22,23-Dihydroergosterol

Simultaneous HPLC determination of ergosterol and 22,23-dihydroergosterol in Flammulina velutipes sterol-loaded microemulsion.[Pubmed:23996456]

Biomed Chromatogr. 2014 Feb;28(2):247-54.

An efficient HPLC method was developed and validated for the simultaneous determination of ergosterol and 22,23-Dihydroergosterol in Flammulina velutipes sterol-loaded microemulsions (FVSMs). The different chromatographic conditions for in vitro and in vivo determinations were investigated, with the application examined by tissue distribution. Chromatographic separation was achieved on an Inertsil ODS-SP (250 x 4.6 mm, 5 microm) analytical column using a mobile phase of 98% methanol (in vitro), and 93% methanol for stomach samples and 96% methanol for other samples (in vivo) at 1.0 mL/min. The sterol content was detected at 282 nm. The established in vitro linearity ranges for ergosterol and 22,23-Dihydroergosterol were 0.58-72.77 microg/mL (r1 = 0.9999) and 0.59-73.25 microg/mL (r2 = 0.9999), respectively, with the biological (in vivo) samples following the same trend. The accuracy of the method was >99% (in vitro) and between 93%-108% (in vivo). The LOQ was 2.15 microg/L for ergosterol and 2.41 microg/L for 22,23-Dihydroergosterol in the in vitro studies. Also, the precisions met the acceptance criterion. These results indicate that the established HPLC method was specific, linear, accurate, precise and sensitive for the separation and simultaneous determination of ergosterol and 22,23-Dihydroergosterol.

Enhanced oral bioavailability and tissue distribution of a new potential anticancer agent, Flammulina velutipes sterols, through liposomal encapsulation.[Pubmed:23721187]

J Agric Food Chem. 2013 Jun 26;61(25):5961-71.

This study innovatively investigated the anticancer effect of Flammulina velutipes sterols (FVSs), the in vivo pharmacokinetics, and the tissue distribution of FVS-loaded liposomes. The FVS consisting of mainly 54.8% ergosterol and 27.9% 22,23-Dihydroergosterol exhibited evident in vitro antiproliferative activity (liver HepG-2, IC50 = 9.3 mug mL(-1); lung A549, IC50 = 20.4 mug mL(-1)). To improve the poor solubility of FVS, F. velutipes sterol liposome (FVSL) was originally prepared. The encapsulation efficiency of ergosterol was 71.3 +/- 0.1% in FVSL, and the encapsulation efficiency of 22,23-Dihydroergosterol was 69.0 +/- 0.02% in FVSL. In comparison to its two free sterol counterparts, the relative bioavailability of ergosterol and 22,23-Dihydroergosterol in FVSL was 162.9 and 244.2%, respectively. After oral administration in Kunming mice, the results of tissue distribution demonstrated that the liposomal FVS was distributed mostly in liver and spleen. The drug was eliminated rapidly within 4 h. These findings support the fact that FVS, a potential nutraceutical and an effective drug for the treatment of liver cancer, could be encapsulated in liposomes for improved solubility and bioavailability.

Cytotoxic effect of novel Flammulina velutipes sterols and its oral bioavailability via mixed micellar nanoformulation.[Pubmed:23524118]

Int J Pharm. 2013 May 1;448(1):44-50.

The aim of this study was to investigate the anti-tumor effect of sterols initially separated from Flammulina velutipes and the pharmacokinetics and tissue distribution after oral administration of F. velutipes sterol nanomicelles (FVSNs). F. velutipes sterol (FVS) consisted of mainly ergosterol (54.78%), 22,23-Dihydroergosterol (27.94%) and ergost-8(14)-ene-3beta-ol (discovered for the first time in F. velutipes). In vitro cytotoxicity assay of FVS against U251 cells and HeLa cells showed that at 72h treatment, the FVS (IC50=23.42mug/mL) exhibited strong inhibitory effect against U251 cells, even overwhelmed the standard anti-tumor drug (5-fluorouracil) to an extent, while the HeLa cells were not significantly susceptible to the FVS. To improve the solubility and bioavailability of FVS, a model for insoluble anti-tumor drugs, FVSNs were prepared. In vitro characterization of FVSNs revealed satisfactory size distribution, loading capacity and encapsulation efficiency. Pharmacokinetic study in SD rats demonstrated that the mixed micellar nanoformulation significantly enhanced the bioavailability of FVS than free drug. Additionly, tissue distribution in mice manifested that the biodistribution of FVSNs as compared to the free FVS suspension were significantly improved. In conclusion, the nanomicelles developed in our study provided a promising delivery system for enhancing the oral bioavailability and selective biodistribution of FVS, a potential anti-tumor agent.

Enhanced oral bioavailability of a sterol-loaded microemulsion formulation of Flammulina velutipes, a potential antitumor drug.[Pubmed:23049254]

Int J Nanomedicine. 2012;7:5067-78.

PURPOSE: To investigate the growth inhibition activity of Flammulina velutipes sterol (FVS) against certain human cancer cell lines (gastric SGC and colon LoVo) and to evaluate the optimum microemulsion prescription, as well as the pharmacokinetics of encapsulated FVS. METHODS: Molecules present in the FVS isolate were identified by gas chromatography/mass spectrometry analysis. The cell viability of FVS was assessed with methyl thiazolyl tetrazolium (MTT) bioassay. Based on the solubility study, phase diagram and stability tests, the optimum prescription of F. velutipes sterol microemulsions (FVSMs) were determined, followed by FVSMs characterization, and its in vivo pharmacokinetic study in rats. RESULTS: The chemical composition of FVS was mainly ergosterol (54.8%) and 22,23-Dihydroergosterol (27.9%). After 72 hours of treatment, both the FVS (half-maximal inhibitory concentration [IC(50)] = 11.99 mug . mL(-1)) and the standard anticancer drug, 5-fluorouracil (IC(50) = 0.88 mug . mL(-1)) exhibited strong in vitro antiproliferative activity against SGC cells, with IC(50) > 30.0 mug . mL(-1); but the FVS performed poorly against LoVo cells (IC(50) > 40.0 mug . mL(-1)). The optimal FVSMs prescription consisted of 3.0% medium chain triglycerides, 5.0% ethanol, 21.0% Cremophor EL and 71.0% water (w/w) with associated solubility of FVS being 0.680 mg . mL(-1) as compared to free FVS (0.67 mug . mL(-1)). The relative oral bioavailability (area-under-the-curve values of ergosterol and 22,23-Dihydroergosterol showed a 2.56-fold and 4.50-fold increase, respectively) of FVSMs (mean diameter ~ 22.9 nm) as against free FVS were greatly enhanced. CONCLUSION: These results indicate that the FVS could be a potential candidate for the development of an anticancer drug and it is readily bioavailable via microemulsion formulations.

Vitamin D4 in mushrooms.[Pubmed:22870201]

PLoS One. 2012;7(8):e40702.

An unknown vitamin D compound was observed in the HPLC-UV chromatogram of edible mushrooms in the course of analyzing vitamin D(2) as part of a food composition study and confirmed by liquid chromatography-mass spectrometry to be vitamin D(4) (22-dihydroergocalciferol). Vitamin D(4) was quantified by HPLC with UV detection, with vitamin [(3)H] itamin D(3) as an internal standard. White button, crimini, portabella, enoki, shiitake, maitake, oyster, morel, chanterelle, and UV-treated portabella mushrooms were analyzed, as four composites each of a total of 71 samples from U.S. retail suppliers and producers. Vitamin D(4) was present (>0.1 microg/100 g) in a total of 18 composites and in at least one composite of each mushroom type except white button. The level was highest in samples with known UV exposure: vitamin D enhanced portabella, and maitake mushrooms from one supplier (0.2-7.0 and 22.5-35.4 microg/100 g, respectively). Other mushrooms had detectable vitamin D(4) in some but not all samples. In one composite of oyster mushrooms the vitamin D(4) content was more than twice that of D(2) (6.29 vs. 2.59 microg/100 g). Vitamin D(4) exceeded 2 microg/100 g in the morel and chanterelle mushroom samples that contained D(4), but was undetectable in two morel samples. The vitamin D(4) precursor 22,23-Dihydroergosterol was found in all composites (4.49-16.5 mg/100 g). Vitamin D(4) should be expected to occur in mushrooms exposed to UV light, such as commercially produced vitamin D enhanced products, wild grown mushrooms or other mushrooms receiving incidental exposure. Because vitamin D(4) coeluted with D(3) in the routine HPLC analysis of vitamin D(2) and an alternate mobile phase was necessary for resolution, researchers analyzing vitamin D(2) in mushrooms and using D(3) as an internal standard should verify that the system will resolve vitamins D(3) and D(4).

Human cytochrome P450scc (CYP11A1) catalyzes epoxide formation with ergosterol.[Pubmed:22106170]

Drug Metab Dispos. 2012 Mar;40(3):436-44.

Cytochrome P450scc (P450scc) catalyzes the cleavage of the side chain of both cholesterol and the vitamin D(3) precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human P450scc to metabolize ergosterol, the vitamin D(2) precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a k(cat) of 53 mol . min(-1) . mol P450scc(-1) and a K(m) of 0.18 mol ergosterol/mol phospholipid, similar to the values observed for cholesterol metabolism. The reaction of ergosterol with P450scc was scaled up to make enough of the two major products for structural analysis. From mass spectrometry, NMR, and comparison of the NMR data to that for similar molecules, we determined the structures of the two major products as 20-hydroxy-22,23-epoxy-22,23-Dihydroergosterol and 22-keto-23-hydroxy-22,23-Dihydroergosterol. Molecular modeling and nuclear Overhauser effect (or enhancement) spectroscopy spectra analysis helped to establish the configurations at C20, C22, and C23 and determine the final structures of major products as 22R,23S-epoxyergosta-5,7-diene-3beta,20alpha-diol and 3beta,23S-dihydroxyergosta-5,7-dien-22-one. It is likely that the formation of the second product is through a 22,23-epoxy (oxirane) intermediate followed by C22 hydroxylation with the formation of strained 22-hydroxy-22,23-epoxide (oxiranol), which is immediately transformed to the more stable alpha-hydroxyketone. Molecular modeling of ergosterol into the P450scc crystal structure positioned the ergosterol side chain consistent with formation of the above products. Thus, we have shown that P450scc efficiently catalyzes epoxide formation with ergosterol giving rise to novel epoxy, hydroxy, and keto derivatives, without causing cleavage of the side chain.

Vitamin D and sterol composition of 10 types of mushrooms from retail suppliers in the United States.[Pubmed:21663327]

J Agric Food Chem. 2011 Jul 27;59(14):7841-53.

Vitamin D(2) (ergocalciferol) and sterols were analyzed in mushrooms sampled nationwide in the United States to update the USDA Nutrient Database for Standard Reference. Vitamin D(2) was assayed using HPLC with [(3)H]-vitamin D(3) internal standard and sterols by GC-FID mass spectrometric (MS) confirmation. Vitamin D(2) was low (0.1-0.3 mug/100 g) in Agaricus bisporus (white button, crimini, portabella) and enoki, moderate in shiitake and oyster (0.4-0.7 mug/100 g), and high in morel, chanterelle, maitake (5.2-28.1 mug/100 g) and UV-treated portabella (3.4-20.9 mug/100 g), with significant variability among composites for some types. Ergosterol (mg/100 g) was highest in maitake and shiitake (79.2, 84.9) and lowest in morel and enoki (26.3, 35.5); the range was <10 mg/100 g among white button composites but 12-50 mg/100 g among samples of other types. All mushrooms contained ergosta-5,7-dienol (22,23-Dihydroergosterol) (3.53-18.0 mg/100 g) and (except morel) ergosta-7-enol. Only morel contained brassicasterol (28.6 mg/100 g) and campesterol (1.23-4.54 mg/100 g) and no ergosta-7,22-dienol. MS was critical in distinguishing campesterol from ergosta-7,22-dienol.

[Search for and structural elucidation of medicinal products from the vegetable kingdom (crude drugs and plant materials)].[Pubmed:18057787]

Yakugaku Zasshi. 2007 Dec;127(12):1975-96.

This review describes chemical and biological studies on natural products achieved by the author for the latest 47 years and its main contents are composed of the following researches of the eight sections, entitled 1) Hopane-type triterpenes from a lichen, Parmelia leucotyliza, 2) Spirostanol and frostanol glycosides from Metanarthecium luteo-viride (Liliaceae), 3) Selective reduction of double bonds: preparation of 22,23-Dihydroergosterol from ergosterol, 4) Compositions and structures of fragrant sesquiterpenes from several types of agarwoods, 5) Triterpenes and other components from Meliaceous plants, 6) Constituents of seeds of crude drugs and medicinal plants, 7) Hopane-type triterpene glycosides from a fern, Diplazium subsinuatum, 8) Search and structural elucidation of biologically active components from American plants obtained from United States of America (Oregon and California), Mexico, Guatemala, and Honduras. In this review, many classes of natural products, i.e., terpenoids (mono-, sesqui-, di-, and triterpenoids), steroids, glycosides, saponins, tannins, phenylpropanoids, lignans, flavonoids (flavones, flavonols, flavanones, biflavones, flavan-3-ols, etc.), etc. are dealt with and referred to.