**3. Results and discussion**

### **3.1. Phenolic profiles**

**Compound name/acronym Peak no.** *Rt* **(min) UV***λ max***(nm) Phenolic class**

Kaempferol or quercetin derivative 1 21 140 262, 346,

316 Organic Fertilizers - From Basic Concepts to Applied Outcomes

Kaempferol or quercetin derivative 3 23 146.3 262, 346,

ANTHO 1 24\* 147.1 278, 518,

FLAVONOL 2 31 160.3 262, 342,

**Table 3.** Phenolic compounds in the radicchio leaves separated by HPLC.

Not detected in var. 'Anivip', 'Castelfranco', and 'Monivip'.

**2.3. Determination of fatty acid levels**

Kaempferol or quercetin derivative 2 22 141.7 262, 346 Flavonol

Apigenin or luteolin derivative 26 149.5 262, 338 Flavone

FLAVONOL 1 30 159.5 262, 346 Flavonol

FLAVONOL 3 33 164.1 262, 266, 342, 346 Flavonol

HCA 5 13 84.9 326 Monomeric hydroxycinnamic acid Cichoric acid (dicaffeoyl tartaric acid) 14 100.5 330 Oligomeric hydroxycinnamic acid HCA 6 15 104.2 330 Oligomeric hydroxycinnamic acid HCA 7 16 112.1 330 Oligomeric hydroxycinnamic acid HCA 8 17 114.4 322 Oligomeric hydroxycinnamic acid UPC 3 18 115.1 262, 266 Unknown phenolic compound Gallic acid derivative 3 19 126.5 262 Unknown phenolic compound HCA 9 20 131.5 326 Oligomeric hydroxycinnamic acid

350

350, 354

522

346

HCA 10 25 149 318, 326 Oligomeric hydroxycinnamic acid

UPC 4 27 149.6 262, 266 Unknown phenolic compound UPC 5 28 155.2 262 Unknown phenolic compound UPC 6 29 159 262, 266 Unknown phenolic compound

Gallic acid derivative 4 32 161 262 Unknown phenolic compound

Fatty acid levels were analyzed using GC with prior prepared fatty acid methyl esters. In the protocol [41], NaOH and BF3 in methanol were used for transesterification and heptadecanoic acid (C17:0) was used as an internal standard for the quantification of fatty acids. The solution of fatty acid methyl esters was quantified on the GC (Agilent 6890N, USA) with flame ionization detector (FID). At the constant flow rate, the separation was performed on a column for analyses of PUFAs as fatty acid methyl esters. The identification and quantification of fatty

Flavonol

Flavonol

Flavonol

Anthocyanin

Thirty-three main phenolic compounds obtained using HPLC detection were selected in all five studied radicchio varieties from six fertilizer managements. Those were grouped accord‐ ing to their absorbance spectra and retention times to UPCs, HCAs, flavonols, flavones, and anthocyanins (**Table 4**). All chromatograms of radicchio samples were similar, but the areas of individual peaks varied considerably. An example of chromatogram for var. 'Castelfranco' is presented in **Figure 4**. Anthocyanins, which are quite unstable, were found in minor quantities in only few radicchio samples.

**Figure 4.** Chromatogram of var. 'Castelfranco' from unfertilized management obtained using HPLC analysis.

The phenolic profile data were comparable to former reports, which also found that chloro‐ genic and cichoric acids are the main phenolic compounds in radicchio leaves [4,6,26,42]. The total phenolic amount (TPA) in the analyzed radicchio leaves under different fertilizer managements varied from 58 to 403 mg/100 fresh weight (FW; **Figure 5**). The results showed large differences between the varieties as well when comparing different fertilizer manage‐ ments. The average levels over all different fertilizer managements for individual variety showed significantly greater TPA for var. 'Treviso' (300 mg/100 g FW) followed by var. 'Verona' (181 mg/100 g FW), var. 'Monivip' (146 mg/100 g FW), var. 'Anivip' (135 mg/100 g FW), and var. 'Castelfranco' (125 mg/100 g FW). The red colored var. 'Treviso' showed two times greater TPA in comparison to red-spotted or green radicchio varieties. A high TPA for var. 'Treviso' was reported by D'evoli et al. [34].

**Figure 5.** TPA (as mg/100 g FW) in the radicchio leaves among varieties and different fertilizer managements.

Across different managements, the highest TPA was seen for unfertilized (CONT) treatment (254 mg/100 g FW) followed by MIN1 (213 mg/100 g FW), combination of ORG1+MIN1 (183 mg/100 g FW), ORG2 (160 mg/100 g FW), ORG1 (129 mg/100 g FW), and MIN2 (126 mg/100 g FW). Significantly greater TPAs were seen for the radicchio varieties grown under unfertilized management and those with mineral fertilizer (MIN1). Crecente-Campo et al. [43] have reported that the organic or conventional cultivation system did not affect the TPA but only the antioxidant compounds. Vinha et al. [10] found greater TPA for organically grown vegetables, whereas Mitchell et al. [8] obtained only greater amounts for quercetin and kaempferol. According to Oliveira et al. [9], organic manner resulted in greater TPA and vitamin C. Some other studies [44,45] reported that the enzyme phenylalanine ammonia-lyase is involved in the biosynthesis of phenolics and is regulated by nitrogen. In general, the availability of soil nitrogen strongly impacts the synthesis of several phenolic compounds [46]. In relation to nitrogen fertilization, the response of radicchio varieties differs, as high and low nitrogen demanding varieties were previously reported [47].

FW), and var. 'Castelfranco' (125 mg/100 g FW). The red colored var. 'Treviso' showed two times greater TPA in comparison to red-spotted or green radicchio varieties. A high TPA for

**Figure 5.** TPA (as mg/100 g FW) in the radicchio leaves among varieties and different fertilizer managements.

Across different managements, the highest TPA was seen for unfertilized (CONT) treatment (254 mg/100 g FW) followed by MIN1 (213 mg/100 g FW), combination of ORG1+MIN1 (183 mg/100 g FW), ORG2 (160 mg/100 g FW), ORG1 (129 mg/100 g FW), and MIN2 (126 mg/100 g FW). Significantly greater TPAs were seen for the radicchio varieties grown under unfertilized management and those with mineral fertilizer (MIN1). Crecente-Campo et al. [43] have reported that the organic or conventional cultivation system did not affect the TPA but only the antioxidant compounds. Vinha et al. [10] found greater TPA for organically grown vegetables, whereas Mitchell et al. [8] obtained only greater amounts for quercetin and kaempferol. According to Oliveira et al. [9], organic manner resulted in greater TPA and vitamin C. Some other studies [44,45] reported that the enzyme phenylalanine ammonia-lyase is involved in the biosynthesis of phenolics and is regulated by nitrogen. In general, the

var. 'Treviso' was reported by D'evoli et al. [34].

318 Organic Fertilizers - From Basic Concepts to Applied Outcomes


The average values (*n*=3) with different lowercase letters in a row are significantly different (*P*<0.001; differences between the fertilizers), and those with different uppercase letters in a column are significantly different (*P*<0.001; differences between the varieties).

**Table 4.** Phenolic classes in the leaves of radicchio varieties derived from different fertilizer managements.

The main classes of phenolic compounds (as mg/100 g FW) among radicchio varieties from different fertilizer managements are presented in **Table 4**. Statistical analysis showed signifi‐ cant differences between both fertilizer managements and the varieties for all of these main classes. HCAs were the greatest represented group of phenolic compounds in radicchios with a range of 60% to 95% followed by unknown phenolics, flavonols, and flavones (**Figure 6**).

Phenolic acids (specifically HCAs) were further on grouped according to their retention times as monomeric (<100 min) and oligomeric (>100 min). HCAs are mostly represented by chlorogenic and cichoric acid in all radicchio samples (**Table 5**). The levels of HCAs varied in a wide range from 47 to 362 mg/100 g FW (**Table 4**). The higher levels of total HCAs were found in var. 'Treviso,' up to two times more than the mean value, whereas var. 'Castelfranco' had the lowest amounts of HCAs. The analysis showed that radicchios contribute a smaller amount of monomeric (27%) in comparison to oligomeric HCAs (56%). Data showed that, across radicchio varieties, var. 'Treviso' had greater total HCA amount compared to other varieties (**Table 4**).

**Figure 6.** Relative phenolic class contents in leaves of radicchio varieties from different fertilizer managements.

The main identified monomeric HCAs were caftaric and chlorogenic acids, whereas the most represented oligomeric was cichoric acid. Cichoric acid was best represented and accounted for 43% of total HCAs, whereas chlorogenic acid with 28% and caftaric acid with 3% were present in lesser quantities (**Table 5**). All three phenolic acids together represent up to 74% of the total HCAs in radicchio samples (**Figure 6**). The HCA quantities were as follow: cichoric acid (16–190 mg/100 g FW), chlorogenic acid (14–89 mg/100 g FW), and caftaric acid (1–14 mg/ 100 g FW). Those levels are in accordance with earlier reports, revealing that the caftaric, chlorogenic, and cichoric acids are the most abundant HCAs in radicchio varieties [4,6,22–26].


The average values (*n*=3) with different lowercase letters in a row are significantly different (*P*<0.001; differences between the fertilizers), and those with different uppercase letters in a column are significantly different (*P*<0.001; differences between the varieties).

**Table 5.** HCA levels in the radicchio leaves.

The main classes of phenolic compounds (as mg/100 g FW) among radicchio varieties from different fertilizer managements are presented in **Table 4**. Statistical analysis showed signifi‐ cant differences between both fertilizer managements and the varieties for all of these main classes. HCAs were the greatest represented group of phenolic compounds in radicchios with a range of 60% to 95% followed by unknown phenolics, flavonols, and flavones (**Figure 6**). Phenolic acids (specifically HCAs) were further on grouped according to their retention times as monomeric (<100 min) and oligomeric (>100 min). HCAs are mostly represented by chlorogenic and cichoric acid in all radicchio samples (**Table 5**). The levels of HCAs varied in a wide range from 47 to 362 mg/100 g FW (**Table 4**). The higher levels of total HCAs were found in var. 'Treviso,' up to two times more than the mean value, whereas var. 'Castelfranco' had the lowest amounts of HCAs. The analysis showed that radicchios contribute a smaller amount of monomeric (27%) in comparison to oligomeric HCAs (56%). Data showed that, across radicchio varieties, var. 'Treviso' had greater total HCA amount compared to other

**Figure 6.** Relative phenolic class contents in leaves of radicchio varieties from different fertilizer managements.

The main identified monomeric HCAs were caftaric and chlorogenic acids, whereas the most represented oligomeric was cichoric acid. Cichoric acid was best represented and accounted for 43% of total HCAs, whereas chlorogenic acid with 28% and caftaric acid with 3% were present in lesser quantities (**Table 5**). All three phenolic acids together represent up to 74% of the total HCAs in radicchio samples (**Figure 6**). The HCA quantities were as follow: cichoric acid (16–190 mg/100 g FW), chlorogenic acid (14–89 mg/100 g FW), and caftaric acid (1–14 mg/

varieties (**Table 4**).

320 Organic Fertilizers - From Basic Concepts to Applied Outcomes

Both flavonols and flavones are chemosystematic markers found in tribe Cichorieae of the Asteraceae family [27]. Total flavonol amounts of studied radicchio varieties were found in the range of 1.7 to 20 mg/100 g FW (**Table 4**). The flavonols represented below 10% of TPA for most of the radicchio samples, except for var. 'Verona' ORG1 (13%) and var. 'Monivip' MIN1 (14%). Flavones represented only small concentrations ranging up to 2 mg/100 g FW (**Table 4**). Arabbi et al. [48] found similar amounts of flavonoids ranging from 18 to 38 mg/100 g FW.

**Figure 7.** LDA plot for 30 radicchio samples according to six fertilizer managements. For legend, see **Table 3**.

Multivariate data analysis by principal component analysis (PCA) and linear discriminant analysis (LDA) was used for plotting the radicchio samples based on their phenolic com‐ pounds. All 60 peaks were included in the analysis. Using PCA, 21 phenolic compounds were selected as the most discriminating variables: 10 HCAs, 3 UPCs, 3 gallic acid derivatives, 3 flavonols, 2 flavones, and a protocatechuic acid (**Table 3**). The LDA scores of the data (30 samples, 21 variables) for first two functions are plotted in **Figure 7**. It should be emphasized that ORG1 and MIN2 fertilizer managements are characterized by slow nitrogen release [49].

#### **3.2. Composition of fatty acids**

The levels of the individual and total fatty acids (mg/100 g FW) of radicchio leaf samples are shown in **Table 6**. Data show significant differences for different varieties and fertilizer managements. Using GC analysis, the following fatty acids were identified and quantified: saturated fatty acids (SFAs) C16:0, C18:0, and C20:0; monounsaturated fatty acid (MUFA); and PUFAs C18:1n9, C18:2n6, and C18:3n3. Linolenic acid (C18:3n3) was represented the most and accounted for 48% to 63% of total fatty acids amount, whereas linoleic acid (C18:2n6) accounted for 16% to 30% and palmitic acid (C16:0) for 14% to 24% (**Table 6**). Stearic (C18:0) and oleic (C18:1n9) fatty acids were less abundant (<5%), and the smallest levels were found for arachidonic acid (C20:0; i.e., <1%). The total fatty acid levels ranged from 173 to 503 mg/100 g FW (**Table 6**). In comparison to other varieties, var. 'Castelfranco' showed greater levels of total fatty acid levels. Between fertilizer managements, there were significantly better total fatty acid levels when the organic fertilizers were used (ORG1 and ORG2). Obtained data are well in accordance to those for forage radicchios [50]. Blanckaert et al. [51] reported almost similar amounts for fatty acid levels of the *Cichorium* '474.'

Impact of Organic Fertilizers on Phenolic Profiles and Fatty Acids Composition: A Case Study for *Cichorium intybus* L. http://dx.doi.org/10.5772/62325 323


C18:3n3; unsaturated

**Figure 7.** LDA plot for 30 radicchio samples according to six fertilizer managements. For legend, see **Table 3**.

**3.2. Composition of fatty acids**

322 Organic Fertilizers - From Basic Concepts to Applied Outcomes

similar amounts for fatty acid levels of the *Cichorium* '474.'

Multivariate data analysis by principal component analysis (PCA) and linear discriminant analysis (LDA) was used for plotting the radicchio samples based on their phenolic com‐ pounds. All 60 peaks were included in the analysis. Using PCA, 21 phenolic compounds were selected as the most discriminating variables: 10 HCAs, 3 UPCs, 3 gallic acid derivatives, 3 flavonols, 2 flavones, and a protocatechuic acid (**Table 3**). The LDA scores of the data (30 samples, 21 variables) for first two functions are plotted in **Figure 7**. It should be emphasized that ORG1 and MIN2 fertilizer managements are characterized by slow nitrogen release [49].

The levels of the individual and total fatty acids (mg/100 g FW) of radicchio leaf samples are shown in **Table 6**. Data show significant differences for different varieties and fertilizer managements. Using GC analysis, the following fatty acids were identified and quantified: saturated fatty acids (SFAs) C16:0, C18:0, and C20:0; monounsaturated fatty acid (MUFA); and PUFAs C18:1n9, C18:2n6, and C18:3n3. Linolenic acid (C18:3n3) was represented the most and accounted for 48% to 63% of total fatty acids amount, whereas linoleic acid (C18:2n6) accounted for 16% to 30% and palmitic acid (C16:0) for 14% to 24% (**Table 6**). Stearic (C18:0) and oleic (C18:1n9) fatty acids were less abundant (<5%), and the smallest levels were found for arachidonic acid (C20:0; i.e., <1%). The total fatty acid levels ranged from 173 to 503 mg/100 g FW (**Table 6**). In comparison to other varieties, var. 'Castelfranco' showed greater levels of total fatty acid levels. Between fertilizer managements, there were significantly better total fatty acid levels when the organic fertilizers were used (ORG1 and ORG2). Obtained data are well in accordance to those for forage radicchios [50]. Blanckaert et al. [51] reported almost


Sign.: levels of significance, \*\*\**P*≤0.001; \*\**P*≤0.01; \**P*≤0.05; Ns, not significant.

The average values (*n*=3) with different lowercase letters in a row are significantly different (*P*<0.001; differences between the fertilizers), and those with different uppercase letters in a column are significantly different (*P*<0.001; differences between the varieties).

**Table 6.** Fatty acid levels (mg/100 g FW) of radicchio varieties produced with different fertilizer managements.

The nutritional information of radicchio varieties for most optimal fertilizer management (ORG2), which signified the uppermost total fatty acid levels, is presented in **Table 7**. PUFAs represent the range from 79% to 81% of total fatty acid levels, SFAs the range from 16% to 19%, and MUFAs the range <3.6%. The ratio of *n*-6/*n*-3 fatty acids was below 0.48 for all radicchio varieties. Simopoulos [52] reported that past human diets had a ratio of *n*-6/*n*-3 fatty acids near 1, whereas modern Western diets have that ratio much higher (up to 20). The optimal ratio of *n*-6/*n*-3 fatty acids is believed to be from 1 to 4 [33,52]. Schreck et al. [53] found a higher ratio of *n*-6/*n*-3 fatty acids for the lettuce seedlings, whereas some prior readings on wild *Cichori‐ um* leaves showed much lower values [54,55]. All analyzed radicchio varieties had the ratio at values are considered as optimal and fully in agreement with current nutritional recommen‐ dations [56].


**Table 7.** Nutritional information of different radicchio varieties derived from organic fertilizer (ORG2) management.
