Menthol based hydrophobic deep eutectic solvent for extraction and purification of ergosterol using response surface methodology.

Vitamin D deficiency has been linked to various diseases, but could be rectified via fortified food stuffs or supplementation. In this study 39 different hydrophobic deep eutectic solvents were evaluated for green extraction of ergosterol from mushroom. Extraction parameters (e.g. time, solvent volume) were optimized using response surface methodology (RSM) and a maximum extraction yield of 6995.00 µg ergosterol/g dry weight mushroom was attained with menthol: pyruvic acid. The extracted ergosterol was purified using a novel methodology and the extraction solvent was reused for six cycles, while retaining extraction efficiency (up to 28%). The ergosterol was exposed to ultra-violet radiation for conversion to ergocalciferol (vitamin D2) resulting in a yield of ergocalciferol that was equivalent to 2142.01 µg/g dry weight mushroom.

Effects of UV-C treatment and ultrafine-grinding on the biotransformation of ergosterol to vitamin D2, physiochemical properties, and antioxidant properties of shiitake and Jew's ear.

In the present study, the impact of ultraviolet (UV)-C treatment and ultrafine grinding on the conversion of ergosterol to vitamin D2, physiochemical properties, and antioxidant properties of shiitake and Jew's ear was assessed. After exposure to UV-C, vitamin D2 contents of both the mushroom samples has increased significantly (p < 0.05). Whereas, ultrafine grinding along with UV-C treatment has a synergistic effect on bioconversion of ergosterol to vitamin D2 and this effect is more prominent in low dose UV-C irradiation groups (2 kJ/m2). Ultrafine grinding significantly (p < 0.05) improved the water holding capacity (WHC), water solubility index (WSI) and polysaccharide dissolution rate (PDR). However, UV-C treatment led to insignificant changes in the physiochemical properties of mushroom samples. A significant improvement was also observed in the antioxidant profiles especially tannin contents of mushrooms followed by the ultrafine grinding and UV-C treatment.

Effect of Vitamin D2 Fortification Using Pleurotus ostreatus in a Whole-Grain Cereal Product on Child Acceptability.

Vitamin D2 deficiency is one of the most common micronutrient insufficiencies among children. Few foods, mainly those derived from animal sources, naturally contain this vitamin. The basidiomycete mushroom Pleurotus ostreatus could be used as an innovative and sustainable ingredient for food fortification with vitamin D2. This study was aimed at exploring children's acceptance of a whole-cereal-based product (breadsticks) combined with increasing concentrations of P. ostreatus powder rich in vitamin D2. The food neophobia trait (fear of trying unfamiliar and new food) on sample acceptability was also investigated. One hundred and three children (47 girls and 56 boys, aged 9-11 years) were recruited, and breadstick-liking was studied in relation to gender and neophobic traits. Results showed that the samples enriched in vitamin D2 were well accepted by children even if liking decreased with increasing concentration of mushroom powder. Generally, neophilic subjects gave higher liking scores compared with the neophobic ones, especially for the modified samples. New, well-accepted fortified products could be developed using an adequate concentration of mushroom powder to deal with the increasing vitamin D2 deficiency among children.

The influence of lipid droplet size on the oral bioavailability of vitamin D2 encapsulated in emulsions: an in vitro and in vivo study.

Vitamin D deficiency is prevalent in some populations leading to adverse health effects, and therefore there is a need to supplement functional foods and beverages with this important micronutrient. In this study, we examined the influence of the initial lipid droplet size on the in vitro bioaccessibility and in vivo absorption of vitamin D2 encapsulated in oil-in-water emulsions. Changes in particle size, charge, and microstructure were measured as vitamin-loaded lipid droplets were passed through a simulated GIT (mouth, stomach, small intestine). The in vitro studies showed that smaller lipid droplets were digested more rapidly than larger ones, thereby leading to the more rapid formation of mixed micelles in the small intestine capable of solubilizing the lipophilic vitamins. This effect may account for the highest vitamin D2 bioaccessibility being observed for the emulsions containing the smallest droplets. In contrast, the in vivo rat feeding studies suggested that the absorption of vitamin D2 was the highest for the emulsions containing the largest droplets. The poor in vitro-in vivo correlation observed in our study may have occurred for a number of reasons: the simulated GIT did not accurately model the complexity of a real GIT; the in vivo approach used did not monitor changes in vitamin levels in the blood over time. Overall, this study suggests that particle size does influence the gastrointestinal fate of encapsulated oil-soluble vitamins, but that further work is needed to establish strong correlations between in vitro and in vivo methods.

Low bioaccessibility of vitamin D2 from yeast-fortified bread compared to crystalline D2 bread and D3 from fluid milks.

The assessment of the efficacy of dietary and supplemental vitamin D tends to be confounded by differences in the serum 25-hydroxyvitamin D response between vitamin D2 and vitamin D3. Serum response differences from these vitamers may be due to differences in bioavailability. To address this specifically, the bioaccessibility was assessed for vitamin D2 from breads fortified with UV-treated yeast, and a benchmark against staple vitamin D3 fortified foods including bovine milks and infant formula, as well as crystalline vitamin D2 fortified bread. Fortified foods were subjected to a three-stage static in vitro digestion model, and vitamin D was analyzed by HPLC-MS. Vitamin D bioaccessibility was significantly greater from bovine milks and infant formula (71-85%) than from yeast-fortified sandwich breads (6-7%). Bioaccessibility was not different between whole wheat and white wheat bread (p > 0.05), but was ∼4× lower from yeast-fortified bread than from crystalline vitamin D2 fortified bread (p < 0.05). Intact yeast cells were observed in the digesta of yeast fortified bread. These results indicate that the low bioavailability of yeast D2 in comparison to other vitamin D2 sources is likely due to entrapment within a less digestible yeast matrix and not only to metabolic differences between vitamins D2 and D3.

Prodifferentiation Activity of Novel Vitamin D2 Analogs PRI-1916 and PRI-1917 and Their Combinations with a Plant Polyphenol in Acute Myeloid Leukemia Cells.

1α,25-dihydroxyvitamin D3 (1,25D3) is a powerful differentiation inducer for acute myeloid leukemia (AML) cells. However, 1,25D3 doses required for differentiation of AML cells may cause lethal hypercalcemia in vivo. There is evidence that vitamin D2 is less toxic than vitamin D3 in animals. Here, we determined the differentiation effects of novel analogs of 1α,25-dihydroxyvitamin D2 (1,25D2), PRI-1916 and PRI-1917, in which the extended side chains of their previously reported precursors (PRI-1906 and PRI-1907, respectively) underwent further 24Z (24-cis) modification. Using four human AML cell lines representing different stages of myeloid maturation (KG-1a, HL60, U937, and MOLM-13), we found that the potency of PRI-1916 was slightly higher or equal to that of PRI-1906 while PRI-1917 was significantly less potent than PRI-1907. We also demonstrated that 1,25D2 was a less effective differentiation agent than 1,25D3 in these cell lines. Irrespective of their differentiation potency, all the vitamin D2 derivatives tested were less potent than 1,25D3 in transactivating the DR3-type vitamin D response elements. However, similar to 1,25D3, both 1,25D2 and its analogs could strongly cooperate with the plant polyphenol carnosic acid in inducing cell differentiation and inhibition of G1-S cell cycle transition. These results indicate that the 24Z modification has contrasting effects on the differentiation ability of PRI-1906 and PRI-1907 and that the addition of a plant polyphenol could result in a similar extent of cell differentiation induced by different vitamin D compounds. The enhanced antileukemic effects of the tested combinations may constitute the basis for the development of novel approaches for differentiation therapy of AML.

25-hydroxyvitamin D circulates in different fractions of calf plasma if the parent compound is vitamin D2 or vitamin D3, respectively.

Vitamin D has become one of the most discussed nutrients in human nutrition, which has led to an increased interest in milk as a vitamin D source. Problems related to fortifying milk with synthetic vitamin D can be avoided by securing a high content of natural vitamin D in the milk by supplying dairy cows with sufficient vitamin D. However, choosing the most efficient route and form of supplementation requires insight into how different vitamin D metabolites are transported in the body of cattle. There are two forms of vitamin D: vitamin D2 (D2) and vitamin D3 (D3). Vitamin D2 originates from fungi on roughage. Vitamin D3 originates either from endogenous synthesis in the skin or from feed supplements. Vitamin D2 is chemically different from, and less physiologically active than, D3. Endogenous and dietary D3 is chemically similar but dietary D3 is toxic, whereas endogenous D3 appears well regulated in the body.

Synthesis and evaluation of geometric analogs of 1α,25-dihydroxyvitamin D2 as potential therapeutics.

An improved convergent strategy was developed for the synthesis of the previously obtained side-chain extended and rigidified analogs of 1α,25-dihydroxyvitamin D2, PRI-1906 and PRI-1907. New (24Z) geometric isomers of the analogs, PRI-1916 and PRI-1917, were also obtained and identified. These side-chain isomers were separable by flash chromatography, as C-25 alcohols, from the synthetic precursors of PRI-1906 and PRI-1907, respectively. The structures of new analogs were determined by advanced techniques of 1H and 13C NMR, including COSY, HSQC and HMBC sequences. Binding affinities of the geometric analogs PRI-1906 and PRI-1916 and their respective C-26, C-27 homologs PRI-1907 and PRI-1917 for the full-length human vitamin D receptor were determined by a fluorescence polarization competition assay. The binding affinity of (24Z) methyl analog PRI-1906 was much higher than that of (24E) analog PRI-1906, while the affinity of (24Z) ethyl analog PRI-1917 was lower than that of the respective PRI-1907. Investigation of the metabolism of these compounds by human CYP24A1 revealed they are much more resistant to CYP24A1 than 1α,25-dihydroxyvitamin D2, indicating they could have longer-term biological effects on target tissues.

Biological evaluation of new vitamin D2 analogues.

1,25-dihydroxyvitamin D3 (1,25D), a steroid hormone which regulates calcium/phosphate homeostasis, has a broad spectrum of anti-cancer activities, including differentiation of acute myeloid leukemia (AML) cells. In order to avoid undesirable side effects such as hypercalcemia, low-calcemic analogues should be produced for therapeutic purposes. In this paper, we describe biological activities of double-point modified analogues of vitamin D2 and we compare them to 1,25D and to paricalcitol, the drug used to treat secondary hyperparathyroidism. In vivo, our new analogues have lower calcemic effects, and lower toxicity in comparison to 1,25D. They have enhanced pro-differentiating and transcription-inducing activities in AML cells. Interestingly, differentiation effects do not correlate with the affinities of the analogues to the vitamin D receptor (VDR).

Extract-filter-shoot liquid chromatography with mass spectrometry for the analysis of vitamin D2 in a powdered supplement capsule and standard reference material 3280.

An "extract-filter-shoot" method for the analysis of vitamin D2, ergocalciferol, in a dry powdered dietary supplement capsule containing rice flour excipient and in a National Institute of Standards and Technology standard reference material 3280 is reported. Quantification of vitamin D2 was done by atmospheric pressure chemical ionization mass spectrometry using selected ion monitoring, two transitions of selected reaction monitoring, and extracted ion chromatograms from full scans. UV detection was used for the quantification of Vitamin D2 in the dry powder capsule, whereas interfering species rendered UV detection unreliable for standard reference material 3280. Average values for standard reference material 3280 ranged from 8.27 ± 0.58 to 8.33 ± 0.57 µg/g using internal standard calibration and response factor approaches, compared to the previous National Institute of Standards and Technology internal value for vitamin D2 of 8.78 ± 0.11 µg/g, and the recently updated reference value of 8.6 ± 2.6 µg/g. The powdered supplement capsule was found to contain 28.19 ± 0.35 to 28.67 ± 0.90 µg/capsule for a capsule labeled to contain 25.00 µg. The triacylglycerol composition of the rice flour excipient in the powdered supplement capsule determined by atmospheric pressure chemical ionization mass spectrometry is also reported.

Vitamin D2-enriched button mushroom (Agaricus bisporus) improves memory in both wild type and APPswe/PS1dE9 transgenic mice.

Vitamin D deficiency is widespread, affecting over 30% of adult Australians, and increasing up to 80% for at-risk groups including the elderly (age>65). The role for Vitamin D in development of the central nervous system is supported by the association between Vitamin D deficiency and incidence of neurological and psychiatric disorders including Alzheimer's disease (AD). A reported positive relationship between Vitamin D status and cognitive performance suggests that restoring Vitamin D status might provide a cognitive benefit to those with Vitamin D deficiency. Mushrooms are a rich source of ergosterol, which can be converted to Vitamin D2 by treatment with UV light, presenting a new and convenient dietary source of Vitamin D2. We hypothesised that Vitamin D2-enriched mushrooms (VDM) could prevent the cognitive and pathological abnormalities associated with dementia. Two month old wild type (B6C3) and AD transgenic (APPSwe/PS1dE9) mice were fed a diet either deficient in Vitamin D2 or a diet which was supplemented with VDM, containing 1±0.2 µg/kg (∼54 IU/kg) vitamin D2, for 7 months. Effects of the dietary intervention on memory were assessed pre- and post-feeding. Brain sections were evaluated for amyloid ß (Aß) plaque loads and inflammation biomarkers using immuno-histochemical methods. Plasma vitamin D metabolites, Aß40, Aß42, calcium, protein and cholesterol were measured using biochemical assays. Compared with mice on the control diet, VDM-fed wild type and AD transgenic mice displayed improved learning and memory, had significantly reduced amyloid plaque load and glial fibrillary acidic protein, and elevated interleukin-10 in the brain. The results suggest that VDM might provide a dietary source of Vitamin D2 and other bioactives for preventing memory-impairment in dementia. This study supports the need for a randomised clinical trial to determine whether or not VDM consumption can benefit cognitive performance in the wider population.

Influence of altitude on vitamin D and bone metabolism of lactating sheep and goats.

This study investigated the influence of alpine grazing on vitamin D (vitD) and bone metabolism in sheep and goats. Two groups of five adult lactating East Friesian milk sheep and Saanen dairy goats were kept on pastures at 2,000 to 2,600 m a.s.l. (SA: sheep alpine; GA: goats alpine) and 400 m a.s.l. (SL: sheep lowland; GL: goats lowland). The animals were milked twice daily and the milk yield was measured. Blood, milk, skin, and forage samples were collected and the left metatarsi were measured with peripheral quantitative computed tomography. The relative humidity and air temperature were recorded and the ultraviolet B (UVB) radiation was measured with a solar meter at both research stations. In addition, animals from the alpine group were equipped with a global positioning system receiver. The UVB radiation was higher at the alpine station (P<0.05) compared to the lowland station. In contrast, both the relative humidity and the air temperature were higher at the lowland station (P<0.04). The group GA produced more milk than GL (P<0.043). No differences in milk production between SA and SL were detected. Only minor differences between the alpine and lowland species groups were found in the total 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D serum concentration and in the 25(OH)D milk concentration. 25-hydroxyvitamin D2 concentration in serum was higher in sheep compared to goats and the 25(OH)D3 concentration in serum increased in all four groups but was higher in the alpine groups during the experiment. In addition, no differences in 7-dehydrocholesterol (7-DHC) concentrations in the skin at high altitude and lowland groups were detectable. However the 7-DHC concentrations in the skin of sheep were less than a tenth of the concentrations in the skin of goats and were nearly not detectable. In both groups SA and SL bone strength index increased during the trial (P=0.043). Bone strength index was lower in GA compared to GL at wk 12 (P=0.047). Mean serum Ca concentrations were higher and P concentrations were lower in the alpine groups than in the lowland groups (P=0.047). In both groups SA and GA the distance travelled increased during the trial. In conclusion, no effect of altitude on vitD status, vitD milk concentration and bone strength could be detected. Both sheep and goats are able to produce vitD in the skin, but sheep depend more on vitD intake with feedstuff, whereas goats rely more on cutaneous vitD production.

Extrarenal vitamin D activation and interactions between vitamin D2, vitamin D3, and vitamin D analogs.

Our understanding of the mechanism of action of vitamin D has been broadened by the discovery of the extrarenal 1α-hydroxylase (CYP27B1) in various vitamin D target tissues around the body and the implications of this for the putative paracrine actions of 1α,25-dihydroxyvitamin D3. This review updates our current knowledge of the cytochrome P450-mediated steps of vitamin D activation (CYP2R1, CYP27B1) and inactivation (CYP24A1, CYP3A4) and the newest physiological roles of vitamin D. The review goes on to examine how well exogenously supplied vitamin D compounds, whether dietary vitamin D2 supplements or prescribed vitamin D analogs, substitute for their natural counterparts; how in some cases vitamin D can be used in conjunction with vitamin D analogs; and the overall impact of these supplements and drugs on the components of the vitamin D signal transduction machinery.

Analytical measurement of serum 25-OH-vitamin D3, 25-OH-vitamin D2 and their C3-epimers by LC-MS/MS in infant and pediatric specimens.

OBJECTIVES: To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D3 (25-OH-D3), 25-OH-vitamin D2 (25-OH-D2), and their C3-epimers. METHODS: Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D3 and C3-epi-25-OH-D3, 407.2→371.3 for d6-25-OH-D3, 413.2→331.2 for 25-OH-D2 and C3-epi-25-OH-D2 and 419.2→337.1 for, d6-25-OH-D2. As a proof-of-principle, 25-OH-D3 and C3-epi-25-OH-D3 were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer. RESULTS: The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D2/3 and <15% for C3-epi-25-OH-D3. C3-epi-25-OH-D3 was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D3 concentrations increased linearly, while C3-epi-25-OH-D3 concentrations remained constant. At 12 months of age, C3-epi-25-OH-D3 concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D3 supplements did not contain appreciable amounts of C3-epi-D3. CONCLUSIONS: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D3 metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.

Characterization of ergocalciferol loaded solid lipid nanoparticles.

UNLABELLED: The use of solid lipid nanoparticles (SLNs) is a technique that has been widely used in the pharmaceutical industry for the last 2 decades and has become of increasing interest to food scientists due to its potential for encapsulation and controlled release. Ergocalciferol (vitamin D2) is a bioactive compound whose deficiency may lead to rickets in children and osteomalacia in adults. In this study, ergocalciferol was encapsulated in tripalmitin SLNs stabilized by polysorbate 20 (Tween 20). SLN dispersions (5% w/w) were prepared by hot homogenization technique using a nozzle-type high-pressure homogenizer. Ergocalciferol at 0%, 5%, 10%, 15%, and 20% (w/w of lipid) was dissolved in the molten lipid at 80 °C, mixed with a 5% (w/w) aqueous solution of polysorbate 20 and homogenized at 138 MPa at 80 °C. Particle size, thermal properties, and microstructure were evaluated by dynamic light scattering (DLS), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM) respectively. As the proportion of ergocalciferol in the SLN increased from 0% to 20%, the Z-average values of the particles gradually decreased (P≤ 0.05) from approximately 120 nm to approximately 65 nm. DSC analysis of freeze dried SLN samples showed gradual decrease in enthalpies of fusion and crystallization for stable ß-subcell whereas for SLN dispersions, the enthalpy of fusion of unstable α-subcell crystal increased with increased ergocalciferol loading. The TEM images of the ergocalciferol loaded SLN samples showed the presence of spherical as well as rod-shaped nanoparticles. It was also observed that the turbidity of the SLN dispersions reduced noticeably with increased ergocalciferol loading. This finding could be useful in terms of fortification of clear juices with ergocalciferol. PRACTICAL APPLICATION: Solid lipid nanoparticles (SLNs) were used in this study to encapsulate vitamin D2, a vitamin important for bone health. It was found that as the concentration of vitamin D2 increased in the lipid phase of SLN dispersion, the clarity of the dispersion increased. Also, with increased vitamin D2 concentration, the stability of lipid crystal structure was affected in a way that indicates higher capacity of lipid to incorporate the vitamin molecules and hence to protect them better from oxygen and light. This vitamin loaded SLNs may offer alternatives to milk and margarine as a source of vitamin D.

[Studies on the chemical constituents of the flowers of Bupleurum chinese].

OBJECTIVE: To study the chemical constituents of the flowers of Bupleurum chinese. METHODS: The compounds were separated by column chromatography with silica gel. Their structures were identified by spectral methods and chemical analysis. RESULTS: Five compounds were isolated and identified as 8-(3',6'-dimethoxy)-4,5-cyclohexadiene-(delta11,12-dioxide-methylene) -dense-dihydrogen-isocoumarin(I), quercetin(II), rutin(III), calcifenol (IV), isorhamnetin (V). CONCLUSION: Compounds I and IV are obtained from the genus for the first time. Compound I is a new compound.

In vivo metabolism of 24R,25-dihydroxyvitamin D3: structure of its major bile metabolite.

In vivo metabolism of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) in female dogs has been studied thoroughly, and its major bile metabolite identified. After single oral administration of 24,25-(OH)2 [6,19,19-3H]D3 the plasma concentrations of radioactive metabolites were monitored for 504 h, and the metabolites in the bile collected and analyzed. The concentration of 24,25-(OH)2D3 in plasma reached a maximum after 6 h and decayed in two distinct phases; a fast-phase with a half-life of 17 h, followed by a slow-phase with a 17-day half-life. The area under the concentration/time curve (AUC) was 78-84% (0-504 h). The only detectable metabolite in the plasma was 25-hydroxy-24-oxovitamin D3 whose AUC was less than 5%. At 504 h, about 50% of administered radioactivity has been excreted, of which about 90% was found in the feces, indicating most of the administered 24,25-(OH)2D3 to be excreted in bile. A major metabolite, which constituted 23% of the total bile radioactivity at 504 h, was found in the bile. This metabolite was efficiently deconjugated by beta-glucuronidase to afford an aglycone which was identified as 23S,25-dihydroxy-24-oxovitamin D3 (23S,25-(OH)2-24-oxo-D3), by co-chromatography on HPLC with synthetic standards. The glucuronide was isolated from the bile of dogs given large doses of 24,25-(OH)2D3, and the structure determined being 23-(beta-glucuronide) of 23S,25-(OH)2-24-oxo-D3, by analyzing its negative ion mass spectrum and the positive ion mass spectrum of its derivatives. Thus it was concluded that, in dogs, 24,25-(OH)2D3 is a long lasting vitamin D metabolite, is mainly excreted in bile when metabolized to 23S,25-(OH)2-24-oxo-D3 and is conjugated at 23-OH as glucuronide.