Application of Direct Analysis in Real Time-High Resolution Mass Spectrometry to Investigations of Induced Plant Chemical Defense Mechanisms-Revelation of Negative Feedback Inhibition of an Alliinase.

Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 µM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.

In vitro antimicrobial activity of total extracts of the leaves of Petiveria alliacea L. (Anamu)

The antimicrobial activity of 13 total extracts was evaluated, 10 soft extracts (B) and 3 blended extracts (E) prepared from dry and fresh leaves of Petiveria alliacea L. Various solvents were used for their preparation: hydroalcoholic solution at 30%, 80% and isopropyl alcohol. The antimicrobial effect of the extracts was tested by means of the method of Kirby-Bauer, using four bacterial strains from the ATCC collection (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa) and a leveduriform fungus (Candida albicans). The following quality control parameters were determined for most active extracts: physical, physical-chemical and chemical parameters. The results were: nine extracts showed antibacterial activity, being the most concentrated (B8 and E3), the ones with the highest activity in the presence of the bacteria tested; the effect of blended extracts (E1, E2 and E3) was greater in the presence of P. aeruginosa. Blended extracts are considered more potent and active than soft extracts. No antifungal activity was obtained for both types of extracts. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for both extracts, with the following results: MIC-soft extracts (>100 mg/mL), blended extracts (>50 mg/mL); MBC-soft extracts (≥400 mg/mL), blended extracts (≥200 mg/mL) based on fresh leaves.

Avaliou-se a atividade antimicrobiana de 13 extratos totais, 10 brandos (B) e 3 batidos (E) elaborados a partir da folha seca e fresca da Petiveria alliacea L. Para sua preparação se empregaram vários solventes: solução hidroalcoólica 30%, 80% e álcool isopropílico. O efeito antimicrobiano dos extratos foi comprovado pelo método do Kirby-Bauer, usando para isso quatro cepas bacterianas da coleção ATCC (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis e Pseudomonas aeruginosa) e um fungo leveduriforme (Candida albicans). Determinaram-se parâmetros de controle da qualidade físicos, físico-químicos e químicos dos extratos mais ativos. Obtiveram-se como resultados: que 9 extratos mostraram atividade antibacteriana, sendo os mais concentrados (B8 e E3) os de maior espectro de ação frente às bactérias provadas; o efeito dos extratos batidos (E1, E2 e E3) foi maior frente a P. aeruginosa; os extratos batidos se consideram mais ativos que os brandos e não se obteve atividade antifúngica caso algum. Determinou-se a Concentração Mínima Inhibitoria e Bactericida para ambos os extratos, resultando a primeira para os extratos brandos maior a 100 mg/mL e para as batidos maior a 50 mg/mL; a segunda para os extratos brandos maior ou igual a 400 mg/mL e para as batidos maior ou igual a 200 mg/mL, em relação à droga fresca.