Antifungal activity screening of fractions from Annona cherimola Mill. leaf extract against Fusarium oxysporum.

The antifungal effect of ethanolic extract fractions of Annona cherimola leaves against the mycelial growth of Fusarium oxysporum was studied. The ethanolic crude extract was solvent partitioned and the ethyl acetate phase was fractionated by column or preparative thin-layer chromatography. All fractions were developed on TLC and analyzed for acetogenins (ACG) with Kedde reagent. The antifungal effect assays were carried out in vitro by the diffusion method on PDA plates. The ethanolic extract of A. cherimola leaves was highly active against F. oxysporum growth; subfractions obtained from the antifungal screening had a significant effect (p < 0.05) on the F. oxysporum growth parameters. The screening showed that as the purification steps progressed, the inhibition of mycelial growth increased. Six bioactive ACG (Annomolon-B, 34-epi annomolon B, almunequin, cherimoline 1, cherimoline 2, and isocherimoline 1) were identified by LC-QTOF-MS/MS. These findings suggested that bioactive ACG from A. cherimola leaves could be an alternative resource of a promising botanical fungicide to control plant diseases caused by F. oxysporum.

Targeted Analysis of the Concentration Changes of Phenolic Compounds in Persian Lime (Citrus latifolia) during Fruit Growth.

Citrus fruits possess a high content of phenolic compounds; however, few studies have focused on the changes occurring during fruit growth. In this study, the changes in the concentration of 20 flavonoids, 4 phenolic acids, and their biosynthetic precursors phenylalanine and tyrosine have been evaluated during fruit maturation (14 weeks). Extracts from all samples, obtained by ultrasound assistance, were analyzed by liquid chromatography coupled to tandem mass spectrometry with a triple quad system (LC-QqQ MS/MS). In general, the concentration of flavanones, which represented over 70% of the studied phenols, and flavones increased during fruit growth, reaching their maximum concentration around week 12. In general, flavanols and phenolic acids exhibited their maximum concentration at week 5 and then decreasing significantly during the rest of maturation. Phenylalanine and tyrosine showed a sinuous behavior during fruit growth. Partial least-squares showed a clear differentiation among fruits belonging to different maturation stages, coumaric acid derivatives being the most influential variables on the projection.

Comparative Study of the Effect of Sample Pretreatment and Extraction on the Determination of Flavonoids from Lemon (Citrus limon).

BACKGROUND: Flavonoids have shown to exert multiple beneficial effects on human health, being also appreciated by both food and pharmaceutical industries. Citrus fruits are a key source of flavonoids, thus promoting studies to obtain them. Characteristics of these studies are the discrepancies among sample pretreatments and among extraction methods, and also the scant number of comparative studies developed so far. OBJECTIVE: Evaluate the effect of both the sample pretreatment and the extraction method on the profile of flavonoids isolated from lemon. RESULTS: Extracts from fresh, lyophilized and air-dried samples obtained by shaking extraction (SE), ultrasound-assisted extraction (USAE), microwave-assisted extraction (MAE) and superheated liquid extraction (SHLE) were analyzed by LC-QTOF MS/MS, and 32 flavonoids were tentatively identified using MS/MS information. ANOVA applied to the data from fresh and dehydrated samples and from extraction by the different methods revealed that 26 and 32 flavonoids, respectively, were significant (p≤0.01). The pairwise comparison (Tukey HSD; p≤0.01) showed that lyophilized samples are more different from fresh samples than from air-dried samples; also, principal component analysis (PCA) showed a clear discrimination among sample pretreatment strategies and suggested that such differences are mainly created by the abundance of major flavonoids. On the other hand, pairwise comparison of extraction methods revealed that USAE and MAE provided quite similar extracts, being SHLE extracts different from the other two. In this case, PCA showed a clear discrimination among extraction methods, and their position in the scores plot suggests a lower abundance of flavonoids in the extracts from SHLE. In the two PCA the loadings plots revealed a trend to forming groups according to flavonoid aglycones. CONCLUSIONS: The present study shows clear discrimination caused by both sample pretreatments and extraction methods. Under the studied conditions, liophilization provides extracts with higher amounts of flavonoids, and USAE is the best method for isolation of these compounds, followed by MAE and SE. On the contrary, the SHLE method was the less favorable to extract flavonoids from citrus owing to degradation.

Comparative study of the effect of auxiliary energies on the extraction of Citrus fruit components.

A comparative study of methods for ultrasound-assisted extraction (USAE), microwave-assisted extraction (MAE) and superheated liquid extraction (SHLE) of compounds from citrus has been performed. The suited conditions for each method were evaluated to maximize the concentration of 10 representative compounds (sugars, carboxylic acids, phenolic acids and flavonoids) by a desirability function approach based on the chromatographic peaks obtained by LC-DAD. Extracts obtained under the suited conditions were analyzed by LC-QTOF MS/MS. The ANOVA on the molecular entities showed 232 significant entities (p<0.01), and pairwise comparison revealed that USAE and MAE methods are the most similar (50 different entities), and USAE and SHLE the most dissimilar (224 different entities). A discrimination test by PCA showed a clear discrimination among the extraction methods, explaining 78.51% of the total variability. Similarities in the abundance of the monitored compounds was tested by ANOVA showing that the extraction of carboxylic acids (malic and citric acids) was equal by all methods; while for each of the other eight compounds, at least one extraction method is different from the others. Under the evaluated conditions the SHLE method is the less favorable to extract metabolites from citrus, being the best the USAE method.