non-destructive-spe-uplc-based-quantification-aflatoxins-stilbenoid

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (<i>Arachis</i> spp.) Seeds

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate ...

U.S. Department of Agriculture

The ionization constant (pKa) and the octanol-water partition coefficient (log P) of the important mycotoxin cyclopiazonic acid (CPA) were determined by means of potentiometric titration, and the lipophilicity profile (log D) was calculated. Under the experimental conditions, pKa of CPA = 2.97 +/- 0.09, log P = 3.83 +/- 0.10, and log D at pH 7.4 = -0.58.

Determination of ionization constant (pKa) and octanol-water partition coefficient (Log P) of cyclopiazonic acid.

A new pigmented, optically active, low molecular weight metabolite has been isolated from peanut (Arachis hypogaea) kernels challenged by four species of Aspergillus. The structure of the new compound, termed SB-1, was elucidated by analysis of 1H NMR, 13C NMR, and mass spectrometric data. The SB-1 molecule bears prenylated benzenoid and but-2-enolide moieties and represents an unusual class of compounds. The closest known analogue to SB-1 was isolated from heartwood of Pericopsis elata. Both A. hypogaea and P. elata belong to the family Leguminosae. The new metabolite was accumulated in different peanut genotypes challenged by five Aspergillus species and may be an important representative of a new class of peanut phytoalexins. SB-1 production often exceeds production of major known stilbenes.

New peanut (Arachis hypogaea) phytoalexin with prenylated benzenoid and but-2-enolide moieties.

A chemical cleanup procedure for low-level quantitative determination of aflatoxins in major economically important agricultural commodities using HPLC has been developed. Aflatoxins were extracted from a ground sample with MeOH/H2O (80:20, v/v), and after a cleanup step on a minicolumn packed with Florisil, aflatoxins were quantified by HPLC equipped with a C18 column, a photochemical reactor, and a fluorescence detector. Water/MeOH (63:37, v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5, 1.7, 5, and 1.7 ng/g were 89.5+/-2.2, 94.7+/-2.5, 90.4+/-1.0, and 98.2+/-1.1, respectively (mean+/-SD, %, n=3). Similar recoveries, precision, and accuracy were achieved for corn, brown and white rice, cottonseed, almonds, Brazil nuts, pistachios, walnuts, and hazelnuts. The quantitation limits for aflatoxins in peanuts were 50 pg/g for aflatoxin B1 and 17 pg/g for aflatoxin B2. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.

Simple, rapid, and inexpensive cleanup method for quantitation of aflatoxins in important agricultural products by HPLC.

In peanuts, a mechanism of resistance to fungal infection is reportedly due to the synthesis of stilbene phytoalexins, which are antibiotic, low molecular weight metabolites. The phytoalexin-associated response of different peanut genotypes to exogenous invasion in the field has not been investigated and may be useful for breeding resistant peanut cultivars. Five peanut genotypes, Georgia Green, Tifton 8, C-99R, GK-7 High Oleic, and MARC I, which differ in resistance to major peanut diseases, were investigated for their ability to produce phytoalexins under field conditions in South Georgia in 2001 and 2002. Five known peanut phytoalexins, trans-resveratrol, trans-arachidin-1, trans-arachidin-2, trans-arachidin-3, and trans-3'-isopentadienyl-3,5,4'-trihydroxystilbene, were quantitated. The phytoalexins were measured in peanuts of different pod maturity (yellow, orange, brown, and black) with or without insect pod damage (externally scarified or penetrated). Kernels from insect-damaged pods of C-99R and Tifton 8 genotypes had significantly higher concentrations of phytoalexins than other genotypes. The same genotypes were the most resistant to tomato spotted wilt virus and late leaf spot, while MARC I, which is highly susceptible to these diseases, produced very low concentrations of phytoalexins. However, there was no significant difference in phytoalexin production by undamaged peanut pods of all tested genotypes. trans-Arachidin-3 and trans-resveratrol were the major phytoalexins produced by insect-damaged peanuts. In damaged seeds, the concentrations of trans-3'-isopentadienyl-3,5,4'-trihydroxystilbene were significantly higher in Tifton 8 as compared to other genotypes. There was an association between total phytoalexin production and published genotype resistance to major peanut diseases. Stilbene phytoalexins may be considered potential chemical markers in breeding programs for disease-resistant peanuts.

Interrelationship of phytoalexin production and disease resistance in selected peanut genotypes.

Peanuts respond to fungal infection by synthesizing phytoalexins, most of which are antibiotic stilbenes. The mechanism and dynamics of phytoalexin formation in the peanut have not been studied. One of the most popular peanut cultivars in the southeastern United States, Georgia Green, was investigated for its ability to produce phytoalexins in response to infection by soil fungal strains. The experimental design allowed for study of phytoalexin production in peanut kernels layer-by-layer. The layers were dissected from different depths of the kernel starting from the infected area down to healthier tissues. Six peanut phytoalexins, trans-resveratrol, trans-arachidin-1, trans-arachidin-2, trans-arachidin-3, trans-3-isopentadienyl-4,3',5'-trihydroxystilbene, and SB-1, were detected in the kernel slices and quantitated. All of the fungal strains triggered phytoalexin production; however, the composition of phytoalexins varied significantly by layer. After incubation for 24 h, tissues remote from the infected area produced almost exclusively trans-resveratrol, whereas closer to the infected area tissues synthesized all six phytoalexins. In all of the experiments, after 48 h of fungal growth, deeper layers produced all tested phytoalexins. There was a significant difference in phytoalexin production elicited by some fungal isolates. No association was observed between phytoalexin production and toxigenic potential of fungal strains that elicited the production in mature peanut kernels.

Localized production of phytoalexins by peanut (Arachis hypogaea) kernels in response to invasion by Aspergillus species.

A new spermidine triamide derivative has been isolated from peanut flowers and identified as N (1)-acetyl- N (5), N (10)-di- p-( EE)-coumaroylspermidine on the basis of detailed analysis of NMR, MS, and UV data. Two other spermidine conjugates, N (1), N (5), N (10)-tri- p-( EEE)-coumaroylspermidine and di- p-( EE)-coumaroylspermidine, as well as four flavonoid conjugates (quercetin-3-glucoside, quercetin-3-glucuronide, isorhamnetin-3-glucoside, and isorhamnetin-3-glucuronide) that have been previously reported in organs of other plants, have been found in this study in peanut ( Arachis hypogaea L.), a representative of the Leguminosae family, for the first time. The dynamics of photoisomerization in the spermidine conjugates have been investigated.

Spermidine and flavonoid conjugates from peanut (Arachis hypogaea) flowers.

The peanut plant (Arachis hypogaea) is known to produce stilbene phytoalexins as a defensive response to fungal invasion; however, the distribution of phytoalexins among different organs of the peanut plant at early stages of growth under axenic conditions has not been studied. Axenic plants produced a stilbenoid, resveratrol, as well as soluble bound and free phenolic acids, including 4-methoxycinnamic acid, which is reported in peanuts for the first time. Neither resveratrol nor phenolic acids were found in the root mucilage; the prenylated stilbenes were restricted to the mucilage and were not found in other organs of the peanut plant. These findings may lead to a better understanding of the defensive role of peanut stilbenes and phenolic acids.

Production of stilbenoids and phenolic acids by the peanut plant at early stages of growth.

Two new stilbene derivatives have been isolated from peanut seeds challenged by an Aspergillus flavus strain, along with chiricanine B, which has not been previously reported from peanuts, as well as a stilbenoid reported previously only as a synthetic product. The structures of these new putative phytoalexins were determined by analysis of 1H and 13C NMR, HRESIMS, MSn, and UV data. The new stilbenoids were named arahypin-13, arahypin-14, and arahypin-15. Together with other known bioactive peanut stilbenoids that were also produced in the challenged seeds, these new compounds may play a defensive role against invasive fungi.

New Monomeric Stilbenoids from Fungus-Challenged Peanut (Arachis hypogaea) Seeds.

Draft genomes of 16 isolates of  Link and  Speare, identified as the predominant genotypes colonizing peanuts in four farming regions in Ethiopia, are reported. These data will allow mining for sequences that could be targeted by RNA interference to prevent aflatoxin accumulation in peanut seeds.

Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia.

Article	Year
Determination of ionization constant (pKa) and octanol-water partition coefficient (Log P) of cyclopiazonic acid. Journal of AOAC International\| PubMed ID: 16385984	2005
New peanut (Arachis hypogaea) phytoalexin with prenylated benzenoid and but-2-enolide moieties. Journal of agricultural and food chemistry\| PubMed ID: 16536583	2006
Simple, rapid, and inexpensive cleanup method for quantitation of aflatoxins in important agricultural products by HPLC. Journal of agricultural and food chemistry\| PubMed ID: 17319689	2007
Interrelationship of phytoalexin production and disease resistance in selected peanut genotypes. Journal of agricultural and food chemistry\| PubMed ID: 17326657	2007
Localized production of phytoalexins by peanut (Arachis hypogaea) kernels in response to invasion by Aspergillus species. Journal of agricultural and food chemistry\| PubMed ID: 18298071	2008
Spermidine and flavonoid conjugates from peanut (Arachis hypogaea) flowers. Journal of agricultural and food chemistry\| PubMed ID: 18412366	2008
Production of stilbenoids and phenolic acids by the peanut plant at early stages of growth. Journal of agricultural and food chemistry\| PubMed ID: 19127717	2006
New Monomeric Stilbenoids from Fungus-Challenged Peanut (Arachis hypogaea) Seeds. Journal of agricultural and food chemistry\| PubMed ID: 26672388	2015
Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia. Microbiology resource announcements\| PubMed ID: 32703834	2020

Victor S Sobolev

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