Introduction

The global area devoted to organic agriculture reached 71.5 million hectares at the end of 2018. Regions most interested in this growing phenomenon are Oceania with 36 million hectares, followed by the European Union which stands at 16.6 million hectares for 2018. The European organic market has grown by 7.8%, with a spending per capita of € 50.5 (IFOAM & FiBL-Organics International 2020). According to the FiBL, organic retail sale has reached another peak in 2018, and globally 1.5% of the world’s agriculture is organic. Two-thirds of the lands devoted to organic agriculture are grassland areas while 13.3 million hectares are arable land, which amounts to an increase of 4.9% compared to 2017. Of these, 13.3 million hectares most are used for cereals, green fodder, oil seed, and 726,845 ha for dry pulse.

Among legumes, Cicer arietinum L., is a valuable source of nitrogen for the soil; it fixes nitrogen from the atmosphere, due to a beneficial interaction with the soil bacteria of the family Rhizobiaceae that induce the formation of nitrogen-fixing radical nodules (Aslam et al. 2000). Chickpea seeds vary in size, shape, and color, and based on this variation, they are classified into two types: “kabuli” and “desi.” The kabuli type has thin seed coat that varies in color from white to cream, with high seeds weight. The desi type has thicker skin and an irregularly shaped seed coat that ranges in color from cream, to black and green.

They are rich in proteins, complex carbohydrates, and vitamins; chickpeas consumption can potentially prevent chronic diseases (Englyst et al. 2003). This potential is related to the presence of phytochemicals, i.e., polyphenols, a heterogeneous group of substances which naturally occur in many fruits, grains, and vegetables. They are derivatives of benzene with one or more hydroxyl groups associated with the benzene ring. This structure allows them to function actively as a scavenger for stabilizing free radicals, as reducing agents, and as a quencher of singlet oxygen formation (Dragsted 2003). The chickpea seed is characterized by both hydrophilic polyphenols (including phenolic acids, flavonols, flavones, isoflavones, anthocyanins, and condensed tannins), as well as lipophilic compounds (tocopherols and carotenoids) (Boschin and Arnoldi 2011; Magalhães et al. 2017). These polyphenols have various biological activities, such as apoptosis, anti-aging, anticancer, anti-inflammation, anti-atherosclerosis, and cardiovascular protection (Amarowicz and Pegg 2008; Bazzano et al. 2008; Campos-Vega et al. 2009).

Despite all these qualities, the chickpea whole seed market has a low commercial value in Europe. Yet, the interest in the cultivation has increased considerably due to the agronomic benefits, the guidelines of the Common Agriculture Policy 2014–2020 (CAP).

Brandt and Mølgaard (2001) estimated that on average, organic vegetables may contain from 10 to 50% higher defense-related secondary metabolites than conventionally cultivated vegetables. Del Amor et al. (2008) observed that sweet peppers grown under organic farming have high levels of phenolic compounds, and peroxidase and capsidiol activity that contribute to disease resistance in organic farming. Balisteiro et al. (2013), in a study on soybean cultivars, showed that those organically cultivated had higher protein content in comparison to those conventionally cultivated, but with lower levels of isoflavones and phenolic compounds. In a recent study on ancient wheat species, Fares et al. (2019) found that the total phenolic content was higher in organic farming, but TEAC and phenolic acids did not differ in the two farming systems.

The aim of this work was to ascertain the effect of organic farming on the bioactive compounds and antioxidant activity of desi and kabuli type chickpeas. We therefore analyzed the nutritional composition, bioactive compounds, and antioxidant activity of a set of chickpea cultivars grown in the same geographic region of Europe (Foggia, South Italy) in order to ascertain which types of chickpea showed the best performance in organic farming and for which characters this occurred.

Material and methods

Field materials

Ten chickpea genotypes were grown during the 2013–2014 planting season in two experimental fields belonging to the Italian Council for Agricultural Research and Economics, Research Centre for Cereal and Industrial Crops of Foggia (southern Italy). The kabuli type consisted of nine varieties (Pascià, Vulcano, Reale, Sultano, Principe, Cairo, Califfo, Calia, and a line named R1). The desi type consisted of a local genotype named Nero lucano.

A complete randomized block design was used, with three replicates, for organic and conventional farming, and in Table 1 are given information about the soil characteristics. For the conventional cultivation only, the plots received an N plus phosphorous fertilizer: 80 kg ha−1 P2O5 at pre-seeding and 30 kg ha−1 N applied at seedbed preparation. Further details of agronomic management of conventional farming are described in De Santis et al. (2021). The soil preparation of the organic and conventional fields consisted of ploughing, hoeing, and harrowing twice before sowing. The plots consisted of three rows of 7.5 m separated of 50 cm each from other, which were sown with 50 seeds m−2 at the beginning of December 2013, under wet conditions. After the harvest, in July 2014, the samples were stored at 4 °C until analysis. Samples (50 g) were ground in a sample mill (Tecator, 1093 Cyclotec; Foss Italia, Padova, Italy) using 1.0-mm sieves, for analysis.

Table 1 Physical and chemical characteristics of organic and conventional soil
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Proximate analysis

Determination of the insoluble dietary fiber (IDF) and the soluble dietary fiber (SDF) was based on the method as proposed by Prosky et al. (1992) using a commercial assay kit (Megazyme International, Bray, Ireland). Protein content was determined using the Dumas combustion nitrogen method, according to the American Association of Cereal Chemists (AACC 2012), Approved Method 46–30.01, and using FP528 (Leco Corp., Saint Joseph, USA). A factor of 5.7 was used to convert the nitrogen content into protein. All the analyses were conducted in triplicate. The seed weight (SW) was determined on 100 seeds (g) three times and expressed as the mean value.

Extraction of phenolic compounds

Phenolic compounds were extracted according to Beta et al. (2005), with minor modifications. The samples (1 g) ground with a 1.0-mm sieve were extracted using 8 mL methanol acidified with 1 N HCl (80:20; v/v) for 30 min in an ultrasonic bath. The mixtures were centrifuged at 2000 g for 15 min. The pellets were extracted with additional 4 mL of acidified methanol and the combined surnatants were used for the determination of the total phenolic content (TPC) and total flavonoid content (TFC).

Determination of total phenolic content

Extracts (0.2 mL) were added to 1.5 mL tenfold diluted Folin–Ciocalteau reagent (Singleton and Rossi 1965). The mixture was allowed to equilibrate for 5 min, and then neutralized with sodium carbonate (from a stock solution of 60 g L−1). After incubation at room temperature for 90 min, the absorbance of the mixtures was measured at 725 nm by a UV/VIS spectrometer (Lambda Bio 20, Perkin Elmer, Germany). Gallic acid was used as the standard, and the data were expressed in µg gallic acid equivalents g−1 (GAE g−1). Determinations were performed in triplicate for each extract and expressed according to the dry matter.

Determination of total flavonoid content

The total flavonoid content (TFC) was determined according to Eberhardt et al. (2000). Catechin was used as the standard, and the data were expressed as µg catechin equivalents g−1(CE g−1). Determinations were performed in triplicate for each extract and expressed according to the dry matter.

Total carotenoid content

The pigment content determination was in accordance with Beleggia et al. (2010). The total carotenoid content (TCC) of the extracts was calculated directly from the absorbance using the conversion factor (1.6632) and was expressed as μg β carotene g−1 according to the dry matter. All of the assays were conducted in triplicate.

Antioxidant activity measured by ABTS assay

The antioxidant activity was determined according to Re et al. (1999), with some modifications: the ABTS was dissolved in water to 7 mM. ABTS radical cations (ABTS•+) were produced before use, by reacting the ABTS stock solution with potassium persulfate (final concentration, 2.45 mM) for 12–16 h in the dark and at room temperature. The ABTS•+ solution was diluted with ethanol to an absorbance of 0.80 ± 0.02 at 734 nm. Ten milligrams ± 1.0 mg was extracted with 10 mL of ABTS•+ solution stirring at room temperature and in the dark for 30 min. The antioxidant activity of the extracts was calculated using a Trolox standard curve, on the basis of the percentage inhibition of the absorbance at 734 nm. These were expressed in μmol Trolox equivalent (TE) g−1 on dry matter. All these tests were conducted in triplicate.

Extraction and determination of tocopherols

Tocopherols were extracted according to Tsochatzis et al. (2012) with some modifications. Each sample (500 mg) was extracted in 10 mL of acetonitrile for 30 min in a 25-mL conical glass flask under magnetic stirring. The sample was collected in a 50-mL centrifuge tube, and the flask was washed twice with 2.5 mL of acetonitrile to ensure complete transfer of the sample into the tube, which was then centrifuged at 3000 g for 15 min at 10 °C and evaporated to dryness. The sample was re-dissolved in 0.5 mL of methanol and vortexed for 30 s, filtered, and used for the analysis. The tocopherols were separated and quantified by HPLC (1100 series chromatography system; Agilent Technologies, Waldbron, Germany) equipped with fluorescence detection (G1321A). Tocopherol separation was carried out with a C18 column (Synergi-Hydro-RP C18; 250 mm × 4.6 mm × 5 µm; Phenomenex). The column temperature was regulated at 30 °C. The mobile phase consisted of acetonitrile/methanol/2-propanol (40:55:5v/v/v) under isocratic conditions at a flow rate of 0.8 mL/min with a total analysis time of 30 min. The injection volume was 20 µL. Tocopherols (α-, β-, γ-, and δ-) were detected by fluorescence with excitation, and emission wavelengths were set at 292 and 335 nm, respectively. Stock solutions were prepared in methanol to a concentration ranging from 2.5 to 25 µg/mL for each compound. All these tests were conducted in triplicate and the data were expressed as µg g−1 according to the dry matter.

Extraction and determination of free and bound phenolic acids

The free and bound phenolic acids were extracted as described previously (Fares and Menga 2012). The quantification of each of the phenolic acids was based on their peak area and those of the phenolic acid standards: p-hydroxybenzoic, p-coumaric, and ferulic acids. The data from the PA free and PA bound (i.e., alkaline plus acid hydrolysates) were summed to give total phenolic acids (TPAs) and were expressed as µg g−1 according to the dry matter.

Extraction and determination of individual flavonoids

The flavonoids were extracted and quantified as described by Zielińska et al. (2012) with some modifications. Each sample (500 mg) was extracted with 5 mL methanol acidified with 1.0 N HCl (85:15; v/v), in an ultrasonic bath for 3 min and subsequently centrifuged for 5 min (13,200 g at 4 °C). The extraction procedure was repeated twice. Supernatants were combined and collected in a 10-mL volumetric flask and the total volume made up to 10 mL. The flavonoids were separated and quantified by the HPLC system. A gradient elution was used, with a mobile phase of aqueous 4% formic acid (solvent A) and acetonitrile containing 4% of formic acid (solvent B) as follows: 9 min = 12% B; 22 min = 22% B; 40 min = 70% B, 45 min = 12% B; 50 min = 12% B. For quantitative analysis, calibration standards were prepared in duplicate within a range of 0.1–40 µM of each compound. The tests were conducted in triplicate and the flavonoid content was expressed as µg g−1 according to the dry matter.

Statistical analysis

Analysis of variance (ANOVA) was carried out with respect to each parameter detected in the chickpea cultivars. Means were separated using Tukey’s multiple test at the probability level of P ≤ 0.05. To obtain a general comprehensive characterization of the samples, the traits were subjected to principal component analysis (PCA) based on correlations. All the statistical analyses were performed using the JMP software, version 8.0 (SAS Institute Inc., Cary, NC, USA).

Results and discussion

Nutritional composition of chickpea cultivars as influenced by farming system

The environmental data (Table 2) of the growing season was collected from an on-site weather station and are compared with the long-term data (last 15 years at Foggia, since 1989). The long-term data showed mean rainfall for the months of the growing season (November 2013 to June 2014) of 357 mm, and mean temperature range from a minimum of 7.6 °C to a maximum of 18.3 °C. In comparison, the cultivation year had more rainfall (445.6 mm), with similar mean minimum and maximum temperatures, of 7.3 °C and 18.1 °C, respectively. The seed weight (SW), Protein content (PC), total polyphenol content (TPC), total carotenoid content (TCC), and antioxidant activity (AA) were significantly affected by the farming system (Table 3). Significant genotypic differences were also observed on all the nutritional compounds analyzed, while the GxF interaction (genotype and farming system) was significant for insoluble dietary fiber (IDF), soluble dietary fiber (SDF), and AA.

Table 2 Monthly rainfall and temperatures for the experimental field during the growing season (November 2013–June 2014)
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Table 3 Differences in nutritional composition of the two groups of chickpeas organically and conventionally cultivated
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Significantly higher values of SW, PC, TCC, and AA were observed in organic farming, with increased values of 7.8%, 9%, 12%, and 6.5% compared to conventional farming (Table 3). The positive effect on PC and TCC was in agreement with Mozafar (1993).The significant increase in protein content and increase in seeds weight in organic farming could be attributed to the excessive spring rainfall and the high temperatures in June (Table 2) which led to a reduction of flower fertilization and consequent reduction of seeds number, increased seed weigh and PC. The TPC was significantly higher in conventional farming but IDF, SDF, and TFC were not significantly different in both organic and conventional farming; this is in agreement with Balisteiro et al. (2013) in relationship to soybean. The significant reduction of TPC in organic farming (of about 26.8%) indicated that more nitrogen is available for crop in organic field. Therefore, the abiotic and biotic stress could be lowered and the biosynthesis of carbon-containing compounds (Shikimato pathway), secondary metabolites, were mitigated.

Significant differences among chickpea genotypes were observed in the nutritional composition, and the desi one showed the highest content for almost all parameters, except for SW, that was similar to those found by Wood et al. (2011). The average PC of kabuli (237.6 g kg−1) and desi (246.7 g kg−1) (Table 3) chickpeas measured in our study were higher than those of Wang et al. (2010), but the ranking between the two groups was the same. In line with Wang et al. (2010), we found that Nero lucano (Supplementary Table 1) had higher IDF and SDF contents than kabuli cultivars with larger seeds. The TPC and TFC of Nero lucano were about twice the mean values of the kabuli cultivars. The considerable differences found in this study for TPC and TFC between desi and kabuli chickpea cultivars, were also observed by Segev et al. (2010).

The antioxidant activity, measured by the ABTS assay, was significantly higher in organic farming of about 6.5% compared to conventional farming (Table 3). The result opposes the trend observed on TPAs and TFs but can be explained by that the measurement was made on methanolic extracts which intercept only the free fraction of antioxidants. Similar conclusion was exposed in a previous work by Fares et al. (2010), where was affirmed that the methanolic extraction was not able to extract the bound phenolic compounds linked to fiber. Moreover, to note that the linkage between polyphenols and antioxidant activity is never univocal as indicated by Faller and Fialho (2010). Thus, the amount and type of polyphenol can modulate the antioxidant capacity analysis.

The superiority of the desi cultivar (23.49 μmol TE g−1) over the other cultivars was established, as suggested by Segev et al. (2010) who had used the FRAP assay. However, the antioxidant activity measured in our samples was higher compared to Marathe et al. (2011), who indicated that white chickpea cultivars with large seeds had a low AA (below 6 μmol TE g−1) while chickpea cultivars with small brown seeds had moderate AA (6.0–12.0 μmol TE g−1). Furthermore, our results (Supplementary Table 1) indicated that chickpea varieties did not show significant differences in organic farming compared to conventional one; only Vulcano had significantly higher SW and IDF in organic and conventional farming, respectively, while Califfo had higher AA in organic than conventional. The excellent response in terms of seed composition of Nero L. was confirmed both in organic and conventional agriculture, with a slightly more positive trend towards organic, in line with what was reported by De Santis et al. (2021) in terms of high adaptability and good productivity in the Mediterranean environment.

Profile of tocopherols

Tocopherols are powerful antioxidants, which prevent the lipid autoxidation of cellular membrane and influence the cellular responses to oxidative stress by modulating the signal-transduction pathways (Bramley et al. 2000). Tocopherols occur in four forms (or vitamers/isomers), named alpha (α-T), beta (β-T), gamma (γ-T), and delta (δ-T), which differ in terms of the methylation pattern of the benzopyran ring (Boschin and Arnoldi 2011). The full tocopherol profile is shown in Table 4. The β-T and tocotrienols were not detected in our samples, in accordance with the study by Boschin and Arnoldi (2011). The total tocopherol content was unaffected by the farming system; however, significant differences were observed in the distribution of vitamers and the α-T was significantly higher in organic farming, while γ-T and δ-T were significantly higher in conventional farming. In organic farming, plants are exposed to various adverse environmental conditions (i.e., pathogen attacks, competition against weeds), which could be the cause of the observed increase not only in α-T, but also in TCC and AA. The isomer γ-T was the most abundant and has been shown to have the highest antioxidant activity in food (Bramley et al. 2000). Amarowicz and Pegg (2008) outlined that the γ-T also plays a role in protection against cardiovascular disease because it decreases platelet aggregation and delays arterial thrombogenesis (in a study conducted on rats). The least abundant was δ-T (mean value of 6.14 μg g−1 and 7.32 μg g−1 for organic and conventional farming respectively). Our results are similar to those of Boschin and Arnoldi (2011) but higher than those of Gül et al. (2008).

Table 4 Mean values of each tocopherol isomer and total tocopherol content (Ts) of the two groups of chickpeas organically and conventionally cultivated
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Regarding to the two groups of cultivars, the significant highest amounts of α-T γ-T and δ-T were found in the mean values of kabuli cultivars (13.37 μg g−1, 99.23 μg g−1 and 7.02 μg g−1, respectively). Only Sultano showed significantly highest content of α-T in organic farming in respect to conventional (Supplementary Table 1). On the best of our knowledge, this result represents a novelty because has indicated that the kabuli cultivars differed significantly from desi for the amounts of all tocopherol isomers especially for α-T, the main contributor to vitamin A activity.

Profile of phenolic acids and flavonoids

Tables 5 and 6 present the profile of flavonoids (TFs) and the phenolic acids (TPAs), respectively. The effect of farming system was significant in terms of total flavonoids (TFs) and total phenolic acids (PAs). They were significant higher in the conventional than in the organic farming, in line with the content of total phenolic compounds. The greatest difference was found in the content of TFs (Table 5) that in the conventionally cultivated chickpeas was 35.7% higher than the average content of the organically cultivated counterpart (201.78 vs. 148.48 μg g−1). Significant, but less incisive differences were found for TPAs (Table 6), where the average content in conventional farming exceeded the average content in organic farming by 2.9%. The average contents of the most active flavonoids (Table 5), quercetin-3-O-glucoside, epicatechin, and catechin increased by about 62.8%, 18.2%, and 37%, respectively, in conventional farming. Kaempferol 3-O-rutinoside showed an increase of 39%; the flavone chrysin showed a mean content of about threefold higher. These results are not in agreement with Brandt and Mølgaard (2001), who estimated that on average, organic vegetables may contain from 10 to 50% higher defense-related secondary metabolites than conventionally cultivated vegetables. However, our findings were in line with Balisteiro et al. (2013) who found lower TPC and flavonoids in organically cultivated soybeans. Confirming this hypothesis, the scarce N availability stimulates biosynthesis of carbon-containing compounds, including secondary metabolites, but this was not the case in this experiment for TPC, TPAs, and TFs.

Table 5 Profile of flavonoids of the two groups of chickpeas organically and conventionally cultivated
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Table 6 Profile of phenolic acids of the two groups of chickpeas organically and conventionally cultivated
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The mean content of genistein in the organically grown chickpea cultivars was around half compared to conventional farming (2.95 μg g−1 in organic farming and 4.45 μg g−1 in conventional farming), and also the mean content of daidzein was 16.9% lower. Lozovaya et al. (2007) highlighted the physiological role of isoflavones in soybeans as defense compounds against pathogens and signal molecules to symbiotic bacteria. Lygin et al. (2010) established the role of the phytoalexin glyceollin, as the molecule involved in the inhibition of pathogen growth, whose precursor is the isoflavone daizein. Therefore, the reduction in daidzein observed in organically cultivated chickpeas was probably caused by the presence of symbiotic bacteria and/or pathogens. To support this hypothesis, in organically cultivated chickpeas, a significant increase of the number of nodules on roots was observed (Supplementary Fig. 1).

Flavonoids were predominant compounds found in our study both in desi and kabuli cultivars compared to phenolic acids. The most complete flavonoid profile was detected in the desi cultivar (Supplementary Table 1), where all flavonoids were significantly the highest, except for chrysin, genistein, and daidzein. The highest concentration of quercetin 3-O-glucoside and its glycoside rutin were obtained. These results are in agreement with Magalhães et al. (2017) who found a complete flavonoid profile in Elmo, the desi cultivar with black seed coat. On the other hand, in the kabuli cultivars, unlike our results, flavonoids were not found. These differences may depend on the seed coat color. In conventional farming, significantly higher content of ( +)-Catechin was detected for Vulcano likewise for Pascià and Principe for the content of Genistein (Supplementary Table 1).

The most prevalent phenolic acid (Table 6) was p-hydroxybenzoic acid in all the cultivars in both the free and bound form, as previously observed by Fares and Menga (2012) and Aguilera et al. (2011). The latter study also underlined the importance of p-hydroxybenzoic acid in terms of its anti-inflammatory and antioxidant properties. In conventional farming, significantly higher content of p-hydroxybenzoic acid free was found in Calia and Vulcano, while in Reale the higher content was in organic farming. Ferulic acid higher content was in Pascià organically cultivated in respect to the counterpart conventionally cultivated (Supplementary Table 1). Aguilera et al. (2011) also found a negligible amount of hydroxycinnamic compounds, which agreed with our results. We found concentrations of p-coumaric acid and ferulic acid below 1 μg g−1 in almost all cultivars, except for the desi cultivar, which had the highest significant ferulic acid content (1.40 μg g−1). The lowest amount of TPAs was detected in desi cultivar, with half of the content (16.92 μg g−1) compared to the average of kabuli cultivars (36.91 μg g−1).

Principal component analysis

In order to ascertain which chickpeas type (desi or kabuli) greatly differed according to the cultivation technique and if so, for which characters this occurred, the principal component analysis (PCA) was performed on the data set of organically and conventionally cultivated chickpea cultivars. The evaluation of PCA determined eigenvalues of 42.12% and 14.68% for PC1 and PC2, respectively, that together explain the 60.81% of total variance found in the data set. PC1 encompassed eigenvectors relative to AA, IDF, TPC, TFC, and TCC, most of phenolic acids and flavonoids except for chrysin, genistein, daidzein, epicatechin, and p-coumaric acid that are encompassed in PC2. Catechin and β –T are described by both components.

The scatter plot (Fig. 1A) showed that chickpea cultivars were well separated based on farming system. In each farming system, also the behavior of the cultivars was homogeneous, and the cultivars organically cultivated were all placed in the lower region of the scatter plot. Differently, the cultivars grown in conventional farming were all in the upper region of the scatter plot. The nine kabuli chickpeas were clearly separated on PC1 from the desi-type cultivar which is highly correlated with factors described on PC1. The loadings (Fig. 1B) showed that the conventionally cultivated kabuli cultivars were separated from organic for the following bioactive compounds: chrysin, genistein, daidzein positively correlated to PC2. The factors α-T and protein content are highly and negatively correlated to PC2 where the organically kabuli cultivars are located. Most probably, the increase of α-T observed in Kabuli organically cultivated is related to the unfavorable weather events. Indeed, the excess of rainfall caused the onset of severe abiotic and biotic stresses (competition with weeds and attacks by pathogens) more severe in organic farming. Kabuli cultivars with light seed do not have powerful antioxidants that protect them in stressful situations, unlike the cultivar with black seed coats.

Fig. 1
figure 1

Principal component analysis (PCA) of the data set. PCA scatter plot (A) and loading plot (B) of PC1 and PC2

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PCA demonstrate to be a helpful tool to summarize characteristics of the different chickpea genotypes, and mostly the important biochemical differences that separate conventional and organic cultivation.

Conclusions

Organic farming enhanced the nutritional composition of all chickpea cultivars in terms of SW, PC, TCC, and AA. All these traits are particularly attractive for consumers in determining the choice for organic products. But significant decrease of TPC, TPAs, and TFs were observed in comparison to conventional farming. In this organic experiment, the increase in root nodules and reduction of the isoflavone daidzein was observed. Abiotic and biotic stress may have been reduced by the presence of N organic of the soil, and therefore, the biosynthesis of carbon-containing compounds (Shikimato pathway), including secondary metabolites, have been mitigated accordingly.

Desi cultivar had the richest flavonoids profile and AA, while the kabuli cultivars provided the most complete profile of Ts and the highest content of phenolic acids. In organic farming, the content of α-T and ferulic acid increased, with differences between the two types of chickpeas. The increase of the α-T content was solely observed in the kabuli cultivars while, differently, the desi cultivar increased the ferulic acid content and AA.