC was similar in smooth- and curly leafed endives (106.3-176.0 mg/kg FW); sitosterol and stigmasterol were major fractions (45-56 versus 38-43%). The stem SC was independent of landrace (101.5-118.6 mg/kg FW); sitosterol prevailed on stigmasterol and fucosterol (73-76 versus 12-14% versus 8-9%); the latter reached 15.7 mg/kg FW, conferring value as potential antidiabetes food. The planting site affected the AC and TPC of endives (893.1-1571.4 µmTE/100 g FW, 30.8-76.1 GAE100/g FW) and chicory stems (729.8-1152.5 µmTE/100 g FW; 56.2-124.4 GAE100/g FW), while the NC was recurrently below dangerous thresholds. PCA showed that environment was the major cause of variation, though it modestly affected these parameters.

Metabolomic analysis can detect the composition of pasta enriched with fibre after cooking.

BACKGROUND: Several studies have demonstrated that metabolomics has a definite place in food quality, nutritional value, and safety issues. The aim of the present study was to determine and compare the metabolites in different pasta samples with fibre, and to investigate the modifications induced in these different kinds of pasta during cooking, using a gas chromatography-mass spectrometry-based metabolomics approach. RESULTS: Differences were seen for some of the amino acids, which were absent in control pasta, while were present both in the commercially available high-fibre pasta (samples A-C) and the enriched pasta (samples D-F). The highest content in reducing sugars was observed in enriched samples in comparison with high-fibre pasta. The presence of stigmasterol in samples enriched with wheat bran was relevant. Cooking decreased all of the metabolites: the high-fibre pasta (A-C) and Control showed losses of amino acids and tocopherols, while for sugars and organic acids, the decrease depended on the pasta sample. The enriched pasta samples (D-F) showed the same decreases with the exception of phytosterols, and in pasta with barley the decrease of saturated fatty acids was not significant as for tocopherols in pasta with oat. Principal component analysis of the metabolites and the pasta discrimination was effective in differentiating the enriched pasta from the commercial pasta, both uncooked and cooked. CONCLUSIONS: The study has established that such metabolomic analyses provide useful tools in the evaluation of the changes in nutritional compounds in high-fibre and enriched pasta, both before and after cooking. © 2015 Society of Chemical Industry.

Removal of stigmasterol from Kraft mill effluent by aerobic biological treatment with steroidal metabolite detection.

Stigmasterol is a phytosterol contained in Kraft mill effluent that is able to increase over 100% after aerobic biological treatment. This compound can act as an endocrine disrupter as its structure is similar to that of cholesterol. The aim of this study was to evaluate the removal of stigmasterol from Kraft mill effluents treated by a moving bed biofilm reactor (MBBR) with steroidal metabolite detection. The MBBR was operated for 145 days, with a hydraulic retention time of 2 days. Stigmasterol and steroidal metabolites were detected by gas chromatography with a flame ionization detector during MBBR operation. The results show that the MBBR removed 87.4% of biological oxygen demand (BOD5), 61.5% of chemical oxygen demand (COD), 24.5% of phenol and 31.5% of lignin, expressed in average values. The MBBR system successfully removed 100% of the stigmasterol contained in the influent (33 µg L(-1)) after 5 weeks of operation. In that case, the organic load rate was 0.343 kg COD m(-3) d(-1). Furthermore, different steroidal compounds (e.g., testosterone propionate, stigmast-4-en-3-one, 5α-pregnan-12-one-20α-hydroxy, 5α-pregnane-3,11,20-trione and 3α-hydroxy-5α-androstane-11,17-dione were detected in the Kraft mill effluent as potential products of phytosterol biotransformation.

Validation of an Isocratic HPLC Method for Simultaneous Estimation of Major Phytosterols in Prunus spinosa L. extracts.

This study aimed to develop a rapid method for separation of stigmasterol, campesterol and ß-sitosterol in Prunus spinosa L. (sloe) fruit extracts by High Performance Liquid Chromatography system. Samples were prepared by Soxhlet extraction method and separated on a high strength silica C18 column using acetonitrile-methanol mobile phase and Photodiode Array Detector. The optimized method resulted in a linear calibration curve ranging from 1.7 ng mL-1 to 130 ng mL-1 for all three phytosterols. Analyses of internal and external phytosterol standards showed good linearity (R2 of 0.998 to 0.999); LOD and LOQ were determined to be 2.33×10-7-2.18×10-4 and 7.07×10-7-6.60×10-4 mg mL-1, respectively. Repeatability and reproducibility precision analyses showed acceptable values of RSD %. ß-sitosterol was the predominant phytosterol (51.53-81.03 % of total) among all samples. Method validation parameters indicated that this analytical method can be applied for accurate and precise determination of campesterol, stigmasterol and ß-sitosterol, in selected extracts.

Evaluation of bioactive potential of an Aloe vera sterol extract.

We prepared a crude gel material from Aloe vera succulent leaf tissues. The ethanolic extract of lyophilized A. vera gel was used for the GC-MS analysis. Hexadecanoic acid (22.22%) was identified as major compound. Sitosterol and stigmasterol were found to be 2.89% and 2.1% in the extract. HPLC analysis was carried out to confirm the presence of stigmasterol. The concentration of sterol extract needed to scavenge DPPH free radical by 50% was calculated as 5.2 mg mL(-1). In the FRAP assay, the sterol extract showed significant hydroxyl radical scavenging in a dose-dependent manner (IC50 value 1.17 µg mL(-1)). Concentration of the sample required to reduce lipid peroxidation was found to be 4.18 µg mL(-1), and the extract also possessed acetylcholinesterase activity (IC50 - 5.26 µg mL(-1)). Catalase activity was 0.196 µM H2 O2 decomposed min(-1) µg(-1) protein, whereas the peroxidase activity was 17.01 µM of pyragallol oxidized min(-1) µg(-1) protein. The extract recorded higher activity against growth of S. greseus and C. albicans in the experiments carried out to determine antibacterial and antifungal activity, respectively.

A novel terpenoid glycoside and other bioactive constituents from the seeds of Cichoriumintybus.

Phytochemical investigation of the seeds of Cichorium intybus L (C. intybus) led to isolate n-hexacosane (CI-1), an aliphatic higher ketone, n-nonacosan-3-one (CI-2), two aliphatic acid esters characterized as n-octacosanyl decanoate (CI-3) and n-tricosanyl hexadecanoate (CI-4), two mixed glycerides identified as as glyceryl-1-(hexadec-7- enoyl)-2- tetradecanoyl-3-hexadecanoate (Cl-5) and glyceryl-1- (eicos-9-enoyl)-2,3, bis-eicosanoate (Cl-6), and three e steroidal constituents and their structures were elucidated as as stigmast-5, 22-dien-3ß -ol-21-oic acids (Cl-7), stigmasterol-3ß-d-glucopyranoside (Cl-8) and stigmast-5, 22-dien-3-ß-ol-3-ß-d-glucuronopyranoside (Cl-9). The dry seeds powder was defatted and finally extracted with ethanol by using a maceration method. The ethanol was evaporated near to dryness and silica gel was added to the extract and a slurry with the help of methanol solvent was prepared. The slurry was loaded to the column by using petroleum ether and was eluted with a mixture of chloroform and methanol. A series of test tubes were collected and each test tube with 2 mL eluents was collected. Based on the thin layer chromatography (TLC) the content of nine test tubes were considered as pure compounds. The solvent was evaporated from the test tube at room temperature. All the nine compounds from the column were characterized by using Infrared (IR), Nuclear Magnetic Resonance (NMR) and Mass spectrometry (MS). Eight compounds were previously isolated from the plant and they showed various biological activities. A new compound was isolated for the first time from the plant kingdoms. Based on the chromatographic and spectroscopic analysis the new compound was characterized as stigmasterol carboxylic acid (CI-9). The isolated new compound could be used to treat liver and cardiac diseases.

An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells.

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, ß-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.

Effect of Costus igneus stem extract on calcium oxalate urolithiasis in albino rats.

The effects of aqueous and ethanolic extracts of Costus igneus (stem) and isolated compounds lupeol and stigmasterol on calcium oxalate urolithiasis have been studied in male albino Wistar rats. Ethylene glycol feeding resulted in hyperoxaluria as well as increased renal excretion of calcium and oxalate. The increased deposition of stone-forming constituents in the urine, serum, and kidney homogenate of urolithic rats was significantly (p < 0.05) lowered by treatment using aqueous and ethanolic extracts of C. igneus (stem), and isolated compounds lupeol and stigmasterol. The calcium oxalate crystal deposition in the kidney was significantly greater in ethylene glycol-induced urolithic rats. After administration of aqueous and ethanolic extract of C. igneus, the deposition of calcium and oxalate was significantly lowered. Treatment with lupeol and stigmasterol significantly reduced the deposition of calcium and oxalate in the kidney, and also in the blood serum; the lipid profile serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels at 50 and 100 mg/kg were significantly (p < 0.05) lowered in urolithiatic rats. From this study, we conclude that both the treatments with aqueous and ethanolic extract of C. igneus (stem) and isolated compounds lupeol and stigmasterol had an inhibitory effect on calcium oxalate urinary stone. Lupeol and stigmasterol were identified from the stem of C. igneus by high-performance thin layer chromatography technique. The isolated compounds were confirmed by Fourier transform infrared (FTIR) and (13)C NMR spectra.

A new isoquinoline and ceramide from the stem barks of Discoglypremna caloneura (Pax) Prain (Euphorbiaceae) with antiproteinase and cytotoxic activities.

Two new compounds, an isoquinoline (1) and caloneuramide (2), a ceramide were isolated from the stem bark of Discoglypremna caloneura together with seven known compounds namely aurantiamide acetate (3), acetylaleuritolic acid (4), 3α-hydroxylaleuritolic acid 2α-p-hydroxybenzoate (5), mixture of stigmasterol (6) and ß-sitosterol (7), mixture of 7-oxo-stigmasterol (8) and 7-oxo-ß-sitosterol (9). Their structures were determined based on data from literature and spectroscopic methods. Derivatization reactions on the isoquinoline led to two new compounds, the methylated (10) and acetylated (11) derivatives. Some compounds and extracts were evaluated for their cytotoxic and antiproteinase activity. Antiproteinase effect of compounds 1, 10 and 11 exhibited IC50 values of 10.77, 1.19 and 3.61 µg/mL respectively; significantly low compared to the standard drug, acetyl salicylic acid (IC50 = 20.28 µg/mL). Ethyl acetate and methanol extract exhibited moderate cytotoxicity activity on Chang liver cells with CC50 values of 167.90 ± 2.20 and 106.30 ± 2.03 µg/mL compared to the reference drug cucurmin (CC50 = 11.05 ± 1.04 µg/mL).

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry.

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

Separation and purification of ergosterol and stigmasterol in Anoectochilus roxburghii (wall) Lindl by high-speed counter-current chromatography.

Ergosterol and stigmasterol are the most common phytosterols in the traditional Chinese medicine. They are two major sterol compounds in Anoectochilus roxburghii (wall) Lindl (A. roxburghii) and have been proved to have many important biological activities. A method by using high-speed counter-current chromatography (HSCCC) has been successfully developed for separation and purification of ergosterol and stigmasterol in A. roxburghii simultaneously in this paper. The optimum conditions used in this method were as follows: The two-phase solvent system consisted of n-hexane-ethylacetate-butanol-methanol-water (3.5:0.3:0.5:2.5:0.3, v/v); the rotation speed was 900 rpm; the flow rate of the lower phase was 1.5 mL/min. About 36.5 mg of ergosterol and 43.6 mg of stigmasterol were obtained from 100 g of A. roxburghii. The purity of ergosterol and stigmasterol was examined to be 92.0 and 95.5%, respectively, by using HPLC. The chemical structures of these components were identified by UV spectra, FT-IR, MS, (1) H-NMR and (13) C-NMR. The results demonstrated that high-speed counter-current chromatography was a feasible method to separate and purify ergosterol and stigmasterol from the herb. This separation and purification method was more effective than many other conventional techniques.

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.

Characterization of seven sterols in five different types of cattle feedstuffs.

This paper provides a method for the quantification of sterols in different types of calf feedstuffs based on soy, sunflower, hay, calf feed and a mixture of all of them. The free fraction and the total sterolic fraction, after saponification and acidic hydrolysis of the samples, are extracted by solvent and the sterols are identified/quantified by reversed phase HPLC coupled to tandem mass spectrometry by atmospheric pressure chemical ionization. After the recovery evaluation, the method is validated in terms of linearity (coefficient of determination R2), repeatability (coefficient of variation RSD), limit of detection and quantification. In most of the cases, the most representative phytosterol is ß-sitosterol, followed by campesterol or stigmasterol and by other minor sterols such as fucosterol, and Δ-5-avenasterol. In addition, also cholesterol and ergosterol, if present, are evaluated in all the samples. As far as we know, very little information is available on the investigated feeds, which are commonly used on farms. The results of this survey were compared to other studies, if present in literature, showing good agreement. The proposed method resulted to be simple, fast and suitable for application to other sterols, feedstuffs and derived foods. The knowledge of the sterolic content and composition is getting more and more important, both in terms of comprehension of the vegetal biochemistry and as basis for sterolomic studies.