4,4-Dimethylsterols are rare bioactive constituents of the unsaponifiable matter of vegetable and animal fats and oils, which are predominantly abundant in lanolin (wool grease). Here we describe the isolation of the 4,4-dimethylsterols dihydrolanosterol and lanosterol from lanolin by countercurrent chromatography (CCC). An initial examination of the extracted unsaponifiable matter of lanolin showed the presence of high shares of fatty alcohols. Prior to CCC, we intended to remove these compounds which were known to co-elute with the analytes in CCC by urea complexation. As anticipated, fatty alcohols were found in the crystalline fraction while cholesterol and other desmethylsterols were detected in the liquid phase. However, contrary to expectation, 4,4-dimethylsterols were also precipitated in the presence of urea. Hence, urea complexation represented a good, novel preparative method for the separation of desmethylsterols and 4,4-dimethylsterols from lanolin. The resulting sample with 4,4-dimethylsterols and fatty alcohols was subjected to shake flask experiments with 14 biphasic solvent systems. Suitable partition coefficients (K values) were obtained with n-hexane/ethanol/water (12:8:1, v/v/v) and n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). After initial tests with conventional CCC, application of CCC in heart-cut recycling mode provided the 4,4-dimethylsterols dihydrolanosterol and lanosterol with purities of 99% and 95%, respectively.
Sterols are produced via complex, multistep biosynthetic pathways involving similar enzymatic conversions in plants, animals and fungi, yielding a variety of sterol metabolites with slightly different chemical properties to exert diverse and specific functions. A tremendously diverse landscape of sterols, and sterol-derived compounds, can be found across the plant kingdom, determining a wide spectrum of functions. Resolving the underlying biosynthetic pathways is thus instrumental to understanding the function and use of these molecules. In only a few plants, sterol biosynthesis has been studied using mutants. In non-model species a pharmacological approach is required. However, this relies on only a few inhibitors. Here, we probed a collection of inhibitors of mammalian cholesterol biosynthesis to identify new inhibitors of plant sterol biosynthesis. We show that imidazole-type fungicides, bifonazole, clotrimazole and econazole inhibit the obtusifoliol 14α-demethylase CYP51 in plants. Moreover, we found that the selective estrogen receptor modulator, clomiphene, inhibits sterol biosynthesis in part by inhibiting the plant-specific cyclopropyl-cycloisomerase CPI1. These results demonstrate that rescreening of inhibitors animal sterol biosynthesis is an easy approach for identifying novel inhibitors of plant sterol biosynthesis. These molecules expand the toolkit for studying and manipulating sterol biosynthesis in the plant kingdom.
Gas chromatography with mass spectrometry (GC/MS) and fractionation steps were used to determine the sterol patterns of red goji berries in detail. Twenty-five sterols were detected in fresh berries of two species (Lycium barbarum and L. chinense) from bushes grown in the botanical garden of the University of Hohenheim, and 20 sterols were identified. The rarely occurring campesta-5,24(25)-dienol, β-sitosterol, Δ5-avenasterol, campesterol, and cycloartenol represented >60 % of the total sterol content. Maturity and drying of fresh red goji berries caused small changes but did not affect the characteristic sterol pattern. This was confirmed by analyzing various commercial dried red goji berry samples from different sources. Separated flesh and seed samples revealed pronounced differences in the sterol pattern. A new method of merging GC/MS chromatograms showed that ∼75 % of the sterols were present in seeds and ∼25 % in flesh. The unique sterol profile may be exploited to authenticate red goji berries.
Abstract Sterols are known for a plethora of 250 different structures. Between 5 and 10% of them usually occur with varying abundance ratios (~ four orders of magnitude) and total amounts (0.4–1000 mg/100 g oil) in samples. Yet, quantitative data are mostly restricted to the few major sterols which are available as reference standards. Here, we developed a gas chromatography with mass spectrometry method operated in selected ion monitoring mode (GC/MS-SIM) that enabled the quantitation of 30 (silylated) sterols although only ten were available as reference standards. This could be obtained by studying the full-scan mass spectra of these ten sterol standards and 20 additional sterols measured in seven oils. In the next step, sterols were assigned to different groups. Values for quantification were then selected on the premise that response factors were constant within a sterol group. The deviation of the response factors within one sterol group was frequently below ± 10% and otherwise about ± 11–12%. Using mean response factors for all sterols, the novel GC/MS-SIM quantification method was superior to GC/FID which was exemplarily applied to two oils. Between eight and 21 of the 30 studied sterols and pentacyclic triterpenols were detected and quantified in 18 vegetable oils and two vegetable fats. The much higher number of sterols that could be quantified resulted in higher sterol amounts and the method and data may be useful for food authentication.
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