Hydroxyl and Methoxyl Derivatives of Benzylglucosinolate in Lepidium densiflorum with Hydrolysis to Isothiocyanates and non-Isothiocyanate Products: Substitution Governs Product Type and Mass Spectral Fragmentation

Abstract

A system of benzylic glucosinolates was found and characterized in common pepperweed, Lepidium densiflorum Schrad. The major glucosinolate was the novel 4-hydroxy-3,5-dimethoxybenzylglucosinolate (3,5-dimethoxysinalbin), present at high levels in seeds, leaves, and roots. Medium-level glucosinolates were 3,4-dimethoxybenzylglucosinolate and 3,4,5-trimethoxybenzylglucosinolate. Minor glucosinolates included benzylglucosinolate, 3-hydroxy- and 3-methoxybenzylglucosinolate, 4-hydroxybenzylglucosinolate (sinalbin), the novel 4-hydroxy-3-methoxybenzylglucosinolate (3-methoxysinalbin), and indole-type glucosinolates. A biosynthetic connection is suggested. NMR, UV, and ion trap MS/MS spectral data are reported, showing contrasting MS fragmentation of p-hydroxyls and p-methoxyls. Additional investigations by GC-MS focused on glucosinolate hydrolysis products. Whereas glucosinolates generally yielded isothiocyanates, the dominating 3,5-dimethoxysinalbin with a free p-hydroxyl group produced the corresponding alcohol and syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde). After thermal deactivation of the endogenous myrosinase enzyme, massive accumulation of the corresponding nitrile was detected. This case study points out how non-isothiocyanate glucosinolate hydrolysis products are prevalent in nature and of interest in both plant-pathogen interactions and human health.

Eruca sativa Meal against Diabetic Neuropathic Pain: An H2S-Mediated Effect of Glucoerucin

“…Fatty acid composition of residual oil was analyzed after trans-methylation in 2N KOH methanol solution. Fatty acid methyl esters (FAMEs) were evaluated by gas chromatography and the internal normalization method [68] was used for determining the fatty acid profile.Glucosinolate content was determined following the ISO 9167-1 method with some minor modifications [69]. Briefly, 250 mg DSM were extracted in 70% ethanol at 80 °C.…”

“…After washing with 25 mM acetate buffer (pH 5.6), GSLs were desulfated by adding purified sulfatase (200 µL, 0.35 U/mL). The desulfo-GSLs were eluted in water (HPLC grade) and detected in HPLC-UV [69] monitoring their absorbance at 229 nm. They were identified with respect to their UV spectra and retention time, according to our library [70], and their amounts were estimated using sinigrin as an internal standard.…”

Glucosinolates in Reseda lutea L.: Distribution in plant tissues during flowering time

“…GSLs were extracted from seeds (250 mg) and from finely powdered freeze-dried herbs, i.e. stems and leaves (350 mg), racemes (200 mg) and roots (500 mg), using boiling 70% ethanol, and they were analyzed by HPLC-UV after enzymatic desulfation following the ISO 9167-1:1992/Amd 1:2013 method with some minor modifications as previously reported (Pagnotta et al, 2017). The desulfated GSLs were detected by monitoring their absorbance at 229 nm and initially identified with respect to their retention times and UV spectra according to our library (Nastruzzi et al, 2000;Galletti et al, 2015;Pagnotta et al, 2017.…”

“…stems and leaves (350 mg), racemes (200 mg) and roots (500 mg), using boiling 70% ethanol, and they were analyzed by HPLC-UV after enzymatic desulfation following the ISO 9167-1:1992/Amd 1:2013 method with some minor modifications as previously reported (Pagnotta et al, 2017). The desulfated GSLs were detected by monitoring their absorbance at 229 nm and initially identified with respect to their retention times and UV spectra according to our library (Nastruzzi et al, 2000;Galletti et al, 2015;Pagnotta et al, 2017. The amounts were estimated using allyl GSL as internal standard; the response factor 0.5 measured for 4-hydroxybenzylGSL (relative to allyl GSL) was arbitrarily adopted for the phenolic GSL and O-glycosylated benzyl GSL detected in our experiments when unavailable in literature (Wathelet et al, 2004).…”

“…This peak was identified as desulfated 2 by means of HPLC-APCI-MS, and NMR techniques as described next in the paragraph 3.2. The assignment of 3-hydroxybenzyl GSL (4), until now never reported in R. lutea, was initially hypothesized by the retention time and UV spectrum of desulfated 4 which overlapped with those characterized in Lepidium densiflorum (Pagnotta et al, 2017), then by HPLC-APCI-MS (APCI --MS m/z measured: 390.1 (100%) [M+HCOO] -, and 194.9 (10%) [Glc-S] -), and finally confirmed on purified desulfated 4 from R. lutea fruits by 1 H-NMR and by 13 C NMR (see https://www.dropbox.com/s/o0gyhy036t37eme/Reseda_lutea_flowers_SI.zip?dl=0 for a temporarily access and https://doi.org/10.5281/zenodo.3262193 for a permanent access).…”