Coumarins from the peel of citrus grown in Colombia: composition, elicitation and antifungal activity

Abstract

The present work analyses the chromatographic profile of the peels from fruits of different citrus cultivated in Colombia: sweet orange (Citrus sinensis [L.] Osbeck var. Valencia), mandarins (Citrus reticulata L. var. Arrayana and Oneco), Key lime (Citrus aurantifolia [Christ.] Swingle var. Pajarito), Mandarine lime (Citrus x limonia, a hybrid between Citrus reticulata and Citrus x limon) and Tahitian lime (C. latifolia Tanaka, syn. Persian lime). Coumarins, furanocoumarins and polymethoxylated flavones are the major compounds. Then, six coumarins were isolated and identified from fruits of Tahitian and Key lime corresponding to 5-geranyloxy-7-methoxycoumarin; 5,7-dimethoxycoumarin (syn. limettin); 5,8-dimethoxypsoralen (syn. isopimpinellin); 5-methoxypsoralen (syn. bergaptene); 5-geranoxypsoralen (syn. bergamottin) and 5-(2,3-dihydroxy-3-methylbutoxy) psoralen (syn. oxypeucedanin hydrate). Coumarins and furanocoumarins were quantified by liquid chromatography (HPLC-DAD). Results show that the prenylated compounds were present in high concentrations in Tahitian and Key lime but in very low amounts in mandarins and sweet orange. Subsequently, the antifungal activity (inhibition of mycelial growth and germination of spores) of the coumarins against the fungus causing the anthracnose, Colletotrichum sp. (isolated from aerial parts of Tahitian lime) was determined. The compounds limettin and bergaptene, as well as mixtures of them, showed significant inhibitory effect (radial growth and spore germination) when compared to the control. Finally, the effect of some recognized elicitors to induce the coumarin production in fruits of C. latifolia was evaluated. The results showed that the chemical profiles are dependent on the applied elicitor and the post-induction time. As a result of the induction, a high concentration of some coumarins and furanocoumarins was maintained in the course of time for the Tahitian lime. In conclusion, isolated coumarins could be involved in the defense mechanisms of C. latifolia, C. aurantifolia and C. limonia and their accumulation may be modulated by the application of elicitors.

Keywords: Antifungal activity; Citrus fruits; Coumarin; Elicitors; Food science; Furanocoumarins; Limettin; Plant elicitor molecule; Rutaceae; Spore germination inhibition; Tahitian lime.

Fig. 1

Structure of coumarin, furanocoumarin and known citrus phytoalexins.

Fig. 2

HPLC-DAD chromatograms of methanolic extracts from citrus fruit peels. Tahitian lime (C. latifolia); Key lime (C. aurantifolia var. Pajarito); Sweet orange (C. sinensis var. Valencia); Mandarine lime (Citrus x limonia) and mandarin (C. reticulata var. Oneco), monitored at 254 nm.

Fig. 3

Structures of isolated compounds from Citrus peels.

Fig. 4

HPLC-DAD chromatogram (monitored at 254 nm) for isolated compounds and some phytoalexins reported in Citrus: 5-geranyloxy-7-methoxycoumarin, 1; bergamottin, 2; isopimpinellin, 3; bergapten, 4; limettin, 5; scoparone, 7; scopoletin, 8; umbelliferone, 9 and xanthyletin, 10.

Fig. 5

HPLC-DAD chromatograms of methanolic extracts from Tahitian lime fruit peels harvested at different times (6:00 to 7:00 a.m. and 12:00 to 2:00 p.m.) during the day and commercialized in local market.

Fig. 6

Time-course accumulation of coumarins and furanocoumarins (A: compound 1; B: compound 5; C: compounds 3 + 4; D: compound 2) in Tahitian lime fruit peels treated with some different elicitors: C1: water (control); T2: hemicelluloses from corn cobs at 400 mg/L; T3 and T4: β-D-glucans from G. lucidum at 50 and 400 mg/L respectively; T5: gentamicin at 400 mg/L; T6: UV radiation (λ = 254 nm) during 30 min.

Fig. 7

Mycelial growth of Colletotrichum sp. in the presence of coumarins and furanocoumarins from peels of Tahitian lime fruit (1 to 6 at 1.0 mM) (A), mixtures of the compounds 5 and 3 (B) (at 1.0 mM), and known citrus phytoalexins (scoparone, scopoletin, and umbelliferone) (C) (at 1.0 mM).

Fig. 8

Inhibition of spore germination of Colletotrichum sp. caused by the individual compounds from Tahitian lime (1, 2, 3 and 5), the mixture of 5 (0.75 mM) and 3 (0.25 mM) and the known phytoalexins (scoparone, scopoletin and umbelliferone) from citrus.