Characterization of Forage Quality, Phenolic Profiles, and Antioxidant Activity in Alfalfa (Medicago sativa L.)

Abstract

Alfalfa (Medicago sativa L.) is one of the most important forage species and is recently more in focus for human consumption mainly due to its content of bioactive phenolics. Samples of the seventeen alfalfa cultivars/populations were collected at the Agricultural Institute Osijek with the aim to evaluate their forage quality, phenolic profiles, and antioxidant potential. Significant differences (p < 0.05) existed among studied alfalfa in all analyzed traits. The cultivar OS 99 and populations L7 and L20 were characterized by high crude protein content (22.5−24.7%) and the lowest neutral (40.2−42.9%) and acid detergent fibres (33−35.5%). The soluble-free phenolics from alfalfa leaves were extracted by methanol while insoluble cell-wall bound phenolics were released by alkaline hydrolysis. The bound phenolic extract showed a stronger DPPH scavenging capacity (20.8 mg TE/g dm) than the soluble (11.4 mg TE/g dm). The HPLC data revealed that more phenolics were found in the bound (3638.0 μg/g dm) than in the soluble form (912.3 μg/g dm). In the soluble extract of the alfalfa leaves, the major compound was catechin (338.3 μg/g dm), while rutin, epicatechin, and ferulic acid were minor ones. In the bound phenolic extract, the most abundant was ferulic (2198.2 μg/g dm) and p-coumaric acid (983.7 μg/g dm), followed by myricetin, apigenin, and quercetin. The principal component analysis revealed that alfalfa cultivars/populations were better discriminated based on the data on phenolics, rather than on forage quality. The cultivars/populations Florida 66, OS 66, L 40, L 42, Seed Force 4, and Torlesse were the most interesting in terms of phenolic health-promoting characteristics.

Keywords: HPLC; alfalfa; antioxidants; nutrition; polyphenols.

Figure 1

Mean values of TPC and AOA of soluble (a) and bound (b) phenolic extracts of alfalfa. Different letters indicate significant difference among accessions at p < 0.05 according to the LSD test. TPC—total phenolic content; AOA—antioxidant activity of soluble (_S) and bound (_B) phenolics.

Figure 2

Mean values of soluble phenolic acids (a) and flavonoids (b) in alfalfa. Different letters indicate significant difference among accessions at p < 0.05 according to the LSD test. 4-HBA—4-hydroxybenzoic; CAT—catechin; CA—caffeic acid; SA—syringic acid; ECAT—epicatechin; p-COA—p-coumaric acid; FA—ferulic acid; RUT—rutin; API—apigenin.

Figure 3

Mean values of bound phenolic acids (a) and flavonoids (b) in alfalfa. Different letters indicate significant difference among accessions at p < 0.05 according to the LSD test. p-COA—p-coumaric acid; FA—ferulic acid; QUE—quercetin; MYR—myricetin; API—apigenin.

Figure 4

Biplot relative to the principal component analysis (factor loadings (a) and scores (b) of first two factors) performed on the nutritive and data on phenolics (CP—crude protein; FAT—crude fat; CF—crude fibre; NDF—neutral detergent fibre; ADF—acid detergent fibre; ADL—acid detergent lignin. TPC—total phenolic content; AOA—antioxidant activity; 4-HBA—4-hydroxybenzoic; CAT—catechin; CA—caffeic acid; SA—syringic acid; ECAT—epicatechin; p-COA—p-coumaric acid; FA—ferulic acid; RUT—rutin; QUE—quercetin; MYR—myricetin; API—apigenin; TP_HPLC—total phenolics obtained by HPLC; _S—soluble phenolics; _B—bound phenolics).