2.1. Content of Total Phenolics and Condensed Tannins
The content of total phenolics in fraction 1 was 3× lower than that determined in the crude extract. The highest amount (290 mg/g) was found in fraction 2 (Table 1
). In fraction 1, the dominant compounds were sugars; these eluted from the column by ethanol together with low-molecular-weight phenolic compounds. The total phenolics content of the crude acetone red lentil extract, was higher than values reported for crude extracts of red bean (55 mg/g) [10
] and pea (23 mg/g) [9
]. A higher quantity was found, however, in the crude extracts of vetch (66 mg/g) [14
], green lentil (68 mg/g) [27
], and adzuki bean (90 mg/g) [11
The marked content of total phenolics in the tannin fraction separated from the crude extract using Sephadex LH-20 column chromatography has been reported for other leguminous seeds (e.g., beach pea, faba bean, pea, vetch, adzuki, bean, green lentil) [9
], canola hulls [30
], and evening primrose [33
The content of tannins, expressed as absorbance units at 500 nm per g, in fraction 2 (129) was nearly 2× greater than that determined in the crude extract (70). For the low-molecular-weight phenolics fraction, a vanillin-positive reaction was noted thereby suggesting that catechin/epicatechin and other flavan-3-ols had eluted from the Sephadex LH-20 column with ethanol [39
]. In numerous papers, the presence of tannins has been reported in leguminous seeds [6
2.7 Identification and Quantification of Phenolic Compounds
In Table 2
the wavelength of UV maximum, molecular ions, typical ion fragments, and the content of each compound separated and detected by the HPLC-PDA and HPLC-ESI-MS systems are given. Sinapic, p
coumaric and trans
-ferulic acids, gallic aldehyde, tryptophan, (+)-catechin, and (−)-epicatechin were identified by comparing their respective retention time and UV spectra with those of corresponding standards, and later confirmed by HPLC-ESI-MS.
Compounds with the same spectral shape and wavelength maximum (λmax 278.9) have been characterized as flavanol monomers and procyanidin oligomers. Among these, compounds 8 and 13 from the HPLC-ESI-MS analysis exhibited a negative molecular ion [M-H]− at an m/z of 451.1 corresponding to a flavanol monomer (either catechin or epicatechin) linked to glucose, and a fragment ion [F-H]− at an m/z of 289 corresponding to either catechin or epicatechin. These compounds were identified as catechin glucoside and epicatechin glucoside, respectively, and the HPLC chromatogram agrees with the sequence of the elution time for the corresponding monomers. From the analysis by HPLC-ESI-MS, compounds 2, 11, and 19 showed a negative molecular ion [M-H]− at an m/z of 577.1 corresponding to a procyanidin dimer, and a negative fragment ion [F-H]− at an m/z of 289.1, which corresponds to either catechin or epicatechin. These peaks were identified as procyanidin dimers. Compounds 5 and 15 from the HPLC-ESI-MS analysis gave a negative molecular ion [M-H]− at an m/z of 865.2 corresponding to a procyanidin trimer and a two fragments [F-H]− at an m/z of 577 and 289.1 from a dimer and a monomer, respectively; these compounds have been identified as procyanidin trimers.
Compounds 3 and 6 exhibited a λmax of 276.4 nm which corresponds to a prodelphinidin. In the HPLC-ESI-MS analysis, an [M-H]− at an m/z of 593.1 from a dimer and two fragment ions [F-H]− at an m/z of 289.1 and 305.1 were found and correspond to epi/catechin and galloepi/catechin. These compounds were classified as prodelphinidin dimers.
By HPLC-ESI-MS analysis, compound 4 showed a negative molecular ion [M-H]− at an m/z of 881.3, which could correspond to a digallate procyanidin dimer, and two fragments [F-H]− at an m/z of 577 and m/z 289 from a procyanidin dimer and a monomer (catechin or epicatechin). Compound 4 was identified as a digallate procyanidin dimer.
Compound 10 had a negative molecular ion [M-H]− at an m/z of 745.1 and two fragments [F-H]− at an m/z of 577 from a procyanidin dimer and 169.1 from gallic acid. Compound 10 was identified as procyanidin gallate.
Three quercetin glycosides (compounds 17, 22, and 23) were tentatively identified by their UV spectra. The quercetin diglycoside (compound 17) was identified because it presented a molecular ion [M-H]− at an m/z of 625.3 corresponding to quercetin linked to a disaccharide (hexose + hexose), and a fragment ion [F-H]− at an m/z of 301.2 from quercetin. Compounds 22 and 23 showed a UV spectrum similar to that of the quercetin glycoside, but possessed a second maximum with a hypsochromic shift to a lower wavelength and a shoulder at 267 nm, both corresponding to an acylated glycoside. Compounds 22 and 23 also had a molecular ion [M-H]− at an m/z of 505.2 and a fragment ion [F-H]− at an m/z of 301.4 from quercetin. Both peaks correspond to two different acylated quercetin hexoses.
A kaempferol derivative was also identified (compound 20) by UV spectra. This compound gave an [M-H]− at an m/z of 447.1 and a fragment [F-H]− at an m/z of 285, corresponding to kaempferol.
Compound 24 was identified by its UV spectrum as an apigenin derivative and confirmed by HPLC-ESI-MS analysis; it yielded an [M-H]− at an m/z of 430.1 and a fragment [F-H]− at an m/z of 269.1, corresponding to apigenin.
Compounds possessing a flavanol structure (monomers, oligomers, and gallates) were the most abundant (~50%) class of compounds detected in the red lentil acetonic extract. It is important to point out that prodelphinidins were in similar abundance (161.6 μg/g) as were the procyanidins (dimers and trimers comprising 161.5 μg/g). The monomers in free and glycosidic forms were plentiful (192.8 μg/g) in the red lentil acetonic extract. Flavonols and flavones were also detected in the samples with quercetin diglycoside being the most abundant for these flavonoid classes. Non-flavonoid compounds (e.g., phenolic acids) were scarce in this type of lentil, and represented a smaller percentage in the totality of the phenolic compounds (20%).
The obtained profile of individual phenolic compounds in the red lentil acetonic extract is in line with those previously reported for leguminous seeds. Catechin and epicatechin glucosides, quercetin glucoside, myricetin, and procyanidin B1
were the main phenolic compounds found in a crude extract of adzuki bean [11
]. Glucosides of flavones and flavonols were determined in the cotyledon of peas (Pisum sativum
]. A high content of quercetin-3-O
-glucoside and myricetin-3-O
-glucoside was also determined in raw cowpeas (Vigna sinensis
]. Caffeic, o
-coumaric, ferulic, and sinapic acids were determined in the crude extract of red bean [10
], whereas vanillic, caffeic, p
-coumaric, sinapic, and ferulic acids, as well as quercetin and kaempferol were found in a pea crude extract [9