Vol.

63, No 2/2016
353358
http://dx.doi.org/10.18388/abp.2015_1178
Regular paper

Isolation and antioxidant activity of flavonoids from Holarrhena

floribunda (G.don) leaves
Jelili A. Badmus1, Okobi E. Ekpo1, Fanie Rautenbach2, Jeanine L. Marnewick2,
Ahmed A. Hussein3 and Donavon C. Hiss1*
1Department of Medical Biosciences, University of the Western Cape, New Life Sciences Building, Private Bag X17 Bellville, Cape Town 7535,
South Africa; 2Oxidative Stress Research Centre, Institute of Biomedical and Microbial Biotechnology, Faculty of Health and Wellness Sciences,
Cape Peninsula University of Technology, Cape Town South Africa; 3Department of Chemistry, University of the Western Cape, Chemical Sci-
ences Building, Private Bag X17 Bellville, Cape Town 7535, South Africa

Bioactive polyphenolics are ubiquitously present in zymes by the flavonoids are essential in preventing im-
plants and may play an important role in the preven- pending damage to important cellular components such
tion and management of certain human diseases. Three as DNA, proteins and lipids (Suganya et al., 2007). Oxi-
known flavonoids viz Kaemperol-3-O-rutinoside (1), dation of lipids for example, leads to lipid peroxidation,
quercetin-3-O-glucoside (2) and kaemperol-3-O-gluco- which is a free-radical mediated propagation of oxida-
side (3) and inseparable mixture (1:1) of quercetin-3-O- tive insult to the polyunsaturated fatty acid component
glucose/galactose (4) were isolated, and identified for of cell membranes (Heim et al., 2002). The potential of
the first time from Holarrhena floribunda. The antioxi- the health benefit of these compounds for the preven-
dant capacity using the ORAC, FRAP and TEAC assays tion and therapeutic uses has led to the investigation and
and inhibition of lipid peroxidation were measured for identification of a wide range of bioactive principles, that
isolated flavonoids. The result showed that compounds include mainly flavonoids and phenolic compounds in
2 and 4 showed significantly increased ORAC, TEAC, and the plants (vegetables, fruits, leaves, seeds, cereal, roots,
FRAP activities with low pro-oxidant potential as well spices and herbs) (Suganya et al., 2007). The protective
as improved lipid peroxidation inhibition levels when effects of flavonoids are attributed to their ability to
compared to compounds 1 and 3. The most active com- transfer electrons, chelate metals, activate antioxidant
pounds were found to be flavonoids with a quercetin enzymes, reduce alpha-tocopherol radicals and inhibit
basic structure. These results imply that the isolated fla- oxidases (Heim et al., 2002). Anti-inflammatory, anti-diar-
vonoid glycosides are responsible for the antioxidant ac- rhoeal, anti-ulcer, anti-viral, anti-allergic and vasodilatory
tivity of the plant leaves and it forms the scientific basis actions have also been attributed to these phytochemi-
for its traditional usage. cals (Proestos & Komaitis, 2006). Previously, the metha-
nolic extract and sub-fractions from the leaves of Holar-
Key words: polyphenolics, plants, Holarrhena floribunda, antioxidant, rhena floribunda was reported to exhibit strong antioxidant
flavonoids activity (Badmus et al., 2010, Badmus et al., 2013). The
Received: 17 August, 2015; revised: 13 January, 2016; accepted: present work sought to isolate and characterize the com-
11 February, 2016; available on-line: 12 May, 2016 pounds responsible for the antioxidant activities.

PLANT MATERIAL
INTRODUCTION
Holarrhena floribunda leaves were collected in Igbajo,
Flavonoids are low molecular weight, structurally re- Osun state, Nigeria during the raining season in July,
lated compounds with basic features of the 2-phenyl- 2011. It was identified and authenticated by the duo of
benzo--prone nucleus consisting of two benzene rings Chukwuma, E.C and Ugbogu O.A at the Federal Re-
linked through a heterocyclic pyran ring (Cushnie & search Institute of Nigeria (FRIN). Herbarium number
Lamb, 2005). The protective health benefits of natural FHI 109764 was issued at the Institute.
flavonoids and its broad pharmacological activity can- Preparation of methanolic leaf extract of Holar-
not be overemphasized and has led to increased in- rhena floribunda. The leaves of Holarrhena floribunda
terest among scientists with bias in functional foods were dried at room temperature. The dried leaves weigh-
(Kahkonen et al., 1999). Flavonoids and other phenolic ing 1.748 kg were extracted with 5.25 L methanol under
compounds with antioxidant activities are reputed to stirring for 48 h (X 2). The filtrate was evaporated till
play a preventive role in the development of oxidative dryness to give 380.70 g representing 21.78% yield (dry
stress-related diseases like certain cancers (Kahkonen et weight).The part of the dried total extract filtrate (50 g)
al., 1999). Reactive oxygen species (ROS) produced dur- was loaded into silica gel column eluted with a gradient
ing normal metabolism or induced by exogenous factors,
have been implicated in the aetiology of several human *
e-mail: [email protected]
diseases (Tapas et al., 2008). ROS is known to contrib- Abbreviations: ABTS, 2,2azabis-(3-ethyl benzothiozoline-6-sul-
ute to cellular aging, mutagenesis, carcinogenesis and fonate); AAPH, 2,2-azobis (2-aminopropane); DCM, dichlorometh-
coronary heart diseases (Sastre et al., 2000, Takabe et al., ane; EGCG, Epigallocatechin gallate; FRAP, ferric reducing antioxi-
dant power; MEOH, methanol; ORAC, oxygen radical absorption
2001, Kawanishi et al., 2001). The scavenging of ROS, capacity; ROS, reactive oxygen species; TE, Trolox equivalent; TEAC,
metal chelating ability or induction of antioxidant en- Trolox equivalent antioxidant capacity
354
J. A. Badmus and others 2016

mixture of ethyl acetate and methanol. The ratio of ethyl in a complex mixture (Arts et al., 2004). The assay was
acetate: methanol used was 9:1, 8:2, 6:4 and 4:6, respec- evaluated in accordance with previously described meth-
tively. A total of 51 eluents (500 ml each) was collected od (Re et al., 1999). Briefly, 25 l of test sample (0.1,
and pooled together according to their TLC profile to 1, 10, 100 g/ml) was added to 300 l of ABTS (1 ml
18 main fractions. of ABTS in 20 ml ethanol) in triplicate. The reaction
Sub-fractions 13 and 14 from the main total extract mixture was incubated for 30 min. Absorbance of the
were shown to contain flavonoids as indicated by TLC reaction was measured with a Multiskan spectrum plate
and selected for further purification. Both fractions (13 reader at a wavelength of 734 nm at 25C against the
& 14) were subjected to silica gel column eluted with blank prepared with ethanol. Results were expressed as
dichloromethane:methanol (DCM: MEOH) mixture 1% micromole Trolox equivalents per milligram dry weight
to 15%. The sub-fractions obtained were purified on Se- (M TE/mgDW) of test samples.
phadex column using 100% ethanol as mobile phase and Inhibition of Fe(II)-induced microsomal lipid
finally high performance liquid chromatography (HPLC) peroxidation. Inhibition of Fe(II)-induced microsomal
using different ratios of acetonitrile and water to yield lipid peroxidation was determined by the method de-
three pure compounds (13) and inseparable mixture scribed by Snijman and co-workers (Snijman et al., 2009).
(1:1) (4). Liver microsomes were prepared from male Fischer 344
Oxygen radical absorption capacity (ORAC). rats. Microsomes were purified from the liver S9 fraction
ORAC generates both qualitative and quantitative meas- using a Sepharose 2B column as previously described
ures which present fast and slow acting antioxidant ac- (Gelderblom et al., 1984). The absorbance was read at
tivity of a test compound (DeLange & Glazer, 1989). 532 nm. The percentage inhibition of sample relative to
Briefly, 12 l of test sample (0.1, 1, 10 and 100 g/ control was calculated by the equation below;
ml) was combined with 138 l of the fluorescein work-
ing solution. The reaction was started by the addition of [(AcontrolAsample)/Acontrol] 100.
50 l reactive species in 96-well plate. The absorbance Where Acontrol and Asample refer to the absorbance of re-
was measured with a Fluoroskan spectrum plate reader acting mixture without the sample and the absorbance in
with excitation/emission wavelengths set at 485/530 nm the presence of sample respectively.
at 37C against a reagent blank prepared with phosphate Statistical analyses. Data are expressed as means
buffer. The method measures the antioxidant scaveng- S.D. of experiments performed in triplicate. The values
ing capacity of thermal decomposition generated by (1) were analyzed by Two-Way ANOVA, followed by Tuk-
peroxyl radical of 2,2-azobis (2-aminopropane) dihydro- eys multiple comparison tests using GraphPad Prism
chloride (AAPH; ORACROO. assay), (2) hydroxyl radical software version 6 for Windows (GraphPad Software, La
(ORACOH. assay), generated by H2O2-Cu2+ (H2O2, 0.3%; Jolla California USA, www.graphpad.com). A P value of
Cu2+ [as CuSO4], 18 M), or (3) Cu2+ [as CuSO4], 18 M less than 0.05 was considered significant.
as a transition metal oxidant at 37C. ORAC values were
expressed as micromole of Trolox equivalents (TE) per
milligram dry weight (M TE/mg DW) of test sample, RESULTS
except when Cu2+ (without H2O2) was used as an oxi-
dant in the assay. In the presence of Cu2+ without H2O2,
test samples acted as prooxidants rather than antioxi- Isolation and characterization of flavonoids from
dants in the ORAC assay. The copper-initiated prooxi- Holarrhena floribunda leaves
dant activity was calculated using [(AreaBlank AreaSample)/ TLC screening of different fractions from the metha-
AreaBlank] 100 and expressed as prooxidant units; one nolic leaf extract of Holarrhena floribunda, showed that
unit equals the prooxidant activity that reduces the area sub-fraction 13 and 14 were rich in flavonoids, which
under the fluorescein decay curve by 1% in the ORAC were submitted for chromatographic purification using
assay (Cao et al., 1997). the combination of silica gel and sephadex column and
Ferric reducing antioxidant power (FRAP). FRAP HPLC. Compound 1 yielded 17.6 mg while 2, 3 and 4
measures a single electron transfer reaction from the an- (inseparable mixture (1:1) of quercetin-3-O-glucose/
tioxidant molecule to the oxidant. The change in absorb- galactose) gave 12.3, 17.3 and 14.72 mg, respectively
ance value of either oxidant or antioxidant is a measure (Fig. 1). The compounds were fully identified based on
of reducing capability of the antioxidant (Ou et al., 2002). 1D and 2D NMR spectra. The data were compared with
A working FRAP reagent was prepared in accordance previously published study (Gudej, 2003; Sikorska &
with previously described method (Benzie and Strain, Matlawska, 2000; Nowak & Wolbis, 2002). These flavo-
1999). The Samples (100 l; [0.1, 1, 10, 100 g/ml]) noids were further subjected to antioxidant capacity test,
were prepared in triplicate followed by the addition of 3 including ORAC, FRAP, TEAC and lipid peroxidation
ml freshly prepared FRAP reagent. The reaction mixture inhibition.
was incubated at 37C for 4 min and absorbance read
at 593 nm against the blank prepared with distilled wa- Evaluation of oxygen radical absorbance capacity
ter. l-Ascorbic acid was used as standard and the results
were expressed as micromole ascorbic acid equivalent The antioxidant evaluation of flavonoid-rich extracts
per milligram dry weight (M AAE/mg DW) of the test using the ORAC assay in the presence of AAPH, Cu2+-
samples.
Trolox equivalent antioxidant capacity (TEAC).
The TEAC assay estimates inhibition of radical cation
production by the antioxidant in the sample. The con-
centration of antioxidant in the sample is inversely
proportional to the absorbance of the radical cation
produced by 2,2-azo-bis-(3-ethyl benzothiozoline-6-sul-
fonate) (ABTS) (Gupta et al., 2009). TEAC assay is a Figure 1. The structure of flavonoids isolated from the Holar-
useful assay for tracking down unknown antioxidants rhena floribunda leaves.
Vol. 63
Flavonoids from Holarrhena floribunda (G.don) leaves 355

20000
****
15
****
15000 ****
M TE/mgDW

pro-oxidant M/mgDW
****
10
10000 ****

5000 5
**

0
C1 C2 C3 C4 T.Extract EGCG
0
C1 C2 C3 C4 T.Extract EGCG

Figure 2. The ORAC values of isolated flavonoids, total extract Figure 4. The percentage of pro-oxidant induction in relation to
and the standard (EGCG) using AAPH as an oxidant. untreated control in the presence of Cu2+ evaluated using ORAC
Data are expressed as mean S.D. M TE/mgDW. ****P<0.0001 assay.
when compared to T.Extract (Total extract). Note that the standard Data are expressed as mean S.D. pro-oxidant M/mgDW.
deviation is very low and as such is not showing in the figure. **P<0.05 when compared to the T.Extract (total extract).

H2O2 and Cu2+ showed that the isolated flavonoids and

total extract have remarkable ORAC activities against the
oxidants AAPH and Cu2+-H2O2 as shown in Figs. 2 and 5000
****
3 below. However, Compounds 2 and 4 were significant-
ly (P<0.05) higher compared with compounds 1, 3 and 4000
the total extract in the AAPH and Cu2+-H2O2 systems.
M AAE/mgDW
Figure 4 shows the percentage pro-oxidant ability of the 3000
compounds in the presence of Cu2+. The result (Fig.4)
showed that none of the isolated compounds and the to- 2000
****
tal extract acted as a pro-oxidant in the presence of the ****

Cu2+. 1000

**** ****
Evaluation of ferric reducing antioxidant power 0
C1 C2 C3 C4 T.Extract EGCG
The ability of compounds to reduce Fe3+ to Fe2+ was
evaluated using the established FRAP method. Figure 5
below shows the ability of each compound and the to-
tal extract to reduce Fe3+ to Fe2+ calculated as micro- Figure 5. Ferric reducing antioxidant power assay (FRAP) of iso-
mole ascorbic acid equivalent per milligram dry weight lated compounds, total extract and the standard (EGCG).
(mol AAE/mgDW). The present investigation showed Data are expressed as mean S.D. M AAE/mgDW. ****P<0.0001
that compounds 1 and 3 have significantly lower FRAP when compared to the T.Extract (total extract).
activity (P<0.0001) when compared with compounds 2, Trolox equivalent antioxidant capacity (TEAC)
4 and the methanolic extract with high activity (1561.37,
1527.63 and 850.06 mol AAE/mgDW respectively). Trolox equivalent antioxidant capacity of compounds
The standard (EGCG; 4754 mol AAE/mgDW) was and the total extract of Holarrhena floribunda are presented
significantly higher than the isolated compounds and the in Fig. 6. The TEAC assay involves evaluation of the
total extract. quenching potential of antioxidants in the presence of

8.0 10 6
**** **** **** 2000

**** ****
6.0 10 6 ****
1500
M TE/mgDW

M TE/mgDW

4.0 10 6
1000
**** ****

2.0 10 6 ****
500 ****

0
0
C1 C2 C3 C4 T.Extract EGCG C1 C2 C3 C4 T.Extract EGCG

Figure 3. The ORAC values of isolated flavonoids, total extract Figure 6. Trolox equivalent capacity (TEAC) of the isolated flavo-
and the standard (EGCG). noids and the total extract in mole trolox equivalent per gram
Cu2+-H2O2 was used to generate OH radical as an oxidant. Data are dry weight.
expressed as mean S.D. M TE/mgDW. ****P<0.0001 when com- Data are expressed as mean .S.D. M TE/mgDW. ****P<0.0001
pared to the T.Extract (total extract). when compared to the T.Extract (total extract).
356
J. A. Badmus and others 2016

long-lived radical cation chromophore 2,2 azinobis-(3- in compounds 1 and 3. The IC50 values of the results
ethylbenzothiazoline-6-sulphonate) ABTS+. The result of were calculated using the Prism 6 statistical software.
the TEAC assay follows the same manner as observed The IC50 values (Table 1) showed that compound 2 has
in the FRAP assay above. Compound 2 shows (1649.4 a 10.4 g/ml while compounds 4 and the extract have
mol TE/mgDW)>compound 4 (1589.9 mol TE/ 9.8 and 7.2 g/ml, respectively and IC50 values for com-
mgDW)>EGCG (1573.5 mol TE/mgDW)> Total ex- pounds 1 and 3 could not be determined because they
tract (1084.4 mol TE/mgDW)>compound 1 (394.8 do not show dose dependent inhibition of lipid peroxi-
mol TE/mgDW)>compound 3 (337.5 mol TE/ dation. There is, however, no significant difference in the
mgDW). The results showed that the TEAC values for IC50 values among the compound 2, 4 and the extract.
compounds 2, 4, Total extract and EGCG were sig-
nificantly (P<0.0001) higher when compared with com- DISCUSSION
pounds 1 and 3.
Dietary antioxidants with potential for therapeutic and
Inhibition of Fe-induced lipid peroxidation in rat liver
prevention use have been a major focus of research in
microsome
recent years. Isolation and characterization of bioactive
The effects of the compounds and the total extract compounds from plants with health-maintaining and dis-
on the induced-lipid peroxidation were evaluated in S9 ease-preventing properties have led to the isolation and
rat liver fraction. The compounds 2, 4 and the extract identification of an array of compounds among which
showed dose dependent inhibition of lipid peroxidation, include flavonoids (Suganya et al., 2007). The present
while dose dependent inhibitions were not observed work isolated flavonoids by subjecting the methanolic
Table 1. Inhibition of Fe-induced lipid peroxidation by isolated compounds and the total extract in rat liver microsome.

Compound Concentration (g/ml) Inhibition (%) IC50 (g/ml) Regression equation

1 0.67 4.500.37 N.D. Y=0.4353*X + 3.093

6.67 6.001.76 R2=0.8370
13.40 7.551.86
16.70 9.272.19
26.70 16.060.98
2 0.67 11.591.62 10.4 Y=2.202*X + 13.13
6.67 20.449.98 R2=0.8193
13.40 55.520.81
16.70 55.371.27
26.70 63.946.45
3 0.67 0.430.7 N.D. Y = 0.4822*X 0.8752
6.67 0.440.4 R2 =0.6700
13.40 4.582.14
16.70 9.751.11
26.70 11.340.55
4 0.67 8.580.64 9.8 Y=2.333*X + 14.05
6.67 31.112.74 R2=0.9074
13.40 50.670.81
16.70 61.21 1.27
26.70 68.340.47
Extract 0.67 2.141.70 7.2 Y=2.742*X + 3.333
6.67 17.335.77 R2=0.9369
13.40 50.131.68
16.70 52.200.84
26.70 70.700.82
Quercetin 0.27 5.311.73 2.95 Y=3.909*X + 11.80
0.53 26.001.70 R2=0.8739
1.13 23.531.73
2.26 51.230.83
4.53 65.280.99
N.D.=Not detected
Vol. 63
Flavonoids from Holarrhena floribunda (G.don) leaves 357

leaf fractions of Holarrhena floribunda to different chroma- flavonoids remarkably correlate with the 3-OH group on
tography techniques. The flavonoids were isolated, puri- the B-ring of flavonoids basic structure which is the only
fied using HPLC, identified with NMR spectroscopy and different between quercetin and kaempferol flavonoids
data compared with that of the literature. Three pure structure. The strong antioxidant activity of the extract,
compounds and an inseparable mixture of two com- however, implies that the isolated compounds 2 and 4
pounds were isolated and identified. The compounds are responsible for the observed antioxidant activity in
showed a dull spot under UV lights, which indicate the the total extract although they are not complementary.
nature of flavonoids. Endogenous and exogenous factors such as metabo-
However, further to the isolation of flavonoids from lism, chemicals and ionizing radiations are linked to the
the leaves, isolated flavonoids were subjected to anti- induction of free radicals in biological tissues (Iqbal et
oxidant evaluation using ORAC, FRAP, TEAC and the al., 2003). Iron is known to be involved in the genera-
in vitro inhibition of lipid peroxidation. The ORAC as- tion of reactive oxygen species (ROS) in vivo which may
say takes into account both inhibition time and the de- attack lipids to form damaged products (Imlay & Linn,
gree of inhibition into a single assay by considering area 1988; Aruoma et al., 1989). Lipid peroxidation is an im-
under the curve (Cao et al., 1995). In this study, in ad- portant product of reaction between free radicals and li-
dition to the commonly used AAPH as an oxidant, pids (DSouza & Prabhu, 2006). The ability of an agent
Cu2+-H2O2 and Cu2+ alone were used as a source of oxi- to protect lipids from oxidative damage by ROS can be
dant. Cu2+-H2O2 is used to mimic the in vivo generation related to its antioxidant ability.
of oxidant as H2O2 and transition metal are available in Iron-induced lipid peroxidation is a reliable biological
vivo and are frequently used in vitro to induce oxidative marker of cellular oxidative stress (Dargel, 1992). The
damage to proteins and nucleic acids (Cao et al., 1997; result, showed that compounds 1 and 3 did not exhibit
Parthasarathy et al., 1989; Sato et al., 1992). In the pres- significant inhibition of lipid peroxidation, as the values
ence of a transition metal like Cu2+ alone, it is believed did not reach up to 50% inhibition in the concentration
that flavonoids act as a pro-oxidant (Cao et al., 1997). used while compounds 2 , 4 and total extract showed
The Cu2+ was used in this study to evaluate pro-oxida- non-significant IC50 values when compared to each oth-
tive potential of the isolated flavonoids. Evaluation of er. The lipid peroxidation inhibitory activities of flavo-
the antioxidant capacity of the isolated compounds using noids are shown to be related to the number of hydroxyl
the ORAC peroxyl radical (ORACROO) assay showed a group, substitution of the hydroxyl group, catechol moi-
high degree of antioxidant capacity for all the isolated ety on the B-ring and double bonds between carbon
compounds. However, compounds 2 and 4 showed sig- 2 and 3 of the C-ring (Cholbi et al., 1991, Mora et al.,
nificantly higher activities compared with the compounds 1990). The presence of a sugar moiety however affects
1 and 3. The ORACOH activity of these compounds the activity of the lipid peroxidation inhibition due to
also followed a similar pattern to what is obtained in steric hindrance between the sugar and adjacent hydrox-
ORACROO activities. The ORACOH activities of the flavo- yl group likewise methoxy group (Cholbi et al., 1991).
noids were several folds higher than what was obtained This can be applied to the results obtained in the pre-
for ORACROO. This could be that the flavonoids isolated sent study when the inhibition of lipid peroxidation of
were more specific to and aimed at OH radical protec- the isolated glycosylated flavonoids was compared with
tion. Chen et al. in their structural-activity relationships the quercetin standard. The result showed that IC50 in-
concluded that phenolic hydroxyls in flavonoids, hy- hibition of peroxidation by the standard is about 3 folds
droxyl groups in A and B rings (ortho-dihydroxyl groups lower than the isolated flavonoids. On the other hand,
in A and B rings), are important to the hydroxyl radical glycosylation of flavonoids increased the hydrophilicity
scavenging activity of flavonoids (Chen et al., 2002). In- and hence enhances bioavailability better than for the
versely however, the pro-oxidant activities using Cu2+ in aglycone flavonoids (Kumar & Pandey, 2013). However,
the ORAC assay showed that compounds 1 and 3 were despite lower activity of glycosylated flavonoids com-
more prone to behave like prooxidants in the presence pared to their aglycone, bioavailability will be a deter-
of the metal than the compounds 2 and 4. This is not mining factor of their bioactivity in vivo (Thilakarathna &
unexpected because the previous reports have correlated Rupasinghe, 2013). Enhancement of bioavailability will
the number of flavonoids OH group substitution to its be an important factor in order to exert an eventual ben-
ORAC activity (Rice-Evans et al., 1996). The observed eficial effect in vivo (Thilakarathna & Rupasinghe, 2013).
results can be related to the basic structure of the fla- In addition, the results obtained in this study is in agree-
vonoids as compounds 2 and 4 have quercetin as their ment with the number of hydroxyl group substitution on
basic structure, while 1 and 3 have kaempferol. Cao et al. the B-ring as compounds 2 and 4 with hydroxyl group
have demonstrated that ORACROO activity of quercetin in B-ring showed effective dose dependent lipid peroxi-
is higher than that of kaempferol (Cao et al., 1997). The dation compared with compounds 1 and 3.
pro-oxidant activity can also be related to the number of In summary, the antioxidant activity of flavonoids,
substituted OH groups in the flavonoids as it showed which involve neutralization of free radicals initiating
in this study that the lower the number of OH groups oxidative-cascade of reactions or termination of the free
the higher the pro-oxidation capacity in the presence of radical chain reaction due to hydrogen donating prop-
Cu2+. erty, can be related to their structures (Cao et al., 1997;
The importance of substitutions such as OH-group, Suganya et al., 2007). Compounds 2 and 4 have an ortho-
methoxy group and O-dihydroxy in the B-ring are relat- dihydroxyl in the B-ring of the flavonoid skeleton (cat-
ed to the high antioxidant activity of the flavonoids us- echol) which plays an essential role in the antioxidant ac-
ing a FRAP assay (Firuzi et al., 2005). The results of the tivity of flavonoids (Bors et al., 1990) and is responsible
FRAP assay of this study show that OH group substitu- for the observed effects in those compounds. The anti-
tion in the compounds 2 and 4 are responsible for the oxidant activity of flavonoids is known to depend largely
significant antioxidant activity compared to compounds 1 on the functional groups attached to the nuclear struc-
and 3. In the same vain, similar activities were shown by ture (Heim et al., 2002). It is noteworthy to observe that
the isolated flavonoids when subjected to TEAC antioxi- in the battery of antioxidant assays done in this study,
dant assay. The result implies that the activities of these no significant differences could be shown when compar-
358
J. A. Badmus and others 2016

ing compounds 2 and 4 and compounds 1 and 3, which ium moniliforme. Biochem Pharmacol 33: 16011603. doi: 10.1016/0006-
showed selective antioxidant activities only. This implies 2952(84)90280-6.
Gudej J (2003) Kaempferol and quercetin glycosides from Rubus idaeus
that the only common factor that is responsible for the L. leaves. Acta Pol Pharm 60: 313315.
observed differences in the antioxidant activities of the Gupta R, Sharma M, Lakshmy R, Prabhakaran D, Reddy KS (2009)
flavonoids in this study is ortho-dihydroxyl in the B-ring. Improved method of total antioxidant assay. Indian J Biochem Biophys
This work has lend scientific credence to the claim 46: 126129.
Heim KE, Tagliaferro AR, Bobilya DJ (2002) Flavonoid antioxidants:
by the tradition that the leave of Holarrhena floribunda has chemistry, metabolism and structure-activity relationships. J Nutr
medicinal values and also that the plant can be a source Biochem 13: 572584. doi:10.1016/S0955-2863(02)00208-5.
of natural antioxidant which is beneficial for maintaining Imlay JA, Linn S (1988) DNA damage and oxygen radical toxicity. Sci-
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Iqbal M, Okazaki Y, Okada S (2003) In vitro curcumin modulates
ferric nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H2O2)-
Acknowledgements induced peroxidation of microsomal membrane lipids and DNA
damage. Teratog Carcinog Mutagen (Suppl 1): 151160.
The authors thank TETFund (Nigeria) for PhD travel Kahkonen MP, Hopia AI, Vuorela HJ, et al. (1999) Antioxidant activity
scholarship granted to the first author. The work was of plant extracts containing phenolic compounds. J Agric Food Chem
sponsored, in part, by the National Research Foundation 47: 39543962. doi:10.1021/jf990146l.
Kawanishi S, Hiraku Y, Oikawa S (2001) Mechanism of guanine-specific
(NRF), South Africa. DNA damage by oxidative stress and its role in carcinogenesis and
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