HPLC Analysis of Grapevine Phytoalexins Coupling Photodiode Array Detection and Fluorometry

Abstract

A reversed-phase HPLC method useful for the analysis of the grapevine phytoalexins resveratrol, its β-d-glucoside, ε-viniferin, and pterostilbene in leaf extracts was developed by coupling diode array detection and fluorometry. Phytoalexins were extracted from UV-irradiated grapevine leaves with methanol/water (80:20) and prepurified on C18 solid phase extraction cartridges. Separation by HPLC was achieved using a C18 column and a gradient elution with acetonitrile and water (from 10 to 85% acetonitrile). Analyses of grapevine leaf extracts were performed by injecting the equivalent of 1 mg of leaf fresh weight. Recovery was nearly 100% for the three stilbenes resveratrol, pterostilbene, and ε-viniferin (ranging from 100.2 to 104.8%), and replicate analyses gave coefficients of variation of 0.6−2.5%. Identification of each phytoalexin was accomplished by line spectral comparisons with known standards, and ε-viniferin was further characterized by MS and GC/MS. Simultaneously, stilbenes were detected by fluorometry, allowing specific identification of these compounds. This procedure provided excellent separation and enabled quantitation of all grapevine phytoalexins present in the extracts. The method can easily be extended to the analysis of wine or biological fluids.

MALDI Mass Spectrometry Imaging for the Simultaneous Location of Resveratrol, Pterostilbene and Viniferins on Grapevine Leaves

“…It suggests that for a susceptible variety, the viniferins may be too far away from infection sites to play a real antifungal role. Their specific locations need to be understood according to the involved biosynthesize pathways and their isomeric composition [21]. Consequently, the investigation of viniferins locations from a range of susceptible to resistant grapevine species is now possible by MALDI-MSI to understand their role in the constitutive and inducible defenses of grapevine against fungi.…”

“…They are also well known for inducing an antifungal activity [13][14][15][16][17][18] and, for example, we observed by MSI experiment a co-localization of resveratrol and pterostilbene (trimethoxystilbene) at the infection site on the leaf abaxial side (Cabernet Sauvignon) [12] after infection by downy mildew. MSI allowed the identification and mapping of resveratrol and pterostilbene but the biosynthesis of some other stilbenes is also expected to be induced in grapevine leaves and even berries in response to pathogen infection or UV irradiation [19][20][21]. Besides the glycosylation of stilbenes (e.g., piceids), oxidative oligomerization of resveratrol catalyzed by plant peroxidases may occur and several of them have been identified in stressed grapevine leaves [22][23][24].…”

Induced Changes in Phenolic Composition of Wines After Foliar Application of Eugenol and Guaiacol Solutions to Grapevines

“…T-resveratrol has antioxidant properties and beneficial effects for human health, with hepatic and cardio protective properties, among others (Rotches-Ribalta, Andres-Lacueva, Estruch, Escribano, & Urpi-Sarda, 2012). In our study the standard of t-resveratrol aglycone allows us to confirm its identity in the samples, but the piceid compound is tentatively identified according to the bibliography and the spectra obtained at 308 nm (Jeandet, 1997). After the alcoholic fermentation, total content of stilbenes ( Figure 1B(a)) was higher in wines from treatments than in control, suggesting that the compounds applied by foliar treatments stimulate the stilbenes synthesis, as well their presence in wines.…”

“…Standard of piceid-t-resveratrol were not available, so it was tentatively identified by spectral parameters (Jeandet, 1997).…”

Determinação de resveratrol e características químicas em sucos de uvas produzidas em sistemas orgânico e convencional

“…Segundo Jeandet et al (1997), a produção de fitoalexinas é maior em cultivares que apresentam maior tolerância à doença do que naqueles mais suscetíveis. As variedades de uvas Concord e Rúbea, utilizadas neste experimento, são altamente vigorosas e resistentes a doenças (Maia & Camargo, 2005), fator intrínseco da variedade que pode ter influenciado o alto teor de resveratrol presente no suco dessas uvas.…”