Article
Article
Article
In this study, solvent extracts were prepared from different parts of Cyclamen mirabile (CM) and their
antioxidant activities were evaluated. Other antioxidant properties of all extracts of CM tubers and
leaves, including free radical scavenging activity, reducing power and total phenolic compound
content, were also determined. Leaves extracts of CM exhibited higher antioxidant activity than tuber
extracts with all the types of solvent used. All concentrations of petroleum ether, acetone, methanol
and water extracts of CM leaves showed higher antioxidant activities than that of 0.5 mg of α-tocopherol
(42%) and close to BHT (99.30%) and had 96.60, 96.00, 96.10 and 97.40% inhibition of lipid peroxidation
of linoleic acid at same doses, respectively. All extracts of CM tubers and leaves had effective free
radical scavenging and reducing power. In addition, total phenolic compounds in all the extracts of CM
tubers and leaves were determined as pyrocatechol equivalents.
Key words: Cyclamen mirabile, antioxidant activity, reducing power, scavenging activity, total phenolic
compound.
INTRODUCTION
One of the principal causes of food quality deterioration is many food products (Andreja et al., 2000). The anti-
the oxidation of unsaturated lipids initiated by free oxidants can be of synthetic or natural origin. The use of
radicals. When lipids are exposed to environmental fac- synthetic antioxidants is restricted in several countries,
tors such as air, light and temperature, oxidation reac- because of their possible undesirable effects on human
tions start to produce undesirable flavours, rancid odours, health (Branen, 1975; Chen et al., 1992; Kahl and
discoloration and other forms of spoilage. The primary Kappus, 1993). Hence, the studies on natural antioxidant
autoxidation products are hydroperoxides, that have no have gained increasingly greater importance.
taste and flavour, but their degradation products (alde- In the search for sources of natural antioxidants, in the
hydes, ketone, etc.) have very potent taste and flavour last few years some medicinal plants have been exten-
modifiers (Gordon, 1991). sively studied for their antioxidant activity and radical
Reactive oxygen species (ROS), such as hydroxyl scavenging activity (De las Heras et al., 1998;
radical, hydrogen peroxide and superoxide anions, are Desmarchelier et al., 2000; Schinella et al., 2002;
produced as by products in aerobic organisms and have VanderJagt et al., 2002). A number of studies on the
been implicated in the pathology of a vast variety of antioxidant activities of various aromatic plants have
human diseases including cancer, atherosclerosis, dia- been reported over the last 20 years (Brraco et al., 1981;
betic mellitus, hypertension, AIDS and aging (Halliwell Herrmann et al., 1981; Kramer, 1985; Lagouri et al.,
and Gutteridge, 1984; Wallace, 1999; Lee et al., 2000a). 1993). Flavonoids and other phenolic compounds of plant
Therefore, antioxidant activity is an important in-view of origin have been reported as scavenger of ROS, thus,
the free radical theory of aging and associated diseases. they are viewed as promising therapeutic drugs for free
To retard or prevent the oxidative deterioration, the radical pathologies (Parshad et al., 1998; Lee et al., 2000b).
antioxidants are added in food. The added antioxidants The antioxidant activity of plant origin is dependent on
then maintain the quality and extend the shelf-life of the type and polarity of the extracting solvent as well as
832 Afr. J. Biotechnol.
on the test system and the substrate to be protected by ferrous chloride, ferric chloride, Folin-Ciocalteu’s reagent (FCR),
the antioxidant (Heinonen et al., 1998; Moure et al., 2000; polyoxyethylenesorbitan monolaurate (Tween-20), methanol, ethyl
acetate and trichloracetic acid (TCA) were obtained from E. Merck
Kang and Lee, 2001). Solvent extraction is frequently
(Darmstadt, Germany). 1,1-diphenyl-2-picrylhydrazyl (DPPH),
used for isolation of the antioxidants and both extraction butylated hydroxytoluene (BHT) and α-tocopherol were obtained
yield and antioxidant activity of the extracts are strongly from Sigma Chemical Co. (St. Lois, MO). All other chemicals and
dependent on the solvent, due to the different antioxidant solvents were of analytical grade.
potentials of compounds with different polarity. For these
reasons, comparative studies for selecting the optimal
Extraction
solvent providing maximum antioxidant activity are
required for each substrate. Although, the use of different 10 g of air-dried parts of C. mirabile were extracted with four
polarity substances can provide more exhaustive infor- different solvents (petroleum ether, acetone, methanol and water).
mation on the properties of the extracts, literature con- 10 g of powdered of C. mirabile leaves and tubers were extracted in
tains few reports of the polarity-based solvent extraction soxhlet apparatus with 100 ml of petroleum ether until the extraction
solvent became colourless. 10 g of powdered C. mirabile leaves
of medicinal plants (Kang et al., 2003). and tubers were extracted with 200 ml of acetone and methanol
Cyclamen mirabile Hildebr, is an endemic species solvents in a shaking incubator at 25 ± 1°C for 24 h. The extraction
grown in South-west Anatolia (C2 Mugla, C3 Isparta), was repeated twice at same condition. These extracts were filtered
Turkey, where it grows in Pinus brutia forests and hill slo- and the organic solvents were removed in vacuum by a rotary eva-
pes with maquis, on limestone, metamorphic and granitic porator (Heidolph Laborota 4010, Germany). For water extraction,
rocks, at altitudes of 400 to 1600 m and flowers from 10 g of powdered of C. mirabile leaves and tubers was mixed with
boiling water (500 ml) for 15 min. The extract was filtered and
September to November (Davis, 1978). evaporated in vacuum below 70°C on a rotary evaporator (Duh and
The tubers of C. mirabile contain glycosides such as Yen, 1997).
starch, glue, organic acids and saponins. In addition, it
has emetic purgative and stimulant effects. The infusions
that are made by fresh tubers cause medium intensity Determination of antioxidant activity
diarrhea. The overdoses that are the medical dosage
The antioxidant activities of extracts from different parts (tubers and
cause dangerous toxication which appears with vomit leaves) of C. mirabile were determined by the thiocyanate method
and strong diarrhea. The tubers are eaten usually by pigs (Mitsuda et al., 1996). 1 ml of extracts at different concentrations
(Baytop, 1984). Calis et al. (1997) had previously repor- were mixed with 2.5 ml of linoleic acid emulsion, including 155 µl
ted the study of the triterpenoid saponins of C. mirabile linoleic acid, 175 µg Tween-20 and potassium phosphate buffer
Hildebr. This resulted in the isolation of six saponins and (0.04 M, pH 7.0) in 50 ml of its solution and 1.5 ml of phosphate
their biological activities (antibacterial and antifungal). buffer (0.04 M, pH 7.0). The mixed solution (5 ml) was incubated at
37°C for 110 h in the dark. 0.1 ml of the sample solution was added
There are no sufficient studies on C. mirabile. Addi- to ethanol (4.7 ml, 75%), ferrous chloride (0.1 ml, 20 mM in 3.5%
tionally, systemic and multi - method evaluations on anti- HCl) and ammonium thiocyanate (0.1 ml, 30%). After the mixture
oxidant activities of solvent extracts of this species has was stirred for 3 min, the peroxide value was determined by mea-
not previously been reported. Therefore, the data suring the absorbance at 500 nm. The percent inhibition of lipid
presented here will be the first record on C. mirabile. peroxidation was calculated using the following equation:
The purpose of this study was to evaluate the anti- Inhibition % = 100- ((A1/A0) x 100)
oxidant activity of various solvent extracts from different
parts (tuber and leaves) of C. mirabile using petroleum Where, A0 is the absorbance of control and A1 is the absorbance of
ether, acetone, methanol and water. Other antioxidant the sample of C. mirabile.
properties of all solvent extracts, including scavenging
activity on 1,1- diphenyl - 2 - picrylhydrazyl, reducing Determination of total phenolic compounds
power and total phenolic content were also determined.
The concentrations of phenolic compounds in all extracts of C.
mirabile, expressed as microgram of pyrocatechol equivalents
MATERIALS AND METHODS (PEs), were determined with Folin-Ciocalteu reagent (FCR)
according to the method of Slinkard and Singleton (1977). 1 ml of
Plant materials the solution extracts (1 mg) was added to 46 ml of distilled water
and 1 ml of FCR and was mixed thoroughly. After 3 min, the mixture
Different parts (leaves and tubers) of C. mirabile were collected was added to 3 ml of sodium carbonate (2%) and shaken
from the natural environment of Mugla city in Turkey in October intermittently for 2 h. The absorbance was read at 760 nm. The
2002 and cleaned to remove any residual compost. The air-dried concentrations of phenolic compounds were calculated to follow the
leaves and tubers were ground to fine powder and then, stored in equation that was obtained from standard pyrocatechol graph:
an air-tight container until further use.
Absorbance = 0.002248 pyrocatechol (µg) + 0.0024 (R2: 0.999)
Chemicals
Free radical scavenging activity
Ammonium thiocyanate, ascorbic acid, potassium ferricyanide, Radical scavenging activity of all extracts of C. mirabile was
Sarikurkcu 833
Table 1. Yield of extracts from C. mirabile leaves thod, peroxides which oxidized ferrous ions to ferric ions
and tubers using various solvents. are formed during the linoleic acid oxidation. Ferric ions
-
form a complex with SCN and this complex has a
Yield (%) maximum absorbance at 500 nm. Therefore, lower absor-
Solvent
Leaf Tuber bance indicates high antioxidant activity.
Petroleum ether 2.34 0.20 The activities of different amounts (0.2 to 1 mg) of the
Acetone 5.88 0.64 extracts of CM tubers and leaves on peroxidation of
Methanol 30.29 28.78 linoleic acid were measured spectrophotometrically by
Water 12.59 7.79 monitoring absorbance at 500 nm. For the sake of
simplification, only the results of extracts of CM tubers
and leaves containing 0.5 mg amounts were given in
Figures 1 and 2, respectively.
determined using DPPH as a reagent (Cuendet et al., 1997; Kirby
and Scmidt, 1997) with slight modification. Different amounts (0.2 to α-Tocopherol and BHT were used as comparison and
1 mg) of extracts from CM in 1 ml of solution were added to 4 ml of positive control. All the extracts from CM tubers and
a 0.004% methanol solution of DPPH. The samples were incubated leaves, except for water extract (34.60%) of CM tubers
for 30 min in the dark at room temperature. Scavenging capacity showed stronger antioxidant activities (>71%) than that of
was read spectrophotometrically by monitoring the decrease in α-tocopherol (42.0%) and lower antioxidant activities than
absorbance at 517 nm using a spectrophotometer (Shimadzu UV-
1601, Japan). The capability to scavenge DPPH. radical was calcu- that of BHT (99.3%).
lated using the following equation: For the extracts of petroleum ether, methanol and
water, the antioxidant activity of the extracts was increa-
DPPH. scavenging effect (%) = 100 – [(A1 /Ao) x 100] sed with increasing amounts of extracts, but the anti-
oxidant activity of acetone extracts of CM tubers and
Where, A0 is the absorbance of control and A1 is the absorbance of
leaves was decreased with increasing amounts of
the sample of C. mirabile (Duh and Yen, 1997).
extracts due to prooxidant activity. This might be explain-
ed by the fact that, at higher amount, the extract served
Reducing power as an oxygen-carrying agent (Holloway and Gainer,
1988) and as a prooxidant in the co-oxidation of linoleic
The reducing power of extracts from C. mirabile was determined acid.
according to the method of Oyaizu (1986). Extracts solution in
methanol and water at different amounts (0.2 to 1 mg) were mixed
with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of
potassium ferricyanide (1%). The mixture was incubated at 50°C for Determination of total phenolic compounds
20 min. After 2.5 ml of TCA (10%) was added, the mixture was
centrifuged at 3000 rpm for 10 min. Supernatant (2.5 ml) was mixed Some investigations have reported that phenolic com-
with distilled water (2.5 ml) and 0.5 ml of ferric chloride (0.1%) and pounds are very important plant materials because of
the absorbance was measured at 700 nm. Higher absorbance of
the reaction mixture indicates greater reducing power. their inhibitory effect on autoxidation of oils (Ramarathnam
et al., 1986) and their radical scavenging ability (Hatano
et al., 1989). Therefore, it is important to determine the
RESULTS AND DISCUSSION effect of the total phenolic compound on the antioxidant
activity of extracts of the different parts of CM. The
Yield of extracts concentration of phenolics in all the extracts of CM tubers
and leaves was expressed as mg pyrocatechol equiva-
The yields of different solvent extracts from C. mirabile lent g of extracts as shown in Table 2.
tubers and leaves are shown in Table 1. For tubers and In petroleum ether, acetone, methanol and water ex-
leaves, the highest yield were obtained with methanol tracts of CM leaves (1 mg), 37.36, 21.06, 17.30 and
(28.78 to 30.29%), followed by water (7.79 to 12.59%). 30.14 µg pyrocatechol equivalent of phenols was detec-
This observation is in agreement with that reported by ted. In addition, the amount of total phenolic compounds
some researchers, that solvents with high polarity are of acetone extract of CM tubers was 10 times more than
effective for extraction of natural antioxidants (Chang et that of methanol extract.
al., 1977; Duh et al., 1992; Economou et al., 1991; Tian
and White, 1994). Results showed that the leaves gave
the highest yield. Free radical scavenging activity
1.2
Control
1.0
Tocopherol
0.8 BHT
Petroleum ether
Absorbance (500 nm)
0.6 Acetone
Methanol
0.4
Water
0.2
0.0
-0.2
0 10 20 30 40 50 60 70 80 90 100 110 120
Time (hours)
Figure 1. Antioxidant activity of solvent extracts (0.5 mg) of C. mirabile tubers using the thiocyanate method. BHT; Butylated
hydroxytoluene (values are means ± standard deviation of three replicate analyses).
1.2
Control
1.0
Tocopherol
0.8 BHT
Absorbance (500 nm)
Petroleum ether
0.6 Acetone
Methanol
0.4
Water
0.2
0.0
-0.2
0 10 20 30 40 50 60 70 80 90 100 110 120
Time (hours)
Figure 2. Antioxidant activity of solvent extracts (0.5 mg) of C. mirabile leaves using the thiocyanate method. BHT; Butylated
hydroxytoluene (values are means ± standard deviation of three replicate analyses).
Table 2. Total phenolic content (µg of PEsa / mg of polyunsaturated fatty acids, proteins and sugars
extract) of extracts from different parts of C. mirabile (Halliwell, 1994).
using various solventsb.
Relatively stable radical, DPPH, has been widely used
in the assessment of radical scavenging activity of some
Solvent Tuber Leaf
natural sources (Imai et al., 1994; Duh and Yen, 1997) of
Petroleum ether 17.42 ± 0.35 37.36 ± 0.87 foods (Yamaguchi et al., 1998) and pure compounds
Acetone 51.76 ± 0.16 21.06 ± 0.46 (Sanchez-Moreno et al., 1998). In this method, the stable
Methanol 5.18 ± 0.03 17.30 ± 0.82 radical DPPH in alcohol is reduced to non-radicalic
Water 6.36 ± 0.19 30.14 ± 0.09 DPPH-H in the presence of a hydrogen-donating anti-
a b oxidant.
PEs, pyrocatechol equivalents; values are means ±
standard deviation of three replicate analyses. Figures 3 and 4 show a significant decrease in the
Sarikurkcu 835
1.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Amounts (mg)
Figure 3. DPPH scavenging activity of tuber extracts of C. mirabile (values are means ± standard
deviation of three replicate analyses).
1.2
Petroleum ether
Acetone
0.9 Methanol
Absorbance (517nm)
Water
0.6
0.3
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Amounts (mg)
Figure 4. DPPH scavenging activity of leaf extracts of C. mirabile (values are means ± standard
deviation of three replicate analyses).
concentration of DPPH radical due to the scavenging and than that of 0.02 mg dose of ascorbic acid (42.44%),
activity of the tuber and leaf extracts of C. mirabile. The but lower than that of 0.1 mg of α-tocopherol (90.87%).
scavenging activity of all extracts increased with The scavenging effect of these extracts followed the
increasing amounts of extracts. The scavenging capacity order: Acetone > petroleum ether > water > methanol for
of 1 mg doses of petroleum ether, acetone, methanol and tuber extracts of CM.
water extracts of CM leaves were found to be 57.51, The results obtained for the free radical scavenging
61.52, 53.07 and 88.25%, respectively and these values activity suggest that, all the extracts of CM possessed the
were greater than that of 0.1 mg dose of BHT (45.13%) ability to quench free radical from reaching biomolecules
836 Afr. J. Biotechnol.
60 0.018
30 0.009
20 0.006
10 0.003
0 0
0 0.1 0.2 0.3 0.4 0.5 0.6
Amount (mg)
Figure 5. The scavenging activity (%) and total phenolic content (mg PEs) of different
amounts of water extract of C. mirabile leaves.
Methanol
2.5 Water
BHT
Tocopherol
1.0
-0.5
0 0.3 0.6 0.9 1.2
Amounts (mg)
Figure 6. Reducing power of solvent extracts of C. mirabile tubers (values are means ± standard
deviation of three replicate analyses).
(polyunsaturated fatty acids, sugars and amino acids vity of water extract of CM leaves. The equation of total
etc.) in susceptible biological and food systems (Halliwell phenolic content (y) and scavenging activity (x) of water
et al., 1995). These activities may be attributed to the extract of CM leaves was y = 0.289792 x + 0.166278 (R2
hydrogen and electron donating abilities of their = 0.99).
phenolics.
In fact, a strong and positive correlation (at 0.1 to 0.5
mg) was found between the scavenging activity of Reducing power
acetone and water extracts with the total phenolic content
2
in C. mirabile tubers and leaves (R = 0.99). Figure 5, As shown in Figures 6 and 7, all the various amount of
shows the total phenolic content and the scavenging acti- extracts from CM tubers and leaves, BHT and α-
Sarikurkcu 837
-0.5
0 0.3 0.6 0.9 1.2
Amounts (mg)
Figure 7. Reducing power of solvent extracts of C. mirabile leaves (values are means ± standard
deviation of three replicate analyses).
1.2 0.060
Reducing power
Phenolic content
0.9 0.045
0.6 0.030
0.3 0.015
0 0.000
0 0.3 0.6 .9 1.2
Amount (mg)
Figure 8. The reducing power (absorbance at 700 nm) and total phenolic content (mg PEs)
of different amounts of acetone extract of C. mirabile tubers.
tocopherol showed higher activities than the control. The shows that the equation of the reducing power (y) and the
reducing power of solvent extracts of CM tuber and total phenolic compounds (x) of different doses of
leaves increased with increasing amounts of extracts and acetone extract of CM tubers was y = 0.016729 x +
decreased in the order acetone > petroleum ether > 0.049617 ( R2: 0.98).
water > methanol, water > acetone > petroleum ether > Many researchers have reported that, the reducing
methanol, respectively. As a whole, the reductive power is generally associated with the reductones (Duh,
capacity of all extracts was less than that of BHT and α- 1998) and might be due to hydrogen-donating ability
tocopherol in the various amounts of the extracts. Like (Shimida et al., 1992). The reducing power of the extracts
the scavenging effect, these results indicate that, there is of CM tubers and leaves might contain reductone, which
a correlation between the reducing power and total could stabilise free radicals and terminate radical chain
phenolic compounds (R2: 0.99). In addition, Figure 8 reactions and this might be due to the hydrogen-donating
838 Afr. J. Biotechnol.
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