1202 Notes Biol. Pharm. Bull. 24(10) 1202—1205 (2001) Vol. 24, No.

10

DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Radical Scavenging Activity of

Flavonoids Obtained from Some Medicinal Plants
Masafumi OKAWA,a Junei KINJO,b Toshihiro NOHARA,*,a and Masateru ONOc
Faculty of Pharmaceutical Sciences, Kumamoto University,a 5–1 Oe-Honmachi, Kumamoto 862–0973, Japan, Faculty of
Pharmaceutical Sciences, Fukuoka University,b 8–19–1 Nanakuma, Fukuoka 814–0180, Japan, and School of Agriculture,
Kyushu Tokai University,c 5435 Choyo, Aso, Kumamoto 869–1404, Japan.
Received March 5, 2001; accepted July 21, 2001

A reactive oxygen species has been implicated in a range of human pathological diseases such as atheroscle-
rosis and certain cancers. Flavonoids are reported to exhibit various biological activities, including antioxidative
and free radical scavenging activities. Several flavonoids obtained from barley leaves, soybean and some medici-
nal plants, Silybum marianum, Sophorae Flos, Cinnamon, Ephedrae Herba and Scutellariae Radix, were tested
for their DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. The structure–activity relationships
suggested that not only the numbers of hydroxy group but also the position of hydroxy group might be important
for mediating potent activity.
Key words flavonoid; flavone; flavonol; DPPH radical scavenging activity; antioxidative activity; ESR

Flavonoids are a group of polyphenolic compounds, which saponarin (6) and 49-glycosyl-6--sinapoylsaponarin (7) were
have the diphenylpropane (C6–C3–C6) skeleton, ubiquitously isolated from young barley leaves.9) A flavone, baicalin10) (8)
found in fruits and vegetables. The flavonoid family includes was obtained from Scutellariae Radix (Scutellaria baicalen-
flavones, flavonols, flavanones, flavanonols, flavans, flava- sis). Flavonols, quercetin (9), kaempferol (10) and rutin (11)
nols, leucoanthocyanidins, anthocyanidins, aurones, chal- were obtained from Sophorae Flos (Sophora japonica).11)
cones, and isofavones. The structural difference in each Flavanols, gallocatechin (12) was obtained from Ephedrae
flavonoid family results from the variation in the number and Herba (Ephedra sinica) and epicatechin (13) and their poly-
arrangement of the hydroxyl groups and the extent of glyco- mers (14—16) were from cinnamon (Cinnamomum cas-
sylation of these groups.1) sia).12) Isoflavones,13) daidzin (17), genistin (18) and glycitin
Epidemiological studies suggest that the consumption of (19), and their metabolites14) (20—25) were obtained from
flavonoids is effective in lowering the risk of coronary heart soybeen (Glycine max). Flavonolignans, silybin (26) and sily-
disease.2—4) In addition, the flavonoids exhibit a wide range christin (27) were isolated from a silymarin group (Silybum
of biological activities, including anticarcinogenic, antiin- marianum).15) (Fig. 1).
flammatory, antiradical, and antioxidant actions. Especially, DPPH Radical Scavenging Activity by ESR The
they may exert antioxidative effects as free radical scav- DPPH radical scavenging activity of a test sample was esti-
engers, hydrogen-donating compounds, singlet oxygen mated by ESR according to the method of Tateyama et al.16)
quenchers, and metal ion chelators, properties attributed to Various concentrations of these flavonoids in DMSO were
the phenolic hydroxyl groups attached to the ring structures. added to 150 m M DPPH radical in ethanol and mixed for 10 s.
Nowadays, it is reported that a reactive oxygen species After further mixing for 60 s, ESR spectra were recorded
(ROS) is implicated in a wide range of human diseases such using a JEOL at 100 KHz field modulation frequency and 1G
as atherosclerosis and certain cancers. When an imbalance modulation amplitude at an output power of 8 mW. Mn(II)
between ROS generation and antioxidants occurs, oxidative was used as standard sample. All experiments were carried
damage will spread over all the cell targets (DNA, lipids, out at room temperature (25 °C). DPPH radical reducing ac-
proteins).5) Antioxidants in foods and medicinal plants (or tivity of the test sample was expressed as I5[(A0/AX)/A0]
herbs) have attracted interest in recent years. Flavonoids 3100%, where A0 and AX were the relative heights of each
given as biological substances in foodstuffs may contribute to radical against the standard signal intensity of the manganese
the prevention of diseases, although they do not have strong oxide marker in a reaction mixture and the test sample, re-
biological activities.6—8) spectively. The results were indicated as the concentration re-
In the preceding paper,9) we reported the antioxidative ef- quired to effect a 50% inhibition of decreasing signal peak
fects of flavon C-glycosides isolated from young green bar- height (IC50) by ESR.
ley (Hordeum vulgare var. nudum) leaves. Herein, we present
antioxidative effects of various types of flavonoids obtained RESULTS AND DISCUSSION
from some medicinal plants, using DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical scavenging activity by ESR. Seven flavones 1—7 isolated from barley leaves showed
similar results (Table 1) to those obtained from a previous in-
MATERIALS AND METHODS vestigation9) by the colorimetric method. Since 5—7 (5:
IC50521.1 m M, 6: IC50551.1 m M, 7: IC50525.5 m M) exhibited
Flavonoids Flavone C-glycosides, isovitexin (1), sapo- stronger DPPH radical scavenging effect than did 1—4 (1:
narin (2), isovitexin 49,7-diglucoside (3), isovitexin 7-rham- IC50.1000 m M, 2: IC505211 m M, 3: IC505291 m M, 4: IC505
nosylglucoside (4), 6--sinapoylsaponarin (5), 6--feruloyl- 299 m M), the importance of sinapoyl and feruloyl groups for
∗ To whom correspondence should be addressed. e-mail: none@[Link] © 2001 Pharmaceutical Society of Japan
October 2001 1203

Fig. 1

the DPPH radical reducing activity was confirmed. Further- isoflavan and flavonolignan) were prepared from some medi-
more, baicalin (8) having 5,6,7-trihydroxy group showed the cinal plants, Sophora japonica, Ephedra sinica, Cinnamo-
strongest activity in the tested flavones (1—8). mum cassia, Glycine max and Silybum marianum, and their
Despite having three hydroxy groups, isovitexin (1) did DPPH radical reducing activity was compared. The results of
not exhibit any activity similarly to the former experiment.9) DPPH radical reducing activity were shown in Table 1.
Since baicalin (8) has no hydroxy group on the B-ring, the The structure of flavonolignans, silybin (26) and sily-
hydroxy group at C-49 may not contribute to scavenge for christin (27), was constitued of a flavanonol unit and a
DPPH radical. Probably, the hydroxy group at C-6 of 8 plays phenylpropanol unit in analog with 5—7 which showed po-
a role in scavenging DPPH radical. tent activity. However, the activity of 26 and 27 was much
Furthermore, nineteen flavonoids (8—27) belonging to six weaker than 5—7. Especially, 26 did not show any activity,
flavonoid classes (flavonol, flavanol, isoflavone, isoflavanone, although silychristin (27) having a hydroxy group at C-39
1204 Vol. 24, No. 10

Table 1. Inhibitory Activity of Various Types of Flavonoids on DPPH (catechol or pyrogallol) was more important than the number
Radical of hydroxy groups.
Compounds No. IC50 (m M)
In the case of isofavone, isoflavanone and isoflavan family
(17—25), a isoflavan (25) exhibited stronger activity than the
Flavones others. Successively, genistin (18) showed stronger activity,
(Glycosides) although the other isoflavone glycosides were much less po-
Isovitexin 1 .1000 tent. Generally, the DPPH radical reducing activity of
Saponarin 2 211
Isovitexin 49,7-diglucoside 3 291 isoflavonoid (isofavone, isoflavanone and isoflavan) was
Isovitexin 7-rhamnosylglucoside 4 299 weaker than those of the other class of flavonoid (flavone,
6--Sinapoylsaponarin 5 21.1 flavonol and flavanol).
6--Feruloylsaponarin 6 51.1 The flavonoids (9, 11—16) which showed potent activity
49-Glucosyl-6--sinapoylsaponarin 7 25.5
have a catechol unit in their structure and 8 has a similar fea-
Baicalin 8 15.5
Flavonols ture carrying an adjacent dihydroxy group (C-5, C-6) in the
(Aglycones) A ring. On the other hand, less potent compounds 26 and 27
Quercetin 9 8.9 have a guaiacyl group instead of a catechol group. Therefore,
Kaempferol 10 41.2 the significance of catechol group on DPPH radical reducing
(Glycoside)
Rutin 11 11.1
activity was thus confirmed.17,18) However, the number of hy-
Flavanols droxy groups was not to be always important when compared
Gallocatechin 12 6.9 with the activity of 12 and 15. The position of hydroxy group
Epicatechin 13 11.7 might be more important for mediating potent DPPH reduc-
Procyanidin B-2 14 11.7 ing activity.
Procyanidin C-1 15 7.3
Procyanidin A-2 16 8.8
Various antioxidants are contained in most foods and med-
Isoflavones icinal plants. They are polyphenols, vitamins, carotenoids,
(Glycosides) and flavonoids in vegetables and fruits. The recent study on
Daidzin 17 642 antioxidative substances in foods and medicinal plants is a
Genistin 18 265 comparatively new province. The digestion, absorption, bio-
Glycitin 19 743
(Aglycones) logical activity and metabolic pathway in each food and med-
Daidzein 20 532 icinal plant is still complicated because a number of sub-
Genistein 21 1000 stances are included in various proportions in one plant.
Glycitein 22 612 Meanwhile, their safety has been established to a certain ex-
Isoflavanones
tent for empirical and traditional use from ancient times.
Dihydrogenistein 23 .1000
Dihydroglycitein 24 311 Therefore, the study of antioxidative substances in foods and
Isoflavan medicinal plants will become important, and such antioxida-
Equol 25 188 tive substances might be applied for treatment and prevention
Flavonolignans of human diseases.
Silybin 26 .1000
Silychristin 27 274
Acknowledgement We are grateful to Dr. G. Nonaka
(Usaien Pharmaceutical Co.) for his helpful suggestion.

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