Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Characterization and Quantitation of Steryl Glycosides in Solanum melongena Philipp Heinz and Marcus A. Glomb* Institute of Chemistry, Food Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 2, 06120 Halle/Saale, Germany
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ABSTRACT: Glycosylated plant sterols or steryl glycosides (SGs) are a small group of glycolipids occurring ubiquitously in plants. In contrast to free sterols, they are insufficiently characterized concerning structural variety, quantity, and biological function. In particular, the type of sugar usually attached to the C-3 hydroxy function of the respective sterol is poorly studied. Eggplants (Solanum melongena) are rich in phytochemicals including SGs. In the present work, the unique glycosylation pattern was investigated by a highly selective LC-MS/MS method that allowed quantitation of the glucosides and galactosides of the most common sterols: cholesterol, β-sitosterol, campesterol, and stigmasterol. The quantitatively most important structure was β-sitosteryl β-D-glucopyranoside, with 54.5 mg/kg fresh weight of total fruit (365.3 mg/kg dry weight) followed by stigmasteryl β-D-glucopyranoside and campesteryl β-D-glucopyranoside. Analyses were performed in different tissues of eggplants (i.e., exocarp and outer mesocarp vs the remaining inner part). Steryl galactosides were determined in eggplants for the first time at significantly lower concentrations by a factor of 100. Furthermore, the rare SG β-sitosteryl β-D-cellobioside (3-β-sitosteryl β-Dglucopyranosyl-(1→4)-β-D-glucopyranoside) was detected in eggplants for the first time. Finally, UV irradiation induced the formation of the vitamin D glucosides 7-dehydrocholesteryl β-D-glucopyranoside and cholecalciferyl β-D-glucopyranoside at very low levels. KEYWORDS: steryl glycosides, eggplant, phytosterol, plant sterol, multilayer countercurrent chromatography, mass spectrometry, β-sitosteryl β-D-cellobioside, 7-dehydrocholesteryl β-D-glucopyranoside, vitamin D glucoside
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INTRODUCTION Steryl glycosides (SGs) are a small group of glycolipids found in a wide range of plant tissues.1 Analogous to phospholipids SGs are located mostly in membranes, where they regulate fluidity and permeability.2 The most important sterol aglycons in plants are β-sitosterol, stigmasterol, and campesterol. Whereas β-sitosterol and stigmasterol are part of cell membranes, campesterol mainly acts as a precursor of brassinosteroids.3 Although native sterols have been characterized in detail, SGs are scarcely studied.1,4 SGs are mainly located in plant plasma membranes, where they are suggested to be important for adaptive responses to environmental changes.3 Other functions, like acting as primer for cellulose biosynthesis or acclimatization to cold conditions are a controversial topic.1 A role in protecting cell-membrane integrity against the disruptive effects of high levels of steroidal glycoalkaloids has been suggested for SGs to explain the relatively high contents found in Solanacae.3 Biosynthetic derivatization of sterols usually occurs on the hydroxyl group at the sterol C-3 position, forming β-glycosides. Although αglycosidic bonds are described in bacteria, they have not been reported for plants.1 The glycosidic headgroups make SGs more polar than free sterols, resulting in a more amphiphilic structure, which explains the poor solubility in organic and aqueous solvents.5 Glucose is the most common sugar moiety in SGs, but other monosaccharides, such as galactose, xylose, and mannose, have also been described.3,6 Solanum species, especially eggplants, contain large amounts of SGs.3 An important point is that biosynthesis of sterols overlaps with © XXXX American Chemical Society
cholesterol metabolism. Cholesterol in Solanacae is converted to steroidal glycoalkaloids, such as solamargine and solasonine or the steroidal saponin diosgenin.7 A specific intermediate of cholesterol biosynthesis is 7-dehydrocholesterol, which can be photochemically converted into cholecalciferol.8 Thus, vitamin D metabolites in free or glycosylated forms are often found in Solanacae.9,10 Analysis of SGs is difficult not only because of their poor solubility. As expected, high-performance liquid chromatography (HPLC) in combination with ultraviolet or lightscattering detection was neither sensitive nor specific.11 Thus, in the literature, SGs are generally determined as their aglycons after enzymatic or, usually, acid hydrolysis.5 As the latter method is highly susceptible to artifact formation, quantitative and structural statements have to be evaluated as very critical. Coupled HPLC tandem mass spectrometry (LCMS/MS) of intact structures is therefore intriguing, but it has been used in very few studies.12−16 Specifically for SGs in eggplants, only glucosides of β-sitosterol, stigmasterol, and poriferasterol were isolated and fully characterized by NMR.17,18 However, from studies on glycoalkaloids it is expected that the glycosylation patterns of SGs should be much more diverse.19 Received: July 28, 2018 Revised: September 28, 2018 Accepted: October 8, 2018
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DOI: 10.1021/acs.jafc.8b04045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
[M + Na]+); stigmasteryl-Cello: m/z 759.4652 (found) m/z 759.4654 (calcd for C41H68NaO11 [M + Na]+); campesteryl-Cello: m/z 747.4650 (found) m/z 747.4654 (calcd for C40H68NaO11 [M + Na]+). Thin-Layer Chromatography (TLC). SGs were monitored on precoated TLC plates (Merck TLC silica gel 60 F254, Darmstadt, Germany) after separation by MLCCC. The plates were developed with a mixture of ethyl acetate, methanol, and water (7:1.5:1.4, v/v/ v). The chromatograms were sprayed with Liebermann−Burchard reagent (0.5 mL of acetic acid anhydride and 0.5 mL of sulfuric acid in 5 mL of ethanol). Sprayed plates were developed at 110 °C for 10 min. The Rf values were 0.78 (D3-Glc), 0.77 (Chol-Glc), 0.75 (sterylGlcs), 0.74 (7-DHC-Glc), 0.72 (steryl-Gals), and 0.58 (steryl-Cellos). Acid Hydrolysis of SGs. Acid hydrolysis of SGs was performed as described in the literature in 2 mL of 1 M methanolic hydrochloric acid for 3 h at 70 °C.27 After the addition of 1 mL of water, free sterols were extracted with n-hexane (2 × 2 mL) and dried with argon. Residues were dissolved in pyridine (50 μL), and N,Obis(trimethylsilyl)-acetamide with 5% trimethylchlorosilane (50 μL) was added. Samples were heated for 15 min at 70 °C prior to injection into the GC-FID system. The authentic sterol aglycons cholesterol, βsitosterol, stigmasterol, and campesterol tested as being stable under the conditions of hydrolysis, whereas 7-DHC and D3 were degraded. GC-FID. Samples were analyzed on an HP 6890N chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a flameionization detector. The column was an HP-5 (30 m × 0.32 mm, film thickness of 0.25 μm, Agilent Technologies, Palo Alto, CA), the injector was at 250 °C, the split ratio was 1:25, and the detector was at 310 °C. Helium 4.6 was used as the carrier gas in constant-flow mode (linear velocity of 41 cm/s, flow of 1.9 mL/min). The oventemperature program was as follows: 250 °C, 10 °C/min to 270 °C (2 min), 270 °C (28 min). The TR values of the silylated sterols were as follows: Chol, 9.9 min; campesterol, 12.0 min; stigmasterol, 12.7 min; and β-sitosterol, 14.2 min. Plant Material. Eggplants (Solanum melongena L., aubergine) were obtained from local markets in Germany. Vegetables (300−400 g per piece) were separated into peel (exocarp and 0.3 cm of mesocarp) and pulp (remaining inner fruit) with a sharp ceramic knife. Material was cut into squares (2 × 2 cm), mixed thoroughly, freeze-dried, and stored at −20 °C until use. Extraction of Plant Material. For isolation of SGs, approximately 47 and 275 g (corresponding to approximately one fruit) of peel or pulp, respectively, was freeze-dried and extracted three times with 150−300 mL of chloroform/methanol (2:1, v/v). Each step included 15 min of ultrasonic treatment and 2 h of shaking at 20 °C. The filtrate was concentrated under reduced pressure (
