- Email: [email protected]
Scavenging capacities of pollen extracts from cistus ladaniferus on autoxidation, superoxide radicals, hydroxyl radicals, and DPPH radicals
Nutrition Research 22 (2002) 519 –526 www.elsevier.com/locate/nutres
Scavenging capacities of pollen extracts from cistus ladaniferus on autoxidation, superoxide radicals, hydroxyl radicals, and DPPH radicals Takeshi Nagaia,*, Reiji Inoueb, Hachiro Inoueb, Nobutaka Suzukia a
Department of Food Science and Technology, National Fisheries University, Yamaguchi 7596595, Japan b Inoue Bee Farm Inc., Hyogo 6693465, Japan Received 18 June 2001; received in revised form 15 November 2001; accepted 19 November 2001
Abstract The antioxidative abilities of pollen extracts were evaluated using lipid peroxidation model system. Ethanol-soluble fraction (ESF) was most active followed by hot-water fraction (HWF). These abilities of pollen extracts were higher than that of 5 mM ascorbic acid and were similar to that of 1 mM ␣-tocopherol. Superoxide-scavenging capacities were decreased in the order water-soluble fraction ⬎ HWF ⬎ ESF. ESF showed the highest hydroxyl radical scavenging ability among these samples. The pollen extracts showed DPPH radical scavenging ability. Particularly the ability of ESF gradually increased with passage of the time (about 80% to 10 min). It suggests that the extracts of the pollen are good scavengers of active oxygen species. This property of the pollen seems to be important in prevention of various diseases such as cancer, cardiovascular diseases, and diabetes. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Cistus ladaniferus; Pollen; Reactive oxygen species; Scavenging capacity
1. Introduction Honeys produced from different floral sources may often have distinctly different aromas and tastes. It has been proposed that the volatile compounds present in honey arises from the plant nectar or via some modification of plant constituents by the bee [1]. To identify honey
* Corresponding author. Tel.: ⫹81-832-86-5111, ext. 407; fax: ⫹81-832-33-1816 E-mail address: [email protected] (T. Nagai). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 4 0 0 - 6
520
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
source and quality, pollen analysis is frequently used. Because honeys are usually classified by the pollen spectra, which can be appropriate for determination of both the botanical and geographical origin of the product. However, a chemical approach to the characterization of the floral sources of honey might prove to be more accurate and more readily available [2]. Honey is the sweet substance produced by honey bees from the nectar of blossom or from secretions of or on living parts of plants, which they collect, transform and combine with specific substances, and store in honey combs. Honey is a highly variable natural product, particulaly in its sensory properties such as colour and odour, water content, ash content, pH value and sugar composition. These attributes depend on the climate, floral type, and individual beekeeping practices. Chemically honey comprises carbohydrates (70 – 80%), water (10 –20%) and other substances such as organic acids, mineral salts, proteins containing enzymes, free amino acids, and vitamins [3–5]. Traditionally, its use in food has been as a sweetening agent. Moreover, it is well-known that honeys posses the several fanctional properties such as antioxidative activity [6]. On the other hand, royal jelly and propolis have been also gone on sale as honey products. Particulally in royal jelly the pollen is the principal ingredient, which contains the large amounts of proteins, vitamins, and minerals. Today, the functional property of pollen is a subject of great interest in apiculture. As a part of studies on the functional properties on some honeys and related products, this paper deal with the evaluation of the scavenging capacities against the autoxidation, the superoxide anion radical, the hydroxyl radical, and DPPH radical. Free radicals and other reactive species (RS) are constantly generated in vivo both by accidents of chemistry and for specific metabolic purposes. The most important reactions of free radicals in aerobic cells involve molecular oxygen and its radical derivates (superoxide anion and hydroxyl radicals), peroxides, and transition metals. RS are thought to play an important role in aging and in the pathogenesis of numerous degenerative or chronic diseases, such as cancer, cardiovascular diseases, diabetes, and atherosclerosis [7–12]. This work should be useful to prevent the various diseases, due to the increasing interest in human health.
2. Materials and methods 2.1. Sample Pollen from Cistus ladaniferus of Spain growth was obtained from Inoue Bee Farm Inc. (Hyogo, Japan) and used in this study. 2.2. Chemicals Xanthine oxidase was obtained from Oriental yeast Co., Ltd. (Tokyo, Japan). Linoleic acid, nitroblue tetrazolium salt, 2,2⬘-azobis(2-amidinopropane) dihydrochloride, ␣-tocopherol, xanthine, 2-deoxy-D-ribose, 2-thiobarbitric acid, and 1,1-diphenyl-2-picrylhydrazyl were purchased from Wako Chemicals Co., Ltd. (Osaka, Japan). Other chemicals were of analytical grade.
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
521
2.3. Preparation of sample solution Sample 1: Pollen was extracted with boiling with 10 volumes of H2O for 1 h, and the extract was centrifuged at 10,000 rpm at 20° for 1 h. The supernatants were collected and used as hot-water fraction (HWF). Sample 2: Pollen was extracted with shaking with 10 volumes of water at 20° for one day, and the extract was centrifuged at 10,000 rpm at 20° for 1 h. Moreover, the supernatants were centrifuged at 13,000 rpm at 20° for 15 min and were used as water-soluble fraction (WSF). Sample 3. Pollen was extracted with shaking with 10 volumes of ethanol at 20° for 1 h, and the extract was centrifuged at 10,000 rpm at 20° for 1 h. The supernatants were as ethanol-soluble fraction (ESF). 2.4. Autoxidation test (FTC method) The autoxidation test was estimated by using a linoleic acid model system. A 0.2 ml of sample solution and 0.5 ml of 0.2 M sodium phosphate buffer (pH 7.0) were mixed with 0.5 ml of 2.5% (w/v) linoleic acid in ethanol. The preoxidation was initiated by the addition of 50l of 0.1 M 2,2⬘-azobis(2-amidinopropane) dihydrochloride (AAPH) and carried out at 37° for 200 min in the dark. The degree of oxidization was measured according to the thiocyanate method [13] for measuring peroxides by reading the absorbance at 500 nm after coloring with FeCl2 and ammonium thiocyanate. A control was performed with linoleic acid but without sample solution. ␣-Tocopherol and ascorbic acid were used as positive control. 2.5. Scavenging capacity of superoxide anion radical Scavenging capacity of superoxide anion radical was evaluated by the method of Nagai et al. [6]. This system contained 0.48 ml of 0.05 M sodium carbonate buffer (pH 10.5), 0.02 ml of 3 mM xanthine, 0.02 ml of 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA), 0.02 ml of 0.15% bovine serum albumin, 0.02 ml of 0.75 mM nitroblue tetrazolium (NBT) and 0.02 ml of sample solution. After at 25° for 10 min, the reaction was started by adding 6 mU xanthine oxidase (XOD) and carried out at 25° for 20 min. After 20 min the reaction was stopped by adding 0.02 ml of 6 mM CuCl. The absorbance of the reaction mixture was measured at 560 nm and the inhibition rate was calculated by measuring the amount of the formazan that was reduced from NBT by superoxide [14]. 2.6. Scavenging capacity of hydroxyl radical The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7.0), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO4-EDTA, 0.15 ml of 10 mM H2O2, 0.525 ml of H2O, and 0.075 ml of sample solution in Eppendorf tube. The reaction was started by the addition of H2O2. After incubation at 37° for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% trichloroacetic acid and 0.75 ml of 1.0% of 2-tribarbitric acid in 50 mM NaOH, the solution was boiled for 10 min, and then cooled in water. The absorbance of the solution was measured at 520 nm. Hydroxyl radical scavenging ability was evaluated as the inhibition rate of 2-deoxyribose oxidation by 䡠 OH [15].
522
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
Fe2⫹⫺EDTA ⫹ H2O2 3 Fe3⫹⫺EDTA ⫹ OH⫺ ⫹ 䡠 OH (Fenton reaction)
heat with TBA 䡠 OH⫹2-deoxy-D-ribose 3 fragments ———————3 plus acid
chromogen
2.7. Scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) Scavenging capacity of DPPH radical was evaluated by the method of Okada and Okada [16] with a slight modification. The assay mixture contained 0.3 ml of 1.0 mM DPPH radical solution, 2.4 ml of 99% ethyl alcohol, and 0.3 ml of sample solution. The solution was rapidly mixed and this scavenging capacity was measured spectrophotometrically by monitoring the decrease in absorbance at 517 nm. ␣-Tocopherol and ascorbic acid were used as positive control.
3. Results 3.1. Antioxidative capacities of pollen extracts The antioxidative capacities of pollen extracts on the peroxidation of linoleic acid were investigated. The antioxidative ability of control was decreased with passage of the time and was suddenly decreased from 100 to 200 min (Fig. 1). This pattern was the same as that of 1 mM ascorbic acid. There were no differences in antioxidative abilities among pollen extracts. In particular, ESF was most active followed by HWF. The abilities of these pollen extracts were higher than that of 5 mM ascorbic acid to 200 min, and were similar to that of 1 mM ␣-tocopherol. Moreover, even after 20 h ESF showed the same ability as well as that of control on 100 min (data not shown). ␣-Tocopherol showed a high antioxidative activity from an initial stage of the peroxidation to 200 min. 3.2. Superoxide-scavenging abilities of pollen extracts Superoxide-scavenging abilities of pollen extracts were estimated using xanthine-xanthine oxidase system (NBT method). These results were indicated as the superoxide productivity. Each sample showed the superoxide-scavenging abilities and this ability was decreased in the order WSF ⬎ HWF ⬎ ESF (Fig. 2). Among them, WSF completely scavenged the superoxide anion radical. 3.3. Scavenging abilities of hydroxyl radical on pollen extracts 2-Deoxyribose is oxidized by 䡠 OH that formed by the Fenton reaction and degraded to malonaldehyde. The scavenging abilities of pollen extracts on hydroxyl radical inhibition was shown in Fig. 3. Each sample showed the hydroxyl radical scavenging abilities and its ability was decreased in the order ESF ⬎ HWF ⬎ WSF (Fig. 3). Specifically, ESF showed the higher ability (91.5%). Moreover, HWF and WSF indicated the abilities about 60%.
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
523
Fig. 1. Antioxidative activities of the pollen extracts as measured by the FTC method. (䊐) hot-water fraction; (F) water-soluble fraction; (‚) ethanol-soluble fraction; (Œ) 1 mM ascorbic acid; (E) 5 mM ascorbic acid; (䊐) 1 mM ␣-tocopherol; (⫹) control.
Fig. 2. Scavenging capacities of the pollen extracts on the superoxide anion radical in xanthine-xanthine oxidase system by the NBT method. (CN) control; (A) hot-water fraction; (B) water-soluble fraction; (C) ethanol-soluble fraction.
524
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
Fig. 3. Hydroxyl radical scavenging capacities of the pollen extracts. (A) hot-water fraction; (B) water-soluble fraction; (C) ethanol-soluble fraction.
3.4. Scavenging abilities of DPPH radical on pollen extracts The scavenging abilities of pollen extracts on DPPH inhibition was shown in Fig. 4. Among these samples, the abilities of HWF and WSF were the same and these scavanged to about 60% against DPPH radical. The ability of the ESF gradually increased with passage of the time and the value was about 80%. The ability of 1 mM ascorbic acid was the highest followed by that of 1 mM ␣-tocopherol. Ascorbic acid (0.1 mM) hardly showed its ability similar to control. 4. Discussion The FTC method was used to measure the amount of peroxide in initial stages of lipid oxidization system. As a result, the high ability was detected in each fraction. In particular, ESF was most active and was higher than 1 mM ␣-tocopherol. Moreover, HWF and WSF was the same ability as 1 mM ␣-tocopherol. Thus it was found that the pollen posses the antioxidative substances having high activity. According to superoxide radical scavenging test, each of the pollen extracts showed high superoxide radical scavenging ability. It was suggested that the pollen extracts have high superoxide dismutase-like activities. Among these samples tested, ESF showed the highest ability against hydroxyl radical. Hydroxyl radical are known to be capable of abstracting hydrogen atoms from membrane and bring about peroxidic reactions of lipids [17]. From this point, it was expected that the pollen
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
525
Fig. 4. DPPH radical scavenging capacities of the pollen extracts. (䊐) hot-water fraction; (F) water-soluble fraction; (‚) ethanol-soluble fraction; (Œ) 0.1 mM ascorbic acid; (E) 1 mM ascorbic acid; (䊐) 1 mM ␣-tocopherol; (⫹) control.
extracts demonstrate the antioxidant effects against lipid peroxidation to scavenge the hydroxyl radicals and superoxide anions at the stage of initiation and termination of peroxyl radicals. From DPPH radical scavenging test, it was found that ESF function effectively and lastingly among these samples. DPPH is a free radical compound and has been widely used to test the free radical scavenging ability of various samples [18 –21]. Amakura et al. [22] reported that DPPH radical scavenging activities of nine berries were associated with the contents of the total phenolics. It suggests that ESF having stronger DPPH radical scavenging activity seems to include high contents of the total phenolics such as quercetin, flavones, isoflavones, flavonones, anthocyanins, catechin, and isocatechin because of easily extraction of phenolic compounds with ethanol. In our recent paper [6], it was reported that some commercially honeys, royal jelly, and propolis possess the antioxidative activities. Particularly it is known that a large amount of pollen contains in honeys and royal jelly. The high antioxidative activities of honeys and royal jelly seem to be attributed to termination of free-radical reactions and quenching of reactive oxygen species (ROS) by the pollen. The scavenging capacities of ROS on the pollen may be apparently due to many factors that are beyond the control of honey keepers, such as differences in soil and atmospheric conditions as well as in the type and physiology of each plant. It also may result the differences of the chemical composition could be attributed to the floral sources and environmental conditions. We are going to examine the scavenging capacities of other species pollens, particularly against ROS, in detail. In conclusion, we demonstrated the extracts of the pollen are good scavengers of active
526
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
oxygen species, including superoxide anion radical and hydroxyl radical. This property of the pollen seems to be important in prevention of various diseases such as cancer, cardiovascular diseases, and diabetes.
References [1] Bonaga G, Giumanini AG. The volatile fraction of chestnut honey. J Apic Res 1986;25:113–20. [2] Tan ST, Wilkins AL, Molan PC, Holland PT, Reid MA. Chemical approach to the determination of floral sources of New Zealand honeys. J Apic Res 1989;28:212–22. [3] Crane E. Honey, a comprehensive survey. New York: Crane Russak, and Co., 1975. [4] Ferreres F, Garciaviguera C, Tomaslorente F, Tomasbarberan FA. Hesperetin C a marker of the floral origin of citrus honey. J Sci Food Agric; 1993;61:121–3. [5] Ioyrish N. Bees and people. Moscow, Russia: MIR Publishers, 1974. [6] Nagai T, Sakai M, Inoue R, Inoue H, Suzuki N. Antioxidative activities of some commercially honeys, royal jelly, and propolis. Food Chem 2001;75:237– 40. [7] Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative disease of aging. Proc Natl Acad Sci USA 1993;90:7915–22. [8] Cerutti P. Oxy-radicals and cancer. Lancet 1994;344:862–3. [9] Dean RT, Gieseg Davis, MJ. Reactive apecies and their accumulation on radical damaged proteins. Trends Biochem Sciences 1993;18:437– 41. [10] Diplock AT, Rice-Evans CA, Burdon RH. Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention. Cancer Res 1994;54:1952s–56s. [11] Gutteridge JMC, Halliwell B. Free radicals and antioxidants in aging and disease: fact or fantasy. Antioxidants in nutrition, health, and disease. Oxford, UK: Oxford University Press 1994. p. 111–35. [12] Willett WC. Diet and health: what should we eat? Science 1994;264:532–7. [13] Mitsuda H., Yasumoto K, Iwami K. Antioxidative action of indole compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo 1966;19:210 – 4. [14] Rosa GD Duncan DS, Keen CL, Hurley LS. Evaluation of negative staining technique for determination of CN–insensitive superoxide dismutase activity. Biochim Biophys Acta 1979;566:32–9. [15] Chung SK, Osawa T, Kawakishi S. Hydroxyl radical-scavenging effects of spices and scavengers from brown mustard (Brassica nigra). Biosci Biotech Biochem 1997;61:118 –23. [16] Okada Y, Okada M. Scavenging effect of water soluble proteins in broad beans on free radicals and active oxygen species. J Agric Food Chem 1998;46:401– 6. [17] Kitada M, Igarashi K, Hirose S, Kitagawa H. Inhibition by polyamines of lipid peroxide formation in rat liver microsomes. Biochem Biophys Res Commun 1979;87:388 –94. [18] Hatano T, Edamatsu R, Hiramatsu M, Mori A, Fujita Y, Yasuhara T, Yoshida T, Okuda T. Effects of the interaction of tannins with coexisting substances. VI. Effects of tannins and related polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chem Pharma Bull 1989;37:2016 – 21. [19] Hatano T. Constituents of natural medicines with scavenging effects on active oxygen species-Tannins and related polyphenols. Nat Med (Tokyo) 1995;49:357– 63. [20] Hatano T, Takagi M, Ito H, Yoshida T. Phenolic constituents of liquorice. VII. A new calcone with a potent radical scavenging activity and accompanying phenolics. Chem Pharma Bull 1997;45:1485–92. [21] Yoshida T, Mori K, Hatano T, Okumura T, Uehara I, Komagoe K, Fujita Y, Okuda T. Studies on inhibition mechanism of autoxidation by tannins and flavonoids. V. Radical-scavenging effects of tannins and related polyphenols on 1,1-diphenyl-2-picrylhydrazyl radical. Chem Pharma Bull 1989;37:1919 –21. [22] Amakura Y, Umino Y, Tsuji S, Tonogai Y. Influence of jam processing on the radical scavenging activity and phenolic content in berries. J Agric Food Chem 2000;48:6292–97.
Scavenging capacities of pollen extracts from cistus ladaniferus on autoxidation, superoxide radicals, hydroxyl radicals, and DPPH radicals Takeshi Nagaia,*, Reiji Inoueb, Hachiro Inoueb, Nobutaka Suzukia a
Department of Food Science and Technology, National Fisheries University, Yamaguchi 7596595, Japan b Inoue Bee Farm Inc., Hyogo 6693465, Japan Received 18 June 2001; received in revised form 15 November 2001; accepted 19 November 2001
Abstract The antioxidative abilities of pollen extracts were evaluated using lipid peroxidation model system. Ethanol-soluble fraction (ESF) was most active followed by hot-water fraction (HWF). These abilities of pollen extracts were higher than that of 5 mM ascorbic acid and were similar to that of 1 mM ␣-tocopherol. Superoxide-scavenging capacities were decreased in the order water-soluble fraction ⬎ HWF ⬎ ESF. ESF showed the highest hydroxyl radical scavenging ability among these samples. The pollen extracts showed DPPH radical scavenging ability. Particularly the ability of ESF gradually increased with passage of the time (about 80% to 10 min). It suggests that the extracts of the pollen are good scavengers of active oxygen species. This property of the pollen seems to be important in prevention of various diseases such as cancer, cardiovascular diseases, and diabetes. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Cistus ladaniferus; Pollen; Reactive oxygen species; Scavenging capacity
1. Introduction Honeys produced from different floral sources may often have distinctly different aromas and tastes. It has been proposed that the volatile compounds present in honey arises from the plant nectar or via some modification of plant constituents by the bee [1]. To identify honey
* Corresponding author. Tel.: ⫹81-832-86-5111, ext. 407; fax: ⫹81-832-33-1816 E-mail address: [email protected] (T. Nagai). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 4 0 0 - 6
520
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
source and quality, pollen analysis is frequently used. Because honeys are usually classified by the pollen spectra, which can be appropriate for determination of both the botanical and geographical origin of the product. However, a chemical approach to the characterization of the floral sources of honey might prove to be more accurate and more readily available [2]. Honey is the sweet substance produced by honey bees from the nectar of blossom or from secretions of or on living parts of plants, which they collect, transform and combine with specific substances, and store in honey combs. Honey is a highly variable natural product, particulaly in its sensory properties such as colour and odour, water content, ash content, pH value and sugar composition. These attributes depend on the climate, floral type, and individual beekeeping practices. Chemically honey comprises carbohydrates (70 – 80%), water (10 –20%) and other substances such as organic acids, mineral salts, proteins containing enzymes, free amino acids, and vitamins [3–5]. Traditionally, its use in food has been as a sweetening agent. Moreover, it is well-known that honeys posses the several fanctional properties such as antioxidative activity [6]. On the other hand, royal jelly and propolis have been also gone on sale as honey products. Particulally in royal jelly the pollen is the principal ingredient, which contains the large amounts of proteins, vitamins, and minerals. Today, the functional property of pollen is a subject of great interest in apiculture. As a part of studies on the functional properties on some honeys and related products, this paper deal with the evaluation of the scavenging capacities against the autoxidation, the superoxide anion radical, the hydroxyl radical, and DPPH radical. Free radicals and other reactive species (RS) are constantly generated in vivo both by accidents of chemistry and for specific metabolic purposes. The most important reactions of free radicals in aerobic cells involve molecular oxygen and its radical derivates (superoxide anion and hydroxyl radicals), peroxides, and transition metals. RS are thought to play an important role in aging and in the pathogenesis of numerous degenerative or chronic diseases, such as cancer, cardiovascular diseases, diabetes, and atherosclerosis [7–12]. This work should be useful to prevent the various diseases, due to the increasing interest in human health.
2. Materials and methods 2.1. Sample Pollen from Cistus ladaniferus of Spain growth was obtained from Inoue Bee Farm Inc. (Hyogo, Japan) and used in this study. 2.2. Chemicals Xanthine oxidase was obtained from Oriental yeast Co., Ltd. (Tokyo, Japan). Linoleic acid, nitroblue tetrazolium salt, 2,2⬘-azobis(2-amidinopropane) dihydrochloride, ␣-tocopherol, xanthine, 2-deoxy-D-ribose, 2-thiobarbitric acid, and 1,1-diphenyl-2-picrylhydrazyl were purchased from Wako Chemicals Co., Ltd. (Osaka, Japan). Other chemicals were of analytical grade.
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
521
2.3. Preparation of sample solution Sample 1: Pollen was extracted with boiling with 10 volumes of H2O for 1 h, and the extract was centrifuged at 10,000 rpm at 20° for 1 h. The supernatants were collected and used as hot-water fraction (HWF). Sample 2: Pollen was extracted with shaking with 10 volumes of water at 20° for one day, and the extract was centrifuged at 10,000 rpm at 20° for 1 h. Moreover, the supernatants were centrifuged at 13,000 rpm at 20° for 15 min and were used as water-soluble fraction (WSF). Sample 3. Pollen was extracted with shaking with 10 volumes of ethanol at 20° for 1 h, and the extract was centrifuged at 10,000 rpm at 20° for 1 h. The supernatants were as ethanol-soluble fraction (ESF). 2.4. Autoxidation test (FTC method) The autoxidation test was estimated by using a linoleic acid model system. A 0.2 ml of sample solution and 0.5 ml of 0.2 M sodium phosphate buffer (pH 7.0) were mixed with 0.5 ml of 2.5% (w/v) linoleic acid in ethanol. The preoxidation was initiated by the addition of 50l of 0.1 M 2,2⬘-azobis(2-amidinopropane) dihydrochloride (AAPH) and carried out at 37° for 200 min in the dark. The degree of oxidization was measured according to the thiocyanate method [13] for measuring peroxides by reading the absorbance at 500 nm after coloring with FeCl2 and ammonium thiocyanate. A control was performed with linoleic acid but without sample solution. ␣-Tocopherol and ascorbic acid were used as positive control. 2.5. Scavenging capacity of superoxide anion radical Scavenging capacity of superoxide anion radical was evaluated by the method of Nagai et al. [6]. This system contained 0.48 ml of 0.05 M sodium carbonate buffer (pH 10.5), 0.02 ml of 3 mM xanthine, 0.02 ml of 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA), 0.02 ml of 0.15% bovine serum albumin, 0.02 ml of 0.75 mM nitroblue tetrazolium (NBT) and 0.02 ml of sample solution. After at 25° for 10 min, the reaction was started by adding 6 mU xanthine oxidase (XOD) and carried out at 25° for 20 min. After 20 min the reaction was stopped by adding 0.02 ml of 6 mM CuCl. The absorbance of the reaction mixture was measured at 560 nm and the inhibition rate was calculated by measuring the amount of the formazan that was reduced from NBT by superoxide [14]. 2.6. Scavenging capacity of hydroxyl radical The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7.0), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO4-EDTA, 0.15 ml of 10 mM H2O2, 0.525 ml of H2O, and 0.075 ml of sample solution in Eppendorf tube. The reaction was started by the addition of H2O2. After incubation at 37° for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% trichloroacetic acid and 0.75 ml of 1.0% of 2-tribarbitric acid in 50 mM NaOH, the solution was boiled for 10 min, and then cooled in water. The absorbance of the solution was measured at 520 nm. Hydroxyl radical scavenging ability was evaluated as the inhibition rate of 2-deoxyribose oxidation by 䡠 OH [15].
522
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
Fe2⫹⫺EDTA ⫹ H2O2 3 Fe3⫹⫺EDTA ⫹ OH⫺ ⫹ 䡠 OH (Fenton reaction)
heat with TBA 䡠 OH⫹2-deoxy-D-ribose 3 fragments ———————3 plus acid
chromogen
2.7. Scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) Scavenging capacity of DPPH radical was evaluated by the method of Okada and Okada [16] with a slight modification. The assay mixture contained 0.3 ml of 1.0 mM DPPH radical solution, 2.4 ml of 99% ethyl alcohol, and 0.3 ml of sample solution. The solution was rapidly mixed and this scavenging capacity was measured spectrophotometrically by monitoring the decrease in absorbance at 517 nm. ␣-Tocopherol and ascorbic acid were used as positive control.
3. Results 3.1. Antioxidative capacities of pollen extracts The antioxidative capacities of pollen extracts on the peroxidation of linoleic acid were investigated. The antioxidative ability of control was decreased with passage of the time and was suddenly decreased from 100 to 200 min (Fig. 1). This pattern was the same as that of 1 mM ascorbic acid. There were no differences in antioxidative abilities among pollen extracts. In particular, ESF was most active followed by HWF. The abilities of these pollen extracts were higher than that of 5 mM ascorbic acid to 200 min, and were similar to that of 1 mM ␣-tocopherol. Moreover, even after 20 h ESF showed the same ability as well as that of control on 100 min (data not shown). ␣-Tocopherol showed a high antioxidative activity from an initial stage of the peroxidation to 200 min. 3.2. Superoxide-scavenging abilities of pollen extracts Superoxide-scavenging abilities of pollen extracts were estimated using xanthine-xanthine oxidase system (NBT method). These results were indicated as the superoxide productivity. Each sample showed the superoxide-scavenging abilities and this ability was decreased in the order WSF ⬎ HWF ⬎ ESF (Fig. 2). Among them, WSF completely scavenged the superoxide anion radical. 3.3. Scavenging abilities of hydroxyl radical on pollen extracts 2-Deoxyribose is oxidized by 䡠 OH that formed by the Fenton reaction and degraded to malonaldehyde. The scavenging abilities of pollen extracts on hydroxyl radical inhibition was shown in Fig. 3. Each sample showed the hydroxyl radical scavenging abilities and its ability was decreased in the order ESF ⬎ HWF ⬎ WSF (Fig. 3). Specifically, ESF showed the higher ability (91.5%). Moreover, HWF and WSF indicated the abilities about 60%.
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
523
Fig. 1. Antioxidative activities of the pollen extracts as measured by the FTC method. (䊐) hot-water fraction; (F) water-soluble fraction; (‚) ethanol-soluble fraction; (Œ) 1 mM ascorbic acid; (E) 5 mM ascorbic acid; (䊐) 1 mM ␣-tocopherol; (⫹) control.
Fig. 2. Scavenging capacities of the pollen extracts on the superoxide anion radical in xanthine-xanthine oxidase system by the NBT method. (CN) control; (A) hot-water fraction; (B) water-soluble fraction; (C) ethanol-soluble fraction.
524
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
Fig. 3. Hydroxyl radical scavenging capacities of the pollen extracts. (A) hot-water fraction; (B) water-soluble fraction; (C) ethanol-soluble fraction.
3.4. Scavenging abilities of DPPH radical on pollen extracts The scavenging abilities of pollen extracts on DPPH inhibition was shown in Fig. 4. Among these samples, the abilities of HWF and WSF were the same and these scavanged to about 60% against DPPH radical. The ability of the ESF gradually increased with passage of the time and the value was about 80%. The ability of 1 mM ascorbic acid was the highest followed by that of 1 mM ␣-tocopherol. Ascorbic acid (0.1 mM) hardly showed its ability similar to control. 4. Discussion The FTC method was used to measure the amount of peroxide in initial stages of lipid oxidization system. As a result, the high ability was detected in each fraction. In particular, ESF was most active and was higher than 1 mM ␣-tocopherol. Moreover, HWF and WSF was the same ability as 1 mM ␣-tocopherol. Thus it was found that the pollen posses the antioxidative substances having high activity. According to superoxide radical scavenging test, each of the pollen extracts showed high superoxide radical scavenging ability. It was suggested that the pollen extracts have high superoxide dismutase-like activities. Among these samples tested, ESF showed the highest ability against hydroxyl radical. Hydroxyl radical are known to be capable of abstracting hydrogen atoms from membrane and bring about peroxidic reactions of lipids [17]. From this point, it was expected that the pollen
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
525
Fig. 4. DPPH radical scavenging capacities of the pollen extracts. (䊐) hot-water fraction; (F) water-soluble fraction; (‚) ethanol-soluble fraction; (Œ) 0.1 mM ascorbic acid; (E) 1 mM ascorbic acid; (䊐) 1 mM ␣-tocopherol; (⫹) control.
extracts demonstrate the antioxidant effects against lipid peroxidation to scavenge the hydroxyl radicals and superoxide anions at the stage of initiation and termination of peroxyl radicals. From DPPH radical scavenging test, it was found that ESF function effectively and lastingly among these samples. DPPH is a free radical compound and has been widely used to test the free radical scavenging ability of various samples [18 –21]. Amakura et al. [22] reported that DPPH radical scavenging activities of nine berries were associated with the contents of the total phenolics. It suggests that ESF having stronger DPPH radical scavenging activity seems to include high contents of the total phenolics such as quercetin, flavones, isoflavones, flavonones, anthocyanins, catechin, and isocatechin because of easily extraction of phenolic compounds with ethanol. In our recent paper [6], it was reported that some commercially honeys, royal jelly, and propolis possess the antioxidative activities. Particularly it is known that a large amount of pollen contains in honeys and royal jelly. The high antioxidative activities of honeys and royal jelly seem to be attributed to termination of free-radical reactions and quenching of reactive oxygen species (ROS) by the pollen. The scavenging capacities of ROS on the pollen may be apparently due to many factors that are beyond the control of honey keepers, such as differences in soil and atmospheric conditions as well as in the type and physiology of each plant. It also may result the differences of the chemical composition could be attributed to the floral sources and environmental conditions. We are going to examine the scavenging capacities of other species pollens, particularly against ROS, in detail. In conclusion, we demonstrated the extracts of the pollen are good scavengers of active
526
T. Nagai et al. / Nutrition Research 22 (2002) 519 –526
oxygen species, including superoxide anion radical and hydroxyl radical. This property of the pollen seems to be important in prevention of various diseases such as cancer, cardiovascular diseases, and diabetes.
References [1] Bonaga G, Giumanini AG. The volatile fraction of chestnut honey. J Apic Res 1986;25:113–20. [2] Tan ST, Wilkins AL, Molan PC, Holland PT, Reid MA. Chemical approach to the determination of floral sources of New Zealand honeys. J Apic Res 1989;28:212–22. [3] Crane E. Honey, a comprehensive survey. New York: Crane Russak, and Co., 1975. [4] Ferreres F, Garciaviguera C, Tomaslorente F, Tomasbarberan FA. Hesperetin C a marker of the floral origin of citrus honey. J Sci Food Agric; 1993;61:121–3. [5] Ioyrish N. Bees and people. Moscow, Russia: MIR Publishers, 1974. [6] Nagai T, Sakai M, Inoue R, Inoue H, Suzuki N. Antioxidative activities of some commercially honeys, royal jelly, and propolis. Food Chem 2001;75:237– 40. [7] Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative disease of aging. Proc Natl Acad Sci USA 1993;90:7915–22. [8] Cerutti P. Oxy-radicals and cancer. Lancet 1994;344:862–3. [9] Dean RT, Gieseg Davis, MJ. Reactive apecies and their accumulation on radical damaged proteins. Trends Biochem Sciences 1993;18:437– 41. [10] Diplock AT, Rice-Evans CA, Burdon RH. Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention. Cancer Res 1994;54:1952s–56s. [11] Gutteridge JMC, Halliwell B. Free radicals and antioxidants in aging and disease: fact or fantasy. Antioxidants in nutrition, health, and disease. Oxford, UK: Oxford University Press 1994. p. 111–35. [12] Willett WC. Diet and health: what should we eat? Science 1994;264:532–7. [13] Mitsuda H., Yasumoto K, Iwami K. Antioxidative action of indole compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo 1966;19:210 – 4. [14] Rosa GD Duncan DS, Keen CL, Hurley LS. Evaluation of negative staining technique for determination of CN–insensitive superoxide dismutase activity. Biochim Biophys Acta 1979;566:32–9. [15] Chung SK, Osawa T, Kawakishi S. Hydroxyl radical-scavenging effects of spices and scavengers from brown mustard (Brassica nigra). Biosci Biotech Biochem 1997;61:118 –23. [16] Okada Y, Okada M. Scavenging effect of water soluble proteins in broad beans on free radicals and active oxygen species. J Agric Food Chem 1998;46:401– 6. [17] Kitada M, Igarashi K, Hirose S, Kitagawa H. Inhibition by polyamines of lipid peroxide formation in rat liver microsomes. Biochem Biophys Res Commun 1979;87:388 –94. [18] Hatano T, Edamatsu R, Hiramatsu M, Mori A, Fujita Y, Yasuhara T, Yoshida T, Okuda T. Effects of the interaction of tannins with coexisting substances. VI. Effects of tannins and related polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chem Pharma Bull 1989;37:2016 – 21. [19] Hatano T. Constituents of natural medicines with scavenging effects on active oxygen species-Tannins and related polyphenols. Nat Med (Tokyo) 1995;49:357– 63. [20] Hatano T, Takagi M, Ito H, Yoshida T. Phenolic constituents of liquorice. VII. A new calcone with a potent radical scavenging activity and accompanying phenolics. Chem Pharma Bull 1997;45:1485–92. [21] Yoshida T, Mori K, Hatano T, Okumura T, Uehara I, Komagoe K, Fujita Y, Okuda T. Studies on inhibition mechanism of autoxidation by tannins and flavonoids. V. Radical-scavenging effects of tannins and related polyphenols on 1,1-diphenyl-2-picrylhydrazyl radical. Chem Pharma Bull 1989;37:1919 –21. [22] Amakura Y, Umino Y, Tsuji S, Tonogai Y. Influence of jam processing on the radical scavenging activity and phenolic content in berries. J Agric Food Chem 2000;48:6292–97.