INTRODUCTION
Constantly developing technology, environmental
pollution, ultraviolet radiation, and many other factors
cause us to be exposed to various toxic substances.
This results in more diseases caused by external
environmental effects, including more pronounced
genetic diseases. Preventing these diseases should
become our priority. Since most of them occur in
people with a weak immune system, we must focus
on strengthening it. For this, we should consume
foods with high antioxidant capacity, especially fruits
and green leafy vegetables that contain antioxidative
phytochemicals [1, 2].
Phytochemicals, or “plant chemicals”, are
compounds of plant origin, mostly polyphenols, that
are essential for human life. They work alongside
macronutrients such as carbohydrates, fats, and proteins,
as well as 13 essential vitamins and 17 minerals [3].
Antioxidant phytochemicals, especially in fruits and
vegetables, combine with free radicals in the human
body to protect cells from the attacks of harmful
radicals [4]. Bioactive compounds in fruits contain
ascorbic acid, organic and phenolic acids, flavonoids,
anthocyanins, and carotenoid substances [5, 6].
Citrus fruits come in different types, varieties, and
flavors and have positive effects on health and nutrition.
Although they have been known as the best sources of
vitamin C for a long time, studies on their use as an
antioxidant substance have recently gained momentum,
due to their richness in phenolic compounds [7]. These
bioactive components are responsible for various health
benefits of citrus fruits, such as prevention of various
diseases or protective effects to lower the risk of various
cancers [8–10].
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Citrus is a fruit group belonging to the genus Citrus,
which is a member of the Aurantioideae subfamily of the
Rutaceae family. The most common citrus varieties are
orange (Citrus sinensis), mandarin (Citrus reticulata),
lemon (Citrus limon), golden ball (Citrus paradisi),
bitter orange (Citrus auranthium), and bergamot (Citrus
bergami) [11]. In addition to fresh table consumption,
citrus fruits are used as jam, marmalade or fruit juice, as
well as raw material in the cosmetics sector [11].
Citrus fruits grow in subtropical climate areas. While
mainland China, Southeast Asia, and India are major
producers of citrus fruits due to suitable ecological
conditions, they are also cultivated in the Mediterranean
and Aegean coastal regions and partly in the Eastern
Black Sea region of Turkey [12, 13]. The distribution
of species and varieties of citrus fruits has gained a
regional identity. For example, Washington navel, as
well as other navel oranges, and Jaffa are harvested in
the Eastern Mediterranean region.
Orange is one of the most produced and consumed
citrus fruits in Turkey due to its preference in the juice
industry and its great potential in the oil industry [14].
Orange is followed by mandarin and lemon products,
respectively. Apart from these species, kumquat,
which is called the “little gem of the citrus family”, has
recently grown in popularity, as well as such species as
Altıntop and citrus, which are lower in production but
can be considered important [15].
Kumquat is also called “citrus fortunella”, taking
its name from the Scottish horticultural expert Robert
Fortune (1812–1880). This species, referred to as
“komquot” in some countries, is also called a “golden
orange” [16]. It is like a tiny lemon in shape and
orangish in color. However, while orange and lemon are
consumed after they are peeled, kumquat is consumed
with its peel. Its scent is reminiscent of bergamot. It
tastes sweet and leaves a lasting scent when you hold it
in your hand.
In addition to fresh consumption, kumquat can be
used in products such as confectionery, marmalade,
liquor, and wine [17, 18]. Essential oil and bioactive
ingredients obtained from its peel are used in the
perfumery, pharmaceutical, and food industries [19].
Kumquat is an excellent source of nutrients containing
minerals, ascorbic acid, carotenoids, flavonoids, and
essential oils [20]. It contains remarkable antioxidant
properties due to its flavonoid content [18]. However,
there are very few studies about kumquat grown in
Turkey.
In this study, we aimed to determine the antioxidant
capacity and the total phenolic and flavonoid contents
of the extracts obtained from fresh and lyophilized
dried fruits and leaves of kumquat and six mutants
from the Mersin Alata Horticultural Research Institute
Directorate.
STUDY OBJECTS AND METHODS
Plant materials. Kumquat leaf and fruit samples
were obtained from the Mersin Alata Horticultural
Research Institute in November 2017 and January 2018,
respectively. We used EP (Old Parcel) with rootstock
species; EP.4, EP.29, EP.31 and YP (New Parcel); YP.117,
YP.141, YP.188 mutants. The leaf samples were dried in
room conditions and in the shade, and stored in a dry
and cool environment for analysis. The fruit samples
were freeze-dried, or lyophilized.
Chemicals and equipment. We used chemicals
and solvents of analytical purity produced by Sigma,
Aldrich, and Riedel-de Haen.
The equipment used in the study included a
lyophilizer (Christ Alpha 1-2 LC plus), a vortex (Fisons),
a rotary evaporator (Laborota 4000-efficient Heidolph),
a spectrophotometer (Shimadzu UV-1601), a shaking
water bath (Clifton 100–400 rpm; with thermostat), an
incubator (EnoLab MB-80), an analytical balance (Gec
Avery), a centrifuge (Nüvefuge CN180), a pH-meter
(WTW pH 330i), a heater and magnetic stirrer (Chiltern
HS31), a disperser and micropipettes (Eppendorf).
Extraction process. Phenolic compounds were
extracted from kumquat fruits and leaves with a Soxhlet
extraction device, using 260 mL of 99, 80, 60, and 50%
methanol and pure water as solvents. In addition, 1 and
0.5% acidified ethanol and hexane solvents were used for
kumquat leaves.
For extraction, 20 g of the samples were weighed into
the cartridge and then placed in the Soxhlet extractor.
The solvent(s) was added to the glass flask and kept
in the Soxhlet device for 8 h. The solvent used for
extraction was concentrated from the obtained phenolic
extracts using a laboratory scale rotary evaporator
under vacuum. The remaining part was removed by
standing in the open air. The extracts were weighed
gravimetrically and stored in dark vials at +4°C in the
refrigerator until analysis.
Determination of free radical capture capacity
(DPPH method). We used 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical to determine the free radical capture
capacity according to the Blois method [21]. This
method is based on the ability of the extracts to donate
a proton or electron and to decolorize the purple colored
DPPH solution (from violet to yellow). A decrease in the
absorbance of the reaction mixture is indicative of high
free radical scavenging activity.
All the extracts, BHA and BHT standards, and
α-tocopherol were dissolved in ethanol at 1 mg/mL.
After taking the samples and standards into 5 different
volumes of 50, 100, 150, 250, and 500 μL, ethanol was
added to a total volume of 3 mL. 1000 μL 0.1 mM DPPH
was added to the tubes and vortexed. The absorbance
of the mixture, which was incubated for 30 min in
the dark at room temperature, was measured in the
UV-visible spectrophotometer at 517 nm. Calculations
were made using the following formula:
% free-radical scavenging activity = 𝐴𝐴C− 𝐴𝐴S/S
𝐴𝐴C
× 100 (1)
y = 0.0292x + 0.0749
0.6
0.8
1.0
1.2
1.4
1.6
Absorbance
where AC is the absorbance of the control reaction;
AS/S is the absorbance of the sample or standard.
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Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66
Determination of reducing capacity. The
Oyaizu method was used to determine the reduction
capacity [22]. According to this method, the reducing
agent in the medium reduces Fe3+ ions to Fe2+ ions
and a complex is formed by adding FeCl3. The
absorbance of the resulting complex is measured in the
UV-visible spectrophotometer at 700 nm. The increase
in absorbance of the reaction mixture is directly
proportional to the reducing power of the sample.
All the extracts, BHA and BHT standards, and
α-tocopherol were dissolved in ethanol at 1 mg/mL.
100, 250, and 500 μL of the samples and standards
were taken into test tubes in three different volumes,
and 3400, 3250, and 3000 μL of pH 6.6 phosphate
buffer was added to them, respectively, to a total
volume of 3500 μL. Then, after adding 2500 μL of 1%
K3 [Fe (CN)6] and vortexing, it was left to incubate for
20 min in a water bath at 50°C. After the incubation,
2500 μL of 10% trichloroacetic acid (TCA) was added
to the test tubes and centrifuged at 3000 rpm for
10 min. 1250 μL of the resulting supernatant was taken
into empty tubes and 1250 μL of distilled water and
500 μL of 0.1% FeCl3 were added to them. The mixture
was vortexed and its absorbance was measured at
700 nm in the UV-visible spectrophotometer.
Determination of iron (II) ions chelating activity.
Antioxidants with metal chelating properties inactivate
free iron by binding it and thus inhibit the formation
of radicals such as hydroxyl and peroxide, which are
formed as a result of Fenton reactions (Fe2+ + H2O2 →
Fe3+ + HO• + HO–) [23]. The Dinis method was
used to determine the activity of chelating iron (II)
ions [24]. All the extracts and EDTA used as control
were dissolved in ethanol to 1 mg/mL. The samples and
standards were taken into 50, 100, 150, 250, and 500
μL test tubes, and 3700, 3650, 3600, 3500, and 3250 μL
of ethanol was added to them, respectively, to a total
volume of 3750 μL. Then, 50 μL of 2mM FeCl2 was
added and vortexed to incubate at room temperature for
10 min. Then, 200 μL of 5mM ferrosine was added. The
resulting purple color was measured in the UV-visible
spectrophotometer at 562 nm after the mixture was kept
at room temperature for 25 min.
Determination of total phenolic content. The
Folin-Ciocalteu method was used to determine the
total phenolic content [25]. The Folin-Ciocalteu reagent
(FCR) used in this method is molybophosphotungstic
heteropolyacid (3H2O·P2O5·13WO3·5MoO3·10H2O). This
method is based on the transfer of electrons from
phenolic compounds and other reducing compounds to
molybdenum. Phenolic compounds only react with the
FCR in basic conditions (pH ~ 10) [26].
Mo(VI) + e– (antioxidant) → Mo(V)
Commercially available 2N Folin-Ciocalteu reagent
was prepared daily by diluting it with purified water
at a ratio of 1/1 (V/V). 500 μL of the extracts (1 mg/
mL) was taken into test tubes and 500 μL of distilled
water was added. After 250 μL of 1N Folin reagent was
added to the mixture, it was incubated for 5 min by
vortexing. 1250 μL of 2% Na2CO3 solution was added to
it, vortexed, and then kept at room temperature for 2 h.
The absorbance of the resulting mixture was measured
at 765 nm in the UV-visible spectrophotometer. The
phenolic content of the extracts was given as mg gallic
acid equivalent (GAE)/g extract.
Determination of total flavonoid content. The
total flavonoid content was measured by an aluminum
chloride colorimetric test according to Zhishen
et al. [27]. All the extracts and a quercetin solution
used as a standard were dissolved in 1 mg/mL ethanol.
500 μL was taken from the extracts prepared in the test
tubes and pure water was added to a total volume of
5000 μL. To this mixture, 300 μL of 5% NaNO2 solution
was added and left to incubate at room temperature
for 5 min, and then 300 μL of 10% AlCl3 solution was
added. After waiting for 6 min, 2 mL of 1.0M NaOH
solution was added and the volume was completed
to 20 mL with distilled water. The absorbance of the
solution was measured at 510 nm in the UV-visible
spectrophotometer. The total flavonoid content of the
extracts was given as mg quercetin equivalent (QUE)/g
extract.
RESULTS AND DISCUSSION
The solubility and distribution of phenolic
compounds in the solvent depend on the polarity of
their structure, so the choice of solvent and method is
one of the most important steps. In our study, for fresh
and lyophilized dried fruits, we preferred methanol and
its aqueous solutions, as well as pure water. For leaves,
we preferred methanol and aqueous solutions, distilled
water, and ethanol acidified with hexane.
Three different methods (DPPH radical scavenging
activity, reducing capacity, and iron (II) ions chelating
activity) were used to determine the antioxidant
capacity. We thought that the extracts could show
activity through different mechanisms depending on
the diversity of phenolic substances. In addition, we
determined the total phenolic content and flavonoid
amounts in all the extracts in order to show that the
antioxidant effect was proportional to the plant content.
Free radical scavenging activity. The DPPH
method is commonly used to evaluate the antioxidant
activity of natural products, as it is easy and highly
sensitive. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a
commercially available stable organic nitrogen radical.
The antioxidant effect is proportional to the removal of
the DPPH radical. The DPPH radical (DPPH•) is purple
in color due to the unpaired nitrogen atom. When
the DPPH solution reacts with an oxygen atom of a
substance (antioxidant chemical) that can give hydrogen
atoms, the initial purple color disappears as the radical
reduces, turning yellow [28]. The reaction takes place
stoichiometrically according to the number of hydrogen
atoms absorbed. Therefore, the antioxidant effect was
easily determined by following the decrease in UV
absorbance at 517 nm until it stabilized.
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We observed that the highest free radical scavenging
activity was in kumquat leaves, and the activity of
kumquat fruit decreased when dried (Table 1). There
was no significant difference between the rootstock
kumquat type and its mutants. The free radical
scavenging activities of the extracts were slightly
below the standards (BHA, BHT, and α-Tocopherol).
The highest activity (81.66%) was seen in the YP.188
hybrid leaf extract using 80% methanol solvent. As for
the fruits, the highest activity (61.37%) was in the EP.4
hybrid extract using a pure methanol solvent.
When we examined all the samples, we associated
high phenolic content with high antioxidant activity. We
found that the total phenolic content was higher in the
samples with high antioxidant activity. As a matter of
fact, the leaf extract with high antioxidant activity also
had a high phenolic content (85.651 ± 0.030 mg GAE/g
extract).
However, when we carefully examined the
results, we saw that having a high amount of phenolic
substances did not give high results in all antioxidant
activity methods. For example, although the YP.188
Leaf 80% methanol and the YP.188 Leaf 50% methanol
extracts contained almost the same amount of phenolic
substances, the former had higher activity in the applied
antioxidant activity methods. This could be explained
by the differences between the phenolic substances they
contained depending on the solvent used.
In fact, other studies have found that the antioxidant
activity of methanol and ethanol extracts, which
generally contained phenolic substances, was higher
than in other solvent systems [29]. For example,
Jayaprakasha et al. extracted powdered kumquat fruit in
5 different solvents and investigated the radical capture
capacities of the extracts, their amounts in total phenolic
matter, and their inhibitory properties for prostate
cancer [30].
In this study, the extracts obtained from EtOAc
and MeOH-water (4:1, v/v) solvents were found to have
the highest and lowest total phenolics, respectively,
according to the Folin-Ciocalteu method. It was also
observed that the EtOAc and MeOH extracts exhibited
the highest and lowest 1,1-diphenyl-2-picyrylhydrazyl
(DPPH) radical scavenging activity, respectively [30].
Table 1 DPPH radical scavenging activity of kumquat fruit and leaf extracts, μg/mL (mean ± SD of triplicate)
Extracts and Standards 12.5* 25.0* 37.5* 62.5* 125*
Rootstock fresh fruit pure methanol 7.22 ± 0.10 11.19 ± 0.2 12.64 ± 0.1 20.94 ± 0.1 30.32 ± 0.3
Rootstock fresh fruit 80% methanol 6.50 ± 0.10 7.94 ± 0.1 9.03 ± 0.2 12.64 ± 0.3 19.49 ± 0.1
Rootstock fresh fruit 60% methanol 4.69 ± 0.10 7.58 ± 0.1 8.66 ± 0.2 13.00 ± 0.3 21.30 ± 0.3
Rootstock fresh fruit 50% methanol 7.03 ± 0.10 9.03 ± 0.0 18.66 ± 0.1 22.02 ± 0.3 28.52 ± 0.1
Rootstock fresh fruit pure water 10.83 ± 0.0 14.08 ± 0.2 14.08 ± 0.2 22.02 ± 0.0 33.57 ± 0.3
Rootstock dry fruit pure methanol 3.09 ± 0.10 4.75 ± 0.2 7.56 ± 0.3 8.25 ± 0.3 9.97 ± 0.1
Rootstock dry fruit 80% methanol 5.15 ± 0.20 6.53 ± 0.0 8.25 ± 0.2 9.28 ± 0.3 12.37 ± 0.3
Rootstock dry fruit 60% methanol 4.81 ± 0.00 7.56 ± 0.1 8.25 ± 0.0 8.93 ± 0.0 9.62 ± 0.2
Rootstock dry fruit 50% methanol 3.78 ± 0.00 6.53 ± 0.1 7.22 ± 0.0 8.25 ± 0.0 10.31 ± 0.2
Rootstock dry fruit pure water 3.78 ± 0.20 4.47 ± 0.1 6.87 ± 0.0 7.22 ± 0.1 8.59 ± 0.2
Rootstock leaf pure methanol 12.46 ± 0.20 23.88 ± 0.3 32.87 ± 0.1 41.87 ± 0.1 57.09 ± 0.1
Rootstock leaf 80% methanol 21.45 ± 0.10 29.76 ± 0.2 37.72 ± 0.2 50.52 ± 0.1 65.74 ± 0.5
Rootstock leaf 60% methanol 13.49 ± 0.20 18.15 ± 0.1 33.91 ± 0.2 46.71 ± 0.0 65.40 ± 0.3
Rootstock leaf 50% methanol 20.76 ± 0.30 31.49 ± 0.0 39.10 ± 0.1 50.87 ± 0.2 66.44 ± 0.3
Rootstock leaf pure water 12.11 ± 0.10 21.11 ± 0.1 30.10 ± 0.3 36.33 ± 0.2 52.25 ± 0.2
Rootstock leaf 0.5% acidified ethanol 3.46 ± 0.10 8.30 ± 0.2 13.84 ± 0.3 20.42 ± 0.1 34.26 ± 0.4
Rootstock leaf 1% acidified ethanol 5.19 ± 0.10 13.15 ± 0.2 15.22 ± 0.1 25.61 ± 0.2 40.83 ± 0.1
Rootstock leaf hexane n.d. n.d. 2.42 ± 0.2 11.07 ± 0.1 12.04 ± 0.1
EP.4 fresh fruit pure methanol 16.97 ± 0.1 20.22 ± 0.3 35.02 ± 0.2 42.60 ± 0.1 61.37 ± 0.3
EP.4 fresh fruit 80% methanol 15.88 ± 0.1 16.61 ± 0.2 21.66 ± 0.2 28.52 ± 0.3 42.96 ± 0.5
EP.4 fresh fruit 60% methanol 13.36 ± 0.1 15.88 ± 0.1 15.88 ± 0.2 22.74 ± 0.3 31.05 ± 0.1
EP.4 fresh fruit 50% methanol 15.88 ± 0.2 18.05 ± 0.1 19.86 ± 0.1 23.10 ± 0.3 33.57 ± 0.2
EP.4 fresh fruit pure water 15.88 ± 0.2 22.38 ± 0.3 28.05 ± 0.1 34.55 ± 0.3 35.38 ± 0.2
EP.4 dry fruit pure methanol 2.06 ± 0.1 2.75 ± 0.3 10.31 ± 0.1 11.37 ± 0.1 14.43 ± 0.1
EP.4 dry fruit 80% methanol 6.25 ± 0.2 7.56 ± 0.0 8.25 ± 0.2 10.31 ± 0.1 14.43 ± 0.2
EP.4 dry fruit 60% methanol 6.53 ± 0.1 8.25 ± 0.1 9.97 ± 0.3 10.97 ± 0.4 13.06 ± 0.1
EP.4 dry fruit 50% methanol 7.56 ± 0.1 8.93 ± 0.1 9.08 ± 0.2 9.97 ± 0.4 13.06 ± 0.3
EP.4 dry fruit pure water 5.15 ± 0.2 8.93 ± 0.2 10.65 ± 0.3 11.68 ± 0.0 15.12 ± 0.3
EP.4 leaf pure methanol 14.88 ± 0.1 23.53 ± 0.0 28.03 ± 0.1 36.33 ± 0.2 54.67 ± 0.7
EP.4 leaf 80% methanol 17.65 ± 0.1 32.53 ± 0.3 39.45 ± 0.2 47.06 ± 0.1 71.63 ± 0.5
EP.4 leaf 60% methanol 16.65 ± 0.1 25.61 ± 0.3 34.95 ± 0.2 44.64 ± 0.1 63.67 ± 0.3
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Extracts and Standards 12.5* 25.0* 37.5* 62.5* 125*
EP.4 leaf 50% methanol 16.61 ± 0.2 26.99 ± 0.3 36.33 ± 0.1 46.02 ± 0.1 65.05 ± 0.1
EP.4 leaf pure water 18.34 ± 0.2 27.68 ± 0.2 34.26 ± 0.2 47.40 ± 0.3 55.36 ± 0.3
EP.4 leaf 0.5% acidified ethanol 1.73 ± 0.0 7.61 ± 0.1 9.69 ± 0.3 17.99 29.41 ± 0.5
EP.4 leaf 1% acidified ethanol 6.92 ± 0.1 12.80 ± 0.2 18.34 ± 0.3 24.57 ± 0.2 35.64 ± 0.3
EP.4 leaf hexane n.d. n.d. n.d. n.d. 7.96 ± 0.5
EP.29 fresh fruit pure methanol 9.42 ± 0.2 15.16 ± 0.2 22.38 ± 0.1 28.88 ± 0.1 37.91 ± 0.1
EP.29 fresh fruit 80% methanol 9.75 ± 0.0 14.08 ± 0.2 17.69 ± 0.1 22.02 ± 0.3 31.77 ± 0.5
EP.29 fresh fruit 60% methanol 12.64 ± 0.1 17.33 ± 0.2 21.30 ± 0.3 25.63 ± 0.3 36.10 ± 0.2
EP.29 fresh fruit 50% methanol 13.72 ± 0.1 17.69 ± 0.1 19.49 ± 0.0 26.71 ± 0.2 36.10 ± 0.4
EP.29 fresh fruit pure water 14.44 ± 0.1 15.16 ± 0.1 17.69 ± 0.1 20.94 ± 0.3 33.21 ± 0.4
EP.29 dry fruit pure methanol 7.90 ± 0.1 9.28 ± 0.1 10.31 ± 0.2 13.06 ± 0.1 15.81 ± 0.5
EP.29 dry fruit 80% methanol 7.56 ± 0.1 11.68 ± 0.1 14.09 ± 0.2 15.43 ± 0.1 19.59 ± 0.2
EP.29 dry fruit 60% methanol 7.90 ± 0.1 10.31 ± 0.1 12.65 ± 0.1 14.43 ± 0.2 19.93 ± 0.4
EP.29 dry fruit 50% methanol 6.80 ± 0.1 9.28 ± 0.1 10.31 ± 0.2 11.68 ± 0.3 14.09 ± 0.5
EP.29 dry fruit pure water 4.81 ± 0.1 5.84 ± 0.1 7.56 ± 0.1 9.28 ± 0.3 12.03 ± 0.1
EP.29 leaf pure methanol 15.57 ± 0.2 26.99 ± 0.1 29.76 ± 0.1 36.33 ± 0.0 52.25 ± 0.3
EP.29 leaf 80% methanol 9.00 ± 0.1 22.15 ± 0.1 31.49 ± 0.1 41.87 ± 0.4 59.86 ± 0.7
EP.29 leaf 60% methanol 12.46 ± 0.2 26.99 ± 0.2 32.53 ± 0.3 46.71 ± 0.1 63.32 ± 0.4
EP.29 leaf 50% methanol 16.96 ± 0.2 28.37 ± 0.1 33.22 ± 0.2 45.67 ± 0.2 60.55 ± 0.4
EP.29 leaf pure water 10.73 ± 0.1 20.7 ± 0.1 26.99 ± 0.1 35.99 ± 0.2 51.21 ± 0.2
EP.29 leaf 0.5% acidified ethanol 3.11 ± 0.1 8.65 ± 0.2 13.84 ± 0.1 20.42 ± 0.2 34.26 ± 0.5
EP.29 leaf 1% acidified ethanol 6.57 ± 0.2 11.07 ± 0.2 15.57 ± 0.1 24.91 ± 0.3 39.79 ± 0.2
EP.29 leaf hexane n.d. n.d. n.d. n.d. 5.54 ± 0.1
EP.31 fresh fruit pure methanol 2.89 ± 0.1 17.69 ± 0.0 22.74 ± 0.3 29.24 ± 0.2 40.7 ± 0.4
EP.31 fresh fruit 80% methanol 12.27 ± 0.2 16.97 ± 0.1 23.10 ± 0.1 33.94 ± 0.2 51.62 ± 0.5
EP.31 fresh fruit 60% methanol 11.91 ± 0.1 22.02 ± 0.1 25.63 ± 0.2 40.43 ± 0.3 54.51 ± 0.1
EP.31 fresh fruit 50% methanol 14.80 ± 0.1 15.52 ± 0.3 21.66 ± 0.1 28.16 ± 0.5 42.96 ± 0.1
EP.31 fresh fruit pure water 8.30 ± 0.2 13.00 ± 0.3 13.72 ± 0.0 18.41 ± 0.1 23.83 ± 0.1
EP.31 dry fruit pure methanol 7.38 ± 0.2 8.72 ± 0.1 30.20 ± 0.2 39.73 ± 0.1 43.42 ± 0.1
EP.31 dry fruit 80% methanol 7.05 ± 0.2 8.39 ± 0.2 8.39 ± 0.1 10.74 ± 0.3 14.43 ± 0.2
EP.31 dry fruit 60% methanol 3.69 ± 0.1 6.38 ± 0.1 8.72 ± 0.2 9.73 ± 0.1 11.07 ± 0.2
EP.31 dry fruit 50% methanol 3.36 ± 0.0 6.04 ± 0.2 6.38 ± 0.1 8.72 ± 0.0 11.41 ± 0.1
EP.31 dry fruit pure water 6.04 ± 0.1 8.39 ± 0.1 3.36 ± 0.1 3.02 ± 0.3 4.36 ± 0.1
EP.31 leaf pure methanol 13.49 ± 0.1 22.15 ± 0.2 28.37 ± 0.1 37.37 ± 0.2 53.98 ± 0.3
EP.31 leaf 80% methanol 20.42 ± 0.1 31.14 ± 0.2 39.45 ± 0.2 50.52 ± 0.3 68.17 ± 0.4
EP.31 leaf 60% methanol 17.99 ± 0.1 30.80 ± 0.1 39.10 ± 0.1 49.83 ± 0.5 65.05 ± 0.4
EP.31 leaf 50% methanol 19.72 ± 0.1 30.45 ± 0.1 33.22 ± 0.2 49.13 ± 0.1 63.67 ± 0.1
EP.31 leaf pure water 12.11 ± 0.1 21.11 ± 0.1 24.91 ± 0.2 36.33 ± 0.3 53.98 ± 0.5
EP.31 leaf 0.5% acidified ethanol 8.30 ± 0.1 17.30 ± 0.2 24.57 ± 0.2 33.56 ± 0.3 51.56 ± 0.5
EP.31 leaf 1% acidified ethanol 10.3 ± 0.2 16.96 ± 0.1 21.45 ± 0.0 32.18 ± 0.3 47.06 ± 0.3
EP.31 leaf hexane n.d. n.d. n.d. n.d. 8.65 ± 0.3
YP.117 fresh fruit pure methanol 10.83 ± 0.2 18.41 ± 0.2 21.66 ± 0.4 33.94 ± 0.1 46.93 ± 0.4
YP.117 fresh fruit 80% methanol 10.11 ± 0.2 15.75 ± 0.1 20.58 ± 0.2 27.08 ± 0.5 41.88 ± 0.4
YP.117 fresh fruit 60% methanol 12.27 ± 0.1 15.88 ± 0.1 18.41 ± 0.3 27.08 ± 0.1 40.7 ± 0.3
YP.117 fresh fruit 50% methanol 12.64 ± 0.1 16.61 ± 0.2 22.38 ± 0.1 30.69 ± 0.1 48.38 ± 0.4
YP.117 fresh fruit pure water 15.88 ± 0.1 13.36 ± 0.3 20.94 ± 0.2 28.16 ± 0.1 41.52 ± 0.4
YP.117 dry fruit pure methanol 2.68 ± 0.1 3.45 ± 0.1 4.68 ± 0.1 6.71 ± 0.0 10.40 ± 0.3
YP.117 dry fruit 80% methanol 3.69 ± 0.1 6.38 ± 0.2 7.05 ± 0.1 8.05 ± 0.1 8.39 ± 0.2
YP.117 dry fruit 60% methanol 5.03 ± 0.1 7.72 ± 0.1 8.71 ± 0.3 8.92 ± 0.1 11.74 ± 0.1
YP.117 dry fruit 50% methanol 4.70 ± 0.1 5.09 ± 0.2 6.38 ± 0.3 6.38 ± 0.1 6.38 ± 0.3
YP.117 dry fruit pure water 5.03 ± 0.3 5.18 ± 0.3 6.04 ± 0.3 7.05 ± 0.2 11.41 ± 0.2
YP.117 leaf pure methanol 13.84 ± 0.2 22.84 ± 0.1 30.45 ± 0.1 42.91 ± 0.5 60.55 ± 0.5
YP.117 leaf 80% methanol 20.70 ± 0.3 26.99 ± 0.2 33.56 ± 0.1 47.40 ± 0.2 67.47 ± 0.7
YP.117 leaf 60% methanol 14.53 ± 0.2 21.80 ± 0.3 39.45 ± 0.2 50.87 ± 0.2 65.40 ± 0.1
YP.117 leaf 50% methanol 19.03 ± 0.3 33.56 ± 0.2 39.10 ± 0.2 52.25 ± 0.1 65.74 ± 0.1
YP.117 leaf pure water 14.53 ± 0.4 20.76 ± 0.2 32.18 ± 0.1 40.83 ± 0.3 57.44 ± 0.4
YP.117 leaf 0.5% acidified ethanol 7.22 ± 0.1 13.15 ± 0.2 19.72 ± 0.1 28.72 ± 0.1 45.67 ± 0.3
Continuation of the Table 1
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Extracts and Standards 12.5* 25.0* 37.5* 62.5* 125*
YP.117 leaf 1% acidified ethanol 7.96 ± 0.1 15.22 ± 0.4 17.99 ± 0.1 28.72 ± 0.1 46.37 ± 0.3
YP.117 leaf hexane n.d. n.d. n.d. 2.69 ± 0.1 14.19 ± 0.2
YP.141 fresh fruit pure methanol 9.03 ± 0.1 11.91 ± 0.1 14.80 ± 0.2 23.47 ± 0.2 35.38 ± 0.2
YP.141 fresh fruit 80% methanol 9.39 ± 0.1 10.83 ± 0.2 16.61 ± 0.3 25.27 ± 0.1 35.74 ± 0.3
YP.141 fresh fruit 60% methanol 11.91 ± 0.1 16.08 ± 0.2 19.86 ± 0.2 26.35 ± 0.2 37.91 ± 0.5
YP.141 fresh fruit 50% methanol 5.05 ± 0.1 8.66 ± 0.1 14.08 ± 0.2 24.55 ± 0.3 41.88 ± 0.2
YP.141 fresh fruit pure water 9.39 ± 0.1 10.11 ± 0.1 15.16 ± 0.2 21.30 ± 0.0 31.05 ± 0.2
YP.141 dry fruit pure methanol 5.03 ± 0.1 5.70 ± 0.1 7.72 ± 0.1 10.40 ± 0.2 12.42 ± 0.2
YP.141 dry fruit 80% methanol 7.72 ± 0.1 9.40 ± 0.0 14.43 ± 0.2 10.40 ± 0.0 11.74 ± 0.4
YP.141 dry fruit 60% methanol 8.05 ± 0.1 8.72 ± 0.1 9.73 ± 0.2 30.87 ± 0.3 32.35 ± 0.1
YP.141 dry fruit 50% methanol 1.01 ± 0.1 6.71 ± 0.1 7.38 ± 0.3 10.40 ± 0.2 12.42 ± 0.5
YP.141 dry fruit pure water 7.05 ± 0.2 16.78 ± 0.1 15.7 ± 0.2 15.37 ± 0.1 16.7 ± 0.2
YP.141 leaf pure methanol 18.34 ± 0.2 28.03 ± 0.2 33.56 ± 0.1 48.79 ± 0.3 64.71 ± 0.1
YP.141 leaf 80% methanol 17.65 ± 0.0 33.56 ± 0.1 43.94 ± 0.2 57.09 ± 0.3 72.66 ± 0.4
YP.141 leaf 60% methanol 18.69 ± 0.2 32.53 ± 0.2 39.79 ± 0.1 53.63 ± 0.6 67.82 ± 0.4
YP.141 leaf 50% methanol 17.65 ± 0.3 31.49 ± 0.2 39.79 ± 0.1 51.90 ± 0.3 63.32 ± 0.5
YP.141 leaf pure water 16.61 ± 0.4 28.03 ± 0.2 32.87 ± 0.2 45.67 ± 0.7 61.59 ± 0.3
YP.141 leaf 0.5% acidified ethanol 7.61 ± 0.1 15.57 ± 0.1 21.45 ± 0.3 32.87 ± 0.2 50.52 ± 0.4
YP.141 leaf 1% acidified ethanol 8.30 ± 0.1 14.88 ± 0.1 19.03 ± 0.3 32.18 ± 0.2 48.79 ± 0.4
YP.141 leaf hexane n.d. n.d. 1.38 ± 0.1 5.88 ± 0.1 15.92 ± 0.1
YP.188 fresh fruit pure methanol 5.42 ± 0.2 10.83 ± 0.1 13.72 ± 0.2 21.66 ± 0.1 36.10 ± 0.6
YP.188 fresh fruit 80% methanol 5.39 ± 0.2 9.42 ± 0.1 11.91 ± 0.3 22.74 ± 0.1 33.57 ± 0.2
YP.188 fresh fruit 60% methanol 9.39 ± 0.2 12.27 ± 0.2 14.08 ± 0.2 23.10 ± 0.1 33.94 ± 0.2
YP.188 fresh fruit 50% methanol 11.05 ± 0.2 11.19 ± 0.2 15.88 ± 0.5 22.74 ± 0.3 32.85 ± 0.4
YP.188 fresh fruit pure water 13.00 ± 0.2 13.72 ± 0.1 22.38 ± 0.3 33.57 ± 0.3 46.93 ± 0.3
YP.188 dry fruit pure methanol 3.09 ± 0.2 4.75 ± 0.2 7.56 ± 0.2 8.25 ± 0.1 9.97 ± 0.1
YP.188 dry fruit 80% methanol 5.15 ± 0.0 6.53 ± 0.2 8.25 ± 0.2 9.28 ± 0.1 12.37 ± 0.1
YP.188 dry fruit 60% methanol 4.81 ± 0.2 7.56 ± 0.2 8.25 ± 0.1 8.93 ± 0.1 9.62 ± 0.2
YP.188 dry fruit 50% methanol 3.78 ± 0.2 6.53 ± 0.1 7.22 ± 0.3 8.25 ± 0.2 10.31 ± 0.5
YP.188 dry fruit pure water 3.78 ± 0.2 4.47 ± 0.2 6.87 ± 0.3 7.22 ± 0.1 8.59 ± 0.1
YP.188 leaf pure methanol 21.11 ± 0.1 31.14 ± 0.4 35.99 ± 0.2 53.98 ± 0.2 73.70 ± 0.1
YP.188 leaf 80% methanol 25.26 ± 0.2 41.18 ± 0.1 47.06 ± 0.3 66.09 ± 0.3 81.66 ± 0.4
YP.188 leaf 60% methanol 29.07 ± 0.0 46.02 ± 0.5 52.25 ± 0.2 66.78 ± 0.4 80.97 ± 0.4
YP.188 leaf 50% methanol 20.42 ± 0.2 33.56 ± 0.1 45.67 ± 0.1 57.09 ± 0.0 73.70 ± 0.1
YP.188 leaf pure water 13.15 ± 0.2 18.69 ± 0.2 21.45 ± 0.1 48.79 ± 0.4 67.82 ± 0.7
YP.188 leaf 0.5% acidified ethanol 7.27 ± 0.1 15.22 ± 0.3 22.49 ± 0.2 37.02 ± 0.2 57.44 ± 0.2
YP.188 leaf 1% acidified ethanol 10.38 ± 0.1 16.96 ± 0.0 21.11 ± 0.2 32.18 ± 0.3 50.17 ± 0.2
YP.188 leaf hexane n.d. n.d. n.d. 4.50 ± 0.2 17.30 ± 0.2
BHA 73.36 ± 0.2 79.58 ± 0.2 80.62 ± 0.1 83.39 ± 0.3 84.43 ± 0.2
BHT 65.74 ± 0.0 72.32 ± 0.1 73.01 ± 0.2 73.36 ± 0.1 72.32 ± 0.0
α-tocopherol 76.12 ± 0.2 76.12 ± 0.1 81.66 ± 0.2 84.78 ± 0.2 84.43 ± 0.0
*It represents the concentrations of the solutions prepared by taking 50, 100, 150, 250, and 500 μL of standard and extract stock solutions prepared
as 1 mg/mL and completing the total volume of 3 mL
n.d.: not detected
The chelating activity of iron (II) ions.
Antioxidants with metal chelating properties inactivate
it by binding free iron and thus inhibit the formation
of radicals such as hydroxyl and peroxide, which are
formed as a result of Fenton reactions. Therefore,
metal chelating plays an important role in determining
antioxidant activity [31].
We evaluated the metal ion chelating activity
according to the competition between plant extracts with
ferrosine in order to bind Fe2+ ions in the solution. We
observed no chelating activity in the extracts obtained
from moist and lyophilized dried fruits (Table 2).
The pure methanol extracts showed weak activity
in kumquat leaves, while the extracts obtained from
aqueous solvents showed no activity at all.
In addition, weak chelating activity was detected
in the 0.5 and 1% acidified ethanol extracts of
kumquat leaves and the hexane solvent extracts. The
highest activity (50.37%) was found in 62.5 μg/mL
concentration of the extract obtained from kumquat
Continuation of the Table 1
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leaves with a hexane solvent. We determined no
correlation between the chelating activity of the extracts
and their concentration. No significant difference was
found between the rootstock kumquat type and its
hybrids.
When we evaluated all the activities, we concluded
that the extracts obtained from kumquat fruits and
leaves were not good at chelating iron (II) ions. The most
important feature that affects the metal chelating activity
depends on the functional groups in the structure of
phenolic compounds and the position and amount of
these functional groups. For this reason, the difference
in the chelating activity of the samples can be explained
by different amounts of phenolic substances, as well as
phenolic substance groups in different structures and
positions [32].
The reducing capacity of the extracts. The
reducing agent in the environment reduces Fe3+ ions
Table 2 Metal chelating capacities of kumquat fruit and leaf extract, μg/mL (mean ± SD of triplicate)
Extracts and Standards 12.5* 25.0* 37.5* 62.5* 125*
Rootstock leaf pure methanol 10.70 ± 0.20 14.95 ± 0.1 20.44 ± 0.1 20.71 ± 0.3 5.76 ± 0.1
Rootstock leaf 0.5% acidified ethanol 4.39 ± 0.10 5.12 ± 0.0 4.39 ± 0.1 5.95 ± 0.1 6.73 ± 0.1
Rootstock leaf 1% acidified ethanol 3.51 ± 0.10 10.10 ± 0.1 11.86 ± 0.2 13.47 ± 0.1 18.59 ± 0.3
Rootstock leaf hexane 2.99 ± 0.0 8.52 ± 0.1 15.10 ± 0.2 18.30 ± 0.1 17.19 ± 0.4
EP.4 leaf pure methanol 10.56 ± 0.10 21.26 ± 0.1 23.32 ± 0.2 28.94 ± 0.1 16.74 ± 0.2
EP.4 leaf 0.5% acidified ethanol 3.51 ± 0.1 10.10 ± 0.2 11.86 ± 0.1 13.47 ± 0.1 18.59 ± 0.3
EP.4 leaf 1% acidified ethanol 4.10 ± 0.1 3.07 ± 0.2 5.42 ± 0.1 6.59 ± 0.3 6.83 ± 0.2
EP.4 leaf hexane 9.87 ± 0.2 14.20 ± 0.1 24.22 ± 0.2 34.08 ± 0.3 25.41 ± 0.3
EP.29 leaf pure methanol 13.03 ± 0.1 25.24 ± 0.1 32.24 ± 0.2 36.90 ± 0.3 19.48 ± 0.1
EP.29 leaf 0.5% acidified ethanol 4.93 ± 0.0 16.29 ± 0.1 23.47 ± 0.1 37.07 ± 0.1 24.96 ± 0.3
EP.29 leaf 1% acidified ethanol 5.38 ± 0.0 10.91 ± 0.2 17.32 ± 0.1 30.19 ± 0.2 31.24 ± 0.1
EP.29 leaf hexane 1.35 ± 0.1 2.54 ± 0.1 6.13 ± 0.1 12.26 ± 0.2 8.97 ± 0.2
EP.31 leaf pure methanol 27.36 ± 0.2 43.84 ± 0.1 44.64 ± 0.1 42.06 ± 0.3 31.20 ± 0.4
EP.31 leaf 0.5% acidified ethanol 2.54 ± 0.2 5.38 ± 0.2 10.46 ± 0.1 15.40 ± 0.3 15.99 ± 0.1
EP.31 leaf 1% acidified ethanol 2.69 ± 0.0 6.43 ± 0.1 9.72 ± 0.2 10.27 ± 0.1 7.92 ± 0.1
EP.31 leaf hexane 8.37 ± 0.2 9.57 ± 0.1 18.22 ± 0.1 20.33 ± 0.2 20.78 ± 0.3
YP.117 leaf pure methanol 11.17 ± 0.2 16.48 ± 0.3 22.35 ± 0.3 24.21 ± 0.2 24.58 ± 0.1
YP.117 leaf 0.5% acidified ethanol 3.44 ± 0.1 9.87 ± 0.0 11.36 ± 0.2 24.66 ± 0.0 19.28 ± 0.3
YP.117 leaf 1% acidified ethanol 5.23 ± 0.2 5.48 ± 0.1 14.20 ± 0.2 14.35 ± 0.2 14.05 ± 0.2
YP.117 leaf hexane 8.67 ± 0.1 20.63 ± 0.4 30.64 ± 0.3 36.32 ± 0.2 38.57 ± 0.3
YP.141 leaf pure methanol 14.79 ± 0.2 30.1 ± 0.2 35.43 ± 0.2 38.53 ± 0.3 35.56 ± 0.1
YP.141 leaf 0.5% acidified ethanol 2.09 ± 0.1 2.64 ± 0.1 6.43 ± 0.2 8.37 ± 0.2 8.74 ± 0.1
YP.141 leaf 1% acidified ethanol 1.20 ± 0.1 5.53 ± 0.1 10.31 ± 0.1 11.96 ± 0.0 14.80 ± 0.2
YP.141 leaf hexane 6.13 ± 0.1 11.36 ± 0.2 13.49 ± 0.1 17.04 ± 0.3 19.73 ± 0.1
YP.188 leaf pure methanol 16.84 ± 0.1 27.38 ± 0.2 31.63 ± 0.3 37.67 ± 0.3 44.39 ± 0.2
YP.188 leaf 0.5% acidified ethanol 2.54 ± 0.0 6.28 ± 0.1 8.07 ± 0.2 11.96 ± 0.0 17.32 ± 0.3
YP.188 leaf 1% acidified ethanol 2.69 ± 0.1 6.88 ± 0.1 10.91 ± 0.1 16.89 ± 0.1 16.35 ± 0.3
YP.188 leaf hexane 13.15 ± 0.1 28.10 ± 0.2 42.75 ± 0.2 50.37 ± 0.1 42.75 ± 0.3
EDTA 3.30 ± 0.0 25.93 ± 0.1 64.18 ± 0.2 91.40 ± 0.1 92.26 ± 0.1
*It represents the concentrations of the solutions prepared by taking 50, 100, 150, 250, and 500 μL of standard and extract stock solutions prepared
as 1 mg/mL and completing the total volume of 3 mL
to Fe2+ ions depending on its antioxidant capacity. The
absorbance of the Prussian blue complex (Fe4[Fe(CN)6])
formed by adding FeCl3 to the reduced product is
measured at 700 nm [22]. The increase in absorbance
of the reaction mixture is directly proportional to the
reducing power of the sample.
We found that the capacity of kumquat leaves to
reduce Fe3+ ions was higher than that of lyophilized
and wet kumquat fruits (Table 3). We observed that
lyophilizing and drying of kumquat fruits did not cause
a significant change in their reducing capacity. The
reducing capacity of the fruit and leaf extracts was lower
than the standards (BHA, BHT and α-tocopherol).
The highest reducing capacity (0.307 ± 0.001) was
observed at a concentration of 29.41 μg/mL of the EP.4
mutant leaf extract obtained with pure water. Among
the fruits, the highest reducing capacity (0.199 ±
0.001) was found at a concentration of 29.41 μg/mL of
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Table 3 The reducing power of extracts and standards, μg/mL (mean ± SD of triplicate)
Extracts and Standards 5.88* 14.7* 29.41*
Rootstock fresh fruit pure methanol 0.104 ± 0.001 0.115 ± 0.003 0.138 ± 0.002
Rootstock fresh fruit 80% methanol 0.105 ± 0.002 0.106 ± 0.001 0.124 ± 0.001
Rootstock fresh fruit 60% methanol 0.120 ± 0.001 0.133 ± 0.001 0.140 ± 0.003
Rootstock fresh fruit 50% methanol 0.096 ± 0.001 0.100 ± 0.002 0.104 ± 0.001
Rootstock fresh fruit pure water 0.082 ± 0.002 0.098 ± 0.003 0.115 ± 0.001
Rootstock dry fruit pure methanol 0.075 ± 0.001 0.082 ± 0.003 0.094 ± 0.001
Rootstock dry fruit 80% methanol 0.074 ± 0.002 0.087 ± 0.006 0.097 ± 0.005
Rootstock dry fruit 60% methanol 0.076 ± 0.001 0.081 ± 0.001 0.089 ± 0.001
Rootstock dry fruit 50% methanol 0.076 ± 0.002 0.082 ± 0.001 0.089 ± 0.003
Rootstock dry fruit pure water 0.078 ± 0.003 0.081 ± 0.001 0.089 ± 0.001
Rootstock leaf pure methanol 0.103 ± 0.002 0.145 ± 0.001 0.241 ± 0.004
Rootstock leaf 80% methanol 0.098 ± 0.001 0.149 ± 0.001 0.227 ± 0.003
Rootstock leaf 60% methanol 0.093 ± 0.001 0.136 ± 0.005 0.218 ± 0.003
Rootstock leaf 50% methanol 0.097 ± 0.002 0.148 ± 0.001 0.240 ± 0.003
Rootstock leaf pure water 0.089 ± 0.001 0.143 ± 0.003 0.209 ± 0.005
Rootstock leaf 0.5% acidified ethanol 0.074 ± 0.001 0.096 ± 0.002 0.128 ± 0.001
Rootstock leaf 1% acidified ethanol 0.076 ± 0.001 0.098 ± 0.003 0.129 ± 0.001
Rootstock leaf hexane 0.091 ± 0.002 0.125 ± 0.003 0.179 ± 0.002
EP.4 fresh fruit pure methanol 0.111 ± 0.002 0.144 ± 0.001 0.199 ± 0.001
EP.4 fresh fruit 80% methanol 0.108 ± 0.001 0.110 ± 0.003 0.100 ± 0.001
EP.4 fresh fruit 60% methanol 0.104 ± 0.002 0.095 ± 0.003 0.112 ± 0.001
EP.4 fresh fruit 50% methanol 0.099 ± 0.003 0.092 ± 0.001 0.143 ± 0.001
EP.4 fresh fruit pure water 0.086 ± 0.001 0.093 ± 0.001 0.115 ± 0.002
EP.4 dry fruit pure methanol 0.070 ± 0.001 0.077 ± 0.001 0.091 ± 0.001
EP.4 dry fruit 80% methanol 0.071 ± 0.002 0.078 ± 0.001 0.089 ± 0.003
EP.4 dry fruit 60% methanol 0.074 ± 0.001 0.076 ± 0.001 0.087 ± 0.003
EP.4 dry fruit 50% methanol 0.071 ± 0.001 0.075 ± 0.003 0.085 ± 0.001
EP.4 dry fruit pure water 0.070 ± 0.002 0.072 ± 0.001 0.081 ± 0.001
EP.4 leaf pure methanol 0.087 ± 0.002 0.134 ± 0.004 0.201 ± 0.001
EP.4 leaf 80% methanol 0.097 ± 0.001 0.145 ± 0.003 0.245 ± 0.004
EP.4 leaf 60% methanol 0.093 ± 0.003 0.139 ± 0.001 0.211 ± 0.003
EP.4 leaf 50% methanol 0.091 ± 0.002 0.149 ± 0.003 0.227 ± 0.005
EP.4 leaf pure water 0.116 ± 0.001 0.193 ± 0.003 0.307 ± 0.001
EP.4 leaf 0.5% acidified ethanol 0.075 ± 0.002 0.093 ± 0.001 0.125 ± 0.001
EP.4 leaf 1% acidified ethanol 0.079 ± 0.001 0.102 ± 0.002 0.133 ± 0.006
EP.4 leaf hexane 0.091 ± 0.003 0.125 ± 0.001 0.179 ± 0.001
EP.29 fresh fruit pure methanol 0.107 ± 0.004 0.118 ± 0.004 0.135 ± 0.006
EP.29 fresh fruit 80% methanol 0.107 ± 0.001 0.114 ± 0.002 0.108 ± 0.002
EP.29 fresh fruit 60% methanol 0.109 ± 0.000 0.109 ± 0.000 0.138 ± 0.000
EP.29 fresh fruit 50% methanol 0.113 ± 0.000 0.117 ± 0.001 0.133 ± 0.000
EP.29 fresh fruit pure water 0.086 ± 0.001 0.092 ± 0.000 0.100 ± 0.001
EP.29 dry fruit pure methanol 0.072 ± 0.000 0.081 ± 0.001 0.098 ± 0.000
EP.29 dry fruit 80% methanol 0.073 ± 0.000 0.080 ± 0.001 0.093 ± 0.000
EP.29 dry fruit 60% methanol 0.072 ± 0.001 0.077 ± 0.001 0.090 ± 0.001
EP.29 dry fruit 50% methanol 0.071 ± 0.001 0.078 ± 0.000 0.088 ± 0.000
EP.29 dry fruit pure water 0.073 ± 0.000 0.076 ± 0.001 0.090 ± 0.000
EP.29 leaf pure methanol 0.090 ± 0.000 0.125 ± 0.001 0.206 ± 0.002
EP.29 leaf 80% methanol 0.093 ± 0.000 0.145 ± 0.001 0.236 ± 0.000
EP.29 leaf 60% methanol 0.106 ± 0.001 0.158 ± 0.000 0.260 ± 0.000
EP.29 leaf 50% methanol 0.103 ± 0.000 0.163 ± 0.000 0.281 ± 0.000
EP.29 leaf pure water 0.101 ± 0.000 0.158 ± 0.001 0.244 ± 0.000
EP.29 leaf 0.5% acidified ethanol 0.086 ± 0.000 0.103 ± 0.001 0.135 ± 0.000
EP.29 leaf 1% acidified ethanol 0.077 ± 0.001 0.094 ± 0.001 0.119 ± 0.001
EP.29 leaf hexane 0.088 ± 0.000 0.136 ± 0.000 0.193 ± 0.000
EP.31 fresh fruit pure methanol 0.091 ± 0.001 0.098 ± 0.001 0.109 ± 0.001
EP.31 fresh fruit 80% methanol 0.087 ± 0.000 0.095 ± 0.000 0.117 ± 0.000
EP.31 fresh fruit 60% methanol 0.081 ± 0.000 0.103 ± 0.001 0.129 ± 0.001
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Extracts and Standards 5.88* 14.7* 29.41*
EP.31 fresh fruit 50% methanol 0.089 ± 0.000 0.115 ± 0.001 0.104 ± 0.000
EP.31 fresh fruit pure water 0.088 ± 0.001 0.094 ± 0.000 0.105 ± 0.001
EP.31 dry fruit pure methanol 0.093 ± 0.000 0.099 ± 0.000 0.125 ± 0.000
EP.31 dry fruit 80% methanol 0.095 ± 0.000 0.102 ± 0.000 0.099 ± 0.000
EP.31 dry fruit 60% methanol 0.099 ± 0.000 0.085 ± 0.001 0.096 ± 0.001
EP.31 dry fruit 50% methanol 0.099 ± 0.000 0.092 ± 0.001 0.097 ± 0.000
EP.31 dry fruit pure water 0.107 ± 0.000 0.100 ± 0.001 0.111 ± 0.001
EP.31 leaf pure methanol 0.089 ± 0.001 0.119 ± 0.001 0.176 ± 0.001
EP.31 leaf 80% methanol 0.093 ± 0.000 0.133 ± 0.001 0.200 ± 0.000
EP.31 leaf 60% methanol 0.101 ± 0.001 0.148 ± 0.001 0.214 ± 0.000
EP.31 leaf 50% methanol 0.100 ± 0.001 0.142 ± 0.000 0.212 ± 0.001
EP.31 leaf pure water 0.094 ± 0.001 0.133 ± 0.000 0.206 ± 0.001
EP.31 leaf 0.5% acidified ethanol 0.089 ± 0.000 0.127 ± 0.000 0.184 ± 0.000
EP.31 leaf 1% acidified ethanol 0.088 ± 0.000 0.113 ± 0.001 0.155 ± 0.000
EP.31 leaf hexane 0.098 ± 0.001 0.119 ± 0.000 0.202 ± 0.001
YP.117 fresh fruit pure methanol 0.099 ± 0.001 0.117 ± 0.000 0.153 ± 0.000
YP.117 fresh fruit 80% methanol 0.096 ± 0.000 0.099 ± 0.000 0.117 ± 0.000
YP.117 fresh fruit 60% methanol 0.100 ± 0.000 0.100 ± 0.001 0.114 ± 0.000
YP.117 fresh fruit 50% methanol 0.107 ± 0.000 0.116 ± 0.001 0.142 ± 0.000
YP.117 fresh fruit pure water 0.088 ± 0.000 0.094 ± 0.000 0.114 ± 0.000
YP.117 dry fruit pure methanol 0.077 ± 0.000 0.082 ± 0.000 0.108 ± 0.001
YP.117 dry fruit 80% methanol 0.074 ± 0.000 0.079 ± 0.001 0.085 ± 0.000
YP.117 dry fruit 60% methanol 0.081 ± 0.000 0.088 ± 0.001 0.093 ± 0.000
YP.117 dry fruit 50% methanol 0.085 ± 0.001 0.080 ± 0.000 0.087 ± 0.000
YP.117 dry fruit pure water 0.079 ± 0.000 0.083 ± 0.000 0.089 ± 0.000
YP.117 leaf pure methanol 0.092 ± 0.001 0.141 ± 0.001 0.206 ± 0.000
YP.117 leaf 80% methanol 0.093 ± 0.000 0.133 ± 0.001 0.201 ± 0.000
YP.117 leaf 60% methanol 0.101 ± 0.001 0.157 ± 0.000 0.235 ± 0.001
YP.117 leaf 50% methanol 0.109 ± 0.001 0.159 ± 0.001 0.262 ± 0.001
YP.117 leaf pure water 0.105 ± 0.000 0.152 ± 0.000 0.242 ± 0.000
YP.117 leaf 0.5% acidified ethanol 0.091 ± 0.000 0.116 ± 0.001 0.165 ± 0.000
YP.117 leaf 1% acidified ethanol 0.087 ± 0.001 0.113 ± 0.001 0.163 ± 0.001
YP.117 leaf hexane 0.072 ± 0.000 0.091 ± 0.000 0.154 ± 0.000
YP.141 fresh fruit pure methanol 0.096 ± 0.001 0.104 ± 0.000 0.124 ± 0.000
YP.141 fresh fruit 80% methanol 0.091 ± 0.000 0.091 ± 0.001 0.105 ± 0.000
YP.141 fresh fruit 60% methanol 0.146 ± 0.000 0.138 ± 0.001 0.139 ± 0.000
YP.141 fresh fruit 50% methanol 0.092 ± 0.000 0.103 ± 0.001 0.142 ± 0.000
YP.141 fresh fruit pure water 0.091 ± 0.000 0.099 ± 0.000 0.117 ± 0.001
YP.141 dry fruit pure methanol 0.092 ± 0.001 0.091 ± 0.001 0.102 ± 0.000
YP.141 dry fruit 80% methanol 0.102 ± 0.000 0.105 ± 0.001 0.120 ± 0.000
YP.141 dry fruit 60% methanol 0.093 ± 0.000 0.090 ± 0.001 0.097 ± 0.000
YP.141 dry fruit 50% methanol 0.097 ± 0.001 0.088 ± 0.001 0.095 ± 0.000
YP.141 dry fruit pure water 0.094 ± 0.001 0.087 ± 0.000 0.098 ± 0.000
YP.141 leaf pure methanol 0.105 ± 0.000 0.155 ± 0.000 0.241 ± 0.001
YP.141 leaf 80% methanol 0.108 ± 0.000 0.165 ± 0.001 0.254 ± 0.000
YP.141 leaf 60% methanol 0.100 ± 0.000 0.154 ± 0.001 0.250 ± 0.000
YP.141 leaf 50% methanol 0.106 ± 0.001 0.162 ± 0.000 0.252 ± 0.002
YP.141 leaf pure water 0.101 ± 0.000 0.141 ± 0.000 0.247 ± 0.001
YP.141 leaf 0.5% acidified ethanol 0.088 ± 0.000 0.123 ± 0.001 0.186 ± 0.001
YP.141 leaf 1% acidified ethanol 0.082 ± 0.000 0.108 ± 0.000 0.148 ± 0.000
YP.141 leaf hexane 0.070 ± 0.001 0.102 ± 0.000 0.162 ± 0.000
YP.188 fresh fruit pure methanol 0.092 ± 0.001 0.111 ± 0.000 0.146 ± 0.000
YP.188 fresh fruit 80% methanol 0.094 ± 0.000 0.107 ± 0.001 0.136 ± 0.001
YP.188 fresh fruit 60% methanol 0.090 ± 0.000 0.104 ± 0.001 0.123 ± 0.000
YP.188 fresh fruit 50% methanol 0.095 ± 0.000 0.096 ± 0.001 0.112 ± 0.000
YP.188 fresh fruit pure water 0.099 ± 0.000 0.103 ± 0.000 0.126 ± 0.000
YP.188 dry fruit pure methanol 0.090 ± 0.001 0.086 ± 0.000 0.110 ± 0.000
Continuation of the Table 3
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Extracts and Standards 5.88* 14.7* 29.41*
YP.188 dry fruit 80% methanol 0.091 ± 0.000 0.088 ± 0.000 0.094 ± 0.001
YP.188 dry fruit 60% methanol 0.089 ± 0.001 0.087 ± 0.000 0.098 ± 0.000
YP.188 dry fruit 50% methanol 0.092 ± 0.000 0.094 ± 0.001 0.099 ± 0.000
YP.188 dry fruit pure water 0.093 ± 0.000 0.087 ± 0.001 0.100 ± 0.000
YP.188 leaf pure methanol 0.102 ± 0.000 0.182 ± 0.001 0.252 ± 0.001
YP.188 leaf 80% methanol 0.115 ± 0.001 0.164 ± 0.000 0.263 ± 0.000
YP.188 leaf 60% methanol 0.116 ± 0.000 0.176 ± 0.000 0.279 ± 0.000
YP.188 leaf 50% methanol 0.109 ± 0.001 0.159 ± 0.000 0.253 ± 0.001
YP.188 leaf pure water 0.111 ± 0.000 0.169 ± 0.000 0.271 ± 0.000
YP.188 leaf 0.5% acidified ethanol 0.098 ± 0.000 0.157 ± 0.001 0.218 ± 0.0001
YP.188 leaf 1% acidified ethanol 0.088 ± 0.000 0.110 ± 0.001 0.147 ± 0.000
YP.188 leaf hexane 0.076 ± 0.001 0.102 ± 0.001 0.167 ± 0.001
BHA 0.690 ± 0.001 1.346 ± 0.000 1.984 ± 0.000
BHT 0.504 ± 0.000 0.939 ± 0.000 1.290 ± 0.002
α-tokeferol 0.234 ± 0.000 0.477 ± 0.001 0.872 ± 0.000
*It represents the concentrations of the solutions prepared by taking 100, 250, and 500 μL of standard and extract stock solutions prepared as
1 mg/mL and completing the total volume of 3.750 μmL
the EP.4 hybrid wet fruit extract obtained with pure
methanol. The reducing capacities of the standards were
1.984 ± 0.001, 1.290 ± 0.002, 0.872 ± 0.001 for BHA,
BHT, and α-toceferol, respectively, at the highest
concentration of 29.41 μg/mL.
No significant difference was observed between the
rootstock kumquat plant and its mutants. Although the
reducing power is an important factor of antioxidant
activity, in our study, the reducing power was lower in
the extracts with high antioxidant activity. Other studies
also show that extracts with high antioxidant activity
may have low reducing power [33, 34]. This is because
in the systems where free iron ions are present in trace
amounts, the net oxidation rate increases with the
Fenton reaction. Substances with high reducing power
may cause further acceleration of oxidation by reducing
Fe(III) to Fe(II). The presence of trace levels of iron ions
in kumquat materials may have caused its low reducing
power and ncreased antioxidant activity [35].
Phenolic and flavonoid content. Since phenolic
and flavonoid compounds contain hydroxyl groups in
their structures and can easily give a hydrogen radical
in hydroxyl groups, they have free radical quenching
properties. Therefore, it is important to know the
total phenolic and flavonoid contents of the samples
to determine their contribution to the antioxidant
activity, including radical scavenging activity tests. For
this, we used the Folin-Ciocalteu method, a standard
method in antioxidant studies. The basis of the method
is that phenolic compounds dissolved in water and
other organic solvents form a colored complex with
a Folin reagent in an alkaline medium. The total
phenolic content of the extracts obtained by Soxhlet
extraction with different solvents was calculated using
the regression equation (y = 0.0292x + 0.0749 and
R² = 0.9994) of the calibration line of the standard gallic
acid solution prepared in the concentration range of
5–50 μg/mL and expressed as gallic acid equivalent (mg
GAE/g extract). The gallic acid standard curve is shown
Figure 1 Standard calibration curve of gallic acid to determine
total phenolic content
Figure 2 Calibration curve of standard quercetin to determine
total flavonoid content
% free-radical scavenging activity = 𝐴𝐴C− 𝐴𝐴S/S
𝐴𝐴C
× 100 (1)
y = 0.0292x + 0.0749
R² = 0.9994
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 10 20 30 40 50
Absorbance
Concentration, μg/mL
y = 0.045x + 0.0305
R² = 0.9913
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Absorbance
% free-radical scavenging activity = 𝐴𝐴C− 𝐴𝐴S/S
𝐴𝐴C
× 100 (1)
y = 0.0292x + 0.0749
R² = 0.9994
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 10 20 30 40 50
Absorbance
Concentration, μg/mL
y = 0.045x + 0.0305
R² = 0.9913
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0 5 10 15 20 25
Absorbance
Concentration, μg/mL
Continuation of the Table 3
61
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Table 4 Total phenolic and total flavonoid contents in kumquat fruit and leaf extracts
Extracts Total Phenolic Substance,
mg GAE/g extract
Total Flavonoid Substance,
mg QUE/g extract
Rootstock fresh fruit pure methanol 16.096 ± 0.045 42.222 ± 0.018
Rootstock fresh fruit 80% methanol 8.432 ± 0.024 24.444 ± 0.014
Rootstock fresh fruit 60% methanol 5.808 ± 0.012 22.222 ± 0.012
Rootstock fresh fruit 50% methanol 7.089 ± 0.018 26.667 ± 0.018
Rootstock fresh fruit pure water 13.747 ± 0.011 41.111 ± 0.020
Rootstock dry fruit pure methanol 8.959 ± 0.038 46.667 ± 0.016
Rootstock dry fruit 80% methanol 9.856 ± 0.033 10.022 ± 0.010
Rootstock dry fruit 60% methanol 5.829 ± 0.011 10.100 ± 0.012
Rootstock dry fruit 50% methanol 5.425 ± 0.010 5.556 ± 0.011
Rootstock dry fruit pure water 3.705 ± 0.011 14.444 ± 0.016
Rootstock leaf pure methanol 66.356 ± 0.034 454.444 ± 0.046
Rootstock leaf 80% methanol 72.548 ± 0.021 258.889 ± 0.024
Rootstock leaf 60% methanol 68.979 ± 0.023 213.333 ± 0.034
Rootstock leaf 50% methanol 67.096 ± 0.018 248.889 ± 0.032
Rootstock leaf pure water 54.062 ± 0.023 174.444 ± 0.024
Rootstock leaf 0.5% acidified ethanol 31.925 ± 0.030 314.444 ± 0.042
Rootstock leaf 1% acidified ethanol 31.062 ± 0.018 308.889 ± 0.014
Rootstock leaf hexane n.d. n.d.
EP.4 fresh fruit pure methanol 20.281 ± 0.013 67.778 ± 0.026
EP.4 fresh fruit 80% methanol 8.678 ± 0.025 32.222 ± 0.024
EP.4 fresh fruit 60% methanol 5.479 ± 0.012 26.667 ± 0.018
EP.4 fresh fruit 50% methanol 7.760 ± 0.021 35.556 ± 0.012
EP.4 fresh fruit pure water 7.534 ± 0.011 25.556 ± 0.010
EP.4 dry fruit pure methanol 11.247 ± 0.013 25.556 ± 0.014
EP.4 dry fruit 80% methanol 11.315 ± 0.022 16.667 ± 0.016
EP.4 dry fruit 60% methanol 14.288 ± 0.023 27.778 ± 0.022
EP.4 dry fruit 50% methanol 9.137 ± 0.014 30.000 ± 0.023
EP.4 dry fruit pure water 7.521 ± 0.021 23.333 ± 0.024
EP.4 leaf pure methanol 63.438 ± 0.015 410.000 ± 0.032
EP.4 leaf 80% methanol 64.797 ± 0.017 271.111 ± 0.023
EP.4 leaf 60% methanol 64.685 ± 0.010 231.111 ± 0.023
EP.4 leaf 50% methanol 65.568 ± 0.022 248.889 ± 0.023
EP.4 leaf pure water 73.034 ± 0.015 255.556 ± 0.023
EP.4 leaf 0.5% acidified ethanol 33.068 ± 0.032 315.556 ± 0.023
EP.4 leaf 1% acidified ethanol 33.952 ± 0.014 355.556 ± 0.023
EP.4 leaf hexane n.d. n.d.
EP.29 fresh fruit pure methanol 14.596 ± 0.011 42.222 ± 0.023
EP.29 fresh fruit 80% methanol 8.884 ± 0.021 31.111 ± 0.023
EP.29 fresh fruit 60% methanol 8.842 ± 0.021 20.000 ± 0.023
EP.29 fresh fruit 50% methanol 11.534 ± 0.018 30.000 ± 0.023
EP.29 fresh fruit pure water 13.404 ± 0.016 21.111 ± 0.023
EP.29 dry fruit pure methanol 12.404 ± 0.012 65.556 ± 0.023
EP.29 dry fruit 80% methanol 12.918 ± 0.012 16.667 ± 0.023
EP.29 dry fruit 60% methanol 9.623 ± 0.018 26.667 ± 0.023
EP.29 dry fruit 50% methanol 9.747 ± 0.017 21.111 ± 0.023
EP.29 dry fruit pure water 7.205 ± 0.013 13.333 ± 0.023
EP.29 leaf pure methanol 60.836 ± 0.022 438.889 ± 0.023
EP.29 leaf 80% methanol 67.589 ± 0.032 223.333 ± 0.023
EP.29 leaf 60% methanol 70.226 ± 0.043 256.667 ± 0.023
EP.29 leaf 50% methanol 64.822 ± 0.023 268.889 ± 0.023
EP.29 leaf pure water 50.390 ± 0.013 184.444 ± 0.023
EP.29 leaf 0.5% acidified ethanol 41.158 ± 0.011 486.667 ± 0.023
EP.29 leaf 1% acidified ethanol 25.856 ± 0.033 242.222 ± 0.023
EP.29 leaf hexane n.d. n.d.
EP.31 fresh fruit pure methanol 6.384 ± 0.014 38.889 ± 0.023
EP.31 fresh fruit 80% methanol 9.952 ± 0.012 20.000 ± 0.023
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Extracts Total Phenolic Substance,
mg GAE/g extract
Total Flavonoid Substance,
mg QUE/g extract
EP.31 fresh fruit 60% methanol 17.500 ± 0.023 42.222 ± 0.023
EP.31 fresh fruit 50% methanol 5.822 ± 0.023 14.444 ± 0.023
EP.31 fresh fruit pure water 8.164 ± 0.013 27.778 ± 0.023
EP.31 dry fruit pure methanol 12.212 ± 0.015 105.556 ± 0.023
EP.31 dry fruit 80% methanol 7.452 ± 0.028 25.556 ± 0.023
EP.31 dry fruit 60% methanol 7.767 ± 0.026 23.333 ± 0.023
EP.31 dry fruit 50% methanol 7.486 ± 0.024 26.667 ± 0.023
EP.31 dry fruit pure water 6.568 ± 0.022 13.333 ± 0.023
EP.31 leaf pure methanol 61.973 ± 0.022 450.000 ± 0.023
EP.31 leaf 80% methanol 64.739 ± 0.018 284.444 ± 0.023
EP.31 leaf 60% methanol 74.082 ± 0.020 260.000 ± 0.023
EP.31 leaf 50% methanol 72.363 ± 0.014 281.111 ± 0.023
EP.31 leaf pure water 50.274 ± 0.024 180.000 ± 0.023
EP.31 leaf 0.5% acidified ethanol 47.699 ± 0.010 454.444 ± 0.023
EP.31 leaf 1% acidified ethanol 43.603 ± 0.018 632.222 ± 0.033
EP.31 leaf hexane n.d. n.d.
YP.117 fresh fruit pure methanol 13.322 ± 0.022 36.667 ± 0.023
YP.117 fresh fruit 80% methanol 8.527 ± 0.012 16.667 ± 0.023
YP.117 fresh fruit 60% methanol 8.486 ± 0.014 17.778 ± 0.023
YP.117 fresh fruit 50% methanol 7.349 ± 0.022 158.889 ± 0.023
YP.117 fresh fruit pure water 8.308 ± 0.018 112.222 ± 0.023
YP.117 dry fruit pure methanol 9.445 ± 0.012 36.667 ± 0.023
YP.117 dry fruit 80% methanol 8.822 ± 0.010 16.667 ± 0.023
YP.117 dry fruit 60% methanol 7.705 ± 0.016 17.778 ± 0.023
YP.117 dry fruit 50% methanol 6.986 ± 0.020 158.889 ± 0.023
YP.117 dry fruit pure water 5.740 ± 0.018 112.222 ± 0.023
YP.117 leaf pure methanol 65.356 ± 0.016 458.889 ± 0.023
YP.117 leaf 80% methanol 70.205 ± 0.014 194.444 ± 0.023
YP.117 leaf 60% methanol 68.514 ± 0.023 298.889 ± 0.023
YP.117 leaf 50% methanol 65.616 ± 0.022 285.556 ± 0.023
YP.117 leaf pure water 55.425 ± 0.020 248.889 ± 0.023
YP.117 leaf 0.5% acidified ethanol 43.603 ± 0.016 312.222 ± 0.023
YP.117 leaf 1% acidified ethanol 41.205 ± 0.022 381.111 ± 0.023
YP.117 leaf hexane n.d. n.d.
YP.141 fresh fruit pure methanol 9.342 ± 0.022 313.333 ± 0.023
YP.141 fresh fruit 80% methanol 7.630 ± 0.020 40.000 ± 0.023
YP.141 fresh fruit 60% methanol 10.740 ± 0.014 40.000 ± 0.023
YP.141 fresh fruit 50% methanol 9.164 ± 0.018 31.111 ± 0.023
YP.141 fresh fruit pure water 8.432 ± 0.012 27.778 ± 0.023
YP.141 dry fruit pure methanol 15.637 ± 0.020 97.778 ± 0.023
YP.141 dry fruit 80% methanol 9.089 ± 0.022 26.667 ± 0.023
YP.141 dry fruit 60% methanol 10.918 ± 0.018 50.000 ± 0.023
YP.141 dry fruit 50% methanol 8.295 ± 0.014 55.556 ± 0.023
YP.141 dry fruit pure water 6.144 ± 0.022 26.667 ± 0.023
YP.141 leaf pure methanol 72.342 ± 0.023 564.444 ± 0.023
YP.141 leaf 80% methanol 76.658 ± 0.010 387.778 ± 0.023
YP.141 leaf 60% methanol 64.322 ± 0.022 354.444 ± 0.023
YP.141 leaf 50% methanol 63.767 ± 0.016 357.778 ± 0.023
YP.141 leaf pure water 60.082 ± 0.014 305.556 ± 0.023
YP.141 leaf 0.5% acidified ethanol 51.048 ± 0.012 470.000 ± 0.023
YP.141 leaf 1% acidified ethanol 32.329 ± 0.012 300.000 ± 0.023
YP.141 leaf hexane n.d. n.d.
YP.188 fresh fruit pure methanol 11.336 ± 0.010 111.111 ± 0.023
YP.188 fresh fruit 80% methanol 8.993 ± 0.012 87.778 ± 0.023
YP.188 fresh fruit 60% methanol 9.986 ± 0.008 86.667 ± 0.023
YP.188 fresh fruit 50% methanol 8.979 ± 0.016 104.444 ± 0.023
YP.188 fresh fruit pure water 20.144 ± 0.022 102.222 ± 0.023
Continuation of the Table 4
63
Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66
in Fig. 1. We found that the kumquat leaf extracts had
the highest total phenolic content (Table 4). In particular,
the highest total phenolic content (86.329 ± 0.022 mg
GAE/g extract) was in the YP.188 mutant extract
obtained with 60% methanol. In the fruit samples, the
highest total phenolic content (20.281 mg GAE/g extract)
was found in the EP.4 mutant extract obtained with pure
methanol. There was no significant difference in total
phenolic contents between the fresh and dried fruit
samples.
Lou et al. compared total phenolic contents in
fresh and dried kumquat fruits [36]. The scientists
investigated changes in total phenolic matter by
changing the drying degree and time. They found that
the total amount of phenolic substances increased with
drying, amounting to 15–17 mg GAE/g extract and 48–
50 mg GAE/g extract in fresh and dried fruit (130°C),
respectively [36].
In another study, Özcan et al. dried kumquat fruit
in hot air, under vacuum, and in a microwave oven [27].
The authors found that the total phenolic content of hot
air-dried fruit was approximately 5 mg GAE/g extract,
but with other drying methods, it varied in the range of
25–30 mg GAE/g extract [37].
Yıldız Turgut et al. studied the functional quality
parameters of the powder obtained from Fortunella
margarita kumquat varieties grown in Turkey. They
reported the total phenolic content of kumquat between
2.62 ± 0.051 – 6.97 ± 0.053 mg GAE/g depending on the
type of drying method [38].
Having determined the total phenolic content, we
measured the total flavonoid content of the samples.
Total flavonoid concentration was determined colorimetrically
using a UV spectrophotometer according to
the method applied by Zhishen et al. [27].
In our study, quercetin was used as a standard and
the results were calculated as quercetin equivalent (mg
QUE/g extract) from the quercetin standard calibration
chart (y = 0.0185x – 0.0019 and R² = 0.9666) (Fig. 2).
The highest amount of total flavonoid substance was
Extracts Total Phenolic Substance,
mg GAE/g extract
Total Flavonoid Substance,
mg QUE/g extract
YP.188 dry fruit pure methanol 9.151 ± 0.014 15.556 ± 0.023
YP.188 dry fruit 80% methanol 8.212 ± 0.028 16.667 ± 0.023
YP.188 dry fruit 60% methanol 7.048 ± 0.014 21.111 ± 0.023
YP.188 dry fruit 50% methanol 7.021 ± 0.012 38.889 ± 0.023
YP.188 dry fruit pure water 5.418 ± 0.008 26.667 ± 0.023
YP.188 leaf pure methanol 72.637 ± 0.010 446.667 ± 0.023
YP.188 leaf 80% methanol 85.651 ± 0.030 330.000 ± 0.023
YP.188 leaf 60% methanol 86.329 ± 0.022 345.556 ± 0.023
YP.188 leaf 50% methanol 75.418 ± 0.022 300.000 ± 0.023
YP.188 leaf pure water 70.849 ± 0.018 313.333 ± 0.023
YP.188 leaf 0.5% acidified ethanol 62.890 ± 0.020 582.222 ± 0.023
YP.188 leaf 1% acidified ethanol 33.226 ± 0.018 275.556 ± 0.023
YP.188 leaf hexane n.d. n.d.
n.d.: not detected
seen in kumquat leaves (Table 4). In particular, the
highest flavonoid content was found in the EP.31 mutant
extract (632.222 ± 0.033 mg QUE/g extract) obtained
with 1% acidified ethanol.
Among the fruit samples, the highest amount
(313.333 ± 0.023 mg QUE/g extract) was found in the
YP.141 mutant extract obtained with pure methanol.
There were no significant differences between the total
flavonoid amounts in the fresh and dried fruits.
Lou et al. reported that the total amount of flavonoid
substance in kumquat varied between 58.23–91.42 mg/g
depending on the drying temperature [36]. In another
study, Lou et al. found that the total phenolic and
flavonoid contents were higher in the extracts from
kumquat and calamondin peel compared to fruit pulp,
and that they were higher in the extracts from unripe
kumquat compared to those from ripe kumquat [39, 40].
CONCLUSION
In antioxidant activity studies, it is common to use a
different polarity solvent system in order to determine
which compound types have the highest activity. There
may be a relationship between phenolic or flavonoid
amounts and antioxidant capacity determination
methods. In particular, a relationship between methods
such as the DPPH, which is based on radical capture,
and total phenolic and flavonoid amounts may be
important in some plant structures. Phenolic acids and
flavonoids are soluble in polar solvents and show strong
activity in polar systems.
In this study, we investigated the effect of different
solvents and their concentrations on the bioactivity
of kumquat fruit and leaf extracts. We found that the
solvent type was extremely important for the extracts’
bioactivity. In particular, the extraction performed with
pure methanol in the fruits and 60 or 80% methanol
in the leaves showed the highest total phenolic and
flavonoid contents, the highest extraction efficiency
(50.18–59.95%), and the highest antioxidant capacity.
Continuation of the Table 4
64
Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66
We found no statistically significant difference
between the total amount of phenolic/flavonoid
substances and % inhibition value in the extraction
performed with 60 and 80% methanol solutions. This
shows that the amount of phenolic substances was
affected by the polarity of the solvent, depending on the
difference in phenolic compounds found in kumquat
fruit and leaves. We concluded that phenolic components
in the structure of a kumquat fruit could be extracted
with a single solvent type, whereas those in the structure
of a kumquat leaf could be extracted better with an
aqueous solution of the relevant solvent, rather than a
single solvent type.
We also observed that the aqueous solutions gave
better results than the pure solutions in the production
of phenolics from kumquat leaves, maximizing at
certain water ratios and showing different distributions
according to the solvents. These results can be explained
by the fact that water increases diffusion by causing
swelling in the leaf structure. In this context, methanol
was the most effective solvent for bioactive component
extraction from the kumquat fruits, whereas methanol +
water was most effective for the leaves.
Having examined the effect of a solvent amount,
we concluded that the extraction with 260 mL solvent
ensured the highest total phenolic content, extraction
efficiency, and antioxidant capacity. In addition, since
methanol is a toxic solvent, it must be removed so that
the obtained extract can be used in foods or consumed as
a food supplement.
Plants are complex systems by nature and have
multiple reaction characteristics and dissolution
properties in different phases. Thus, it is not possible
for a single method to reveal all of their radical sources
or antioxidants [41–43]. For these reasons, we used
a combination of methods, namely the DPPH, metal
chelation, and iron reduction. In addition, we used the
Folin-Ciocalteu method and the aluminum chloride
method to determine the total phenol and flavonoid
contents, respectively. The results clearly showed that
the differences in the phenolic contents affected the
plants’ antioxidant properties.
We found that having a high phenolic content or
high radical scavenging activity did not yield high
results in all antioxidant activity studies. Thus, we
concluded that determining the antioxidant activity with
a single method was not the right approach and that it
would be more accurate to simulate biochemical events
in living systems by using a variety of methods. In
summary, antioxidant structures can demonstrate their
antioxidant activities by different mechanisms such as
binding transition metal ions, breaking down peroxides,
preventing hydrogen absorption, and removing radicals.
Our study revealed that the kumquat leaf extracts
had a higher DPPH radical scavenging power than
the fruit extracts. However, both the fruit and leaf
extracts showed high levels of free radical scavenging
activity with high antioxidant activity at a 125 μg/mL
concentration. Due to high antioxidant activity,
kumquat leaves can be recommended to be used as
food, just as kumquat fruit, against many diseases –
from gastrointestinal to infertility, from cardiovascular
to respiratory and excretory disorders, especially to
prevent cell damage caused by free radicals in human
and animal bodies.
CONTRIBUTION
The authors were equally involved in writing the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors have declared no conflicts of interest for
this manuscript.
ACKNOWLEDGMENTS
The authors are thankful to Mr. M. Murat HOCAGİL
for his helpful contribution with the plant material
collection.

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