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Electron spin resonance of free radicals in some legumes, cereals and their aqueous solutions under photolysis
Rodior.Phys. Chem. Vol. 38, No. 1, pp. 17-21, 1991 Int. J. Radiat. Appt. Instrum., Part C Printed in Great Britain. All rights reserved
OM-5724/91 $3.00+ 0.00
Copyright 0 1991 F’crgamon Press pk
ELECTRON SPIN RESONANCE OF FREE RADICALS IN SOME LEGUMES, CEREALS AND THEIR AQUEOUS SOLUTIONS UNDER PHOTOLYSIS S. CAKIR, F. K~KSAL, R. TAPRAMAZand 0. CAKIR Ondokuz Mayis University, Faculty of Arts and Sciences, Samsun, Turkey
(Received 25 September 1990)
Abstrae-In this study free radicals produced by U.V.photolysis in some legwnes and cereals have been investigated by electron spin resonance (ESR) technique. The ESR spectra of small grains and Powdered legumes and cereals samples have been investigated at room temperature before and during photolysis. Before photolysis barley, wheat, rye, oat and lentil samples exhibit the well known spectra of Mn2+ ions in addition to a central signal of g = 2.0045which we attributed to the melanin radical. The melanin signal has been observed also more clearly, in the samples of bean, chick-pea and maize shells before photolysis. The melanin signal has been observed during photolysis in wheat, barley, oat, ma&, rice, bean, lentil and chick-pea samples at room temperature. Furthermore, it has been observed that the aqueous solutions of all the cereals and legumes samples investigated in this study gave HCO and CO; radicals at 123 K under photolysis. It has been shown that HCO and CO,- radicals originate from the glucose molecules in the carbohydrate chains of these samples.
INTRODUCTION
main components of foodstuffs (Raffi et al., 1981; Matsuyama et al., 1988; Ramarathnam et al., 1989; Toyo’oka et al., 1989). But most of the studies do not report any chemical structure which are responsible for the recorded ESR spectra. Therefore, we have undertaken this study to investigate photolysis products of some cereals and legumes seeds.
Ionizing radiation may affect the compositions of foodstuffs as well as their sensorial properties like colour, taste and odour in negative manner. These can be exemplified as irradiated peaches become dense red depending on the carotenoid antocyanin production rate, and vegetables like lettuces and cabbages become abnormally pale (Urbain, 1973), and green tomatoes become red (Bums and Desrosier, 1957). Furthermore, ionizing radiation increases some enzyme activities as well as speeding metabolic phenomena. The negative effect of ionizing radiation is not limited to changes of sensorial properties of foodstuff, since depending on the deterioration of proteins, lipids, carbohydrates, enzymes and vitamins in foodstuffs, it can initiate a series of chemical reactions and cause undesired changes (Taub, 1984). Free radicals, produced by the interaction of ionizing radiation with foodstuffs, are reactive and by making reactions with other components of foodstuffs they produce undesired new species. ESR is the most convenient method for investigating irradiation products as they are mostly unpaired spin carrying free radicals (Kiiksal and Yiiksel, 1975; Kiiksal and Celik, 1988; Kiiksal et al., 1989; Kiiksal et al., 1990). During the last years ESR spectroscopy has been used in the tests of irradiation (Rat% and Agnel, 1989) and in the determination of dosages in foodstuffs (Desrosiers and Shnic, 1988; Desrosiers, 1989), in the determination of the effect of irradiation on sprouts times of cereals and legumes (Hepburn et al., 1986; Ramarathnam et al., 1987) and in the investigation of the effects of irradiation on the RPC 38,1-B
EXPERIMENTAL
All the cereals and the legumes samples in this study are in domestic origin and were obtained from markets, Small grains, powders and their partly dissolved aqueous solutions of the samples were used in ESR tubes. The ESR spectra were recorded before and under U.V. photolysis at room temperature and for the aqueous samples at 123 K. The spectrometer used was a Varian E 109 C ESR spectrometer equipped with a Varian temperature control unit at our faculty. The U.V. source used was a Conrad Hanovia 1 kW xenon lamp. The g factors were determined by comparison with a DPPH sample (g = 2.0036). RESULTS AND DIWURSION
The ESR spectra of barley, wheat, oat, lentil and rye exhibit the ESR lines of Mn2+ ions superimposed with a central signal of g = 2.0045 as in Fig. 1 at room temperature before U.V. photolysis. However, the shell parts of maize, bean and chick-pea exhibit only the central signal as shown in Fig. 2, and rice samples do not give any ESR signal before photolysis 17
S.
18
rJ
,‘I
CA-
et al.
IA)
’ IA J\
Fig. 1. ESR spectra of rye (A), barley (B), wheat (C), lentil (D), oat (E), under photolysis (-) photolysis (---) respectively at room temperature.
at room temperature. Under U.V. photolysis all the samples (barley, wheat, oat, rye, maize, rice, bean, lentil, chick-pea) gave the central signal with increased intensity at room temperature as shown in Figs 1 and 2. The g factors were found to be the same 2.0045, in the limits of experimental errors of +0.0005 as given in Table 1. This signal is similar to the ones obtained in melanin type of compounds (Hepburn et al., 1986; Jahan et al., 1987). The seeds of cereals are lively biological systems and include carbohydrates, lipids, proteins and enzymes. Therefore the sextet signals in Fig. 1 are the characteristic for MnZf ions which is known to appear as a trace element in enzymes. Almost all proteins contain 26% tyrosine, which produces dark colour unsaturated polymer products
and before
called melanin by the effect of phenyloxidase enzyme over dihidroxyphenyl-alanine (DOPA, 3-hydroxythrosine). On the other hand since the U.V. light and ionizing radiation increase the melanin type of compounds in foodstuffs (Commoner et al., 1954) increase of the central signal is in agreement with photochemical increase in melanine concentration. According to the ESR study by Hepburn and his coworkers on free radical contents of seeds by the signals of Mn*+ ions and a central signal have been observed and identified as melanin radical. Therefore our results are in agreement with that study. Furthermore, the aqueous solutions of all the samples in this study have been investigated at 123 K under U.V. photolysis. For wheat, rye, lentil, rice, maize, bean and chick-pea samples the spectra shown
ESR of free radicals in legumes, cereals and their aqueous solutions
(___)
I-_)
19
z
et
I
i
\ \-“’ /\ \
\ \
f
1.
I\
WI
.
~ Fig. 2. ESR spectra of rice (A), bean (B), chick-pea (C), maize. (D) under photolysis (-) photolysis (---) respectively at room temperature. in Fig. 3 have been obtained. When the temperature is raised the mid signal disappears leaving only a doublet. The separation between the lines of the doublet is a = 125 gauss and the g = 2.000. For the singlet g = 2.003. Therefore we attribute the doublet to HCO radical and the singlet to CO; radical since these values are in agreement with the data reported early workers (Miyagawa and Gordy, 1961; Atkins et al., 1962). Table I. The g valuer for the single lines of some photolysed, and unphotolyscd cereals and legumes samples at room temperature spccimcn
%t3T oat Rye
Maize Rice &an Chick-pea Lentil
Photolwd 2.0046 :%z 2:ooM 2.0045 2.0042 2.0042 2.0046
In order to test whether the origins of these radicals are carbohydrates, which are included 60-70% in cereal seeds, we photolysed aqueous glucose at 123 K and obtained the same signals principally as shown in Fig. 4. The a and g values of these spectra are given in Table 2. Similar experiments were also performed with pure starch, lactose, maltose and fructose samples and any signals belonging to HCO radical Table 2. ESR parameters for He0 and CO, radic& in some cereals, legumes and ~~ICOSC samples under photolysis at 123K Doublet Specimen
Radical
D (gauss)
uUDhot&%ed 2.0046 2.0044 2.0049 2.0046 2.0052 2.0051 2.0045 2.0047 2.0049
and before
Singlet g
whcai oat
Hz0
130*2 -
He0 He0 HtO
122i4 130*2 127zt2
RYC Maize
Rice &all
Chick-pea LAMi Glucose
2.009 2.0018 2.ooo5 2.0019 2.0005 1.9995 1.9991 2.0020
Radical
B
-
co;
2.0025
co,COi co;
2.0018 2.0030 2.0025 2.0030
co;
20
S. Cam
et al.
(8)
” 100 t-l
(G)
4-r ‘“H”
Fig. 3. ESR spectrum of aqueous chick-pea (A), rye (B), wheat (C), maize (D), rice (E), bean (F), lentil (G) solution under photolysis at 123K. could not be obtained. However, in the hydrolysed lactose samples to glucose and glactose weak signals belonging to the He0 radical are obtained. Therefore, we state that the origins of He0 and CO;
Table 3. ESR parametersfor He0 and CO; radicals
Specimen Form? a+ ~~~~~ z$ Tin oxsalate Potassium formate
Radical
co;
I&) a(gauss) Ref. 126.0 (Holmerg, 1969) 2.002 133.0 (Brivati ef al., 1962) 135.0 (Miyagawa and Gordy, 1961) 2.0022 (Atkins e? al., 1962)
co;
2.0048
He0 He0 HCO
-
(Atkins ef al., 1962)
radicals are glucose molecules. It can also be stated that the free aldehyde group in glucose molecule is effective in forming He0 radical in solution. Since in solid glucose the carbonyl group is not free and therefore solid glucose do not give any ESR signal under U.V. photolysis, but there might be factors other than molecular structure which may influence the radical formation (Table 3). study was supported partly by the Research Fund of Ondokuz Mayls University.
Acknowledgement-This
REFERENCES Fig. 4. ESR spectrum of aqueous glucose solution under photolysis at 123 K.
Atkins P. W., Keen N. and Symons M. C. R. (1962) J. Chem. Sot. 2873.
ESR of free radicals in legumes, cereals and their aqueous solutions Brivati J. A., Keen N. and Symons M. C. R. (1962) J. Chem. Sot. 231. Bums E. E. and Desrosier N. W. (1957) Food Tech. 11,313. Commoner B., Townsend J. and Pake G. E. (1954) Nature 174 689. Desrosiers M. F. (1989) J. Agric. Food Chem. 37, 96. Desrosiers M. F. and Simic M. G. (1988) J. Agric. Food Chem. 36,601. Hepburn H. A., Goodman B., McPhail D. B., Matthews S. and Powel A. A. (1986) J. Exn Botow 37, 1675. Hohnerg R. W. (1969) J.pChem.-Phys. Si, 3%. Jahan M. S., Drouin T. R. and Sayre R. M. (1987) Photo&m. Photobiol. 34, 891. Kiiksal F. and Yilksel H. (1975) Z. Nuturfirsch. 3Oa, 1044. KBksal F. and Celik F. (1988) J. Chem. Sot. Far&y Trans. 1. g4, 2305. Kiiksal F., Qlik F. and Qku 0. (1989) Rodiut. Pbys. Chem. 33, 135. K&al F., Birey M., Tapramaz R. and elik F. (1990) J. Molec. Structure 211, 51.
21
Matsuyama T., Menhofer H. and Heusinger H. (1988) Radiat. Phys. Chem. 32, 735. Miyagawa I. and Gordy W. (1961) J. Am. Chem. Sot. 83, 1036. Ra5 J. J. and Agnel J-P. L. (1989) Rudiut. Phys. Chem. 34, 891. I&l% J. J., Agnel J-P. L., Thiery C. J., Frejaville C. M. and Saint-Lebe L. R. (1981) J. Agric. Food Chem. 29, 1227. Ramarathnam N., Gsawa T., Kawakishi S. and Namiki M.’ (1987) J. Agric. Food Chem. 3S, 8. Ramacathnam N., Gsawa T., Namiki M. and Kawakishi S. (1989) JAOCS. 66, 105. Taub I. A. (1984) J. Chem. Educ. 61, 313. Toyo’oka T., Uchiyama S. and Saito Y. (1989) J. Agic. Food Cbem. 37, 769. Urbain W. M. (1973) Food Irradiution, Bmejits and Limitations. Factors Injbencing the Economical Application of Food Irradiation. Proc. of panel IAEA. Vienna, p. 101.
OM-5724/91 $3.00+ 0.00
Copyright 0 1991 F’crgamon Press pk
ELECTRON SPIN RESONANCE OF FREE RADICALS IN SOME LEGUMES, CEREALS AND THEIR AQUEOUS SOLUTIONS UNDER PHOTOLYSIS S. CAKIR, F. K~KSAL, R. TAPRAMAZand 0. CAKIR Ondokuz Mayis University, Faculty of Arts and Sciences, Samsun, Turkey
(Received 25 September 1990)
Abstrae-In this study free radicals produced by U.V.photolysis in some legwnes and cereals have been investigated by electron spin resonance (ESR) technique. The ESR spectra of small grains and Powdered legumes and cereals samples have been investigated at room temperature before and during photolysis. Before photolysis barley, wheat, rye, oat and lentil samples exhibit the well known spectra of Mn2+ ions in addition to a central signal of g = 2.0045which we attributed to the melanin radical. The melanin signal has been observed also more clearly, in the samples of bean, chick-pea and maize shells before photolysis. The melanin signal has been observed during photolysis in wheat, barley, oat, ma&, rice, bean, lentil and chick-pea samples at room temperature. Furthermore, it has been observed that the aqueous solutions of all the cereals and legumes samples investigated in this study gave HCO and CO; radicals at 123 K under photolysis. It has been shown that HCO and CO,- radicals originate from the glucose molecules in the carbohydrate chains of these samples.
INTRODUCTION
main components of foodstuffs (Raffi et al., 1981; Matsuyama et al., 1988; Ramarathnam et al., 1989; Toyo’oka et al., 1989). But most of the studies do not report any chemical structure which are responsible for the recorded ESR spectra. Therefore, we have undertaken this study to investigate photolysis products of some cereals and legumes seeds.
Ionizing radiation may affect the compositions of foodstuffs as well as their sensorial properties like colour, taste and odour in negative manner. These can be exemplified as irradiated peaches become dense red depending on the carotenoid antocyanin production rate, and vegetables like lettuces and cabbages become abnormally pale (Urbain, 1973), and green tomatoes become red (Bums and Desrosier, 1957). Furthermore, ionizing radiation increases some enzyme activities as well as speeding metabolic phenomena. The negative effect of ionizing radiation is not limited to changes of sensorial properties of foodstuff, since depending on the deterioration of proteins, lipids, carbohydrates, enzymes and vitamins in foodstuffs, it can initiate a series of chemical reactions and cause undesired changes (Taub, 1984). Free radicals, produced by the interaction of ionizing radiation with foodstuffs, are reactive and by making reactions with other components of foodstuffs they produce undesired new species. ESR is the most convenient method for investigating irradiation products as they are mostly unpaired spin carrying free radicals (Kiiksal and Yiiksel, 1975; Kiiksal and Celik, 1988; Kiiksal et al., 1989; Kiiksal et al., 1990). During the last years ESR spectroscopy has been used in the tests of irradiation (Rat% and Agnel, 1989) and in the determination of dosages in foodstuffs (Desrosiers and Shnic, 1988; Desrosiers, 1989), in the determination of the effect of irradiation on sprouts times of cereals and legumes (Hepburn et al., 1986; Ramarathnam et al., 1987) and in the investigation of the effects of irradiation on the RPC 38,1-B
EXPERIMENTAL
All the cereals and the legumes samples in this study are in domestic origin and were obtained from markets, Small grains, powders and their partly dissolved aqueous solutions of the samples were used in ESR tubes. The ESR spectra were recorded before and under U.V. photolysis at room temperature and for the aqueous samples at 123 K. The spectrometer used was a Varian E 109 C ESR spectrometer equipped with a Varian temperature control unit at our faculty. The U.V. source used was a Conrad Hanovia 1 kW xenon lamp. The g factors were determined by comparison with a DPPH sample (g = 2.0036). RESULTS AND DIWURSION
The ESR spectra of barley, wheat, oat, lentil and rye exhibit the ESR lines of Mn2+ ions superimposed with a central signal of g = 2.0045 as in Fig. 1 at room temperature before U.V. photolysis. However, the shell parts of maize, bean and chick-pea exhibit only the central signal as shown in Fig. 2, and rice samples do not give any ESR signal before photolysis 17
S.
18
rJ
,‘I
CA-
et al.
IA)
’ IA J\
Fig. 1. ESR spectra of rye (A), barley (B), wheat (C), lentil (D), oat (E), under photolysis (-) photolysis (---) respectively at room temperature.
at room temperature. Under U.V. photolysis all the samples (barley, wheat, oat, rye, maize, rice, bean, lentil, chick-pea) gave the central signal with increased intensity at room temperature as shown in Figs 1 and 2. The g factors were found to be the same 2.0045, in the limits of experimental errors of +0.0005 as given in Table 1. This signal is similar to the ones obtained in melanin type of compounds (Hepburn et al., 1986; Jahan et al., 1987). The seeds of cereals are lively biological systems and include carbohydrates, lipids, proteins and enzymes. Therefore the sextet signals in Fig. 1 are the characteristic for MnZf ions which is known to appear as a trace element in enzymes. Almost all proteins contain 26% tyrosine, which produces dark colour unsaturated polymer products
and before
called melanin by the effect of phenyloxidase enzyme over dihidroxyphenyl-alanine (DOPA, 3-hydroxythrosine). On the other hand since the U.V. light and ionizing radiation increase the melanin type of compounds in foodstuffs (Commoner et al., 1954) increase of the central signal is in agreement with photochemical increase in melanine concentration. According to the ESR study by Hepburn and his coworkers on free radical contents of seeds by the signals of Mn*+ ions and a central signal have been observed and identified as melanin radical. Therefore our results are in agreement with that study. Furthermore, the aqueous solutions of all the samples in this study have been investigated at 123 K under U.V. photolysis. For wheat, rye, lentil, rice, maize, bean and chick-pea samples the spectra shown
ESR of free radicals in legumes, cereals and their aqueous solutions
(___)
I-_)
19
z
et
I
i
\ \-“’ /\ \
\ \
f
1.
I\
WI
.
~ Fig. 2. ESR spectra of rice (A), bean (B), chick-pea (C), maize. (D) under photolysis (-) photolysis (---) respectively at room temperature. in Fig. 3 have been obtained. When the temperature is raised the mid signal disappears leaving only a doublet. The separation between the lines of the doublet is a = 125 gauss and the g = 2.000. For the singlet g = 2.003. Therefore we attribute the doublet to HCO radical and the singlet to CO; radical since these values are in agreement with the data reported early workers (Miyagawa and Gordy, 1961; Atkins et al., 1962). Table I. The g valuer for the single lines of some photolysed, and unphotolyscd cereals and legumes samples at room temperature spccimcn
%t3T oat Rye
Maize Rice &an Chick-pea Lentil
Photolwd 2.0046 :%z 2:ooM 2.0045 2.0042 2.0042 2.0046
In order to test whether the origins of these radicals are carbohydrates, which are included 60-70% in cereal seeds, we photolysed aqueous glucose at 123 K and obtained the same signals principally as shown in Fig. 4. The a and g values of these spectra are given in Table 2. Similar experiments were also performed with pure starch, lactose, maltose and fructose samples and any signals belonging to HCO radical Table 2. ESR parameters for He0 and CO, radic& in some cereals, legumes and ~~ICOSC samples under photolysis at 123K Doublet Specimen
Radical
D (gauss)
uUDhot&%ed 2.0046 2.0044 2.0049 2.0046 2.0052 2.0051 2.0045 2.0047 2.0049
and before
Singlet g
whcai oat
Hz0
130*2 -
He0 He0 HtO
122i4 130*2 127zt2
RYC Maize
Rice &all
Chick-pea LAMi Glucose
2.009 2.0018 2.ooo5 2.0019 2.0005 1.9995 1.9991 2.0020
Radical
B
-
co;
2.0025
co,COi co;
2.0018 2.0030 2.0025 2.0030
co;
20
S. Cam
et al.
(8)
” 100 t-l
(G)
4-r ‘“H”
Fig. 3. ESR spectrum of aqueous chick-pea (A), rye (B), wheat (C), maize (D), rice (E), bean (F), lentil (G) solution under photolysis at 123K. could not be obtained. However, in the hydrolysed lactose samples to glucose and glactose weak signals belonging to the He0 radical are obtained. Therefore, we state that the origins of He0 and CO;
Table 3. ESR parametersfor He0 and CO; radicals
Specimen Form? a+ ~~~~~ z$ Tin oxsalate Potassium formate
Radical
co;
I&) a(gauss) Ref. 126.0 (Holmerg, 1969) 2.002 133.0 (Brivati ef al., 1962) 135.0 (Miyagawa and Gordy, 1961) 2.0022 (Atkins e? al., 1962)
co;
2.0048
He0 He0 HCO
-
(Atkins ef al., 1962)
radicals are glucose molecules. It can also be stated that the free aldehyde group in glucose molecule is effective in forming He0 radical in solution. Since in solid glucose the carbonyl group is not free and therefore solid glucose do not give any ESR signal under U.V. photolysis, but there might be factors other than molecular structure which may influence the radical formation (Table 3). study was supported partly by the Research Fund of Ondokuz Mayls University.
Acknowledgement-This
REFERENCES Fig. 4. ESR spectrum of aqueous glucose solution under photolysis at 123 K.
Atkins P. W., Keen N. and Symons M. C. R. (1962) J. Chem. Sot. 2873.
ESR of free radicals in legumes, cereals and their aqueous solutions Brivati J. A., Keen N. and Symons M. C. R. (1962) J. Chem. Sot. 231. Bums E. E. and Desrosier N. W. (1957) Food Tech. 11,313. Commoner B., Townsend J. and Pake G. E. (1954) Nature 174 689. Desrosiers M. F. (1989) J. Agric. Food Chem. 37, 96. Desrosiers M. F. and Simic M. G. (1988) J. Agric. Food Chem. 36,601. Hepburn H. A., Goodman B., McPhail D. B., Matthews S. and Powel A. A. (1986) J. Exn Botow 37, 1675. Hohnerg R. W. (1969) J.pChem.-Phys. Si, 3%. Jahan M. S., Drouin T. R. and Sayre R. M. (1987) Photo&m. Photobiol. 34, 891. Kiiksal F. and Yilksel H. (1975) Z. Nuturfirsch. 3Oa, 1044. KBksal F. and Celik F. (1988) J. Chem. Sot. Far&y Trans. 1. g4, 2305. Kiiksal F., Qlik F. and Qku 0. (1989) Rodiut. Pbys. Chem. 33, 135. K&al F., Birey M., Tapramaz R. and elik F. (1990) J. Molec. Structure 211, 51.
21
Matsuyama T., Menhofer H. and Heusinger H. (1988) Radiat. Phys. Chem. 32, 735. Miyagawa I. and Gordy W. (1961) J. Am. Chem. Sot. 83, 1036. Ra5 J. J. and Agnel J-P. L. (1989) Rudiut. Phys. Chem. 34, 891. I&l% J. J., Agnel J-P. L., Thiery C. J., Frejaville C. M. and Saint-Lebe L. R. (1981) J. Agric. Food Chem. 29, 1227. Ramarathnam N., Gsawa T., Kawakishi S. and Namiki M.’ (1987) J. Agric. Food Chem. 3S, 8. Ramacathnam N., Gsawa T., Namiki M. and Kawakishi S. (1989) JAOCS. 66, 105. Taub I. A. (1984) J. Chem. Educ. 61, 313. Toyo’oka T., Uchiyama S. and Saito Y. (1989) J. Agic. Food Cbem. 37, 769. Urbain W. M. (1973) Food Irradiution, Bmejits and Limitations. Factors Injbencing the Economical Application of Food Irradiation. Proc. of panel IAEA. Vienna, p. 101.