342
Activity of Polyphenolic Crude Extracts as Scavengers of Superoxide Radicals and Inhibitors of Xanthine Oxidase Luca C'ostantino 1.2 Albano Albasini', Giulio Rastelli', and Stefania I3envenuti' Dipartimento di Scienze Farmaceutiche della Università di Modena. Via Campi 183. 1-411 00-Modena. Italy 2 Address for correspondence
Received: December 13. 1991
In view of the pharmacological interest in
radical activity against chemically-generated superoxide
radicals of the crude extracts as well as the inhibitory effects on xanthine oxidase, the enzyme which generates superoxide radicals (7). were studied.
phenolic substances, we have determined the total amount of anthocyanins and polyphenols present in the
berries of several cultivars of Ribes. Rubus, and Vaccinium genera. The in vitro antiradical activity of the
crude extracts on chemically-generated superoxide radicals as well as the inhibitory activity towards the
Four cultivars were tested for each species mentioned, except for Rubus occidentalis for which only one cv. was available, and their scavenging activity was compared with that of reference substances (delphinidine chloride and gallic acid).
enzyme xanthine oxidase were studied. All the crude ex-
tracts examined showed a remarkably high activity towards chemically-generated superoxide radicals. The activities were greater than those expected on the basis of the quantities of anthocyanins and polyphenols present in the samples. Furthermore, the extracts showed a
certain inhibitory activity towards xanthine oxidase. Ribes nigrum extracts exhibit the highest activity, being the richest in both anthocyanins and polyphenols. On the other hand, Ribes rubrum extracts seem to contain more active substances than the other crude extracts.
Materials and Methods The berries were purchased from the Istituti di Coltivazioni Arboree delle Università di Torino e di Perugia, from the Istituto Sperimentale per Ia Frutticultura Sezione operativa di
Trento, and from Azienda Regionale Foreste (ARF) Emilia Romagna, Modena, Italy.
Delphinidine chloride (IIPLC purity) (Roth); superoxide dismutase (SOD) (from bovine erythrocytes, 3010 U/mg),
gallic acid, l-nicotinamide-adenine dinucleotide (/-NADIB, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS),
Key words
radicals, free radicals scavengers, xanthine oxidase inhibitors, polyphenolic crude extracts, Ribes species. Rubus species, Vaccinium Superoxide
species.
xanthine, xanthine oxidase (from buttermilk. 1.36 U/mg protein), and Folin-Ciocalteu's phenol reagent were all purchased from
Fluka; allopurinol was from Sigma. The scavenging activity against superoxide radicals and the inhibitory activity against xanthine oxidase were determined in phosphate buffer at pH 7.6 (Normex, Carlo Erba). All the spectrophotometric measurements were made using a Perkin-Elmer A 15 UV/Vis spectrophotometer.
Preparation of crude extracts
Introduction It is well known that plant anthocyanosidic and polyphenolic extracts act as free radical scavengers (1,
2) and as antilipoperoxidants (2) and are helpful in protecting collagen from degradation caused by superoxide anion radicals (3); they are also able to diminish capillary
fragility and permeability and exert a powerful antiphlogistic effect (4—6).
Thus, we have determined the total anthocyanins and polyphenols contents of the berries of Ribes nigrum (black currant), Ribes rubrum (red currant), Rubus idaeus (red raspberry), Rubus occidentalis (black raspberry), Rubusfruticosus (blackberry), and Vaccinium corymbosum (highbush blueberry). Then the in vitro anti-
50 ml of methanol/HCI 2% (95 : 5 v/v) were added to 20 g of berries, deep-frozen at picking-time and kept at —18 °C; after 60 mill, the berries were homogenized at 11 000 rpm
for 1 mill, then centrifuged for 15 mm at 3000rpm.
Determination oftotal anthocyanosidic content of crude extracts
The total anthocyanin amount, calculated as % of
delphinidine chloride present in the berries, was determined by means of the pH differential method (8).
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Abstract
Planta Med. 58(1992) 343
Activity of Polyphenolic Crude Extracts as Scavengers of Superoxide Radicals
Determination of total polyphenols content of crude extracts The method based on Folin-Ciocalteu's phenol
chloride (3.1 O M) and quercetin (7. iO M) in methanoi/I-ICI 95: 5 (v/v). Methanol at the concentrations used (max. 2% v/v) had no effect on NBT reduction.
reagent and spectrophotometric determination at 680 nm was
Xanthine oxidase enzyme assay
employed (9). The results are expressed as % of gallic acid present in the berries.
Xanthine oxidase activity was assayed spectro-
photometrically at 20°C by determining uric acid formation at 295nm (11) with xanthine (21zM) as substrate and xanthine oxidase enzyme sufficient to obtain an average reaction rate for the control reaction (in presence of 1% methanol) of 0.015
Superoxide anion scavenging effect of crude polyphenolic extracts The antiradical activity of crude extracts was de-
termined according to (10) in which superoxide radicals were generated in the system /3-NADH/PMS (NBT 25 fLM. PMS 10 iiM.
-NADH 1561zM). Crude extracts and deiphinidine chloride were present in the reaction mixture as well as in the reference blanks to compensate for their absorbance. The crude extracts and the reference substances
0.001 absorbance units/mm. The enzyme-catalyzed reaction was triggered by the addition of the enzyme. The inhibitory activity of
the extracts was determined by including 10;l/ml of extract in both the reaction and the reference mixtures, to compensate for their absorbance. The percentage inhibition of each crude extract examined was calculated by comparing the reaction rate of the
solution containing inhibitor with that of the control solution. Three determinations were made for each extract.
were assayed at three concentrations with three repetitions value after I mm with that of the control reaction (without in-
As reference, we determined the IC50 value (concentration required for the inhibition of uric acid formation by 50%) for allopurinol (lC5: 0.65 MM).
hibitor), IC50 (concentrations required to inhibit NBT reduction by 50%) values were calculated from log dose-inhibition curves. For calibration of the method, we determined also the activity of SOD (1C50: 6.71 U/mI) and quercetin (1C50: 29.71 ptM). SOD (1mg/mi) and gallic acid (12.10-s M) were dissolved in water, deiphinidine
Extract of
Anthocyaninsa
Polyphenolsb
1C50c
(g/loogbernes) (g/loogberries) (iI/mI)
Anthocyanlnsd
(pM)
Polyphenols° (pM)
Ribes nigrum cv.: BlackDawn
0.34
1.14
1.18
3.58
21.61
P1 73013
0.25
Ben Lomon
0.20
1.42 1.82
Black Reward
0.15
0.66 0.78 0.90
2.28
3.17 3.25 3.06
22.80 33.01
Rotespatelese
0.02
0.28
5.95
1.06
26.84
Red Lake
0.02 0.01 0.02
0.32 0.28 0.19
5.96 8.26
1.07 0.74 1.64
30.73 37.26 28.00
0.04 0.04 0.05 0.02
0.28
7.88 5.69
0.28
7.09
2.82 2.04 2.52 1.27
35.52 32.98 32.72 32.00
0.70
1.21
1.01
6.34
19.72
0.09
0.42
0.13 0.19 0.05
0.41
4.10 3.16 2.40
27.76 20.85
5.87
3.30 3.67 4.08 2.63
0.21
0.42
1.99
3.74
0.08
0.25 0.31
3.47 2.85
13.47 19.53
0.06 0.13
4.85 5.30
26.47
0.34
3.33
3.80
18.25
15.08
Ribes rubrum cv.:
Heros Perfection
9.15
Rubusidaeuscv.: Glen Moy Haida Meeker Rumiloba
0.36 0.36
5.64
Rubus occidentalis cv.:
Bristol Rubus fruticosus cv.:
Loganberry HulIthornless BedfordGiant Hullthornless8
Vaccinium corymbosum cv.: Collins Berkeley Bluecrop Atlantic
Controls: Deiphinidine chloride Gallic acid
0.89 0.31
34.38 29.33
25.75 146.39
a Expressed in terms of delphinidine chloride. Expressed in terms of gallic acid. Amount (pl/ml) of crude extract which causes 50% inhibition of NBT reduction. Anthocyanins (as delphinidine chloride) molar conc. present in the reaction mixture inhibited by 50%. Polyphenols (as gallic acid) molar concentration present in the reaction mixture inhibited by 50%. i,g Harvested in Modena and Trento, Italy.
Table 1 Anthocyanins, polyphenols and super. oxide radical scavenging activity of crude extracts.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
(inhibition between 20 and 80%). After the percentage inhibition had been calculated from the difference between the absorbance
Planta Med. 58(1992)
Results and Discussion
Luca Costantino et a!. Table 2 Anthocyanins, polyphenols and inhibitory activity of crude extracts against xanthine oxidase enzyme.
Table I sets out the anthocyanins and polyphenols contents, expressed as % of deiphinidine chloride and gallic acid, respectively, for every cv. examined. The IC50 values (MI of crude extract/mi of reaction mixture
Extract of
Anthocyaninsa Polyphenols1' (g/ 100 g berries) (g/ 100 g berries)
Ribes nigrum cv.: BlackDawn
% inhibition (S.D.)c
Ben Lomon BlackReward
0.36 0.28 0.20 0.17
1.16 0.72 0.83 1.01
58.46 (0.87) 39.94 (2.45) 50.73 (2.16) 47.89 (0.59)
Ribes rubrum cv.: Rotespatelese Red Lake
0.01 0.02
these are among the most active natural substances (1. 12).
Heros Perfection
0.02 0.02
0.26 0.30 0.29 0.20
41.97 (3.52) 38.42 (4.21) 44.97 (0.38) 28.25 (4.59)
From the data of Table I, a correlation between crude extracts contents and scavenging activity is
clearly apparent. Among the species examined, Ribes nigrum and Rubus occidentalis are the richest in both
Rubusidaeuscv.: Glen Moy Haida Meeker Rumiloba
0.04
0.25 0.25
0.04
0.31
0.03
0.28
16.50 (0.84) 18.37 (1.66) 20.73 (1.80) 18.25 (1.53)
polyphenols and anthocyanins and show the highest activ-
Rubus occidentalis cv.:
0.64
1.11
26.45 (3.43)
0.12
0.45 0.37 0.89 0.38
26.86 (2.15) 21.07 (1.34) 42.50 (1.59) 15.00 (1.70)
0.45 0.25
14.57 (2.96)
0.28 0.34
4.04 (0.84) 6.66 (0.23)
that causes a 50% decrease in NBT reduction) for the scavenging activity against chemically-generated superoxide radicals, the anthocyanins and the polyphenols molar concentrations present in the reaction mixture inhibited by 50% were also reported. We chose deiphinidine
chloride and gallic acid as reference compounds since
ity. As regards the actual contents of anthocyanins and polyphenols present in the reaction mixture inhibited by 50%, it should be noted that these substances are present in a lower amount than that expected on the basis of the ac-
tivity of gallic acid or delphinidine chloride (Table I). Although the use of single reference compounds implies oversimplification, and hence inaccuracy, the above comparisons show quite clearly that the anti-radical activity of crude extracts cannot be accounted for simply in terms of their anthocyanins and polyphenols content; they might contain substances that are more active than delphinidine chloride and gallic acid or that contribute to an overall synergic effect.
P173013
0.04
Bristol Rubus fruticosus cv.:
Loganberry Hullthornless4 Bedford Giant
0.10
0.23 0.05
Hullthornless Vaccinium corymbosum cv.: Collins Berkeley Bluecrop Atlantic
0.19 0.09 0.07 0.12
736 (1.15)
& b See footnotes of Table 1. Each value represent the mean and the standard deviation (S.D.) of three experirnents. Harvested in Modena and Trento, Italy.
As regards the inhibition of xanthine oxidase enzyme (Table 2), the data show the existence of a
modest correlation between the contents of methanolic crude extracts and the enzyme inhibitory activity of the genera examined; however, given the capacity of Ribes
rubrum to inhibit xanthine oxidase as well as the
scavenging of the superoxide ion, it emerges as a very promising species for research into substances that are effective against pathological conditions involving free radicals; it appears to contain substances that are more active than those present in the other extracts, for it has a lower anthocyanins and polyphenols content, yet is pro-
References Salvayre, H., Braquet, P.. Perruchot, T., Douste-Blazy, L. (1982) Stud. Org. Chem. 11,437—442. 2 Meunier, M. 1., Duroux. E., Bastide, P. (1989) P1. Med. et Phytother. 23. 267—274. Bonboisse,
J. C.. Braquet. P., Randoux, A.. Borel, J. P. (1983)
Biochem. Pharmacol. 32. 53—58.
Serrano, J. J.. Mongold. J. J.. Susplusgas. P. (1990) Revue de Phytotherapie Pratique 2, 13—15. Flarborne, J. B., Grayer, R. J. (1988) in: The Flavonoids; Advances in Research since 1980, (Harborne, J. B., ed), pp. 17—18, Chapman and Hall. London. A.lcaraz, M. J., Jimenez. M. J. (1988) Fitoterapia 59, 25—38.
portionally more active.
Acknowledgements
Jones, H. P., Mc Cord, J. M. (1987) Ann. Rep. Med. Chem. 22.
The autors wish to thank Prof. G. Bounous,
Vampa, G., Pecorari, P., Albasini, A., Rinaldi, M.. Melegari. M.,
Istituto di Coltivazioni Arboree dell'Università di Torino, Prof. A. Bergamini, Istituto Sperimentale per Ia Frutticoltura Sezione op-
erativa di Trento, Prof. A. Standardi, Istituto di Coltivazioni Arboree dell'Università di Perugia and Dott. P. Corsinotti, ARF Modena, for kindly providing the fruits.
253— 257.
Losacco. S. (1978) Atti Soc. Nat. Mat. di Modena 109, l—XlI. ' Singleton, V. L., Rossi, J. A. (1965) Am. J. Enol. Viticult. 16,
144—158. ° Robak, J., Gryglewski, R. J. (1988) Biochem. Pharmacol. 37,
'
837—841. White, G. J. (1981)AgentsActions 11.503—509. Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fujita, Y.,
Yasuhara, T., Yoshida, T., Okuda, T. (1989) Chem. Pharm. Bull. 37, 2016—2021.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
344
Activity of Polyphenolic Crude Extracts as Scavengers of Superoxide Radicals and Inhibitors of Xanthine Oxidase Luca C'ostantino 1.2 Albano Albasini', Giulio Rastelli', and Stefania I3envenuti' Dipartimento di Scienze Farmaceutiche della Università di Modena. Via Campi 183. 1-411 00-Modena. Italy 2 Address for correspondence
Received: December 13. 1991
In view of the pharmacological interest in
radical activity against chemically-generated superoxide
radicals of the crude extracts as well as the inhibitory effects on xanthine oxidase, the enzyme which generates superoxide radicals (7). were studied.
phenolic substances, we have determined the total amount of anthocyanins and polyphenols present in the
berries of several cultivars of Ribes. Rubus, and Vaccinium genera. The in vitro antiradical activity of the
crude extracts on chemically-generated superoxide radicals as well as the inhibitory activity towards the
Four cultivars were tested for each species mentioned, except for Rubus occidentalis for which only one cv. was available, and their scavenging activity was compared with that of reference substances (delphinidine chloride and gallic acid).
enzyme xanthine oxidase were studied. All the crude ex-
tracts examined showed a remarkably high activity towards chemically-generated superoxide radicals. The activities were greater than those expected on the basis of the quantities of anthocyanins and polyphenols present in the samples. Furthermore, the extracts showed a
certain inhibitory activity towards xanthine oxidase. Ribes nigrum extracts exhibit the highest activity, being the richest in both anthocyanins and polyphenols. On the other hand, Ribes rubrum extracts seem to contain more active substances than the other crude extracts.
Materials and Methods The berries were purchased from the Istituti di Coltivazioni Arboree delle Università di Torino e di Perugia, from the Istituto Sperimentale per Ia Frutticultura Sezione operativa di
Trento, and from Azienda Regionale Foreste (ARF) Emilia Romagna, Modena, Italy.
Delphinidine chloride (IIPLC purity) (Roth); superoxide dismutase (SOD) (from bovine erythrocytes, 3010 U/mg),
gallic acid, l-nicotinamide-adenine dinucleotide (/-NADIB, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS),
Key words
radicals, free radicals scavengers, xanthine oxidase inhibitors, polyphenolic crude extracts, Ribes species. Rubus species, Vaccinium Superoxide
species.
xanthine, xanthine oxidase (from buttermilk. 1.36 U/mg protein), and Folin-Ciocalteu's phenol reagent were all purchased from
Fluka; allopurinol was from Sigma. The scavenging activity against superoxide radicals and the inhibitory activity against xanthine oxidase were determined in phosphate buffer at pH 7.6 (Normex, Carlo Erba). All the spectrophotometric measurements were made using a Perkin-Elmer A 15 UV/Vis spectrophotometer.
Preparation of crude extracts
Introduction It is well known that plant anthocyanosidic and polyphenolic extracts act as free radical scavengers (1,
2) and as antilipoperoxidants (2) and are helpful in protecting collagen from degradation caused by superoxide anion radicals (3); they are also able to diminish capillary
fragility and permeability and exert a powerful antiphlogistic effect (4—6).
Thus, we have determined the total anthocyanins and polyphenols contents of the berries of Ribes nigrum (black currant), Ribes rubrum (red currant), Rubus idaeus (red raspberry), Rubus occidentalis (black raspberry), Rubusfruticosus (blackberry), and Vaccinium corymbosum (highbush blueberry). Then the in vitro anti-
50 ml of methanol/HCI 2% (95 : 5 v/v) were added to 20 g of berries, deep-frozen at picking-time and kept at —18 °C; after 60 mill, the berries were homogenized at 11 000 rpm
for 1 mill, then centrifuged for 15 mm at 3000rpm.
Determination oftotal anthocyanosidic content of crude extracts
The total anthocyanin amount, calculated as % of
delphinidine chloride present in the berries, was determined by means of the pH differential method (8).
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Abstract
Planta Med. 58(1992) 343
Activity of Polyphenolic Crude Extracts as Scavengers of Superoxide Radicals
Determination of total polyphenols content of crude extracts The method based on Folin-Ciocalteu's phenol
chloride (3.1 O M) and quercetin (7. iO M) in methanoi/I-ICI 95: 5 (v/v). Methanol at the concentrations used (max. 2% v/v) had no effect on NBT reduction.
reagent and spectrophotometric determination at 680 nm was
Xanthine oxidase enzyme assay
employed (9). The results are expressed as % of gallic acid present in the berries.
Xanthine oxidase activity was assayed spectro-
photometrically at 20°C by determining uric acid formation at 295nm (11) with xanthine (21zM) as substrate and xanthine oxidase enzyme sufficient to obtain an average reaction rate for the control reaction (in presence of 1% methanol) of 0.015
Superoxide anion scavenging effect of crude polyphenolic extracts The antiradical activity of crude extracts was de-
termined according to (10) in which superoxide radicals were generated in the system /3-NADH/PMS (NBT 25 fLM. PMS 10 iiM.
-NADH 1561zM). Crude extracts and deiphinidine chloride were present in the reaction mixture as well as in the reference blanks to compensate for their absorbance. The crude extracts and the reference substances
0.001 absorbance units/mm. The enzyme-catalyzed reaction was triggered by the addition of the enzyme. The inhibitory activity of
the extracts was determined by including 10;l/ml of extract in both the reaction and the reference mixtures, to compensate for their absorbance. The percentage inhibition of each crude extract examined was calculated by comparing the reaction rate of the
solution containing inhibitor with that of the control solution. Three determinations were made for each extract.
were assayed at three concentrations with three repetitions value after I mm with that of the control reaction (without in-
As reference, we determined the IC50 value (concentration required for the inhibition of uric acid formation by 50%) for allopurinol (lC5: 0.65 MM).
hibitor), IC50 (concentrations required to inhibit NBT reduction by 50%) values were calculated from log dose-inhibition curves. For calibration of the method, we determined also the activity of SOD (1C50: 6.71 U/mI) and quercetin (1C50: 29.71 ptM). SOD (1mg/mi) and gallic acid (12.10-s M) were dissolved in water, deiphinidine
Extract of
Anthocyaninsa
Polyphenolsb
1C50c
(g/loogbernes) (g/loogberries) (iI/mI)
Anthocyanlnsd
(pM)
Polyphenols° (pM)
Ribes nigrum cv.: BlackDawn
0.34
1.14
1.18
3.58
21.61
P1 73013
0.25
Ben Lomon
0.20
1.42 1.82
Black Reward
0.15
0.66 0.78 0.90
2.28
3.17 3.25 3.06
22.80 33.01
Rotespatelese
0.02
0.28
5.95
1.06
26.84
Red Lake
0.02 0.01 0.02
0.32 0.28 0.19
5.96 8.26
1.07 0.74 1.64
30.73 37.26 28.00
0.04 0.04 0.05 0.02
0.28
7.88 5.69
0.28
7.09
2.82 2.04 2.52 1.27
35.52 32.98 32.72 32.00
0.70
1.21
1.01
6.34
19.72
0.09
0.42
0.13 0.19 0.05
0.41
4.10 3.16 2.40
27.76 20.85
5.87
3.30 3.67 4.08 2.63
0.21
0.42
1.99
3.74
0.08
0.25 0.31
3.47 2.85
13.47 19.53
0.06 0.13
4.85 5.30
26.47
0.34
3.33
3.80
18.25
15.08
Ribes rubrum cv.:
Heros Perfection
9.15
Rubusidaeuscv.: Glen Moy Haida Meeker Rumiloba
0.36 0.36
5.64
Rubus occidentalis cv.:
Bristol Rubus fruticosus cv.:
Loganberry HulIthornless BedfordGiant Hullthornless8
Vaccinium corymbosum cv.: Collins Berkeley Bluecrop Atlantic
Controls: Deiphinidine chloride Gallic acid
0.89 0.31
34.38 29.33
25.75 146.39
a Expressed in terms of delphinidine chloride. Expressed in terms of gallic acid. Amount (pl/ml) of crude extract which causes 50% inhibition of NBT reduction. Anthocyanins (as delphinidine chloride) molar conc. present in the reaction mixture inhibited by 50%. Polyphenols (as gallic acid) molar concentration present in the reaction mixture inhibited by 50%. i,g Harvested in Modena and Trento, Italy.
Table 1 Anthocyanins, polyphenols and super. oxide radical scavenging activity of crude extracts.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
(inhibition between 20 and 80%). After the percentage inhibition had been calculated from the difference between the absorbance
Planta Med. 58(1992)
Results and Discussion
Luca Costantino et a!. Table 2 Anthocyanins, polyphenols and inhibitory activity of crude extracts against xanthine oxidase enzyme.
Table I sets out the anthocyanins and polyphenols contents, expressed as % of deiphinidine chloride and gallic acid, respectively, for every cv. examined. The IC50 values (MI of crude extract/mi of reaction mixture
Extract of
Anthocyaninsa Polyphenols1' (g/ 100 g berries) (g/ 100 g berries)
Ribes nigrum cv.: BlackDawn
% inhibition (S.D.)c
Ben Lomon BlackReward
0.36 0.28 0.20 0.17
1.16 0.72 0.83 1.01
58.46 (0.87) 39.94 (2.45) 50.73 (2.16) 47.89 (0.59)
Ribes rubrum cv.: Rotespatelese Red Lake
0.01 0.02
these are among the most active natural substances (1. 12).
Heros Perfection
0.02 0.02
0.26 0.30 0.29 0.20
41.97 (3.52) 38.42 (4.21) 44.97 (0.38) 28.25 (4.59)
From the data of Table I, a correlation between crude extracts contents and scavenging activity is
clearly apparent. Among the species examined, Ribes nigrum and Rubus occidentalis are the richest in both
Rubusidaeuscv.: Glen Moy Haida Meeker Rumiloba
0.04
0.25 0.25
0.04
0.31
0.03
0.28
16.50 (0.84) 18.37 (1.66) 20.73 (1.80) 18.25 (1.53)
polyphenols and anthocyanins and show the highest activ-
Rubus occidentalis cv.:
0.64
1.11
26.45 (3.43)
0.12
0.45 0.37 0.89 0.38
26.86 (2.15) 21.07 (1.34) 42.50 (1.59) 15.00 (1.70)
0.45 0.25
14.57 (2.96)
0.28 0.34
4.04 (0.84) 6.66 (0.23)
that causes a 50% decrease in NBT reduction) for the scavenging activity against chemically-generated superoxide radicals, the anthocyanins and the polyphenols molar concentrations present in the reaction mixture inhibited by 50% were also reported. We chose deiphinidine
chloride and gallic acid as reference compounds since
ity. As regards the actual contents of anthocyanins and polyphenols present in the reaction mixture inhibited by 50%, it should be noted that these substances are present in a lower amount than that expected on the basis of the ac-
tivity of gallic acid or delphinidine chloride (Table I). Although the use of single reference compounds implies oversimplification, and hence inaccuracy, the above comparisons show quite clearly that the anti-radical activity of crude extracts cannot be accounted for simply in terms of their anthocyanins and polyphenols content; they might contain substances that are more active than delphinidine chloride and gallic acid or that contribute to an overall synergic effect.
P173013
0.04
Bristol Rubus fruticosus cv.:
Loganberry Hullthornless4 Bedford Giant
0.10
0.23 0.05
Hullthornless Vaccinium corymbosum cv.: Collins Berkeley Bluecrop Atlantic
0.19 0.09 0.07 0.12
736 (1.15)
& b See footnotes of Table 1. Each value represent the mean and the standard deviation (S.D.) of three experirnents. Harvested in Modena and Trento, Italy.
As regards the inhibition of xanthine oxidase enzyme (Table 2), the data show the existence of a
modest correlation between the contents of methanolic crude extracts and the enzyme inhibitory activity of the genera examined; however, given the capacity of Ribes
rubrum to inhibit xanthine oxidase as well as the
scavenging of the superoxide ion, it emerges as a very promising species for research into substances that are effective against pathological conditions involving free radicals; it appears to contain substances that are more active than those present in the other extracts, for it has a lower anthocyanins and polyphenols content, yet is pro-
References Salvayre, H., Braquet, P.. Perruchot, T., Douste-Blazy, L. (1982) Stud. Org. Chem. 11,437—442. 2 Meunier, M. 1., Duroux. E., Bastide, P. (1989) P1. Med. et Phytother. 23. 267—274. Bonboisse,
J. C.. Braquet. P., Randoux, A.. Borel, J. P. (1983)
Biochem. Pharmacol. 32. 53—58.
Serrano, J. J.. Mongold. J. J.. Susplusgas. P. (1990) Revue de Phytotherapie Pratique 2, 13—15. Flarborne, J. B., Grayer, R. J. (1988) in: The Flavonoids; Advances in Research since 1980, (Harborne, J. B., ed), pp. 17—18, Chapman and Hall. London. A.lcaraz, M. J., Jimenez. M. J. (1988) Fitoterapia 59, 25—38.
portionally more active.
Acknowledgements
Jones, H. P., Mc Cord, J. M. (1987) Ann. Rep. Med. Chem. 22.
The autors wish to thank Prof. G. Bounous,
Vampa, G., Pecorari, P., Albasini, A., Rinaldi, M.. Melegari. M.,
Istituto di Coltivazioni Arboree dell'Università di Torino, Prof. A. Bergamini, Istituto Sperimentale per Ia Frutticoltura Sezione op-
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