Free Radical Biology & Medicine, Vol. 9, pp. 19-21, 1990 Printedin the USA.All rightsreserved.
0891-5849/90 $3.00+ .00 © 1990PergamonPressplc
Original Contribution FLAVONOIDS AS SUPEROXIDE SCAVENGERS A N D A N T I O X I D A N T S CHEN YUTING,* ZHENG RONGLIANG,*t JIA ZHONGJIAN,:~ and Ju YONG:~ Department of Biology,* Institute of Organic Chemistry,* Lanzhou University, Lanzhou, 730000, Gansu Province, China (Received 20 April 1989; Revised 27 December 1989; Rerevised and Accepted 28 March 1990)
Abstract--The superoxide anions scavenging activity and antioxidation of seven flavonoids--quercetin, rutin,
morin, acacetin, hispidulin, hesperidin, and naringin--were studied. The superoxide anions were generated in a phenazin methosulphate-NADH system and were assayed by reduction of nitroblue tetrazolium. The scavenging activity ranked: rutin was the strongest, and quercetin and naringin the second, while morin and hispidulin were very weak. The concentration values yielding 50% inhibition of lipid peroxidation in mouse liver homogenate were in order of 10-6 M for quercetin, rutin, and morin; and of 10-5 M for acacetin and hispidulin, while naringin and hesperidin had no antioxidative action. In comparison with the antioxidative and scavenging activities of flavonoids, there are no correlations. Keywords--Flavonoid, Superoxide, Scavenger, Antioxidation, Free radicals
chased from Sigma Chemical Company. N-methylphenazonium methosulphate(PMS) was produced by Shanghai Biochemistry Institute, Academia Sinica. Nitroblue tetrazolum chloride(NBT) was a product of Shanghai Qianjing Chemical Company. Quercetin and rutin were products of Beijing Medical and Biological Products Inspection Institute. Hesperidin was purchased from Shanghai Second Reagent Company. Naringin dihydrate was purchased from Kock-Light Laboratories Ltd. Morin was produced by Kunming Plant Institute, Academia Sinica. Acacetin 1° and hispidulin 1' were isolated from Saussurea stella Maxim and Saussurea involucrata Kar et Kir, respectively, by the Institute of Organic Chemistry at Lanzhou University. All other reagents were graded analytically on purity.
INTRODUCTION
Flavonoids are a group of naturally occurring, benzo~/-pyrone derivatives with low molecular weights and are ubiquitous in photosynthesizing cells. They are widespread in the plant kingdom, of course, including herbs used as folk medicines in China for centuries. Flavonoids have been reported by many authors to have anti-inflammatory, antiallergic, antiviral, t'2 and catecholamine activities, 3 inhibiting DNA synthesis in tumor cells 4 and inhibiting tumor promotion. 5 Many flavonoids are also found to be strong free radical scavengers and antioxidants.6.7,8.9 It is known that excessive free radical production and lipid peroxidation in vivo may cause many kinds of diseases. Thus, the antioxidation of flavonoids may be related to their pharmacological actions and so they may be used as protective agents in a number of diseases. In the present paper, the superoxide anions scavenging activity and the antioxidative action of seven flavonoids have been tested.
Lipid peroxidation in mouse liver homogenate Mouse liver homogenate 5% w/v in saline 1.5 mL was incubated in a water bath at 37°C for 1.5 h with shaking, and was stopped by TCA 20% 1.5 mL. Five concentrations of each flavonoid, dissolved in DMSO, 0.1 mL was added to inhibit lipid peroxidation. DMSO of 0.1 mL was added for control. The lipid peroxidation was assayed by TBA test at room temperature and read at 532 nm against a blank sample. Their effect on the generation of MDA was used to calculate regression
MATERIALS AND METHODS
Chemicals and flavonoids 2-thiobarbituric acid(TBA), 13-nicotinamide adenine dinucleotide(reduced form, NADH) were purThis project was supportedby National ScienceFoundationof China. tAuthor to whom correspondence should be addressed. 19
20
C. YUTING et al.
OH HO
0 I
Oil
CT°,TC °2
OH
0
Quercetin: R=H Rutin: R=(C6H1 00LI.)0(C6HI 1 04)
Acacetin: Rq=H, ~2=CH 3 Hispidulin: Rfl=0CH~, R2=H
H0 (/,I
(~I
o
Norin
Ctt
0
Naringin
lines and half inhibition concentration values(ICs0). Every test has three replicates.
Superoxide anions generation Superoxide anions were generated in samples which contained 73 pM NADH, 15 pM phenazine methosulphate, 50 gM NBT, and flavonoids of various concentrations in 0.016 M Tris-HC1 buffer pH8.0 at room temperature. After absorbance at 560 nm O.D. reached a maximum and became stable in the control tube, the color was read.'2 Every test has three replicates, too. Naringin and rutin were dissolved in buffer solution. For these two flavonoids, the absorbance of the control tube reached a maximum 2 min after the initiation of reaction. Quercetin and morin were dissolved in ethanol and hispidulin was dissolved in acetone. For these three flavonoids, the absorbances of the control tubes, which contained 50% ethanol or acetone, reached a maximum 5 min after the initiation of reaction. ICs0 values for the inhibition of the generation of superoxide anions by the investigated flavonoids were calculated as described above. R E S U L T S AND DISCUSSION
The half inhibition concentrations of flavonoids on superoxide anion production are presented in Table 1. Five flavonoids scavenged superoxide anions concentration-dependently. Among them, rutin was the strongest savenger, naringin and quercetin ranked second, and hispidulin and morin were the weakest. It was
OH
0
Hesperidin
suggested that the 3',4'-diphenolic group was required for flavonoids to be effective free radical scavengers. 13.14Rutin and quercetin possess 3',4'-diphenolic group while morin and hispidulin do not. However, without this group, naringin also presented a rather strong activity. It seems that even if the 3',4'-diphenolic group is very important for the effectiveness of superoxide anions scavenging activity, it is not a prerequisite for flavonoids to scavenge superoxide anions. Among the seven tested flavonoids, quercetin, rutin, morin, hispidulin, and acacetin inhibited lipid peroxidation of mouse liver homogenate very strongly and concentration-dependently. Their ICs0 values are presented in Table 1, as well. The IC50 of quercetin, morin, and rutin, which possessed two phenol groups in ring B, were within the range of 10 - 6 M , while that of hispidulin and acacetin ranged in 10 -5 M. Naringin and hesperidin did not inhibit lipid peroxidation even
Table 1. Half Inhibition Concentrations(ICs0, #M) of Flavonoids on Superoxide Anions Generation and on Lipid Peroxidation
(n = 3). Flavonoid
Scavenging of Superoxide
Rutin Morin Quercetin Acacetin Hispidulin Naringin Hesperidin
56 - 3.5 1400 - 152.6 207 --- 15.9 500* 192 --- 6.7
*Only 23.5% inhibited at 500 #M.
Inhibition of LP 3.2 4.4 5.2 13.8 64.0 inactive inactive
--- 0.4 -+ 0.2 --- 0.3 - 1.7 - 2.1 at 2000.0 at 1000.0
Antioxidation of flavonoids
when their concentrations were as high as 2000 or 1000 /tM, respectively. Although the inhibitions of quercetin and rutin, which have 3',4'-diphenolic groups were stronger than those of hispidulin and acacetin, which have no such groups, morin, which has two phenol groups in C-2' and C-4' positions instead of C-3' and C-4', still exhibited strong inhibition of lipid peroxidation. Therefore, the inhibition of flavonoids on lipid peroxidation is not only contributed by 3',4'-diphenolic group but by two phenol groups, no matter what positions they assume. In comparison with the antioxidative and scavenging activities of flavonoids, there is no correlation. Morin and hispidulin have very strong inhibition of lipid peroxidation while their scavenging activity is very weak. On the other hand, naringin, as it has strong scavenging activity, does not inhibit lipid peroxidation at all. Thus, the scavenging of superoxide anions is not the only mechanism for the inhibition of lipid peroxidation. Some flavonoids, such as quercetin, are known as "OH scavengers 8'9 and metal ion chelators.6'15 These properties of flavonoids may also contribute to their inhibition of lipid peroxidation. REFERENCES
1. Middleton, E. The flavonoids. TIPS (August):335-338; 1984. 2. Havsteen, B. Flavonids, a class of natural products of high pharmacological potency. Biochem. Pharmacol. 32(7):11411148; 1983. 3. Kuhnan, J. WorldRev. Nutr. Diet 24:117-191; 1976.
21
4. Xiao, X.; Wang, X.; Liu, L.; Zhang, L.; Zheng, R. Inhibition of DNA synthesis in leukemia 7712 cells and induction of sister chromatid exchange in human lymphocytes induced by quercetin. Chinese J. of Pharmacol. & Toxicol. 2(3):317; 1988, 5. Nakamura, Y.; Colhurn, H. N.; Ginhart, T. D. Role of reactive oxygen in tumor promotion: implication of superoxide anion in promotion of neoplastic transformation in JB-6 cells by TPA. Carcinogenesis 6(2):229-235; 1985. 6. Scott, R.; Slater, T. E Free radical scavenging activity of (+)catechin and other flavonoids. In: Slater, T. E; Garner, A., eds. Recent advances in lipid peroxidation and tissue injury. London: Brunel University; 1981: 233-244. 7. Robak, J.; Gryglewski, R. J. Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 37:837-841; 1988. 8. Rafat Husain, S.; Cillard, J.; Cillard, P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 26:2489-2491; 1987. 9. Chen, Y.; Li, X.; Zhao, B.; Zheng, R.; Xin, W. Superoxide and hydroxyl radical scavenging activities of rutin and other natural products studied by ESR. Acta Biophysica Sinica. 5:253-240; 1989. 10. Jia, Z.; Ju, Y.; Chu, T. Studies on the chemical constituents of Saussurea stella Maxim(l), J. Lanzhou University 25:64-67; 1989. 11. Jia, Z.; Li, Y.; Du, M.; Chu, T. Studies on the constituents of Saussurea involucrata Kar. et Kir.(1). Chemical J. Chinese Universities 4:581-584; 1983. 12. Ponti, V.; Dianzani, M. U.; Cheeseman, K.; Slater, T. E Studies on the reduction of NBT mediated through the action of NADH and PMS. Chem-Biol. Interact. 23:281; 1978. 13. Latan, A. The relation of structure to antioxidant activity of quercetin and some of its derivatives: I. Primary activity. J. Food Sci. 31:395-399; 1966. 14. Younes, M.; Siegers, C. P. Inhibitory action of some flavonoids on enhanced spontaneous lipid peruxidation following glutathione depletion. Planta Med. 43:240-243; 1981. 15. Latan, A. The relation of structure to antioxidant activity of quercetin and some of its derivatives: II. Secondary(metal-complexing) activity. J. Food Sci. 31:518-522; 1966.
0891-5849/90 $3.00+ .00 © 1990PergamonPressplc
Original Contribution FLAVONOIDS AS SUPEROXIDE SCAVENGERS A N D A N T I O X I D A N T S CHEN YUTING,* ZHENG RONGLIANG,*t JIA ZHONGJIAN,:~ and Ju YONG:~ Department of Biology,* Institute of Organic Chemistry,* Lanzhou University, Lanzhou, 730000, Gansu Province, China (Received 20 April 1989; Revised 27 December 1989; Rerevised and Accepted 28 March 1990)
Abstract--The superoxide anions scavenging activity and antioxidation of seven flavonoids--quercetin, rutin,
morin, acacetin, hispidulin, hesperidin, and naringin--were studied. The superoxide anions were generated in a phenazin methosulphate-NADH system and were assayed by reduction of nitroblue tetrazolium. The scavenging activity ranked: rutin was the strongest, and quercetin and naringin the second, while morin and hispidulin were very weak. The concentration values yielding 50% inhibition of lipid peroxidation in mouse liver homogenate were in order of 10-6 M for quercetin, rutin, and morin; and of 10-5 M for acacetin and hispidulin, while naringin and hesperidin had no antioxidative action. In comparison with the antioxidative and scavenging activities of flavonoids, there are no correlations. Keywords--Flavonoid, Superoxide, Scavenger, Antioxidation, Free radicals
chased from Sigma Chemical Company. N-methylphenazonium methosulphate(PMS) was produced by Shanghai Biochemistry Institute, Academia Sinica. Nitroblue tetrazolum chloride(NBT) was a product of Shanghai Qianjing Chemical Company. Quercetin and rutin were products of Beijing Medical and Biological Products Inspection Institute. Hesperidin was purchased from Shanghai Second Reagent Company. Naringin dihydrate was purchased from Kock-Light Laboratories Ltd. Morin was produced by Kunming Plant Institute, Academia Sinica. Acacetin 1° and hispidulin 1' were isolated from Saussurea stella Maxim and Saussurea involucrata Kar et Kir, respectively, by the Institute of Organic Chemistry at Lanzhou University. All other reagents were graded analytically on purity.
INTRODUCTION
Flavonoids are a group of naturally occurring, benzo~/-pyrone derivatives with low molecular weights and are ubiquitous in photosynthesizing cells. They are widespread in the plant kingdom, of course, including herbs used as folk medicines in China for centuries. Flavonoids have been reported by many authors to have anti-inflammatory, antiallergic, antiviral, t'2 and catecholamine activities, 3 inhibiting DNA synthesis in tumor cells 4 and inhibiting tumor promotion. 5 Many flavonoids are also found to be strong free radical scavengers and antioxidants.6.7,8.9 It is known that excessive free radical production and lipid peroxidation in vivo may cause many kinds of diseases. Thus, the antioxidation of flavonoids may be related to their pharmacological actions and so they may be used as protective agents in a number of diseases. In the present paper, the superoxide anions scavenging activity and the antioxidative action of seven flavonoids have been tested.
Lipid peroxidation in mouse liver homogenate Mouse liver homogenate 5% w/v in saline 1.5 mL was incubated in a water bath at 37°C for 1.5 h with shaking, and was stopped by TCA 20% 1.5 mL. Five concentrations of each flavonoid, dissolved in DMSO, 0.1 mL was added to inhibit lipid peroxidation. DMSO of 0.1 mL was added for control. The lipid peroxidation was assayed by TBA test at room temperature and read at 532 nm against a blank sample. Their effect on the generation of MDA was used to calculate regression
MATERIALS AND METHODS
Chemicals and flavonoids 2-thiobarbituric acid(TBA), 13-nicotinamide adenine dinucleotide(reduced form, NADH) were purThis project was supportedby National ScienceFoundationof China. tAuthor to whom correspondence should be addressed. 19
20
C. YUTING et al.
OH HO
0 I
Oil
CT°,TC °2
OH
0
Quercetin: R=H Rutin: R=(C6H1 00LI.)0(C6HI 1 04)
Acacetin: Rq=H, ~2=CH 3 Hispidulin: Rfl=0CH~, R2=H
H0 (/,I
(~I
o
Norin
Ctt
0
Naringin
lines and half inhibition concentration values(ICs0). Every test has three replicates.
Superoxide anions generation Superoxide anions were generated in samples which contained 73 pM NADH, 15 pM phenazine methosulphate, 50 gM NBT, and flavonoids of various concentrations in 0.016 M Tris-HC1 buffer pH8.0 at room temperature. After absorbance at 560 nm O.D. reached a maximum and became stable in the control tube, the color was read.'2 Every test has three replicates, too. Naringin and rutin were dissolved in buffer solution. For these two flavonoids, the absorbance of the control tube reached a maximum 2 min after the initiation of reaction. Quercetin and morin were dissolved in ethanol and hispidulin was dissolved in acetone. For these three flavonoids, the absorbances of the control tubes, which contained 50% ethanol or acetone, reached a maximum 5 min after the initiation of reaction. ICs0 values for the inhibition of the generation of superoxide anions by the investigated flavonoids were calculated as described above. R E S U L T S AND DISCUSSION
The half inhibition concentrations of flavonoids on superoxide anion production are presented in Table 1. Five flavonoids scavenged superoxide anions concentration-dependently. Among them, rutin was the strongest savenger, naringin and quercetin ranked second, and hispidulin and morin were the weakest. It was
OH
0
Hesperidin
suggested that the 3',4'-diphenolic group was required for flavonoids to be effective free radical scavengers. 13.14Rutin and quercetin possess 3',4'-diphenolic group while morin and hispidulin do not. However, without this group, naringin also presented a rather strong activity. It seems that even if the 3',4'-diphenolic group is very important for the effectiveness of superoxide anions scavenging activity, it is not a prerequisite for flavonoids to scavenge superoxide anions. Among the seven tested flavonoids, quercetin, rutin, morin, hispidulin, and acacetin inhibited lipid peroxidation of mouse liver homogenate very strongly and concentration-dependently. Their ICs0 values are presented in Table 1, as well. The IC50 of quercetin, morin, and rutin, which possessed two phenol groups in ring B, were within the range of 10 - 6 M , while that of hispidulin and acacetin ranged in 10 -5 M. Naringin and hesperidin did not inhibit lipid peroxidation even
Table 1. Half Inhibition Concentrations(ICs0, #M) of Flavonoids on Superoxide Anions Generation and on Lipid Peroxidation
(n = 3). Flavonoid
Scavenging of Superoxide
Rutin Morin Quercetin Acacetin Hispidulin Naringin Hesperidin
56 - 3.5 1400 - 152.6 207 --- 15.9 500* 192 --- 6.7
*Only 23.5% inhibited at 500 #M.
Inhibition of LP 3.2 4.4 5.2 13.8 64.0 inactive inactive
--- 0.4 -+ 0.2 --- 0.3 - 1.7 - 2.1 at 2000.0 at 1000.0
Antioxidation of flavonoids
when their concentrations were as high as 2000 or 1000 /tM, respectively. Although the inhibitions of quercetin and rutin, which have 3',4'-diphenolic groups were stronger than those of hispidulin and acacetin, which have no such groups, morin, which has two phenol groups in C-2' and C-4' positions instead of C-3' and C-4', still exhibited strong inhibition of lipid peroxidation. Therefore, the inhibition of flavonoids on lipid peroxidation is not only contributed by 3',4'-diphenolic group but by two phenol groups, no matter what positions they assume. In comparison with the antioxidative and scavenging activities of flavonoids, there is no correlation. Morin and hispidulin have very strong inhibition of lipid peroxidation while their scavenging activity is very weak. On the other hand, naringin, as it has strong scavenging activity, does not inhibit lipid peroxidation at all. Thus, the scavenging of superoxide anions is not the only mechanism for the inhibition of lipid peroxidation. Some flavonoids, such as quercetin, are known as "OH scavengers 8'9 and metal ion chelators.6'15 These properties of flavonoids may also contribute to their inhibition of lipid peroxidation. REFERENCES
1. Middleton, E. The flavonoids. TIPS (August):335-338; 1984. 2. Havsteen, B. Flavonids, a class of natural products of high pharmacological potency. Biochem. Pharmacol. 32(7):11411148; 1983. 3. Kuhnan, J. WorldRev. Nutr. Diet 24:117-191; 1976.
21
4. Xiao, X.; Wang, X.; Liu, L.; Zhang, L.; Zheng, R. Inhibition of DNA synthesis in leukemia 7712 cells and induction of sister chromatid exchange in human lymphocytes induced by quercetin. Chinese J. of Pharmacol. & Toxicol. 2(3):317; 1988, 5. Nakamura, Y.; Colhurn, H. N.; Ginhart, T. D. Role of reactive oxygen in tumor promotion: implication of superoxide anion in promotion of neoplastic transformation in JB-6 cells by TPA. Carcinogenesis 6(2):229-235; 1985. 6. Scott, R.; Slater, T. E Free radical scavenging activity of (+)catechin and other flavonoids. In: Slater, T. E; Garner, A., eds. Recent advances in lipid peroxidation and tissue injury. London: Brunel University; 1981: 233-244. 7. Robak, J.; Gryglewski, R. J. Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 37:837-841; 1988. 8. Rafat Husain, S.; Cillard, J.; Cillard, P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 26:2489-2491; 1987. 9. Chen, Y.; Li, X.; Zhao, B.; Zheng, R.; Xin, W. Superoxide and hydroxyl radical scavenging activities of rutin and other natural products studied by ESR. Acta Biophysica Sinica. 5:253-240; 1989. 10. Jia, Z.; Ju, Y.; Chu, T. Studies on the chemical constituents of Saussurea stella Maxim(l), J. Lanzhou University 25:64-67; 1989. 11. Jia, Z.; Li, Y.; Du, M.; Chu, T. Studies on the constituents of Saussurea involucrata Kar. et Kir.(1). Chemical J. Chinese Universities 4:581-584; 1983. 12. Ponti, V.; Dianzani, M. U.; Cheeseman, K.; Slater, T. E Studies on the reduction of NBT mediated through the action of NADH and PMS. Chem-Biol. Interact. 23:281; 1978. 13. Latan, A. The relation of structure to antioxidant activity of quercetin and some of its derivatives: I. Primary activity. J. Food Sci. 31:395-399; 1966. 14. Younes, M.; Siegers, C. P. Inhibitory action of some flavonoids on enhanced spontaneous lipid peruxidation following glutathione depletion. Planta Med. 43:240-243; 1981. 15. Latan, A. The relation of structure to antioxidant activity of quercetin and some of its derivatives: II. Secondary(metal-complexing) activity. J. Food Sci. 31:518-522; 1966.
