Analysis of Gamma-Irradiation Effect on Radicals Formation and on Antiradical Capacity of Horse Chestnut (Aesculus hippocastanum L.) Seeds

Abstract

The irradiation by gamma-rays is a widely used technique for disinfection in the pharmaceutical and cosmetic industries. In view of growing concerns by consumers about this technique, further investigation of the effects of radiation is required. In this work electron paramagnetic resonance (EPR) spectroscopy was applied to study the free radicals in irradiated horse chestnut (Aesculus hippocastanum L.) seeds and to evaluate the free radical scavenging activity (FRSA) using the stable DPPH radical. In order to evaluate the antiradical potential, a spectrophotometric study was also used. The identification and quantification of some individual polyphenol compounds before and after irradiation by 1, 5, and 10 kGy gamma rays of peeled and shell seeds were obtained by high performance liquid chromatography (HPLC). The EPR spectrum recorded on irradiated horse chestnut is a typical signal for irradiated cellulose-contained substances. The results show that the signal is stable, and it can be found in the samples irradiated with a dose of 1 kGy, 45 days after treatment, whereas for samples irradiated by 5 and 10 kGy, it is even found 250 days later. The study showed that free radical scavenging activity increases in shell seeds, while it decreases in peeled seed extracts after irradiation depending on the dosage, which corresponds to the total phenolic content. Shell seed extracts have significantly stronger antiradical activity than that of peeled seeds. Regarding the HPLC analysis, some polyphenolics were degraded and others were formed as a result of irradiation. The irradiation by 5 kGy dosage has a most significant positive effect on the antioxidant potential of shell chestnut seeds.

1. Introduction

The enormous interest in natural sources of phytochemicals, which have potential benefits for improving health and combating some socially significant diseases, is noticeable. Horse chestnut (Aesculus hippocastanum L.) from the family Hippocastanaceae, which is a native to Europe is known to have various medicinal and health benefits obtained from the numerous phytoconstituents present in its seed [1]. Hippocastanum L. has been widely used for centuries in traditional medicines as a cure for several diseases and disorders, such as chronic venous insufficiency, hemorrhoid disease, traumatic edema, etc. [2,3]. Its extracts, whose main bioactive components are saponins, flavonoids, tannins, alkaloids, glycosides, triterpenoids, steroids, and phenolic substances, have various beneficial effects, including anti-inflammatory, venotonic, and vascular protective effects, as well as immune-modulatory, anticarcinogenic, antibacterial and antioxidant activity [2,3,4,5]. The biological activity of horse chestnut allows its widespread use in the pharmaceutical and cosmetic industry. Moreover, the horse chestnut finds various applications as a valuable resource in industry [2,5].

Some studies evaluated the biochemical composition and the biological activity of horse chestnut from different areas. Hippocastanum L. seeds are natural products whose chemical composition is very complex, since they contain a lot of different molecules. The majority of them are polysaccharides (both starches and non-starches), lipids, proteins, and essential mineral elements, such as nitrogen, phosphorus, calcium, copper, iron, zinc, etc., and many minor components among others [2,6]. According to the literature, significant differences in the phytochemical composition and antioxidant activity of extracts obtained from different organs of the plant (bark, fruits, leaves, flowers, seeds), and also between separate parts of the seed-coat and endosperm were observed. The data obtained on the content of polyphenols are the following: in horse chestnuts leaves (7.81–24.48 mg/g FW), seeds (24.24–70.40 mg/g FW), flowers (8.17 mg/g), and bark (363.58 mg/g FW) [7]. Previously published data indicated relatively strong antioxidant properties of horse chestnut seed coat extracts and significant absorbance in the ultraviolet range, suggesting their potential utility against UVB-induced DNA damage [8,9].

On the other hand, in order to prevent natural products from developing pathogenic microorganisms and thus prolonging their shelf-life time, they require disinfection. In the past, the fresh chestnut fruits were fumigated with methyl bromide after harvest to comply with the international phytosanitary regulations for pest quarantine [10]. But its use has been prohibited in Europe (following a 239 European Scientific Journal December 2013/SPECIAL/edition vol.3 ISSN: 1857–7881). The treatment of chestnut by gamma rays could be a feasible alternative for disinfection, since the technology has proven itself to be in practice easy, fast, cheap, reliable, and reducing spoilage losses and it was approved by international organizations of food (FAO—Food and Agriculture Organization) and health (WHO—World Health Organization). The increasing application of gamma treatment to natural products in order to improve their hygienic qualities requires study of the impact of high-energy radiation on their chemical composition and bioactivity. Previous studies of plant-based products established a correlation between gamma treatment, chemical composition, and antioxidant capacity [11,12]. It is known that as a result of the irradiation various radical structures are formed in the substances. Some radicals quickly recombine, but others are relatively stable because of the trapping in the solid phase of substances. These radicals and their decay could be detected and analyzed by EPR spectroscopy, which is a highly sensitive technique for the detection of paramagnetic organic and inorganic substances. EPR spectroscopy is widely used also for studying the free radical scavenging activity of substances using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical. Because of a strong absorption band at 520 nm and the changes in the optical spectra, DPPH is a common antioxidant assay also for spectrophotometric study [13]. For detection and quantification of the individual antioxidant compounds, HPLC is widely used. However, up to now there are no data about the impact of gamma treatment on the free radicals generated in horse chestnut seeds. In the studies reported in the literature up to now the following have been analyzed: the biochemical composition and bioactivity of unprocessed Aesculus hippocastanum L. samples and their dependence on different factors, such as geographical and environmental conditions, the extraction procedure, used seed fractions, etc. [1,8]. To the best of our knowledge, there are no scientific investigations about the influence of gamma irradiation on the antioxidant properties of horse chestnuts. According to the literature data, it is known that irradiation might affect differently the polyphenolic composition and antioxidant capacity of plant samples. So, this defined the aim of the present research work, namely, to study the gamma irradiation effect of horse chestnut seeds (peeled and shell seeds) on the formation of radical components, as well as on their polyphenol content and antiradical activity, using EPR, spectrophotometric, and HPLC analyses.

2. Materials and Methods

2.1. Materials

The seeds of horse chestnut were harvested from trees located in the South Park in Sofia, Bulgaria during the month of October. They were cleaned and dried at room temperature for several days and stored in a dry, cool, and dark place until further processing. The seeds were opened, and their shell seed (SS) and endosperm (peeled seeds PS) parts were separated and ground (Scheme 1).

The used reagents, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox, Folin-Ciocâlteu reagent (FCR), methyl and ethyl alcohol, acetone, gallic acid, sodium carbonate (Na₂CO₃), sodium acetate, aluminum trichloride (AlCl₃), sodium nitrite (NaNO₂), sodium hydroxide (NaOH), and standards for HPLC analysis (catechin, chlorogenic acid, p-coumaric acid, caffeic acid, ferulic acid, rutin, hesperidin, morin, luteolin, and quercetin) were purchased from Sigma Aldrich. Distilled water was used. All the reagents used in the study were of analytical grade purity.

2.2. Preparation of Extracts

In order to determine the most appropriate solvent for the preparation of the PS and SS extracts, three types of extractants were tested as follows: (1) acetone/ethanol/water (1:0.7:0.3 v/v/v); (2) ethanol/water (6:4 v/v), and (3) water/acetone (1:1). The obtained results showed better antiradical activity using the extractant water/acetone for both chestnut samples (PS and SS). In view of this this extractant was used for all further extractions.

Extracts for all analyses were prepared as 1 g of non-irradiated and irradiated shell seeds, and peeled seeds were suspended in 10 mL aqueous acetone solution (1:1). The mixtures were extracted for 18 h at room temperature in the dark without air access, and then they were filtered off. For HPLC analysis the resulting extracts were further centrifuged in a Beckman Model J2-21M (Brea, CA, USA) apparatus at −7 °C, 10,000 rpm, for 10 min. Before being subjected to subsequent chromatographic examination, the samples underwent additional filtration using a syringe filter with a pore size of 0.22 μm. Only the shell seed extracts were diluted (1 mL extract:1 mL aqueous acetone solution) for the EPR analysis. The supernatants thus obtained were used for further experiments, and these were prepared fresh daily.

2.3. Sample Irradiation

The shell seed and peeled seed samples were packed in polyethylene bags, and they were irradiated in a cobalt-60 (⁶⁰Co) source with 8200 Ci activity at 1, 5, and 10 kGy by a semi-industrial radiation system, “NIGU-7”, using four liters of volume working chamber. The irradiation treatments were performed at room temperature in the air at the National Centre of Radiobiology and Radiation Protection, Sofia, Bulgaria. For the study of the absorbed dose distribution alanine dosimeters (Kodak BioMax, Eastman Kodak Company, Rochester, NY, USA) were used. They were measured by an ESR spectrometer, E-scan Bruker and calibrated in units of absorbed dose in water.

The samples processed by gamma rays were stored in closed polyethylene bags in the dark at room temperature before subsequent analysis.

2.4. Instrumentation

The EPR spectra were detected as a first derivative of the absorption signal on a JEOL JES-FA 100 EPR, JEOL, Tokyo, Japan spectrometer equipped with a standard TE₀₁₁ cylindrical resonator operating in the X-band (9.3 GHz). The measurements were carried out at room temperature. The solid samples were placed in a quartz standard EPR tube of inner diameter 4 mm and positioned in the EPR cavity. Three independent measurements were used for every solid sample, including a procedure, where inserting–removing–inserting of the sample was performed in the cavity of the EPR spectrometer. The data were averaged, and in that way, the error of the measurement was determined to be 5%. Before and after each series of measurements under the same conditions, a reference sample, magnetic Mn diluted in MgO (an internal standard for the above-mentioned spectrometer), was analyzed in the same way as those used for the sample measurements, in order to normalize the signal intensity of the samples and to minimize the error resulting from any instability in the spectrometer.

2.5. Estimation of DPPH Free Radical Scavenging Activity—FRSA (Antiradical Activity)

Free radical scavenging activity of non-irradiated and irradiated horse chestnut extracts (from peeled seeds and shell seeds) was determined by EPR spectroscopy and spectrophotometry using the stable DPPH radical.

EPR determination: Briefly, a specified amount of the extract was added to a 1 mL 0.002 M DPPH solution (ethanol or acetone). After completing the reaction, the mixture was transferred into a capillary tube, positioned inside in the cavity of the EPR spectrometer.

For calculation of the percent DPPH radicals scavenged by the studied extracts, the following equation was used:

Scavenged DPPH radicals (%) = [(I₀ − I)/I₀] × 100

The “I₀” was the intensity of the second DPPH peak of the control samples. The same procedure was used to prepare the DPPH control, which did not contain any extract sample. The “I” was the intensity of the second peak of the same EPR spectrum after the addition of the measured extract.

A previously used method, based on EPR spectroscopy, for determination of Trolox equivalent antioxidant capacity (TEAC) was applied [14,15] for investigation of horse chestnut samples. Specified volumes of Trolox solution (0.4 mL for PS and 0.06 mL for SS determinations) at different concentrations were added to 1 mL 0.002 M DPPH to construct the calibration curves for both PS and SS samples (y = 6.18x − 39.29 and correspondingly y = 0.907x − 24.74). The regression equations (given in

Table 1. Regression equations and TEAC values for non-irradiated and irradiated with 1, 5, and 10 kGy peeled (PS) and shell seed (SS) horse chestnut samples determined by EPR measurements.

Dose (kGy)	Sample	Regression Equation	TEAC 1 (µmol/mL)
0	PS	y = 151.1x − 0.06	30.8 a ± 0.22
1	PS	y = 135.8x + 0.42	28.4 b ± 0.28
5	PS	y = 131.5x + 3.2	28.15 b ± 0.23
10	PS	y = 123.5x − 0.7	26.2 c ± 0.36
0	SS	y = 951x − 14.58	1056.2 a ± 2.68
1	SS	y = 959.5x − 7.99	1083.8 b ± 2.85
5	SS	y = 998.5x − 7.67	1116.0 c ± 1.69
10	SS	y = 986x − 9.32	1100.5 d ± 2.9

¹ Trolox equivalent antioxidant capacity. Different superscript letters (a–d) indicate significant differences between values (p < 0.05).

) were determined on the basis of the linear dependence between the percentage of scavenged DPPH radicals and the concentration of Trolox. The obtained data were expressed as μmol Trolox per mL of studied extract.

Spectrophotometric determination: The antioxidant capacity was determined by the method proposed by Brand-Williams et al. [16] with some slight modifications. Briefly, to 0.6 mL of DPPH solution (0.2 mM), there were added 0.9 mL of methanol and 0.5 mL of the respective dilutions of the tested extracts. The resulting solution was homogenized, and it was left in the dark at room temperature for 60 min. Similarly, a blank control sample was prepared with 80% methanol instead of extract. The absorbance was read at 517 nm on a Lybra S22 spectrophotometer (Biochrom Ltd., Cambridge, UK). The DPPH radical scavenging capacity was calculated using a formula derived from the standard Trolox calibration curve (y = 0.0319x + 0.0189; R² = 0.992), and it was expressed as TEAC in µg/mL.

2.6. Total Phenolic and Flavonoid Contents

The total phenolic content (TPC) of the horse chestnut extracts was evaluated according to the Folin–Ciocalteu method proposed by Valyova et al. [17], with some slight modifications. An aliquot of 0.5 mL horse chestnut solution was mixed with 3.0 mL distilled H₂O, 0.25 mL of Folin–Ciocalteu reagent, and 0.75 mL of Na₂CO₃ (20% w/v). After incubation, the absorbance of the prepared mixture was measured at 765 nm against a blank sample. Gallic acid was used for calibration of the standard curve (y = 0.0101x − 0.0178, R² = 0.9991). The results for total content of phenolics were expressed as mg of gallic acid equivalents (GAE/mL of sample).

The total flavonoid content (TFC) was evaluated by the aluminium chloride method. Two procedures according to quercetin and catechin standards were used for estimation of TFC. For the first procedure, 0.5 mL measured extract was mixed with 1.5 mL 95% EtOH, 0.1 mL 10% AlCl₃ solution, 0.1 mL Na acetate, and 2.8 mL distilled H₂O. After 40 min incubation, the samples were measured at 415 nm wavelength. The total flavonoid content was evaluated using the standard curve of quercetin (20–100 μg/mL). For another procedure, TFC was determined as follows: to the mixture of 0.25 mL horse chestnut extract, 1.25 mL distilled H₂O, and 0.075 mL of a 5% NaNO₂ solution, there were added 0.15 mL 10% AlCl₃ solution, 0.5 mL 1 M NaOH, and 0.775 mL distilled H₂O. The calibration curve using catechin as a standard (50–400 μg/mL) was then constructed [18], and then the absorbance was read at 510 nm wavelength. Analyses were carried out in triplicate. TFC was expressed as mg of quercetin equivalents (QE) and catechin equivalents (CE) per mL of sample.

2.7. High Performance Liquid Chromatography (HPLC) Analysis

The analyses of flavonoids, present in horse chestnut seeds, were performed using an Agilent 1260 Infinity Quaternary LC, DAD (UV/VIS), Agilent Technologies, Waldbronn, Germany apparatus. The column used for the separation of compounds was Agilent Poroshell 120 HPH-C18 (4.6 × 100 mm, 2.7 µm particles). In order to achieve sufficient resolution of the peaks, mixtures of methanol/ultrapure water/acetic acid were used in the ratios 5:93:2 for Phase A and 86:12:2 for Phase B. The injection volume was 10 µL. The following mobile phase gradient was applied in the chromatographic separation: 0–3 min B increases to 20%, 3–7 min B increases from 20% up to 55%, and finally 7–12 min A increases to 100%. Mobile phase flow was set at 1 mL/min and the detection was carried out at 280 nm, at 35 °C.

Flavonoids compounds were detected based on their chromatographic data (retention time and UV–Vis spectra) by comparison with the available commercial standards. The concentrations of detected phenolic compounds in the PS and SS extracts were expressed in mg/g.

2.8. Statistical Analysis

All the measurements and analyses were performed in triplicate. The data are expressed as the mean value ± standard deviation (SD). The statistical significance was evaluated by Students’ t-test (MS Excel, 2016). The values at p < 0.05 were considered statistically significant.

3. Results

3.1. EPR Study

3.1.1. Detection of Free Radicals in Solid Samples of Horse Chestnuts (Shell and Peeled Seeds)

The ESR spectra of irradiated chestnuts having three different radiation doses, 3 days after irradiation, as well as the spectrum of a non-irradiated sample are shown in Figure 1a. There is no difference in the shape of the spectra between the samples taken from the shell seeds and peeled seeds. Figure 1b shows the EPR spectra at the same scale of the same samples, but 250 days after irradiation and multiplied by two (except for non-irradiated samples). As can be seen, the spectra consist of a central line and two satellite lines and they are stable within the time. The only changeable parameter is the intensity of the line. The time dependence decay of the central and the satellite lines for each dosage is shown in Figure 2 and Figure 3, respectively.

3.1.2. Features of EPR Spectra of Horse Chestnut Extracts

The spectra of non-irradiated PS and SS horse chestnut extracts exhibit a narrow asymmetric EPR signal at g = 2.00 and ∆H = 0.359 mT, whose intensity slightly decreases after irradiation, while the value of the g-factor did not change. The same signal was observed in other cellulose-containing products from plant origin, like rosehip seeds [19]. According to the literature data, this g-value is very close to those of carbon-centered radicals [20]. Since Aesculus hippocastanum L. contains compounds having antioxidant activity, this suggests the possibility that the recorded signal is due to radicals formed as a result of scavenging activities [21]. Lignin, which is a heterogeneous, natural polymer involved in the construction of cell walls in plants could react with a DPPH• molecule (used to detect antiradical activity). It is supposed that lignin captures and stabilizes DPPH radicals [22], and during the process, stable C-centered radicals are being formed.

3.1.3. Antiradical Activity Assay

The antiradical activity of non-irradiated and irradiated PS and SS samples was evaluated by EPR using the stable DPPH radical due the transfer of labile hydrogen atoms to it. The method is based on the transfer of a labile H atom to the radical in the presence of substances with radical scavenging ability, and as a result the amount, respectively the EPR signal intensity of the DPPH, decreases, which is proportional to the radical scavenging activity of the tested sample. The relationship between the percentages of scavenged DPPH radicals and the volume of non-irradiated and irradiated peeled and shelled horse chestnut seed extracts is represented in Figure 4. The experimental results of the PS samples show that FRSA was reduced upon increasing the irradiation dose, as in the samples irradiated by 10 kGy, the decrease is approximately 15%. The antiradical activity in the samples irradiated by 1 and 5 kGy decreases by approximately 7%. The opposite results concerning FRSA were observed for shelled seeds. As the radiation dose increased, the FRSA was enhanced, with the highest value at 5 kGy (the increase was 11%). In addition, the observed antiradical activity of SS horse chestnut samples was significantly higher than that of PS samples.

The calculated values of Trolox equivalent antioxidant capacity for PS and SS samples confirmed these experimental data. The data are represented in Table 1.

3.2. Spectrophotometric Study

3.2.1. Total Phenolic and Flavonoid Content Assay

The obtained data for TPC and TFC of shell seeds and peeled seeds samples are represented in

Table 2. Summarized data from spectrophotometric analysis: TEAC, TPC, and TFC of non-irradiated and irradiated by 1, 5, and 10 kGy peeled (PS) and shell seed (SS) horse chestnut samples.

Dose (kGy)	Sample	TEAC 1 (μg/mL)	TPC 2 (mg GAE 4/mL)	TFC 3 (mg QE 5/mL)	TFC 3 (mg CE 6/mL)
0	PS	5.176 a ± 0.002	0.183 a ± 0.002	1.458 a ± 0.02	0.699 a ± 0.004
1	PS	5.160 b ± 0.006	0.169 a,b ± 0.005	1.513 b ± 0.02	0.684 b ± 0.004
5	PS	5.034 c ± 0.002	0.174 a ± 0.005	1.505 b ± 0.006	0.725 c ± 0.001
10	PS	4.721 d ± 0.002	0.161 b ± 0.002	1.362 c ± 0.01	0.628 d ± 0.006
0	SS	56.770 a ± 0.01	1.272 a ± 0.007	0.228 a ± 0.003	9.895 a ± 0.05
1	SS	57.870 b ± 0.04	1.363 b ± 0.01	0.295 b ± 0.003	10.228 a ± 0.01
5	SS	61.000 c ± 0.02	1.521 c ± 0.02	0.323 c ± 0.001	11.567 b ± 0.02
10	SS	55.990 d ± 0.03	1.397 d ± 0.01	0.252 d ± 0.004	10.762 c ± 0.05

¹ Trolox equivalent antioxidant capacity; ² Total phenolic content; ³ Total flavonoid content; ⁴ gallic acid equivalents; ⁵ quercetin equivalents; ⁶ catechin equivalents. Each value of PC and FC is represented as arithmetic mean ± SD, n ≥ 3. Different superscript letters (a–d) within each column indicate significant differences between values (p < 0.05).

. A slight decrease in TPC values of peeled seeds was observed after irradiation, which was the most visible at 10 kGy, while in shell seeds the values increased, and the most significant growth was observed after irradiation by 5 kGy. Regarding the TFC results, in the case of QE after irradiation of the peeled seeds by doses of 1 and 5 kGy, the values increased, while at 10 kGy, the value decreased. The TFC assay of peeled seeds according to CE shows a decrease of the obtained values after irradiation by doses of 1 and 10 kGy with the exception of the sample irradiated by 5 kGy, where the value increased. In comparison to non-irradiated samples, TFC results, according to QE and CE of gamma treated shell seeds, exhibit an increase of the obtained values as the most enhancements were observed at the sample irradiated by a dose of 5 kGy. The general trend observed is that the 5 kGy irradiated samples show the highest values of TPC and TFC.

3.2.2. Trolox Equivalent Antioxidant Capacity Assay

The results obtained from the Trolox equivalent antioxidant capacity assay show that in comparison to non-irradiated samples of peeled seeds in the irradiated ones, upon increase of the dose, the TEAC decreased. A different effect was observed in shell seeds. There, after gamma treatment by doses of 1 and 5 kGy, their antioxidant capacity increased, whereas in the samples irradiated by 10 kGy, the values of TEAC slightly decreased (Table 2).

3.3. HPLC Analysis

The experimental data of the concentration of peeled and shell chestnut seeds before and after irradiation by 1, 5, and 10 kGy gamma rays are represented in

Table 3. Concentration of individual phenolic compounds in non-irradiated and irradiated peeled (PS) and shell seed (SS) horse chestnut samples.

	Sample C 3	PS 1 0 kGy	PS 1 1 kGy	PS 1 5 kGy	PS 1 10 kGy	SS 2 0 kGy	SS 2 1 kGy	SS 2 5 kGy	SS 2 10 kGy
(μg/mL)
Catechin		0.45 a ± 0.02	3.19 b ± 0.12	3.28 b ± 0.13	143.02 c ± 5.46	641.47 a ± 24.50	594.53 a ± 22.71	585.55 a,b ± 22.37	0.52 c ± 0.02
Chlorogenic acid		6.04 a ± 0.25	5.14 b ± 0.22	7.21 c ± 0.30	8.55 d ± 0.36	9.52 a ± 0.40	7.21 b ± 0.30	7.92 c ± 0.33	35.89 d ± 1.51
Caffeic acid		74.72 a ± 3,27	49.39 b ± 2.16	57.52 c ± 2.52	45.27 b ± 1.98	<LQ a	690.06 b ± 30.22	<LQ a	644.68 b ± 28.24
p-Coumaric acid		2.84 a ± 0.11	14.46 b ± 0.57	2.97 a ± 0.12	2.13 c ± 0.08	<LQ a	<LQ a	27.66 b ± 1.10	227.90 c ± 9.05
Ferulic acid		0.41 a ± 0.02	0.31 b ± 0.01	0.42 a ± 0.02	0.27 c ± 0.01	<LQ a	<LQ a	0.95 b ± 0.04	<LQ a
Rutin		185.94 a ± 7.96	185.74 a ± 7.95	202.93 a ± 8.69	185.40 a ± 7.94	4285.08 a ± 183.40	2564.87 b ± 109.78	1742.38 c ± 74.57	897.66 d ± 38.42
Hesperidin		7.11 a ± 0.31	8.49 b ± 0.37	8.51 b ± 0.37	8.20 b ± 0.35	<LQ a	<LQ a	1425.30 b ± 61.72	<LQ a
Morin		<LQ a	0.77 b ± 0.03	0.66 c ± 0.03	<LQ a	<LQ a	<LQ a	0.70 b ± 0.03	1.92 c ± 0.08
Luteolin		<LQ a	<LQ a	<LQ a	1.97 b ± 0.08	1.22 a ± 0.05	1.06 b ± 0.05	0.38 c ± 0.02	0.50 d ± 0.02
Quercetin		4.77 a ± 0.21	4.82 a ± 0.21	5.30 a ± 0.23	1.53 b ± 0.07	1.73 a ± 0.08	1.35 b ± 0.06	2.08 c ± 0.09	1.45 b ± 0.06

¹ Peeled seeds; ² Shell seeds; ³ Concentration (μg/mL); LQ, limit of quantification; Each value of concentration is represented as mean value ± standard deviation (SD), n ≥ 3. Different superscript letters (a–d) on each line indicate significant differences between PS and SS values (p ≤ 0.05).

. The results showed the presence of rutin and caffeic acid in significant amounts in the peeled seeds. Hesperidin, chlorogenic acid, and quercetin in moderate amounts were also found. Catechin, p-coumaric acid, and ferulic acid were determined only in small or trace amounts. According to the literature data, the phenolic fraction of the seed kernel extracts of horse chestnut was constituted mainly of O-glycosylated quercetin, kaempferol, and isorhamnetin derivatives, but caffeic acid and catechin derivatives were also present. The four aescin saponins were also detected [1]. In the current study it was established, after gamma irradiation, that the concentration of some of the detected substances changed significantly depending on the applied radiation dose. An increase in catechin concentration was observed after irradiation, most significantly at 10 kGy, opposite caffeic acid decreases. Quercetin and ferulic acid concentration decreased, while chlorogenic acid concentration was enhanced after 10 kGy irradiation. A significant increase of p-coumaric acid was observed after gamma treatment by 1 kGy. It is interesting to note that morin and luteolin were not detected in non-irradiated samples. However, after irradiation by 1 and 5 kGy, morin was detected, while after irradiation by 10 kGy, luteolin was detected.

Different concentrations of individual polyphenolic substances were observed in the shell seeds compared to the peeled seeds (Table 3). Significant amounts of rutin and catechin were detected in shell chestnut seeds, many times higher than those in PS. Chlorogenic acid, luteolin, and quercetin were detected in moderate and small amounts in SS. The absence of hydroxycinnamic acids (caffeic acid, p-coumaric acid, and ferulic acid), hesperidin, and morin were observed in the non-irradiated SS samples, while after irradiation, depending on the dose of high-energy radiation, these substances were detected in some of the samples. Ferulic acid, p-coumaric acid, hesperidin, and morin were detected in the samples irradiated by 5 kGy. Moreover, caffeic acid, p-coumaric acid, and morin were detected in SS after 10 kGy irradiation.

In the literature it was reported that horse chestnut seed shell is rich in polyphenolic components, including dimers, trimers, tetramers, oligomers, and monomeric flavonols, such as catechins and epicatechin derivatives, as these substances determine the strong radical-scavenging activity detected for reactive oxygen species (ROS) induced by UVB irradiation [9]. In Japanese horse chestnut seed coats, flavonols, condensed tannins named proanthocyanidins and their glycosides were identified [8]. Kedzierski et al. [23] established the presence of two isomer groups of escin saponins, sterols, flavonoids—kaempferol and quercetin, glucosides, and epicatechin and its dimer procyanidin in whole chestnut seeds from trees growing in Poland. Rutin was the most abundant phenolic compound in Serbian chestnut samples from different regions in Serbia, as in our study of Bulgarian horse chestnut seeds. In addition, quercetin, kaempferol, proanthocyanidin, and coumarins (esculin and fraxin) were identified [24].

4. Discussion

4.1. Features of EPR Spectra of Solid Samples of Horse Chestnuts

Usually, a broad line at g = 2.004 appears before being exposed to radiation in most spices and herbs, as well as in other foods. It is commonly accepted that this strong signal is derived from semiquinone radicals, produced by oxidation of plant phenolic groups present in polyphenols or in lignin [25]. It is seen that the intensity of this signal increases significantly upon irradiation, however, it cannot be used to prove or disprove irradiation using high energy beams. Instead of that, the accepted protocols [26] use the pair of lines, which appear on both sides of the central peak after irradiation (see insets in Figure 1a). The protocol shows that a pair of EPR lines appears on both sides of the central signal in EPR spectra due to the cellulose radicals formed by ionizing radiation. According to the literature [27] these lines are a triplet with a 3 mT coupling constant arising from the interactions between the two hydrogen atoms at the C6 position of the glucose unit with the unpaired electron, formed by the removal of the hydrogen atom at the C5 position of the glucose unit by irradiation. Moreover, the central line of the triplet overlaps with the strong central peak. The signal is known as “cellulose-like”. Figure 1b shows the ESR spectra at the same scale of the same samples, but 250 days after irradiation and multiplied by two (except for non-irradiated samples). The intensities of the central peaks are very similar for all samples, however, the pair of lines upfield and downfield from the central line, which are indicative of irradiation, is still visible especially on the left part in the spectra (denoted by asterix). On the other hand, an additional pair of lines in the EPR spectrum of the chestnut sample irradiated with 10 kGy is observed (denoted by arrows in Figure 1a). This signal might be attributed to free radicals of starch, known as the “carbohydrate” spectrum [28]. The starch free radicals were not observed in the EPR spectra of 1 and 5 kGy irradiated chestnut samples, probably because of lower doses of irradiation. The results show that usually this signal is observed at higher doses. It is interesting that it is also not recorded in the EPR spectrum of 10 kGy irradiated sample storage under normal conditions for 250 days. The reason for this is the fact that the concentration of starch radicals decreased in the time. The kinetic evolution of the spectrum shows that it cannot be recorded after more than 3 weeks [29]. Three weeks after the irradiation, it is observed as a narrower signal which probably overlaps with the “cellulose-like” signal recorded in the chestnut. That is why it is not visible in the spectrum in Figure 1b.

4.2. Time Stability of the Radiation Induced Free Radicals Studied by EPR Spectroscopy

It is known that EPR spectroscopy can be applied for the identification of previous radiation treated products from plant origin (herbs, spices, nuts, etc.). This depends on the life time of the free radicals induced after irradiation. The storage conditions of the samples, as the temperature, humidity and access to light, have an impact on the shelf life of the radicals. It was discovered that at normal storage conditions the “cellulose-like” EPR signal can be observed within a period of 60–80 days after irradiation. However, in the current study this period is longer. Figure 2a exhibits the decay of the central EPR peak with time for the peeled seed samples irradiated by different doses, whereas Figure 2b shows the decay of the shell seeds. It was observed that in the first days after irradiation the intensity increases, and then begins to slowly decrease. Usually when some mechanical treatments are carried out on the samples, it leads to additional creation of free radicals. Very often the radicals are similar to these induced upon irradiation. The horse chestnuts samples were ground before gamma treatment. Probably, the grinding process generates the same kind of free radicals as these occurring in irradiated samples. The natural oxidation of chestnut ingredients, triggered by the exposure of fresh surfaces to atmospheric oxygen, also leads to formation of radicals. So, probably the reason for the observed increase in the intensity of the EPR signals in the first days is just that these radicals, which are more unstable in comparison to the radiation induced radicals, decay for several days. The results show that the EPR signal intensities of irradiated by 1, 5, and 10 kGy peeled seeds decreased with 84%, 86%, and 82%, respectively, for a period of 250 days. For the same time period the intensity of the signal of horse chestnut shells decreased with 35%, 42%, and 37% for 1, 5, and 10 kGy irradiated samples, respectively. It is interesting to mention that in the samples from peeled seeds the intensity of the line recorded at irradiation with 5 kGy is higher in comparison those at 10 kGy. This could be explained by the saturation of the free radicals at higher doses due to recombination. On the other hand, on the whole, higher quantities of free radicals are recorded with shell seeds than peeled seeds. The evidence is the more intensive EPR signal arising from cellulose in the EPR spectrum. Also, in the shell samples, the saturation of the EPR signal at 10 kGy of irradiated samples is not observed. The different behavior of the EPR signal with various kinds of horse chestnut samples (peeled seeds and shell) is probably due to their different composition and water content.

Figure 3 shows the decay over time of the two satellite peaks attributed to the cellulose radical formed upon irradiation. As it was mentioned before, these are the peaks used to prove that cellulose-containing foods were subjected to high energy ionizing radiation. After the irradiation, the signal intensity decays exponentially with time. For the samples irradiated by 1 kGy, the signal becomes difficult to detect 45 days after the irradiation in both case (peels seeds and shell seeds). For this time period the EPR signal intensity of the samples irradiated by 1 kGy decreased with 85% for peeled seeds and 73% for shell seeds. The samples irradiated by 5 and 10 kGy gamma rays show higher stability. Their intensity decreased with 82% and 71% for peeled seeds and 91% and 92% for shell seeds, respectively, but for the 250 days period.

On the basis of the obtained results, it can be concluded, that for time periods longer than 250 days after irradiation, the identification of the radiation treatment of horse chestnut is very difficult because of the disappeared “satellite-lines” of the “cellulose-liked” EPR spectrum. For the samples irradiated by lower doses (at this case 1 kGy), identification of previous irradiation is not possible for a time period more than 45 days.

4.3. Gamma Irradiation Effect on Polyphenolic Content

Polyphenol compounds are common secondary metabolites in plants. They are produced in plants as a response to biotic and abiotic environmental stress conditions. The bioactive compounds, having antioxidant properties present in horse chestnut seeds, could vary depending on their genotype, geographical and environmental conditions, used part of the seed (coat, endosperm, or whole seed), treatment of the sample, extraction procedure, and used solvent, etc. [1,7,8,24]. In previous study, the dependence of total phenolic content and DPPH radical scavenging capacity on the maturation process of the seeds was determined [23]. However, the exert of gamma irradiation on polyphenolic content has not been studied. The present investigation shows that the effect of gamma irradiation depends on the absorbed dosage of high-energy radiation and also on the type of sample. The observed slight decrease in TPC of irradiated peeled seeds is probably due to the degradation of some phenolic compounds compared to those with a non-phenolic structure or with tannins, decomposed as a result of cleavage of glycosidic bonds by the gamma rays [30,31]. Some studies also described the negative effect of irradiation on the polyphenol content of irradiated products [31].

The opposite effect is observed in the shell seeds, as the phenolic content increases, most significantly after irradiation by 5 kGy. The probable reason for it is that more polyphenolic substances are being formed upon irradiation as a result of the breakdown of some larger phenolic molecular structures into smaller ones. A similar behavior of enhancement of phenolic content and antioxidant capacity after irradiation of almond skin extracts was reported by Harrison and Were [32]. On the other hand, in defatted almonds irradiated by 10 and 25 kGy, an increase in total polyphenols was observed only at 25 kGy [11].

Significant differences were found in polyphenolic compounds between peeled and shell chestnut seeds by HPLC analysis. The plant seed is a multicomponent system in which substances interact with each other as a result of the absorbed additional energy, in the case of the gamma radiation. In the process of irradiation, when a certain amount of ionizing energy is introduced into the system, some polyphenolic structures are degraded with the intermediate participation of free radicals. It is possible that new phenolic compounds could be formed as a result of the degradation of higher molecular ones or to be formed as a result of the recombination of radical fragments, depending on the applied radiation dose. This is the case for the formation of morin and luteolin after irradiation of PS by certain doses of high-energy radiation, as well as for caffeic acid, p-coumaric acid, ferulic acid, hesperidin, and morin in SS.

4.4. Gamma Irradiation Effect on Antiradical Capacity

Previous studies of plant products established a relationship between the content of phytochemicals in the samples and the antioxidant properties which they exhibit. In this study, a significant correlation was also established between the data on the total phenolic and flavonoid content and the antiradical activity exhibited by the tested non-irradiated and irradiated samples of peeled and shell chestnut seeds. This indicates that the major contribution to the antioxidant activity of horse chestnut has its polyphenolic content.

The experimental results from the current study show that the radical scavenging activity of both kinds of horse chestnut samples (peeled and shell seeds) differs and also the irradiation effect on them, obtained by both EPR and spectrophotometric measurements. Other researchers also reported a higher amount of phenolic substances in A. hippocastanum seed shell extract, and which exhibited higher DPPH radical-scavenging activity than peeled seed extract [8,9].

The generation of free radicals is one of the main effects of gamma irradiation because of the high energy ionizing rays releasing electrons from molecules. These radiation induced free radicals could cause structural and chemical changes on the irradiated samples. Furthermore, irradiation effects are strongly dependent on the chemical structure of the sample, the amount of compounds having antioxidant properties and their activity (i.e., polyphenolic compounds), and on the applied dose of high-energy radiation [33]. On the other hand, antioxidant molecules found in plant extracts could scavenge the gamma-generated radicals and, in the course of the process, they become exhausted. The combination of these factors will influence the gamma radiation behavior, the changes of composition, and the biological activity of the A. hippocastanum seeds.

In this relation, a slight decrease in the TEAC values in peeled seeds was found after irradiation (see Section 3.1.3 and Section 3.2.1). As mentioned, the polyphenolic composition is mainly responsible for the antiradical ability of plant products. Although the amount of some of the individual polyphenolic components increased according to the HPLC analysis after gamma processing of PS, a decrease in total phenolics was observed. The reduction of total phenolics and flavonoids was most noticeable for samples irradiated by 10 kGy. Probably, this is the reason for the observed greatest decrease in FRSA after irradiation of PS at this dose. The significant decreases in concentration of quercetin, which is a strong free radical scavenger [34], and also in caffeic and ferulic acids are probably responsible for reduced scavenging activity at 10 kGy for irradiated peeled chestnut seeds. On the other hand, gamma generated free radicals from the degradation of cellulose and starch in chestnut seeds could be reducing the antioxidant activity, since the phenolic compounds can take part in the process of free radicals inactivation.

The positive effect of gamma irradiation on antiradical activity of chestnut shell seeds was established in our study, most pronounced in the 5 kGy irradiated samples. This result is in agreement with the observed increase in both the total phenolic and flavonoid content of SS. Probably, the reasons are complex for enhanced free radical scavenging activity after gamma treatment. It is known that the radiation induced free radicals could lead to the breakage of some glycosidic bonds in the polyphenolic constituents, resulting in the formation of more compounds having antioxidant ability. The various phenolic derivatives possess different contributions to antioxidant activity [34]. In the case of irradiated shell chestnut seeds, probably the presence of p-coumaric acid, caffeic acid, and morin (see Section 3.3) are responsible for enhanced FRSA compared to non-irradiated samples (these compounds were not available in non-irradiated SS). It was reported that morin, p-coumaric acid, and caffeic acid, which is a metabolite of caffeoylquinic acid (with FRSA equivalent to those of ascorbic acid and Trolox), exhibit strong DPPH radical scavenging activity [34,35,36]. Moreover, ferulic acid and a significant amount of hesperidin were detected in 5 kGy irradiated SS. These compounds could contribute to the highest TEAC values obtained here and for the strong radical-scavenging activity. Ferulic acid (FA) is the most abundant hydroxycinnamic acid, followed to a lesser extent by coumaric acid found in plant cell walls, which are covalently linked to polysaccharides and lignin. Probably FA was bound on the cell wall matrix of shell seeds and after application of 5 kGy dosage gamma radiation, the bond between FA and α-L–arabinofuranose in hemicellulose released FA and degraded the crosslinking between hemicellulose and lignin [37]. As a result of irradiation with certain doses of radiation, the release of various hydroxycinnamic acids (HCAs) from the biomass of shell chestnut seeds was observed. The current study showed that hydroxycinnamic acids have the potential to provide a significant source of antioxidant characteristics. A wide range of lignin depolymerization approaches have been investigated including enzymatic, catalytic, thermal, and alkaline pretreatment, but information on the irradiation application for this purpose was not found in the literature [38]. These are the preliminary investigations on the application of gamma irradiation as a method for partial degradation of lignin and the release of biologically active HCAs from plant biomass, used as an excellent renewable resource for phytochemicals possessing bioactive properties.

On the other hand, other studies reported increase of the amount of reducing sugars and better antioxidants extractability from the plant source during irradiation [39].

5. Conclusions

In this study it was determined that gamma irradiation leads to the formation of a larger amount of radicals in shell chestnut seeds than in peeled seeds. The EPR signal recorded in irradiated horse chestnut can be used as unambiguous evidence that the samples have been irradiated. It is in accordance with the CEN protocol EN 1787 for detection of irradiated food containing cellulose. On the other hand, the signal is stable, and it can be found in the samples irradiated with a dose of 1 kGy, 45 days after treatment, whereas for the samples irradiated by 5 and 10 kGy it can be found even 250 days later. Regarding the evaluation of the effect of gamma-irradiation of Bulgarian horse chestnut seeds on their polyphenolic content and free radical scavenging activity, significant correspondence was established between them. Gamma treatment has a positive effect on phenolic content and antioxidant capacity of shell seeds, while the effect was negative for peeled seeds. Results showed that ionizing rays induced changes in phenolic content and the antiradical capability of PS and SS, depending on the dose. No clear tendency was found with changing the content of the different phenolic compounds by varying the dosage. However, the general trend was an increase in TPC and TFC or without any change after irradiation of horse chestnut seeds by 5 kGy.

Since shell chestnut seeds exhibited strong antioxidant capability, the extraction of major polyphenolic compounds from this plant could be important for the development of value-added products from renewable by-products. In conclusion, gamma irradiation could be used as a potential method for disinfection of horse chestnut seeds on the one hand, and on the other hand for a partial delignification process of plants for recovering hydroxycinnamic acids as a value-added product from the lignin.