Composition and Radical Scavenging Capacity of Phenolic Compounds in Wheat-Chickpea Dough

Abstract

The content, composition, and radical scavenging capacity of phenolic compounds in wheat-chickpea (60:40, w/w) flour and dough were studied in this research. The content of phenolic compounds in wheat-chickpea dough was almost twice as high as in the flour from which the dough was made. The addition of chickpea flour to wheat flour contributed to the improved scavenging capacity of dough. The wheat-chickpea dough had a lower maximal achieved scavenging capacity than wheat-chickpea flour, but higher than that of the wheat dough. The quercetin, genkwanin, and apigenin glucosides could be considered as stable components during the mixing of the wheat-chickpea dough. The wheat-chickpea bread retained the radical scavenging capacity which the dough had.

Keywords:

• Chickpea
• Phenolic compounds
• Scavenging capacity
• Dough
• Mixing

INTRODUCTION

Plant phenolic compounds are a very diversified group of phytochemicals which include simple phenolics, phenolic acids, coumarins, flavonoids, hydrolysable and condensed tannins, lignans, and lignins.^[1] They are secondary plant metabolites synthesized by plants during their normal development or in response to stress conditions such as infection, wounding, and UV radiation.^[23] The distribution of phenolic compounds in plants at the tissue, cellular, and sub-cellular levels is not uniform. Insoluble phenolic compounds are found in cell walls, while soluble ones are present within the plant cell vacuoles.^[4] Phenolic compounds have free radical scavenging abilities, anti-mutagenic and anti-carcinogenic activities, and the ability to reduce the risk of cardiovascular and carcinogenic diseases.^[5] Many studies have confirmed the relationship between the dietary intake of phenolic compounds, especially flavonoids, and the effects of these abilities.^[67] Recently it was determined that the antiproliferative effect of legumes can be associated with the presence of phenolic compounds.^[89] Spectrochemic analyses are used for phenolic compounds quantification and chromatographic methods for their identification.^[10] In addition, several methods have been developed for the determination of free radical scavenging abilities. The most commonly used methods are based on spectrometric measurements of the disappearance of free radicals, such as 2, 2 diphenil-1-picrylhydrazyl radical-DPPH radical.^[11,12]

Legumes such as the chickpea are readily available and relatively inexpensive. They are a rich sources of nutritional protein and dietary fiber and contribute to the phenolic compounds intake. Among legumes, the free radical scavenging abilities of soybeans were also studied extensively, but there are also data on the antioxidant activity and phenolic content of lentils, chickpeas, and peas as well.^[13,14] Chickpea seeds are usually consumed raw, in their green and tender stage or in the form of mature dry seeds after parching as a popular snack food. The flour from decorticated chickpea seeds is used in several dishes and as a supplement in weaning food mixes, bread, and biscuits.^[15] In order to improve the protein quality by the destruction or inactivation of the heat labile anti-nutritional factors, legumes are usually cooked before used for human consumption.^[16] The process including decortification, cooking, and soaking affects the overall phenolic compounds content (PCC) and antioxidant activity in some legumes. For example, the antioxidant activity and total phenolics content was reduced by 80% through the removal of the hull in lentils, by 16–41% through cooking lentils, chickpeas and peas and by 22–42% through soaking lentils.^[1417] In order to improve the nutritional quality of chickpeas, protein digestibility and the retention rates of B vitamins and minerals, Saleh and Tarek^[18] recommended microwave cooking and reduction of cooking time. In literature there is data on the physicochemical, cooking, and textural properties of seeds of different chickpea cultivars^[19] and data about the effect of legume proteins on baking properties.^[20] It was found that when caffeic acid was added to bread, the dough retained its antioxidant activity during baking, and the recovery of caffeic acid was 74–80%.^[21] However, no data about the changes in the PCC and composition affected by mixing dough from wheat-chickpea flour (WCpF) mixture. Starting from the fact that the addition other types of flour to wheat flour (WF) changes the chemical composition of the flour mixture,^[22,^23] the idea was to obtain food products enriched with chickpea nutritional constituents.

In this article, the effect of mixing the PCC, composition and radical scavenging capacity (SC) in WF, CpF, wheat dough (WD) and wheat-chickpea dough (WCpD), in a ratio of 60:40 (w/w) was investigated. The PCC was determined by using the spectrometric method, their composition means of the HPLC method, and radical SC by applying the DPPH method. The correlation coefficients between the studied parameters were determined using statistical analysis.

MATERIALS

Flour, Dough Mixing, Bread Making

The WF type 400, Kikinda Mlin, Serbia, was mixed with the flour from decorticated and fried chickpea seeds, originated from Iran. The seeds had been bought from the local market in Leskovac, Serbia and the CpF flour was obtained by milling chickpea seeds and sieving them through a 0.25 mm riddle. The protein content (PC) in the flour was determined by the Kjeldahl method (Nx5.95).

WD and dough made of a mixture of wheat and CpF (60:40, w/w) (WCpD) were obtained by mixing on a farinograph (Brabender Model 8 10 101, Duisburg, Germany), according to the ISO 5530-1 test procedure. The obtained dough was cut into slices approximately 1.5 × 0.5 × 0.5 cm in size, dried at 30^oC, for 4 h and then milled. The home-made breads were prepared with in the following manner: WF (300 g), CpF (200 g), salt (10 g), sugar (10 g), yeast (5 g), and water (270 mL) for wheat-chickpea bread (WCpB), and WF (500 g) and the same amount of salt, sugar, yeast, and water for wheat bread (WB). The duration of the bread-making process was: 15 min of mixing, 25 min of rising at 30^oC, 10 min of mixing, 60 min or rising at 30^oC, and 5 min of mixing. Then the dough was shaped into round balls, approximately 30 cm in diameter and 2.5 cm in height and baked at 180^oC for 50 min in an oven (Candy, FPP403/1). After baking, the bread was allowed to cool down to room temperature. Then the bread was sliced (the size of the slices was approximately of 25 × 1.5 cm). The slices dried for 3 h at 50^oC, were milled and sieved through a 0.25 mm riddle.

METHODS

Extracts Preparation

For the measurements of PCC in WF, CpF, WD, and WCpD, 5 g of the sample was measured and 100 mL of 80% (v/v) ethanol was added. The mixture was stirred by MR1 magnetic stirrer (IKA-Werke, Staufen, Germany) for 10 min at 200 min⁻¹ and vacuum filtered through No. 54 Wathman filter paper (GE Healthcare, Brondby, Denmark). The solids were re-extracted with 50 mL of 80% (v/v) ethanol, and the filtrates combined to a final volume of 150 mL. For the measurement of PCC, 10 mL of each extract was filtered through a 0.45 μm membrane filter (Agilent Technologies, Wilimington, Delaware, USA). For the radical SC measurements and the HPLC analysis, 140 mL of each extract were evaporated in vacuum at 45^oC until dry and dissolved in 96% (v/v) ethanol, prior to analysis.

PCC

For PCC measurement, a standard curve for five chlorogenic acid (Sigma Chemical, St. Louis, Missouri, USA) concentrations, covering a range from 50 to 500 μM/L, was determined. A total of 0.25 mL of 0.1 g/mL HCl in 95% (v/v) ethanol, 4.50 mL of 2 g/mL (w/v) HCl, and 0.25 mL of chlorogenic acid standard solutions were added to a test tube, mixed by a vortex and allowed to stand for 15 min. Then the absorbance (A) was read at 280 nm using a UV 21000 Spectrophotometer (Cole Parmer Instruments Company, Vernon Hills, Illinois, USA). For measuring PCC in the WF, CpF and WCpD extracts, 0.25 mL of 0.1 g/mL HCl in 95% (v/v) ethanol, 4.50 mL of 2 g/mL HCl, and 0.25 mL of filtered extracts were added into test tube, and further treated as standard solutions of chlorogenic acid.^[24] The PCC (mM/L) was determined as follows:

Radical SC

The radical SC of the extract, diluted by ethanol to concentrations ranging from 0.2–6 mg/mL, was determined by the DPPH test.^[10^] The ethanol solution of DPPH radical concentration of 0.1 mM/L (1 mL) was added to a 2.5 mL ethanol solution of given concentration of investigated extract and allowed to react at room temperature for 30 min. Then the Ab value was measured at 518 nm on the UV 21000 Spectrophotometer (Cole Parmer Instruments Company) and converted into the percentage of radical SC by using the equation previously defined^[25] as:

where A_sample is the absorbance at 518 nm of the ethanol solution of the extract treated with the DPPH radical solution; A_blank is absorbance at 518 nm of the ethanol solution of the extract (1 mL of ethanol added to 2.5 mL of extract), and A_control is absorbance at 518 nm of ethanol solution of the DPPH radical (1 mL of a 0.3 mM/L added to 2.5 mL of ethanol). The final results are presented as EC₅₀ value, i.e., concentration of the studied extracts that causes a decrease in the initial DPPH concentration by 50%.

HPLC Analysis

The HPLC analyses were performed on an Agilent 1100 Series HPLC system (Agilent Technologies) consisting of a micro vacuum degasser, binary pump, thermostated column compartment and variable wavelength detector, using the Viet et al. method.^[26] The column was Agilent Eclipse XDB-C18 4.6 mm ID × 150 mm (5 μm) with a pore size of 8 nm. The mobile phase was composed of solvent (A) 0.15 g/mL phosphoric acid in H₂O:MeOH (77:23 (v/v), pH = 2.0) and solvent (B) methanol as follows: isocratic 03.6 min 100% A + 0% B; linear gradient in 24 min 80.5% A + 19.5% B; isocratic with 80.5% A + 19.5% B up to 30 min; linear gradient in 60 min 51.8% A + 48.2% B; linear gradient in 67.2 min 0% A + 100% B; followed by isocratic elution with 100% B for last 5 min. The flow rate was 1 mL/min. The dosing volume of ethanol extract, diluted to a concentration of 5 mg/mL, was 20 μL. A spectrophotometer Agilent 1100 G1316A (Agilent Technologies), and the UV region at 350 nm were used; therefore, all the used solvents had to have sufficient light permeability at this wavelength. In order to eliminate variations with the injection volume, precise amount of naringenin as a reference compound was added to a known volume of the extract and the areas of the peaks were compared. Peak identity was checked by comparison of their relative retention indices with the reference compounds (chlorogenic acid, caffeic acid methyl ester, luteolin 5-O-(6^”-O-malonylglucoside and apigenin 5-O-(6^”-O-malonylglucoside purchased from Sigma Chemical, St. Louis, Missouri, USA). The mass of these compounds in the samples was calculated by introducing the areas of the peaks into the corresponding calibration curves. The mass of other compounds present in the samples was quantified with the calibration curves of the most similar compound by structure or by retention time.

Statistical Analysis

Statistica version 5.0 Software (StatSoft, Tulsa, Oklahoma, USA) was used to perform the statistical analysis: the mean, standard deviations, the correlation coefficients, and statistical dependence. The mean and standard deviations were obtained by using Descriptive Statistics, marking the Median and Quartiles and Confirm Limits for Means. The correlation coefficients were obtained by a correlations matrix analysis with displaying p and N value, based on eight parameters (extract yield-EY; PPC; the concentration of the extracts that causes a decrease in the initial DPPH concentration by 50%-EC₅₀; total quercetin glucosides content-TQg; total kaempferol glucosides content-TKg; total luteolin glucosides content-TLg; total genkwanin glucosides content-TGg and total apigenin glucosides content-TAg). The sample size was eight (N = 8, four samples and maximal and minimal values of three determinations). Only the correlations which were above the absolute value of 0.8, were taken into consideration. Statistical dependence was determined by t-test for dependent samples (p < 0.05).

Table 1  Dry residue content, yield, phenolic compounds content, and radical scavenging capacity of extracts obtained from flour and dough

Parameter/sample	WF	CpF	WFCpF (60:40, w/w)	WD	WCpD
Dry residue content (g per 100 g of extract)	6.6 ± 0.5	11.1 ± 0.6	8.4*	6.7 ± 0.4	8.4 ± 0.5
EY (g of dry residue content per 100 g of flour or dough)	10.7 ± 0.8	17.9 ± 1.4	13.6*	10.7 ± 0.6	13.7 ± 0.8
PCC (μg of chlorogenic acid per g of flour or dough)	798 ± 23	365 ± 16	624.8*	1603 ± 52	1147 ± 39
SC (%)	61.8 ± 4.4	98.3 ± 1.6	76.5	46.2 ± 2.3	58.3 ± 3.6
EC50 (μg of chlorogenic acid per g of flour or dough)	263.6 ± 28	84.8 ± 11	192.1	1836.4 ± 126	763.6 ± 49

Values are the means and standard deviation (N = 3) obtained by descriptive statistics and marking the median and quartiles and confirm limits for means.

*Data obtained based on results for WF and CpF, considering the flour ratio of 60:40, w/w.

RESULTS AND DISCUSSION

Characterization of the Extract and Content of Phenolic Compounds

The characteristics of the obtained extracts from the WF, CpF, WD, and WCpD, dry residue content expressed as g per 100 g of extract and EY in g of dry residue content per 100 g of flour or dough are shown in Table 1. The results of maximal achieved radical SC (%) and concentration of the extracts that causes a decrease in the initial DPPH concentration by 50%, EC₅₀ (μg/g) are also presented in Table 1. The results are mean value of three determinations followed by a standard deviation. The results show that CpF had the highest dry residue content of 11.1% and EY of 17.9% while WF and WD had the lowest values. These values in WF and WD are almost the same and this is understandable, as only water was added during dough mixing. The PCC obtained by the applied method was the highest in the dough made from WF only (1603 μg/g) and this content was twice as high as the one in WF where it was 798 μg/g. The same dependence can be noticed between the WCpF and WCpD (the PCC in the flour mixture, considering the PCC in wheat and CpF and their ratio of 60:40 (w/w) in the flour mixture, was 625 μg/g and it is almost half the value of that of the sample of the WCpD, where it was 1147 μg/g). Based only on these results it is difficult to explain how the PCC content appeared to be higher in the sample of dough than in corresponding samples of flour. However, as water was added during dough mixing, hydration reactions of phenolic compounds probably occurred. Then phenolic compounds exist in their hydrate state and this probably increases the extractability of phenolic compounds^[27] and causes higher PCC in the dough samples. Increases in the total phenolic content of soaked chickpeas, peas and soybeans, which were obtained by a process similar to that of dough mixing, with the addition of water, were also observed by Han and Baik.^[14]

The DPPH radical SC depended on the phenolic compounds concentrations in the extract. The maximal achieved SC of the studied samples ranged from 46.2–98.5% and the EC₅₀ values were from 84.8–1836.4 μg of chlorogenic acid per g of sample. Generally speaking, the lower EC₅₀ value indicates higher antioxidant capacity. The CpF appeared to have the highest, while the dough from the WF (WD) the lowest SC, although the PCC was higher in the WF than in the CpF. This could be explained by different phenolic compounds composition and their activity in DPPH radicals or by the presence of non-phenolic compounds with a radical scavenging ability, such as proteins. The PC in the CpW was 22.4 g/100 g and it was higher than in the WF, where it was found to be 9.8 g/100 g. Proteins have excellent antioxidant potential through different pathways, including scavenging free radicals.^[28]

Comparing the results for flour to those for dough, EC₅₀ value of dough was higher than the value of flour. The explanation for this could be found in the report of MacRitchie et al.^[29] who found that high speed mixing breaks disulfide bonds and creates thiol free radicals in gluten which react with reducing compounds in the flour, i.e., phenolic compounds, causing a decrease in the antioxidant activity. The reason for this might also be the oxidation processes of phenolic compounds which can occur during mixing. Han and Koh^[21] also found lower total antioxidant activity, measured as the inhibition of beta carotene bleaching activity, in dough into which the phenolic acids were added, than in the flour from which they were made. According to these authors, the decrease of antioxidant activity can be explained by interactions between the thiol free radicals of gluten and added phenolic acids, in which the phenolic acids act as a reducing agent. Based on a comparison of the results of SC and EC₅₀ for analyzed dough, the WCpD had better antioxidant activity than WD, so the addition of CpF to WF contributed to the improvement of antioxidant activity.

Results obtained with breads, showed the content of phenolic compounds in WB and WCpB was 1268.4 ± 34 and 961.8 ± 28 μg of chlorogenic acid per g of bread sample, respectively. Value of EC₅₀ of WB and WCpB, was 1784.3 ± 32 and 738.2 ± 29 μg of chlorogenic acid per g of bread, respectively, so WCpB had higher SC than WD. Between results for WB and WCpB and appropriate results for WD and WCpD (Table 1), there are no statistical significant differences, which means the bread retained radical SC which dough had had. These results are in accordance with results that dough retained the antioxidant activity during baking, reported by Han and Koh.^[21]

Phenolic Compounds Composition in Flour and Dough

The results of the HPLC analysis of 80% (v/v) ethanol extracts obtained from WF and WD are presented in Table 2, and from CpW and WCpD in Table 3. As there are many compounds, only those that could be identified and had a content of over 20 μg/100 g are presented and considered. As several derivates of phenolic compounds have been found in the studied samples, in order to simplify the results overview, the total content of these compounds is presented and discussed in Tables 2 and 3. The compounds with retention a time of 2.9, 3.1, 3.2, 6.5, and 66.4 min, in Table 3, unfortunately could not been identified, although they were presented in samples of CpF or WCpD with a content from 26–116 μg/100 g. In a chromatographic analysis including raw chickpea, Aguilera et al.^[30] identified 24 phenolic compounds, where the isoflavones were the main ones. These compounds might be a kind of isoflavones, such as genistein hexoside, biochanin A 7-O-glucoside, biochanin A derivate, biochanin B, biochanin B hexsoide or biochanin B derivate. In chickpea cotyledon, in addition to quercetin and kaempferol, the presence of myrcetin, diadzein, and genistein has also been observed.^[31]

In WD the content of kaempferol glucosides (713.2 μg/100 g) and in WCpD the content of quercetin glucosides (321.3 μg/100 g) was the highest. By comparing the samples of WD to WF, it can be seen that the content of quercetin, kaemferol, genkwanin, and apigenin glucosides content is more than twice the value of that in the dough than in the flour, probably due to hydration reactions, and these compounds could be considered as stable during mixing, while luteolin glucosides are unstable compounds during WD mixing. Taking into consideration the content of glucosides in WF and CpF mixtures (60:40, w/w) the total content of quercetin, kaempferol, luteolin, genkwanin, and apigenin glucosides was calculated to be 137.3, 193.2, 56.5, 20.8, and 14.3 μg/100 g, respectively. By comparing these contents to the corresponding ones in the WCpD sample, it can be seen that the content of quercetin, genkwanin, and apigenin glucosides are higher in the dough than in the flour, and they could be considered stable, while kaemferol and luteolin glucosides could be considered unstable compounds during WCpD mixing. The different stability of kaemferol glucosides in WD and WCpD might be explained by their different behavior in mediums with different content of proteins.

Table 2  Phenolic compounds in 80% (v/v) ethanol extract from WF and WD detected by HPLC analysis

Compound/content (μg/100 g of flour or dough)	WF	WD
1. Quercetin 3-O-sophoroside	200.9	57.5
2. Kaempferol 3-O-(6”-O-malonylglucoside )-7-O-glucoside	147.6	ND
3. Caffeic acid methylester	42.7	57.5
4. Kaempferol 3-O-(6”-O-malonylglucoside )-7-O-glucoside	46.1	ND
5. Protogenkwanin-4^’-O-glucoside	ND	64.3
6. Dihydrokaempferol	ND	78.5
7. Quercetin 3-O- rutinoside -7-O-rhamnoside	23.9	ND
8. Kaempferol 3-O-sophoroside	23.3	ND
9. Quercetin 3-O-(6”-O-malonylglycoside)-7-O-rhamnoside	ND	455.5
10. Luteolin 5-O-glucoside	94.2	58.6
11. Kaempferol 3-O-glucoside -7-O-rhamnoside	36.1	137.5
12. Kaempferol 3-O-rutinoside -7-O-rhamnoside	ND	189.7
13. Quercetin 3-O- glucoside	ND	37.2
14. Apigenin 4^’-O-glucoside	23.9	ND
15. Kaempferol 3-O-rhamnoside-(6”-O-malonylglycoside)	ND	145.5
16. Apigenin 5-O-(6”-O- malonylglycoside)	ND	127.1
17. Kaempferol 3-O-rhamnoside	7.1	116.1
18. Kaempferol 7-O-rhamnoside	24.5	45.9
19. Genkwanin 4^’-O-glucoside	26.1	46.3
Total Quercetin glucosides	224.8	550.2
Total Kaempferol glucosides	284.7	713.2
Total Luteolin glucosides	94.2	58.6
Total Genkwanin glucosides	26.1	110.6
Total Apigenin glucosides	23.9	127.1

NIC: Not identified compound; ND: not detected.

Table 3  Phenolic compounds in 80% (v/v) ethanol extract from CpF and WCpD (60:40, w/w) detected by HPLC analysis

Compound/content (μg/100 g of flour or dough)	CpF	WCpD
1. NIC (Rt 2.9 min)	27.8	115.9
2. NIC (Rt 3.1 min)	44.7	95.7
3. NIC (Rt 3.2 min)	56.1	60.7
4. NIC (Rt 6.5 min)	43.1	28.9
5. Chlorogenic acid	26.2	29.3
6. Hydroxycinnamic acid ”C1”	ND	27.2
7. 5-O-Caffeoylshikimic acid	28.9	30.4
8. Quercetin 3-O-(6”-O-malonylglycoside)-7-O-rhamnoside	ND	249.8
9. Luteolin 5-O-glucoside	ND	27.0
10. Quercetin 3-O- glucoside	6.0	38.7
11. Kaempferol 3-O-glucoside-7-O-rhamnoside	ND	26.5
12. Quercetin 3-O- (6”-O-malonylglucoside)	ND	32.8
13. Kaempferol 3-O-rutinoside	ND	26.9
14. Genkwanin 5-O-glucoside	ND	57.7
15. Apigenin 5^’-O-(6”-O-malonylglucoside)	ND	33.8
16. Kaempferol 3-O-rhamnoside	34.2	59.9
17. Kaempferol 7-O-rhamnoside	21.7	30.0
18. Genkwanin 4^,-O-glucoside	12.9	25.3
19. NIC (Rt 66.4 min)	30.5	105.3
Total Quercetin glucoside	6.0	321.3
Total Kaempferol glucoside	55.9	143.3
Total Luteolin glucoside	−	27.0
Total Genkwanin glucoside	13.0	83.0
Total Apigenin glucoside	−	33.8

NIC: Not identified compound; ND: not detected.

Table 4  Correlation matrix for extract yield, phenolic compounds content, scavenging capacity, and the total glucosides content for flour and dough (N = 8, p ≤ 0.05)

	EY	PCC	EC50	TQg	TKg	TLg	TGg
PCC	−0.72
EC50	−0.56	0.85
TQg	−0.77	0.99	0.87
TKg	−0.78	0.81	0.89	0.86
TLg	−0.90	0.38	0.27	0.46	0.61
TGg	−0.53	0.97	0.82	0.94	0.67	0.13
TAg	−0.63	0.92	0.97	0.93	0.93	0.34	0.87

The correlation coefficients obtained based on eight parameters are presented in Table 4. They show that a higher PCC content obtained by 80% ethanol extraction, does not mean a higher antioxidant activity (lower EC₅₀ values indicate higher antioxidant activity). With a higher EY, the content of luteolin glucosides in the extract was lower. Also, during extraction, when the higher content of quercetin was extracted, a higher kaempferol, genkwanin, and apigenin glucosides content was extracted as well. The higher content of apigenin glucosides in the extract meant a higher content of kaempferol and genkwanin glucosides. The positive correlations between EC₅₀ on the one hand, and quercetin, kaempferol, genkwanin, and apigenin glucosides on the other show that the higher content of these compounds does not contribute to higher antioxidant activity. The higher content of some others compounds, such as those extracted by methanol-water solutions^[3031] or proteins,^[28] probably contribute to higher radical SC. Similar results were reported by Wojdyło et al.^[32] based on a study of 32 selected herbs, when the positive correlation between the total antioxidant capacity and the total phenolic content was not found in all the family groups, and by Souri et al.^[33] based on a study of the PCC and antioxidant capacity of 24 medicinal plants, when no significant correlation was found between these parameters.

CONCLUSION

The PCC was higher in the WCpD than in the WCpF mixture probably due to the hydration reactions during mixing, which increase the extractability of phenolic compounds. The SC is decreased through dough mixing, and the addition of CpF to the WF contributes to the better SC of the WCpD than that of the WD. The WCpB retained the radical SC which the dough had.

FUNDING

This work was supported under the project Grant No.172047 by the Ministry of Education, Sciences and Technological Development of Republic of Serbia.