3. Results and discussion

Feature Description

Pconv Convex Perimeter Convex perimeter of the seed (mm) PCrof Crofton Perimeter Crofton perimeter of the seed (mm)

Dmax Max diameter Maximum diameter of the seed (mm) Dmin Min diameter Minimum diameter of the seed (mm)

Pconv/PCrof Perimeter ratio Ratio between convex and Crofton's perimeters

Dmin/Dmax Feret ratio Ratio between minimum and maximum diameters

Ecd Eq. circular diameter Diameter of a circle with equivalent area (mm)

Com Compactness degree Seed compactness descriptor = [√(4/ π) A]/Dmax EAmax Maximum ellipse axis Maximum axis of an ellipse with equivalent area (mm) EAmin Minimum ellipse axis Minimum axis of an ellipse with equivalent area (mm) Rmean Mean red channel Red channel mean value of seed pixels (gray levels) Rsd Red std. deviation Red channel standard deviation of seed pixels Gmean Mean green channel Green channel mean value of seed pixels (gray levels) Gsd Green std. deviation Green channel standard deviation of seed pixels Bmean Mean blue channel Blue channel mean value of seed pixels (gray levels) Bsd Blue std. deviation Blue channel standard deviation of seed pixels Hmean Mean hue channel Hue channel mean value of seed pixels (gray levels) Hsd Hue std. deviation Hue channel standard deviation of seed pixels

Lmean Mean lightness ch. Lightness channel mean value of seed pixels (gray levels) Lsd Lightness std. dev. Lightness channel standard deviation of seed pixels Smean Mean saturation ch. Saturation channel mean value of seed pixels (gray levels) Ssd Saturation std. dev. Saturation channel standard deviation of seed pixels Dmean Mean density Density channel mean value of seed pixels (gray levels) Dsd Density std. deviation Density channel standard deviation of seed pixels

S Skewness Asymmetry degree of intensity values distribution (gray levels) K Kurtosis Peakness degree of intensity values distribution (densit. units) H Energy Measure of the increasing intensity power (densitometric units)

according to Hâruta [22] and the Haralick's descriptors reported in Table 2.

Table 3. List of morphometric features measured on seeds, excluding the elliptic Fourier descriptors (EFDs) calculated

F Fiber length Seed length along the fiber axis C Curl degree Ratio between Dmax and F Conv Convessity degree Ratio between PCrof and P Sol Solidity degree Ratio between A and convex area

)

Sf Shape factor Seed shape descriptor = (4 <sup>π</sup> area)/perimeter<sup>2</sup> (normalized value)

) (normalized value)

Rf Roundness factor Seed roundness descriptor = (4 area)/(<sup>π</sup> max diameter<sup>2</sup>

A Area Seed area (mm<sup>2</sup>

30 Rediscovery of Landraces as a Resource for the Future

P Perimeter Seed perimeter (mm)

#### 3.1. Phenolic profile in wheat landraces

Phenolics are mainly concentrated in the outer layers of kernel and contribute to the wheat flour nutraceutical value owing to their antioxidant, anti-inflammatory and anticancer properties [35]. In literature, ca. 70 different phenolic compounds, including coumarins, phenolic acids, anthocyanins, flavones, isoflavones, proanthocyanidins, stilbenes and lignans, were identified in durum wheat genotypes [14].

Referring to flavones, whose interest has grown enormously due to their putative beneficial effects against atherosclerosis, osteoporosis, diabetes mellitus and certain cancers [36] 5,7,4<sup>0</sup> trihydroxyflavone (apigenin) and 5,7,3<sup>0</sup> ,40 -tetrahydroxyflavone (luteolin) are the main representatives in wheat, where they accumulate as 6-C and/or 8-C-glycosidic conjugates. The 8-Cglucosides of apigenin and luteolin are also known as vitexin and orientin, respectively.

Hydro-alcoholic extracts from wheat grains were exhaustively analyzed by means of HPLC/ DAD and HPLC/ESI-MS. Although the major portion of phenolics in grains exist in the bound form [37], there is a general trend for studying polyphenols in the free form when dealing with chemotaxonomic studies [38, 39]. The chromatograms relating to free phenolics profile of durum wheat grains showed ca. 20 different signals, eluting in the range from 7 to 30 min. Among these, 13 signals were tentatively identified: a preliminary analysis of the UV–VIS (in terms of spectrum shape and absorption maximum, see Table 4) spectra of the peaks revealed the presence of compounds belonging to the chemical subclasses of hydroxycinnamic acids and organic acids; several peaks showing the typical spectrum of apigenin derivatives were also detected.

The use of mass spectrometry as detector was helpful in tentatively identifying wheat metabolites (Table 4); peak assignments were further confirmed by comparison with literature data [14, 40, 41] and co-injection with pure reference standards when available (see material and methods).

According to Lo Bianco et al. [11], three hydroxycinnamic acids were identified in wheat grains: caffeic acid (peak 4), ferulic acid (peak 10) and another member of this class (peak 1) for which unfortunately the MS spectrum was not determined. Vanillic acid (peak 3) was identified for its diagnostic UV–VIS and mass spectrum; the assignment was confirmed with co-injection with the corresponding standard. Peaks 2 and 6, showing almost identical UV–VIS spectra (a symmetrical absorption with λ max = 317 nm) were tentatively identified as coumarins; furthermore, peak 2 showed a clear mass spectrum with a pseudomolecular ion at 145.14 m/z (M-H)�. Presence of coumarins in durum wheat has been reported by other authors [14, 40]. The UV–VIS spectrum of peak 5 (λ max = 268, 35 nm) was typical of that of luteolin derivatives; the corresponding mass spectrum exhibited a base peak of 609.52 m/z (pseudomolecular ion) with no signals ascribable to fragments generated by the loss of sugars. The peak corresponding to luteolin aglycone was


Some of the phenolic markers identified in the free form, were quantitatively quite different among the genotypes studied. For example, coumarin (peak 6), ranging from 2.57 μg/g in Tumminia SG3 to 0.09 μg/g in Tripolino, and vanillic acid (peak 3) from 1.34 μg/g in Manto di Maria to 0.25 μg/g in Tumminia SG3. The apigenin C-hexoside-C-pentoside (peak 7) content was significantly different among wheat grains, recording values above 21 μg/g for Tumminia

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33

Luteolin di-C-hexoside (lucenin-2 isomer), present in all genotypes in low concentration (mean value 0.33 μg/g, excluding the extremes of the interval, Tumminia SG3 and Manto di Maria) was about 50-times more abundant in Tumminia SG3 (18.15 μg/g) than in other genotypes.

Free ferulic acid content resulted almost 3-times higher in Tumminia SG3 (5.81 μg/g) with

Total phenolics concentration ranged from 65.65 μg/g of grain in Russello SG8 to 104.84 μg/g of grain in Scavuzza, and a mean value of 82.78 μg/g was recorded. Three landraces

In general, genotype has been demonstrated to affect the phenolic content of wheat grains. Previous investigations reported on highly significant differences of polyphenol content among different wheat cultivars, suggesting the genotype-specificity of this characteristic [9, 14]. Moreover, the comparison of wheat cultivars grown at different locations showed that environmental and growing conditions may have a certain effect on the biosynthesis and

With regard to the bound phenolic fraction subjected to alkaline hydrolysis, the main component is undoubtedly ferulic acid, as already observed by other authors [43], and confirmed by coinjection with the corresponding analytical standard; this metabolite is present ubiquitously in all the genotypes considered with a mean value of 543.20 μg/g. The landrace Ruscia showed the highest level of ferulic acid content (673.58 μg/g), while Scavuzza the lowest (375.13 μg/g)

In order to discriminate among the studied wheat landraces, a statistical classification system was implemented using the data from the 15 analyzed chemical variables and the 138 measured morpho-colorimetric parameters. An overall percentage of correct identification of 100.0% was achieved, proving the peculiarity of the nine studied Sicilian wheat landraces and, on the other

Finally, in the evaluation of the parameters that more than other influenced the discrimination process of the studied landraces, none of the assessed variables chosen by the stepwise LDA highlighted particular statistical weight, proving that a high amount of quantitative information is necessary to distinguish and characterize botanical entities so heterogeneous, under

This work represent the first attempt of wheat landraces identification based on glume phenotypic characters, applying image analysis techniques, coupled with phenolic fingerprinting.

hand, the absolute effectiveness of the proposed method (Table 6).

chemical, phenotypical and genetic profile, such as landraces.

(Tumminia SG3, Tripolino, Scavuzza) showed a content higher than the average.

SG3 and about 6 μg/g for Trentino.

respect to the mean value (1.84 μg/g).

accumulation of phenolic compounds [42].

3.3. Landraces statistical comparison

(Table 5).

Table 4. Phenolic compounds detected in the free form extracts from durum wheat grains.

absent as well. These data are usually diagnostic of the presence of C-bound glycosides; the peak was then tentatively identified as luteolin di-C-hexoside (lucenin-2 isomer). This is in discordance to what was reported by Dinelli et al. [14, 40] who found in durum wheat grains several isomers of lucenin 1/3, the C-hexoside-C-pentoside derivative of luteolin. Peaks 7, 8, 9, 11, 12 and 13 showed UV–VIS spectra whose shapes and absorption maxima clearly recalled apigenin (Table 4); in this case mass analysis was determinant in the assignments. Peaks 7, 8 and 9 all exhibited a mass spectrum with a base peak of 563.14 m/z units, corresponding to the pseudomolecular ion of an hexoside- pentoside derivative; absence of intermediate fragments lead us to assign the peaks as C-hexoside C-pentoside derivatives of apigenin (Table 4). Similarly, peak 13 was tentatively identified as apigenin C-hexoside, whilst peaks 11 and 12, both showing a base peak of 769.18 m/z units, were tentatively identified as apigenin C-hexoside C-hexoside O-glucuronide.

#### 3.2. Phenolic content in wheat landraces

The determination of free phenolics in whole grains extracted by a hydroalcoholic solution (see experimental) was carried out through calibration curves obtained via HPLC/DAD triplicate injection of standard solutions. In Table 5 the concentration of 13 phenolic markers and total free phenolics for the all investigated wheat genotypes is given.

Some of the phenolic markers identified in the free form, were quantitatively quite different among the genotypes studied. For example, coumarin (peak 6), ranging from 2.57 μg/g in Tumminia SG3 to 0.09 μg/g in Tripolino, and vanillic acid (peak 3) from 1.34 μg/g in Manto di Maria to 0.25 μg/g in Tumminia SG3. The apigenin C-hexoside-C-pentoside (peak 7) content was significantly different among wheat grains, recording values above 21 μg/g for Tumminia SG3 and about 6 μg/g for Trentino.

Luteolin di-C-hexoside (lucenin-2 isomer), present in all genotypes in low concentration (mean value 0.33 μg/g, excluding the extremes of the interval, Tumminia SG3 and Manto di Maria) was about 50-times more abundant in Tumminia SG3 (18.15 μg/g) than in other genotypes.

Free ferulic acid content resulted almost 3-times higher in Tumminia SG3 (5.81 μg/g) with respect to the mean value (1.84 μg/g).

Total phenolics concentration ranged from 65.65 μg/g of grain in Russello SG8 to 104.84 μg/g of grain in Scavuzza, and a mean value of 82.78 μg/g was recorded. Three landraces (Tumminia SG3, Tripolino, Scavuzza) showed a content higher than the average.

In general, genotype has been demonstrated to affect the phenolic content of wheat grains. Previous investigations reported on highly significant differences of polyphenol content among different wheat cultivars, suggesting the genotype-specificity of this characteristic [9, 14]. Moreover, the comparison of wheat cultivars grown at different locations showed that environmental and growing conditions may have a certain effect on the biosynthesis and accumulation of phenolic compounds [42].

With regard to the bound phenolic fraction subjected to alkaline hydrolysis, the main component is undoubtedly ferulic acid, as already observed by other authors [43], and confirmed by coinjection with the corresponding analytical standard; this metabolite is present ubiquitously in all the genotypes considered with a mean value of 543.20 μg/g. The landrace Ruscia showed the highest level of ferulic acid content (673.58 μg/g), while Scavuzza the lowest (375.13 μg/g) (Table 5).

#### 3.3. Landraces statistical comparison

absent as well. These data are usually diagnostic of the presence of C-bound glycosides; the peak was then tentatively identified as luteolin di-C-hexoside (lucenin-2 isomer). This is in discordance to what was reported by Dinelli et al. [14, 40] who found in durum wheat grains several isomers of lucenin 1/3, the C-hexoside-C-pentoside derivative of luteolin. Peaks 7, 8, 9, 11, 12 and 13 showed UV–VIS spectra whose shapes and absorption maxima clearly recalled apigenin (Table 4); in this case mass analysis was determinant in the assignments. Peaks 7, 8 and 9 all exhibited a mass spectrum with a base peak of 563.14 m/z units, corresponding to the pseudomolecular ion of an hexoside- pentoside derivative; absence of intermediate fragments lead us to assign the peaks as C-hexoside C-pentoside derivatives of apigenin (Table 4). Similarly, peak 13 was tentatively identified as apigenin C-hexoside, whilst peaks 11 and 12, both showing a base peak of 769.18 m/z

13 28.99 270, 334 [M-H] 431.10 Apigenin C-hexoside Flavone-C-glycoside

The determination of free phenolics in whole grains extracted by a hydroalcoholic solution (see experimental) was carried out through calibration curves obtained via HPLC/DAD triplicate injection of standard solutions. In Table 5 the concentration of 13 phenolic markers and total

units, were tentatively identified as apigenin C-hexoside C-hexoside O-glucuronide.

free phenolics for the all investigated wheat genotypes is given.

3.2. Phenolic content in wheat landraces

Peak #

Retention time (min)

(sh) is for shoulder.

λ ass. (nm) Selected ion

32 Rediscovery of Landraces as a Resource for the Future

m/z calculated

5 16.57 268, 348 [M-H] 609.52 Luteolin di-C-hexoside (lucenin-2

11 25.18 272, 332 [M-H] 769.18 Apigenin C-hexoside-C-hexoside

12 26.00 272, 332 [M-H] 769.18 Apigenin C-hexoside-C-hexoside

Table 4. Phenolic compounds detected in the free form extracts from durum wheat grains.

2 14.24 317 [M-H] 145.14 Coumarin Coumarin

6 17.10 316 n.d Coumarin Coumarin

1 13.66 295(sh), 316 n.d Hydroxycinnamic acid Hydroxycinnamic acid

3 14.57 258, 291 [M-H] 167.04 Vanillic acid Hydroybenzoic acid 4 14.98 290 (sh), 323 [M-H] 179.16 Caffeic acid Hydroxycinnamic acid

7 17.99 270, 334 [M-H] 563.14 Apigenin C-hexoside-C-pentoside Flavone-C-glycoside 8 18.95 270, 335 [M-H] 563.14 Apigenin C-hexoside-C-pentoside Flavone-C-glycoside 9 19.52 271, 335 [M-H] 563.14 Apigenin C-hexoside-C-pentoside Flavone-C-glycoside 10 22.54 295 (sh), 323 [M-H] 193.05 Ferulic acid Hydroxycinnamic acid

isomer)

O-glucuronide

O-glucuronide

Tentative identification Phenolic subclass

Flavone-C-glycoside

Flavone-C-glycoside

Flavone-C-glycoside

In order to discriminate among the studied wheat landraces, a statistical classification system was implemented using the data from the 15 analyzed chemical variables and the 138 measured morpho-colorimetric parameters. An overall percentage of correct identification of 100.0% was achieved, proving the peculiarity of the nine studied Sicilian wheat landraces and, on the other hand, the absolute effectiveness of the proposed method (Table 6).

Finally, in the evaluation of the parameters that more than other influenced the discrimination process of the studied landraces, none of the assessed variables chosen by the stepwise LDA highlighted particular statistical weight, proving that a high amount of quantitative information is necessary to distinguish and characterize botanical entities so heterogeneous, under chemical, phenotypical and genetic profile, such as landraces.

This work represent the first attempt of wheat landraces identification based on glume phenotypic characters, applying image analysis techniques, coupled with phenolic fingerprinting.


The achieved results here discussed allowed to demonstrate the usefulness of this discrimination system for the identification and classification wheat landraces, notoriously very difficult to do. The technique here proposed, conveniently sustained by a conspicuous database, can be undoubtedly considered a helpful identification tool both for commercial varieties and for no

Overall 100.0%

Ruscia Russello SG8

Phenolic Fingerprinting and Glumes Image Analysis as an Effective Approach for Durum Wheat Landraces…

(192)

Urrìa — — —— — — — 100.0%

(192)

Tumminia SG3 — — —— — — 100.0%

Percentages refer to the classification performance; in parentheses, the number of analyzed glumes.

Trentino Tripolino Tumminia

—— — — — — 100.0%

(192)

— —— — — 100.0%

— —— — — — — 100.0%

(282)

SG3

http://dx.doi.org/10.5772/intechopen.79595

—— — — 100.0%

(192)

—— — 100.0%

— — 100.0%

(192)

Urrìa Total

(192)

35

(192)

(192)

(192)

(282)

(192)

100.0% (192)

(1626)

— 100.0% (192)

Considering the heterogeneous nature of the wheat landrace samples used in this study, in order to validate these preliminary achievements, further trials will have to be conducted focusing on the collection of new data, enriching the database with new and accurate informa-

, Sebastiano Blangiforti

2 Italian National Research Council - Institute of Biomolecular Chemistry ICB, Catania, Italy

2

1 Stazione Sperimentale di Granicoltura per la Sicilia, Caltagirone (CT), Italy

1

, Laura Siracusa

2 ,

genetically defined samples, such as populations or landraces.

Table 6. Percentages identification among the studied landraces.

tion, allowing to the system to give results more and more reliable.

1

<sup>1</sup> and Giuseppe Ruberto

\*Address all correspondence to: oscar.grillo.mail@gmail.com

Author details

Gianfranco Venora

1

\*, Marisol Lo Bianco

Margherito Manto di Maria

— 100.0%

Ruscia — — 100.0%

(192)

Trentino — — —— 100.0%

Tripolino — — —— — 100.0%

Russello SG8 —— — 100.0%

Margherito 100.0%

Manto di Maria

(192)

Oscar Grillo

Phenolic Fingerprinting and Glumes Image Analysis as an Effective Approach for Durum Wheat Landraces… http://dx.doi.org/10.5772/intechopen.79595 35


Percentages refer to the classification performance; in parentheses, the number of analyzed glumes.

Table 6. Percentages identification among the studied landraces.

The achieved results here discussed allowed to demonstrate the usefulness of this discrimination system for the identification and classification wheat landraces, notoriously very difficult to do. The technique here proposed, conveniently sustained by a conspicuous database, can be undoubtedly considered a helpful identification tool both for commercial varieties and for no genetically defined samples, such as populations or landraces.

Considering the heterogeneous nature of the wheat landrace samples used in this study, in order to validate these preliminary achievements, further trials will have to be conducted focusing on the collection of new data, enriching the database with new and accurate information, allowing to the system to give results more and more reliable.
