INTRODUCTIONConstantly developing technology, environmentalpollution, ultraviolet radiation, and many other factorscause us to be exposed to various toxic substances.This results in more diseases caused by externalenvironmental effects, including more pronouncedgenetic diseases. Preventing these diseases shouldbecome our priority. Since most of them occur inpeople with a weak immune system, we must focuson strengthening it. For this, we should consumefoods with high antioxidant capacity, especially fruitsand green leafy vegetables that contain antioxidativephytochemicals [1, 2].Phytochemicals, or “plant chemicals”, arecompounds of plant origin, mostly polyphenols, thatare essential for human life. They work alongsidemacronutrients such as carbohydrates, fats, and proteins,as well as 13 essential vitamins and 17 minerals [3].Antioxidant phytochemicals, especially in fruits andvegetables, combine with free radicals in the humanbody to protect cells from the attacks of harmfulradicals [4]. Bioactive compounds in fruits containascorbic acid, organic and phenolic acids, flavonoids,anthocyanins, and carotenoid substances [5, 6].Citrus fruits come in different types, varieties, andflavors and have positive effects on health and nutrition.Although they have been known as the best sources ofvitamin C for a long time, studies on their use as anantioxidant substance have recently gained momentum,due to their richness in phenolic compounds [7]. Thesebioactive components are responsible for various healthbenefits of citrus fruits, such as prevention of variousdiseases or protective effects to lower the risk of variouscancers [8–10].52Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Citrus is a fruit group belonging to the genus Citrus,which is a member of the Aurantioideae subfamily of theRutaceae family. The most common citrus varieties areorange (Citrus sinensis), mandarin (Citrus reticulata),lemon (Citrus limon), golden ball (Citrus paradisi),bitter orange (Citrus auranthium), and bergamot (Citrusbergami) [11]. In addition to fresh table consumption,citrus fruits are used as jam, marmalade or fruit juice, aswell as raw material in the cosmetics sector [11].Citrus fruits grow in subtropical climate areas. Whilemainland China, Southeast Asia, and India are majorproducers of citrus fruits due to suitable ecologicalconditions, they are also cultivated in the Mediterraneanand Aegean coastal regions and partly in the EasternBlack Sea region of Turkey [12, 13]. The distributionof species and varieties of citrus fruits has gained aregional identity. For example, Washington navel, aswell as other navel oranges, and Jaffa are harvested inthe Eastern Mediterranean region.Orange is one of the most produced and consumedcitrus fruits in Turkey due to its preference in the juiceindustry 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”, hasrecently grown in popularity, as well as such species asAltıntop and citrus, which are lower in production butcan be considered important [15].Kumquat is also called “citrus fortunella”, takingits name from the Scottish horticultural expert RobertFortune (1812–1880). This species, referred to as“komquot” in some countries, is also called a “goldenorange” [16]. It is like a tiny lemon in shape andorangish in color. However, while orange and lemon areconsumed after they are peeled, kumquat is consumedwith its peel. Its scent is reminiscent of bergamot. Ittastes sweet and leaves a lasting scent when you hold itin your hand.In addition to fresh consumption, kumquat can beused in products such as confectionery, marmalade,liquor, and wine [17, 18]. Essential oil and bioactiveingredients obtained from its peel are used in theperfumery, pharmaceutical, and food industries [19].Kumquat is an excellent source of nutrients containingminerals, ascorbic acid, carotenoids, flavonoids, andessential oils [20]. It contains remarkable antioxidantproperties due to its flavonoid content [18]. However,there are very few studies about kumquat grown inTurkey.In this study, we aimed to determine the antioxidantcapacity and the total phenolic and flavonoid contentsof the extracts obtained from fresh and lyophilizeddried fruits and leaves of kumquat and six mutantsfrom the Mersin Alata Horticultural Research InstituteDirectorate.STUDY OBJECTS AND METHODSPlant materials. Kumquat leaf and fruit sampleswere obtained from the Mersin Alata HorticulturalResearch Institute in November 2017 and January 2018,respectively. We used EP (Old Parcel) with rootstockspecies; EP.4, EP.29, EP.31 and YP (New Parcel); YP.117,YP.141, YP.188 mutants. The leaf samples were dried inroom conditions and in the shade, and stored in a dryand cool environment for analysis. The fruit sampleswere freeze-dried, or lyophilized.Chemicals and equipment. We used chemicalsand solvents of analytical purity produced by Sigma,Aldrich, and Riedel-de Haen.The equipment used in the study included alyophilizer (Christ Alpha 1-2 LC plus), a vortex (Fisons),a rotary evaporator (Laborota 4000-efficient Heidolph),a spectrophotometer (Shimadzu UV-1601), a shakingwater bath (Clifton 100–400 rpm; with thermostat), anincubator (EnoLab MB-80), an analytical balance (GecAvery), a centrifuge (Nüvefuge CN180), a pH-meter(WTW pH 330i), a heater and magnetic stirrer (ChilternHS31), a disperser and micropipettes (Eppendorf).Extraction process. Phenolic compounds wereextracted from kumquat fruits and leaves with a Soxhletextraction device, using 260 mL of 99, 80, 60, and 50%methanol and pure water as solvents. In addition, 1 and0.5% acidified ethanol and hexane solvents were used forkumquat leaves.For extraction, 20 g of the samples were weighed intothe cartridge and then placed in the Soxhlet extractor.The solvent(s) was added to the glass flask and keptin the Soxhlet device for 8 h. The solvent used forextraction was concentrated from the obtained phenolicextracts using a laboratory scale rotary evaporatorunder vacuum. The remaining part was removed bystanding in the open air. The extracts were weighedgravimetrically and stored in dark vials at +4°C in therefrigerator until analysis.Determination of free radical capture capacity(DPPH method). We used 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical to determine the free radical capturecapacity according to the Blois method [21]. Thismethod is based on the ability of the extracts to donatea proton or electron and to decolorize the purple coloredDPPH solution (from violet to yellow). A decrease in theabsorbance of the reaction mixture is indicative of highfree radical scavenging activity.All the extracts, BHA and BHT standards, andα-tocopherol were dissolved in ethanol at 1 mg/mL.After taking the samples and standards into 5 differentvolumes of 50, 100, 150, 250, and 500 μL, ethanol wasadded to a total volume of 3 mL. 1000 μL 0.1 mM DPPHwas added to the tubes and vortexed. The absorbanceof the mixture, which was incubated for 30 min inthe dark at room temperature, was measured in theUV-visible spectrophotometer at 517 nm. Calculationswere made using the following formula:% free-radical scavenging activity = 𝐴𝐴C− 𝐴𝐴S/S𝐴𝐴C× 100 (1)y = 0.0292x + 0.07490.60.81.01.21.41.6Absorbancewhere AC is the absorbance of the control reaction;AS/S is the absorbance of the sample or standard.53Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Determination of reducing capacity. TheOyaizu method was used to determine the reductioncapacity [22]. According to this method, the reducingagent in the medium reduces Fe3+ ions to Fe2+ ionsand a complex is formed by adding FeCl3. Theabsorbance of the resulting complex is measured in theUV-visible spectrophotometer at 700 nm. The increasein absorbance of the reaction mixture is directlyproportional to the reducing power of the sample.All the extracts, BHA and BHT standards, andα-tocopherol were dissolved in ethanol at 1 mg/mL.100, 250, and 500 μL of the samples and standardswere taken into test tubes in three different volumes,and 3400, 3250, and 3000 μL of pH 6.6 phosphatebuffer was added to them, respectively, to a totalvolume of 3500 μL. Then, after adding 2500 μL of 1%K3 [Fe (CN)6] and vortexing, it was left to incubate for20 min in a water bath at 50°C. After the incubation,2500 μL of 10% trichloroacetic acid (TCA) was addedto the test tubes and centrifuged at 3000 rpm for10 min. 1250 μL of the resulting supernatant was takeninto empty tubes and 1250 μL of distilled water and500 μL of 0.1% FeCl3 were added to them. The mixturewas vortexed and its absorbance was measured at700 nm in the UV-visible spectrophotometer.Determination of iron (II) ions chelating activity.Antioxidants with metal chelating properties inactivatefree iron by binding it and thus inhibit the formationof radicals such as hydroxyl and peroxide, which areformed as a result of Fenton reactions (Fe2+ + H2O2 →Fe3+ + HO• + HO–) [23]. The Dinis method wasused to determine the activity of chelating iron (II)ions [24]. All the extracts and EDTA used as controlwere dissolved in ethanol to 1 mg/mL. The samples andstandards were taken into 50, 100, 150, 250, and 500μL test tubes, and 3700, 3650, 3600, 3500, and 3250 μLof ethanol was added to them, respectively, to a totalvolume of 3750 μL. Then, 50 μL of 2mM FeCl2 wasadded and vortexed to incubate at room temperature for10 min. Then, 200 μL of 5mM ferrosine was added. Theresulting purple color was measured in the UV-visiblespectrophotometer at 562 nm after the mixture was keptat room temperature for 25 min.Determination of total phenolic content. TheFolin-Ciocalteu method was used to determine thetotal phenolic content [25]. The Folin-Ciocalteu reagent(FCR) used in this method is molybophosphotungsticheteropolyacid (3H2O·P2O5·13WO3·5MoO3·10H2O). Thismethod is based on the transfer of electrons fromphenolic compounds and other reducing compounds tomolybdenum. Phenolic compounds only react with theFCR in basic conditions (pH ~ 10) [26].Mo(VI) + e– (antioxidant) → Mo(V)Commercially available 2N Folin-Ciocalteu reagentwas prepared daily by diluting it with purified waterat a ratio of 1/1 (V/V). 500 μL of the extracts (1 mg/mL) was taken into test tubes and 500 μL of distilledwater was added. After 250 μL of 1N Folin reagent wasadded to the mixture, it was incubated for 5 min byvortexing. 1250 μL of 2% Na2CO3 solution was added toit, vortexed, and then kept at room temperature for 2 h.The absorbance of the resulting mixture was measuredat 765 nm in the UV-visible spectrophotometer. Thephenolic content of the extracts was given as mg gallicacid equivalent (GAE)/g extract.Determination of total flavonoid content. Thetotal flavonoid content was measured by an aluminumchloride colorimetric test according to Zhishenet al. [27]. All the extracts and a quercetin solutionused as a standard were dissolved in 1 mg/mL ethanol.500 μL was taken from the extracts prepared in the testtubes and pure water was added to a total volume of5000 μL. To this mixture, 300 μL of 5% NaNO2 solutionwas added and left to incubate at room temperaturefor 5 min, and then 300 μL of 10% AlCl3 solution wasadded. After waiting for 6 min, 2 mL of 1.0M NaOHsolution was added and the volume was completedto 20 mL with distilled water. The absorbance of thesolution was measured at 510 nm in the UV-visiblespectrophotometer. The total flavonoid content of theextracts was given as mg quercetin equivalent (QUE)/gextract.RESULTS AND DISCUSSIONThe solubility and distribution of phenoliccompounds in the solvent depend on the polarity oftheir structure, so the choice of solvent and method isone of the most important steps. In our study, for freshand lyophilized dried fruits, we preferred methanol andits aqueous solutions, as well as pure water. For leaves,we preferred methanol and aqueous solutions, distilledwater, and ethanol acidified with hexane.Three different methods (DPPH radical scavengingactivity, reducing capacity, and iron (II) ions chelatingactivity) were used to determine the antioxidantcapacity. We thought that the extracts could showactivity through different mechanisms depending onthe diversity of phenolic substances. In addition, wedetermined the total phenolic content and flavonoidamounts in all the extracts in order to show that theantioxidant effect was proportional to the plant content.Free radical scavenging activity. The DPPHmethod is commonly used to evaluate the antioxidantactivity of natural products, as it is easy and highlysensitive. DPPH (2,2-diphenyl-1-picrylhydrazyl) is acommercially available stable organic nitrogen radical.The antioxidant effect is proportional to the removal ofthe DPPH radical. The DPPH radical (DPPH•) is purplein color due to the unpaired nitrogen atom. Whenthe DPPH solution reacts with an oxygen atom of asubstance (antioxidant chemical) that can give hydrogenatoms, the initial purple color disappears as the radicalreduces, turning yellow [28]. The reaction takes placestoichiometrically according to the number of hydrogenatoms absorbed. Therefore, the antioxidant effect waseasily determined by following the decrease in UVabsorbance at 517 nm until it stabilized.54Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66We observed that the highest free radical scavengingactivity was in kumquat leaves, and the activity ofkumquat fruit decreased when dried (Table 1). Therewas no significant difference between the rootstockkumquat type and its mutants. The free radicalscavenging activities of the extracts were slightlybelow the standards (BHA, BHT, and α-Tocopherol).The highest activity (81.66%) was seen in the YP.188hybrid leaf extract using 80% methanol solvent. As forthe fruits, the highest activity (61.37%) was in the EP.4hybrid extract using a pure methanol solvent.When we examined all the samples, we associatedhigh phenolic content with high antioxidant activity. Wefound that the total phenolic content was higher in thesamples with high antioxidant activity. As a matter offact, the leaf extract with high antioxidant activity alsohad a high phenolic content (85.651 ± 0.030 mg GAE/gextract).However, when we carefully examined theresults, we saw that having a high amount of phenolicsubstances did not give high results in all antioxidantactivity methods. For example, although the YP.188Leaf 80% methanol and the YP.188 Leaf 50% methanolextracts contained almost the same amount of phenolicsubstances, the former had higher activity in the appliedantioxidant activity methods. This could be explainedby the differences between the phenolic substances theycontained depending on the solvent used.In fact, other studies have found that the antioxidantactivity of methanol and ethanol extracts, whichgenerally contained phenolic substances, was higherthan in other solvent systems [29]. For example,Jayaprakasha et al. extracted powdered kumquat fruit in5 different solvents and investigated the radical capturecapacities of the extracts, their amounts in total phenolicmatter, and their inhibitory properties for prostatecancer [30].In this study, the extracts obtained from EtOAcand MeOH-water (4:1, v/v) solvents were found to havethe highest and lowest total phenolics, respectively,according to the Folin-Ciocalteu method. It was alsoobserved that the EtOAc and MeOH extracts exhibitedthe 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 ± 0.3Rootstock fresh fruit 80% methanol 6.50 ± 0.10 7.94 ± 0.1 9.03 ± 0.2 12.64 ± 0.3 19.49 ± 0.1Rootstock fresh fruit 60% methanol 4.69 ± 0.10 7.58 ± 0.1 8.66 ± 0.2 13.00 ± 0.3 21.30 ± 0.3Rootstock fresh fruit 50% methanol 7.03 ± 0.10 9.03 ± 0.0 18.66 ± 0.1 22.02 ± 0.3 28.52 ± 0.1Rootstock fresh fruit pure water 10.83 ± 0.0 14.08 ± 0.2 14.08 ± 0.2 22.02 ± 0.0 33.57 ± 0.3Rootstock dry fruit pure methanol 3.09 ± 0.10 4.75 ± 0.2 7.56 ± 0.3 8.25 ± 0.3 9.97 ± 0.1Rootstock dry fruit 80% methanol 5.15 ± 0.20 6.53 ± 0.0 8.25 ± 0.2 9.28 ± 0.3 12.37 ± 0.3Rootstock dry fruit 60% methanol 4.81 ± 0.00 7.56 ± 0.1 8.25 ± 0.0 8.93 ± 0.0 9.62 ± 0.2Rootstock dry fruit 50% methanol 3.78 ± 0.00 6.53 ± 0.1 7.22 ± 0.0 8.25 ± 0.0 10.31 ± 0.2Rootstock dry fruit pure water 3.78 ± 0.20 4.47 ± 0.1 6.87 ± 0.0 7.22 ± 0.1 8.59 ± 0.2Rootstock leaf pure methanol 12.46 ± 0.20 23.88 ± 0.3 32.87 ± 0.1 41.87 ± 0.1 57.09 ± 0.1Rootstock leaf 80% methanol 21.45 ± 0.10 29.76 ± 0.2 37.72 ± 0.2 50.52 ± 0.1 65.74 ± 0.5Rootstock leaf 60% methanol 13.49 ± 0.20 18.15 ± 0.1 33.91 ± 0.2 46.71 ± 0.0 65.40 ± 0.3Rootstock leaf 50% methanol 20.76 ± 0.30 31.49 ± 0.0 39.10 ± 0.1 50.87 ± 0.2 66.44 ± 0.3Rootstock leaf pure water 12.11 ± 0.10 21.11 ± 0.1 30.10 ± 0.3 36.33 ± 0.2 52.25 ± 0.2Rootstock leaf 0.5% acidified ethanol 3.46 ± 0.10 8.30 ± 0.2 13.84 ± 0.3 20.42 ± 0.1 34.26 ± 0.4Rootstock leaf 1% acidified ethanol 5.19 ± 0.10 13.15 ± 0.2 15.22 ± 0.1 25.61 ± 0.2 40.83 ± 0.1Rootstock leaf hexane n.d. n.d. 2.42 ± 0.2 11.07 ± 0.1 12.04 ± 0.1EP.4 fresh fruit pure methanol 16.97 ± 0.1 20.22 ± 0.3 35.02 ± 0.2 42.60 ± 0.1 61.37 ± 0.3EP.4 fresh fruit 80% methanol 15.88 ± 0.1 16.61 ± 0.2 21.66 ± 0.2 28.52 ± 0.3 42.96 ± 0.5EP.4 fresh fruit 60% methanol 13.36 ± 0.1 15.88 ± 0.1 15.88 ± 0.2 22.74 ± 0.3 31.05 ± 0.1EP.4 fresh fruit 50% methanol 15.88 ± 0.2 18.05 ± 0.1 19.86 ± 0.1 23.10 ± 0.3 33.57 ± 0.2EP.4 fresh fruit pure water 15.88 ± 0.2 22.38 ± 0.3 28.05 ± 0.1 34.55 ± 0.3 35.38 ± 0.2EP.4 dry fruit pure methanol 2.06 ± 0.1 2.75 ± 0.3 10.31 ± 0.1 11.37 ± 0.1 14.43 ± 0.1EP.4 dry fruit 80% methanol 6.25 ± 0.2 7.56 ± 0.0 8.25 ± 0.2 10.31 ± 0.1 14.43 ± 0.2EP.4 dry fruit 60% methanol 6.53 ± 0.1 8.25 ± 0.1 9.97 ± 0.3 10.97 ± 0.4 13.06 ± 0.1EP.4 dry fruit 50% methanol 7.56 ± 0.1 8.93 ± 0.1 9.08 ± 0.2 9.97 ± 0.4 13.06 ± 0.3EP.4 dry fruit pure water 5.15 ± 0.2 8.93 ± 0.2 10.65 ± 0.3 11.68 ± 0.0 15.12 ± 0.3EP.4 leaf pure methanol 14.88 ± 0.1 23.53 ± 0.0 28.03 ± 0.1 36.33 ± 0.2 54.67 ± 0.7EP.4 leaf 80% methanol 17.65 ± 0.1 32.53 ± 0.3 39.45 ± 0.2 47.06 ± 0.1 71.63 ± 0.5EP.4 leaf 60% methanol 16.65 ± 0.1 25.61 ± 0.3 34.95 ± 0.2 44.64 ± 0.1 63.67 ± 0.355Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Extracts 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 ± 0.1EP.4 leaf pure water 18.34 ± 0.2 27.68 ± 0.2 34.26 ± 0.2 47.40 ± 0.3 55.36 ± 0.3EP.4 leaf 0.5% acidified ethanol 1.73 ± 0.0 7.61 ± 0.1 9.69 ± 0.3 17.99 29.41 ± 0.5EP.4 leaf 1% acidified ethanol 6.92 ± 0.1 12.80 ± 0.2 18.34 ± 0.3 24.57 ± 0.2 35.64 ± 0.3EP.4 leaf hexane n.d. n.d. n.d. n.d. 7.96 ± 0.5EP.29 fresh fruit pure methanol 9.42 ± 0.2 15.16 ± 0.2 22.38 ± 0.1 28.88 ± 0.1 37.91 ± 0.1EP.29 fresh fruit 80% methanol 9.75 ± 0.0 14.08 ± 0.2 17.69 ± 0.1 22.02 ± 0.3 31.77 ± 0.5EP.29 fresh fruit 60% methanol 12.64 ± 0.1 17.33 ± 0.2 21.30 ± 0.3 25.63 ± 0.3 36.10 ± 0.2EP.29 fresh fruit 50% methanol 13.72 ± 0.1 17.69 ± 0.1 19.49 ± 0.0 26.71 ± 0.2 36.10 ± 0.4EP.29 fresh fruit pure water 14.44 ± 0.1 15.16 ± 0.1 17.69 ± 0.1 20.94 ± 0.3 33.21 ± 0.4EP.29 dry fruit pure methanol 7.90 ± 0.1 9.28 ± 0.1 10.31 ± 0.2 13.06 ± 0.1 15.81 ± 0.5EP.29 dry fruit 80% methanol 7.56 ± 0.1 11.68 ± 0.1 14.09 ± 0.2 15.43 ± 0.1 19.59 ± 0.2EP.29 dry fruit 60% methanol 7.90 ± 0.1 10.31 ± 0.1 12.65 ± 0.1 14.43 ± 0.2 19.93 ± 0.4EP.29 dry fruit 50% methanol 6.80 ± 0.1 9.28 ± 0.1 10.31 ± 0.2 11.68 ± 0.3 14.09 ± 0.5EP.29 dry fruit pure water 4.81 ± 0.1 5.84 ± 0.1 7.56 ± 0.1 9.28 ± 0.3 12.03 ± 0.1EP.29 leaf pure methanol 15.57 ± 0.2 26.99 ± 0.1 29.76 ± 0.1 36.33 ± 0.0 52.25 ± 0.3EP.29 leaf 80% methanol 9.00 ± 0.1 22.15 ± 0.1 31.49 ± 0.1 41.87 ± 0.4 59.86 ± 0.7EP.29 leaf 60% methanol 12.46 ± 0.2 26.99 ± 0.2 32.53 ± 0.3 46.71 ± 0.1 63.32 ± 0.4EP.29 leaf 50% methanol 16.96 ± 0.2 28.37 ± 0.1 33.22 ± 0.2 45.67 ± 0.2 60.55 ± 0.4EP.29 leaf pure water 10.73 ± 0.1 20.7 ± 0.1 26.99 ± 0.1 35.99 ± 0.2 51.21 ± 0.2EP.29 leaf 0.5% acidified ethanol 3.11 ± 0.1 8.65 ± 0.2 13.84 ± 0.1 20.42 ± 0.2 34.26 ± 0.5EP.29 leaf 1% acidified ethanol 6.57 ± 0.2 11.07 ± 0.2 15.57 ± 0.1 24.91 ± 0.3 39.79 ± 0.2EP.29 leaf hexane n.d. n.d. n.d. n.d. 5.54 ± 0.1EP.31 fresh fruit pure methanol 2.89 ± 0.1 17.69 ± 0.0 22.74 ± 0.3 29.24 ± 0.2 40.7 ± 0.4EP.31 fresh fruit 80% methanol 12.27 ± 0.2 16.97 ± 0.1 23.10 ± 0.1 33.94 ± 0.2 51.62 ± 0.5EP.31 fresh fruit 60% methanol 11.91 ± 0.1 22.02 ± 0.1 25.63 ± 0.2 40.43 ± 0.3 54.51 ± 0.1EP.31 fresh fruit 50% methanol 14.80 ± 0.1 15.52 ± 0.3 21.66 ± 0.1 28.16 ± 0.5 42.96 ± 0.1EP.31 fresh fruit pure water 8.30 ± 0.2 13.00 ± 0.3 13.72 ± 0.0 18.41 ± 0.1 23.83 ± 0.1EP.31 dry fruit pure methanol 7.38 ± 0.2 8.72 ± 0.1 30.20 ± 0.2 39.73 ± 0.1 43.42 ± 0.1EP.31 dry fruit 80% methanol 7.05 ± 0.2 8.39 ± 0.2 8.39 ± 0.1 10.74 ± 0.3 14.43 ± 0.2EP.31 dry fruit 60% methanol 3.69 ± 0.1 6.38 ± 0.1 8.72 ± 0.2 9.73 ± 0.1 11.07 ± 0.2EP.31 dry fruit 50% methanol 3.36 ± 0.0 6.04 ± 0.2 6.38 ± 0.1 8.72 ± 0.0 11.41 ± 0.1EP.31 dry fruit pure water 6.04 ± 0.1 8.39 ± 0.1 3.36 ± 0.1 3.02 ± 0.3 4.36 ± 0.1EP.31 leaf pure methanol 13.49 ± 0.1 22.15 ± 0.2 28.37 ± 0.1 37.37 ± 0.2 53.98 ± 0.3EP.31 leaf 80% methanol 20.42 ± 0.1 31.14 ± 0.2 39.45 ± 0.2 50.52 ± 0.3 68.17 ± 0.4EP.31 leaf 60% methanol 17.99 ± 0.1 30.80 ± 0.1 39.10 ± 0.1 49.83 ± 0.5 65.05 ± 0.4EP.31 leaf 50% methanol 19.72 ± 0.1 30.45 ± 0.1 33.22 ± 0.2 49.13 ± 0.1 63.67 ± 0.1EP.31 leaf pure water 12.11 ± 0.1 21.11 ± 0.1 24.91 ± 0.2 36.33 ± 0.3 53.98 ± 0.5EP.31 leaf 0.5% acidified ethanol 8.30 ± 0.1 17.30 ± 0.2 24.57 ± 0.2 33.56 ± 0.3 51.56 ± 0.5EP.31 leaf 1% acidified ethanol 10.3 ± 0.2 16.96 ± 0.1 21.45 ± 0.0 32.18 ± 0.3 47.06 ± 0.3EP.31 leaf hexane n.d. n.d. n.d. n.d. 8.65 ± 0.3YP.117 fresh fruit pure methanol 10.83 ± 0.2 18.41 ± 0.2 21.66 ± 0.4 33.94 ± 0.1 46.93 ± 0.4YP.117 fresh fruit 80% methanol 10.11 ± 0.2 15.75 ± 0.1 20.58 ± 0.2 27.08 ± 0.5 41.88 ± 0.4YP.117 fresh fruit 60% methanol 12.27 ± 0.1 15.88 ± 0.1 18.41 ± 0.3 27.08 ± 0.1 40.7 ± 0.3YP.117 fresh fruit 50% methanol 12.64 ± 0.1 16.61 ± 0.2 22.38 ± 0.1 30.69 ± 0.1 48.38 ± 0.4YP.117 fresh fruit pure water 15.88 ± 0.1 13.36 ± 0.3 20.94 ± 0.2 28.16 ± 0.1 41.52 ± 0.4YP.117 dry fruit pure methanol 2.68 ± 0.1 3.45 ± 0.1 4.68 ± 0.1 6.71 ± 0.0 10.40 ± 0.3YP.117 dry fruit 80% methanol 3.69 ± 0.1 6.38 ± 0.2 7.05 ± 0.1 8.05 ± 0.1 8.39 ± 0.2YP.117 dry fruit 60% methanol 5.03 ± 0.1 7.72 ± 0.1 8.71 ± 0.3 8.92 ± 0.1 11.74 ± 0.1YP.117 dry fruit 50% methanol 4.70 ± 0.1 5.09 ± 0.2 6.38 ± 0.3 6.38 ± 0.1 6.38 ± 0.3YP.117 dry fruit pure water 5.03 ± 0.3 5.18 ± 0.3 6.04 ± 0.3 7.05 ± 0.2 11.41 ± 0.2YP.117 leaf pure methanol 13.84 ± 0.2 22.84 ± 0.1 30.45 ± 0.1 42.91 ± 0.5 60.55 ± 0.5YP.117 leaf 80% methanol 20.70 ± 0.3 26.99 ± 0.2 33.56 ± 0.1 47.40 ± 0.2 67.47 ± 0.7YP.117 leaf 60% methanol 14.53 ± 0.2 21.80 ± 0.3 39.45 ± 0.2 50.87 ± 0.2 65.40 ± 0.1YP.117 leaf 50% methanol 19.03 ± 0.3 33.56 ± 0.2 39.10 ± 0.2 52.25 ± 0.1 65.74 ± 0.1YP.117 leaf pure water 14.53 ± 0.4 20.76 ± 0.2 32.18 ± 0.1 40.83 ± 0.3 57.44 ± 0.4YP.117 leaf 0.5% acidified ethanol 7.22 ± 0.1 13.15 ± 0.2 19.72 ± 0.1 28.72 ± 0.1 45.67 ± 0.3Continuation of the Table 156Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Extracts 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 ± 0.3YP.117 leaf hexane n.d. n.d. n.d. 2.69 ± 0.1 14.19 ± 0.2YP.141 fresh fruit pure methanol 9.03 ± 0.1 11.91 ± 0.1 14.80 ± 0.2 23.47 ± 0.2 35.38 ± 0.2YP.141 fresh fruit 80% methanol 9.39 ± 0.1 10.83 ± 0.2 16.61 ± 0.3 25.27 ± 0.1 35.74 ± 0.3YP.141 fresh fruit 60% methanol 11.91 ± 0.1 16.08 ± 0.2 19.86 ± 0.2 26.35 ± 0.2 37.91 ± 0.5YP.141 fresh fruit 50% methanol 5.05 ± 0.1 8.66 ± 0.1 14.08 ± 0.2 24.55 ± 0.3 41.88 ± 0.2YP.141 fresh fruit pure water 9.39 ± 0.1 10.11 ± 0.1 15.16 ± 0.2 21.30 ± 0.0 31.05 ± 0.2YP.141 dry fruit pure methanol 5.03 ± 0.1 5.70 ± 0.1 7.72 ± 0.1 10.40 ± 0.2 12.42 ± 0.2YP.141 dry fruit 80% methanol 7.72 ± 0.1 9.40 ± 0.0 14.43 ± 0.2 10.40 ± 0.0 11.74 ± 0.4YP.141 dry fruit 60% methanol 8.05 ± 0.1 8.72 ± 0.1 9.73 ± 0.2 30.87 ± 0.3 32.35 ± 0.1YP.141 dry fruit 50% methanol 1.01 ± 0.1 6.71 ± 0.1 7.38 ± 0.3 10.40 ± 0.2 12.42 ± 0.5YP.141 dry fruit pure water 7.05 ± 0.2 16.78 ± 0.1 15.7 ± 0.2 15.37 ± 0.1 16.7 ± 0.2YP.141 leaf pure methanol 18.34 ± 0.2 28.03 ± 0.2 33.56 ± 0.1 48.79 ± 0.3 64.71 ± 0.1YP.141 leaf 80% methanol 17.65 ± 0.0 33.56 ± 0.1 43.94 ± 0.2 57.09 ± 0.3 72.66 ± 0.4YP.141 leaf 60% methanol 18.69 ± 0.2 32.53 ± 0.2 39.79 ± 0.1 53.63 ± 0.6 67.82 ± 0.4YP.141 leaf 50% methanol 17.65 ± 0.3 31.49 ± 0.2 39.79 ± 0.1 51.90 ± 0.3 63.32 ± 0.5YP.141 leaf pure water 16.61 ± 0.4 28.03 ± 0.2 32.87 ± 0.2 45.67 ± 0.7 61.59 ± 0.3YP.141 leaf 0.5% acidified ethanol 7.61 ± 0.1 15.57 ± 0.1 21.45 ± 0.3 32.87 ± 0.2 50.52 ± 0.4YP.141 leaf 1% acidified ethanol 8.30 ± 0.1 14.88 ± 0.1 19.03 ± 0.3 32.18 ± 0.2 48.79 ± 0.4YP.141 leaf hexane n.d. n.d. 1.38 ± 0.1 5.88 ± 0.1 15.92 ± 0.1YP.188 fresh fruit pure methanol 5.42 ± 0.2 10.83 ± 0.1 13.72 ± 0.2 21.66 ± 0.1 36.10 ± 0.6YP.188 fresh fruit 80% methanol 5.39 ± 0.2 9.42 ± 0.1 11.91 ± 0.3 22.74 ± 0.1 33.57 ± 0.2YP.188 fresh fruit 60% methanol 9.39 ± 0.2 12.27 ± 0.2 14.08 ± 0.2 23.10 ± 0.1 33.94 ± 0.2YP.188 fresh fruit 50% methanol 11.05 ± 0.2 11.19 ± 0.2 15.88 ± 0.5 22.74 ± 0.3 32.85 ± 0.4YP.188 fresh fruit pure water 13.00 ± 0.2 13.72 ± 0.1 22.38 ± 0.3 33.57 ± 0.3 46.93 ± 0.3YP.188 dry fruit pure methanol 3.09 ± 0.2 4.75 ± 0.2 7.56 ± 0.2 8.25 ± 0.1 9.97 ± 0.1YP.188 dry fruit 80% methanol 5.15 ± 0.0 6.53 ± 0.2 8.25 ± 0.2 9.28 ± 0.1 12.37 ± 0.1YP.188 dry fruit 60% methanol 4.81 ± 0.2 7.56 ± 0.2 8.25 ± 0.1 8.93 ± 0.1 9.62 ± 0.2YP.188 dry fruit 50% methanol 3.78 ± 0.2 6.53 ± 0.1 7.22 ± 0.3 8.25 ± 0.2 10.31 ± 0.5YP.188 dry fruit pure water 3.78 ± 0.2 4.47 ± 0.2 6.87 ± 0.3 7.22 ± 0.1 8.59 ± 0.1YP.188 leaf pure methanol 21.11 ± 0.1 31.14 ± 0.4 35.99 ± 0.2 53.98 ± 0.2 73.70 ± 0.1YP.188 leaf 80% methanol 25.26 ± 0.2 41.18 ± 0.1 47.06 ± 0.3 66.09 ± 0.3 81.66 ± 0.4YP.188 leaf 60% methanol 29.07 ± 0.0 46.02 ± 0.5 52.25 ± 0.2 66.78 ± 0.4 80.97 ± 0.4YP.188 leaf 50% methanol 20.42 ± 0.2 33.56 ± 0.1 45.67 ± 0.1 57.09 ± 0.0 73.70 ± 0.1YP.188 leaf pure water 13.15 ± 0.2 18.69 ± 0.2 21.45 ± 0.1 48.79 ± 0.4 67.82 ± 0.7YP.188 leaf 0.5% acidified ethanol 7.27 ± 0.1 15.22 ± 0.3 22.49 ± 0.2 37.02 ± 0.2 57.44 ± 0.2YP.188 leaf 1% acidified ethanol 10.38 ± 0.1 16.96 ± 0.0 21.11 ± 0.2 32.18 ± 0.3 50.17 ± 0.2YP.188 leaf hexane n.d. n.d. n.d. 4.50 ± 0.2 17.30 ± 0.2BHA 73.36 ± 0.2 79.58 ± 0.2 80.62 ± 0.1 83.39 ± 0.3 84.43 ± 0.2BHT 65.74 ± 0.0 72.32 ± 0.1 73.01 ± 0.2 73.36 ± 0.1 72.32 ± 0.0α-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 preparedas 1 mg/mL and completing the total volume of 3 mLn.d.: not detectedThe chelating activity of iron (II) ions.Antioxidants with metal chelating properties inactivateit by binding free iron and thus inhibit the formationof radicals such as hydroxyl and peroxide, which areformed as a result of Fenton reactions. Therefore,metal chelating plays an important role in determiningantioxidant activity [31].We evaluated the metal ion chelating activityaccording to the competition between plant extracts withferrosine in order to bind Fe2+ ions in the solution. Weobserved no chelating activity in the extracts obtainedfrom moist and lyophilized dried fruits (Table 2).The pure methanol extracts showed weak activityin kumquat leaves, while the extracts obtained fromaqueous solvents showed no activity at all.In addition, weak chelating activity was detectedin the 0.5 and 1% acidified ethanol extracts ofkumquat leaves and the hexane solvent extracts. Thehighest activity (50.37%) was found in 62.5 μg/mLconcentration of the extract obtained from kumquatContinuation of the Table 157Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66leaves with a hexane solvent. We determined nocorrelation between the chelating activity of the extractsand their concentration. No significant difference wasfound between the rootstock kumquat type and itshybrids.When we evaluated all the activities, we concludedthat the extracts obtained from kumquat fruits andleaves were not good at chelating iron (II) ions. The mostimportant feature that affects the metal chelating activitydepends on the functional groups in the structure ofphenolic compounds and the position and amount ofthese functional groups. For this reason, the differencein the chelating activity of the samples can be explainedby different amounts of phenolic substances, as well asphenolic substance groups in different structures andpositions [32].The reducing capacity of the extracts. Thereducing agent in the environment reduces Fe3+ ionsTable 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 ± 0.1Rootstock leaf 0.5% acidified ethanol 4.39 ± 0.10 5.12 ± 0.0 4.39 ± 0.1 5.95 ± 0.1 6.73 ± 0.1Rootstock leaf 1% acidified ethanol 3.51 ± 0.10 10.10 ± 0.1 11.86 ± 0.2 13.47 ± 0.1 18.59 ± 0.3Rootstock leaf hexane 2.99 ± 0.0 8.52 ± 0.1 15.10 ± 0.2 18.30 ± 0.1 17.19 ± 0.4EP.4 leaf pure methanol 10.56 ± 0.10 21.26 ± 0.1 23.32 ± 0.2 28.94 ± 0.1 16.74 ± 0.2EP.4 leaf 0.5% acidified ethanol 3.51 ± 0.1 10.10 ± 0.2 11.86 ± 0.1 13.47 ± 0.1 18.59 ± 0.3EP.4 leaf 1% acidified ethanol 4.10 ± 0.1 3.07 ± 0.2 5.42 ± 0.1 6.59 ± 0.3 6.83 ± 0.2EP.4 leaf hexane 9.87 ± 0.2 14.20 ± 0.1 24.22 ± 0.2 34.08 ± 0.3 25.41 ± 0.3EP.29 leaf pure methanol 13.03 ± 0.1 25.24 ± 0.1 32.24 ± 0.2 36.90 ± 0.3 19.48 ± 0.1EP.29 leaf 0.5% acidified ethanol 4.93 ± 0.0 16.29 ± 0.1 23.47 ± 0.1 37.07 ± 0.1 24.96 ± 0.3EP.29 leaf 1% acidified ethanol 5.38 ± 0.0 10.91 ± 0.2 17.32 ± 0.1 30.19 ± 0.2 31.24 ± 0.1EP.29 leaf hexane 1.35 ± 0.1 2.54 ± 0.1 6.13 ± 0.1 12.26 ± 0.2 8.97 ± 0.2EP.31 leaf pure methanol 27.36 ± 0.2 43.84 ± 0.1 44.64 ± 0.1 42.06 ± 0.3 31.20 ± 0.4EP.31 leaf 0.5% acidified ethanol 2.54 ± 0.2 5.38 ± 0.2 10.46 ± 0.1 15.40 ± 0.3 15.99 ± 0.1EP.31 leaf 1% acidified ethanol 2.69 ± 0.0 6.43 ± 0.1 9.72 ± 0.2 10.27 ± 0.1 7.92 ± 0.1EP.31 leaf hexane 8.37 ± 0.2 9.57 ± 0.1 18.22 ± 0.1 20.33 ± 0.2 20.78 ± 0.3YP.117 leaf pure methanol 11.17 ± 0.2 16.48 ± 0.3 22.35 ± 0.3 24.21 ± 0.2 24.58 ± 0.1YP.117 leaf 0.5% acidified ethanol 3.44 ± 0.1 9.87 ± 0.0 11.36 ± 0.2 24.66 ± 0.0 19.28 ± 0.3YP.117 leaf 1% acidified ethanol 5.23 ± 0.2 5.48 ± 0.1 14.20 ± 0.2 14.35 ± 0.2 14.05 ± 0.2YP.117 leaf hexane 8.67 ± 0.1 20.63 ± 0.4 30.64 ± 0.3 36.32 ± 0.2 38.57 ± 0.3YP.141 leaf pure methanol 14.79 ± 0.2 30.1 ± 0.2 35.43 ± 0.2 38.53 ± 0.3 35.56 ± 0.1YP.141 leaf 0.5% acidified ethanol 2.09 ± 0.1 2.64 ± 0.1 6.43 ± 0.2 8.37 ± 0.2 8.74 ± 0.1YP.141 leaf 1% acidified ethanol 1.20 ± 0.1 5.53 ± 0.1 10.31 ± 0.1 11.96 ± 0.0 14.80 ± 0.2YP.141 leaf hexane 6.13 ± 0.1 11.36 ± 0.2 13.49 ± 0.1 17.04 ± 0.3 19.73 ± 0.1YP.188 leaf pure methanol 16.84 ± 0.1 27.38 ± 0.2 31.63 ± 0.3 37.67 ± 0.3 44.39 ± 0.2YP.188 leaf 0.5% acidified ethanol 2.54 ± 0.0 6.28 ± 0.1 8.07 ± 0.2 11.96 ± 0.0 17.32 ± 0.3YP.188 leaf 1% acidified ethanol 2.69 ± 0.1 6.88 ± 0.1 10.91 ± 0.1 16.89 ± 0.1 16.35 ± 0.3YP.188 leaf hexane 13.15 ± 0.1 28.10 ± 0.2 42.75 ± 0.2 50.37 ± 0.1 42.75 ± 0.3EDTA 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 preparedas 1 mg/mL and completing the total volume of 3 mLto Fe2+ ions depending on its antioxidant capacity. Theabsorbance of the Prussian blue complex (Fe4[Fe(CN)6])formed by adding FeCl3 to the reduced product ismeasured at 700 nm [22]. The increase in absorbanceof the reaction mixture is directly proportional to thereducing power of the sample.We found that the capacity of kumquat leaves toreduce Fe3+ ions was higher than that of lyophilizedand wet kumquat fruits (Table 3). We observed thatlyophilizing and drying of kumquat fruits did not causea significant change in their reducing capacity. Thereducing capacity of the fruit and leaf extracts was lowerthan the standards (BHA, BHT and α-tocopherol).The highest reducing capacity (0.307 ± 0.001) wasobserved at a concentration of 29.41 μg/mL of the EP.4mutant leaf extract obtained with pure water. Amongthe fruits, the highest reducing capacity (0.199 ±0.001) was found at a concentration of 29.41 μg/mL of58Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Table 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 ± 0.002Rootstock fresh fruit 80% methanol 0.105 ± 0.002 0.106 ± 0.001 0.124 ± 0.001Rootstock fresh fruit 60% methanol 0.120 ± 0.001 0.133 ± 0.001 0.140 ± 0.003Rootstock fresh fruit 50% methanol 0.096 ± 0.001 0.100 ± 0.002 0.104 ± 0.001Rootstock fresh fruit pure water 0.082 ± 0.002 0.098 ± 0.003 0.115 ± 0.001Rootstock dry fruit pure methanol 0.075 ± 0.001 0.082 ± 0.003 0.094 ± 0.001Rootstock dry fruit 80% methanol 0.074 ± 0.002 0.087 ± 0.006 0.097 ± 0.005Rootstock dry fruit 60% methanol 0.076 ± 0.001 0.081 ± 0.001 0.089 ± 0.001Rootstock dry fruit 50% methanol 0.076 ± 0.002 0.082 ± 0.001 0.089 ± 0.003Rootstock dry fruit pure water 0.078 ± 0.003 0.081 ± 0.001 0.089 ± 0.001Rootstock leaf pure methanol 0.103 ± 0.002 0.145 ± 0.001 0.241 ± 0.004Rootstock leaf 80% methanol 0.098 ± 0.001 0.149 ± 0.001 0.227 ± 0.003Rootstock leaf 60% methanol 0.093 ± 0.001 0.136 ± 0.005 0.218 ± 0.003Rootstock leaf 50% methanol 0.097 ± 0.002 0.148 ± 0.001 0.240 ± 0.003Rootstock leaf pure water 0.089 ± 0.001 0.143 ± 0.003 0.209 ± 0.005Rootstock leaf 0.5% acidified ethanol 0.074 ± 0.001 0.096 ± 0.002 0.128 ± 0.001Rootstock leaf 1% acidified ethanol 0.076 ± 0.001 0.098 ± 0.003 0.129 ± 0.001Rootstock leaf hexane 0.091 ± 0.002 0.125 ± 0.003 0.179 ± 0.002EP.4 fresh fruit pure methanol 0.111 ± 0.002 0.144 ± 0.001 0.199 ± 0.001EP.4 fresh fruit 80% methanol 0.108 ± 0.001 0.110 ± 0.003 0.100 ± 0.001EP.4 fresh fruit 60% methanol 0.104 ± 0.002 0.095 ± 0.003 0.112 ± 0.001EP.4 fresh fruit 50% methanol 0.099 ± 0.003 0.092 ± 0.001 0.143 ± 0.001EP.4 fresh fruit pure water 0.086 ± 0.001 0.093 ± 0.001 0.115 ± 0.002EP.4 dry fruit pure methanol 0.070 ± 0.001 0.077 ± 0.001 0.091 ± 0.001EP.4 dry fruit 80% methanol 0.071 ± 0.002 0.078 ± 0.001 0.089 ± 0.003EP.4 dry fruit 60% methanol 0.074 ± 0.001 0.076 ± 0.001 0.087 ± 0.003EP.4 dry fruit 50% methanol 0.071 ± 0.001 0.075 ± 0.003 0.085 ± 0.001EP.4 dry fruit pure water 0.070 ± 0.002 0.072 ± 0.001 0.081 ± 0.001EP.4 leaf pure methanol 0.087 ± 0.002 0.134 ± 0.004 0.201 ± 0.001EP.4 leaf 80% methanol 0.097 ± 0.001 0.145 ± 0.003 0.245 ± 0.004EP.4 leaf 60% methanol 0.093 ± 0.003 0.139 ± 0.001 0.211 ± 0.003EP.4 leaf 50% methanol 0.091 ± 0.002 0.149 ± 0.003 0.227 ± 0.005EP.4 leaf pure water 0.116 ± 0.001 0.193 ± 0.003 0.307 ± 0.001EP.4 leaf 0.5% acidified ethanol 0.075 ± 0.002 0.093 ± 0.001 0.125 ± 0.001EP.4 leaf 1% acidified ethanol 0.079 ± 0.001 0.102 ± 0.002 0.133 ± 0.006EP.4 leaf hexane 0.091 ± 0.003 0.125 ± 0.001 0.179 ± 0.001EP.29 fresh fruit pure methanol 0.107 ± 0.004 0.118 ± 0.004 0.135 ± 0.006EP.29 fresh fruit 80% methanol 0.107 ± 0.001 0.114 ± 0.002 0.108 ± 0.002EP.29 fresh fruit 60% methanol 0.109 ± 0.000 0.109 ± 0.000 0.138 ± 0.000EP.29 fresh fruit 50% methanol 0.113 ± 0.000 0.117 ± 0.001 0.133 ± 0.000EP.29 fresh fruit pure water 0.086 ± 0.001 0.092 ± 0.000 0.100 ± 0.001EP.29 dry fruit pure methanol 0.072 ± 0.000 0.081 ± 0.001 0.098 ± 0.000EP.29 dry fruit 80% methanol 0.073 ± 0.000 0.080 ± 0.001 0.093 ± 0.000EP.29 dry fruit 60% methanol 0.072 ± 0.001 0.077 ± 0.001 0.090 ± 0.001EP.29 dry fruit 50% methanol 0.071 ± 0.001 0.078 ± 0.000 0.088 ± 0.000EP.29 dry fruit pure water 0.073 ± 0.000 0.076 ± 0.001 0.090 ± 0.000EP.29 leaf pure methanol 0.090 ± 0.000 0.125 ± 0.001 0.206 ± 0.002EP.29 leaf 80% methanol 0.093 ± 0.000 0.145 ± 0.001 0.236 ± 0.000EP.29 leaf 60% methanol 0.106 ± 0.001 0.158 ± 0.000 0.260 ± 0.000EP.29 leaf 50% methanol 0.103 ± 0.000 0.163 ± 0.000 0.281 ± 0.000EP.29 leaf pure water 0.101 ± 0.000 0.158 ± 0.001 0.244 ± 0.000EP.29 leaf 0.5% acidified ethanol 0.086 ± 0.000 0.103 ± 0.001 0.135 ± 0.000EP.29 leaf 1% acidified ethanol 0.077 ± 0.001 0.094 ± 0.001 0.119 ± 0.001EP.29 leaf hexane 0.088 ± 0.000 0.136 ± 0.000 0.193 ± 0.000EP.31 fresh fruit pure methanol 0.091 ± 0.001 0.098 ± 0.001 0.109 ± 0.001EP.31 fresh fruit 80% methanol 0.087 ± 0.000 0.095 ± 0.000 0.117 ± 0.000EP.31 fresh fruit 60% methanol 0.081 ± 0.000 0.103 ± 0.001 0.129 ± 0.00159Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Extracts and Standards 5.88* 14.7* 29.41*EP.31 fresh fruit 50% methanol 0.089 ± 0.000 0.115 ± 0.001 0.104 ± 0.000EP.31 fresh fruit pure water 0.088 ± 0.001 0.094 ± 0.000 0.105 ± 0.001EP.31 dry fruit pure methanol 0.093 ± 0.000 0.099 ± 0.000 0.125 ± 0.000EP.31 dry fruit 80% methanol 0.095 ± 0.000 0.102 ± 0.000 0.099 ± 0.000EP.31 dry fruit 60% methanol 0.099 ± 0.000 0.085 ± 0.001 0.096 ± 0.001EP.31 dry fruit 50% methanol 0.099 ± 0.000 0.092 ± 0.001 0.097 ± 0.000EP.31 dry fruit pure water 0.107 ± 0.000 0.100 ± 0.001 0.111 ± 0.001EP.31 leaf pure methanol 0.089 ± 0.001 0.119 ± 0.001 0.176 ± 0.001EP.31 leaf 80% methanol 0.093 ± 0.000 0.133 ± 0.001 0.200 ± 0.000EP.31 leaf 60% methanol 0.101 ± 0.001 0.148 ± 0.001 0.214 ± 0.000EP.31 leaf 50% methanol 0.100 ± 0.001 0.142 ± 0.000 0.212 ± 0.001EP.31 leaf pure water 0.094 ± 0.001 0.133 ± 0.000 0.206 ± 0.001EP.31 leaf 0.5% acidified ethanol 0.089 ± 0.000 0.127 ± 0.000 0.184 ± 0.000EP.31 leaf 1% acidified ethanol 0.088 ± 0.000 0.113 ± 0.001 0.155 ± 0.000EP.31 leaf hexane 0.098 ± 0.001 0.119 ± 0.000 0.202 ± 0.001YP.117 fresh fruit pure methanol 0.099 ± 0.001 0.117 ± 0.000 0.153 ± 0.000YP.117 fresh fruit 80% methanol 0.096 ± 0.000 0.099 ± 0.000 0.117 ± 0.000YP.117 fresh fruit 60% methanol 0.100 ± 0.000 0.100 ± 0.001 0.114 ± 0.000YP.117 fresh fruit 50% methanol 0.107 ± 0.000 0.116 ± 0.001 0.142 ± 0.000YP.117 fresh fruit pure water 0.088 ± 0.000 0.094 ± 0.000 0.114 ± 0.000YP.117 dry fruit pure methanol 0.077 ± 0.000 0.082 ± 0.000 0.108 ± 0.001YP.117 dry fruit 80% methanol 0.074 ± 0.000 0.079 ± 0.001 0.085 ± 0.000YP.117 dry fruit 60% methanol 0.081 ± 0.000 0.088 ± 0.001 0.093 ± 0.000YP.117 dry fruit 50% methanol 0.085 ± 0.001 0.080 ± 0.000 0.087 ± 0.000YP.117 dry fruit pure water 0.079 ± 0.000 0.083 ± 0.000 0.089 ± 0.000YP.117 leaf pure methanol 0.092 ± 0.001 0.141 ± 0.001 0.206 ± 0.000YP.117 leaf 80% methanol 0.093 ± 0.000 0.133 ± 0.001 0.201 ± 0.000YP.117 leaf 60% methanol 0.101 ± 0.001 0.157 ± 0.000 0.235 ± 0.001YP.117 leaf 50% methanol 0.109 ± 0.001 0.159 ± 0.001 0.262 ± 0.001YP.117 leaf pure water 0.105 ± 0.000 0.152 ± 0.000 0.242 ± 0.000YP.117 leaf 0.5% acidified ethanol 0.091 ± 0.000 0.116 ± 0.001 0.165 ± 0.000YP.117 leaf 1% acidified ethanol 0.087 ± 0.001 0.113 ± 0.001 0.163 ± 0.001YP.117 leaf hexane 0.072 ± 0.000 0.091 ± 0.000 0.154 ± 0.000YP.141 fresh fruit pure methanol 0.096 ± 0.001 0.104 ± 0.000 0.124 ± 0.000YP.141 fresh fruit 80% methanol 0.091 ± 0.000 0.091 ± 0.001 0.105 ± 0.000YP.141 fresh fruit 60% methanol 0.146 ± 0.000 0.138 ± 0.001 0.139 ± 0.000YP.141 fresh fruit 50% methanol 0.092 ± 0.000 0.103 ± 0.001 0.142 ± 0.000YP.141 fresh fruit pure water 0.091 ± 0.000 0.099 ± 0.000 0.117 ± 0.001YP.141 dry fruit pure methanol 0.092 ± 0.001 0.091 ± 0.001 0.102 ± 0.000YP.141 dry fruit 80% methanol 0.102 ± 0.000 0.105 ± 0.001 0.120 ± 0.000YP.141 dry fruit 60% methanol 0.093 ± 0.000 0.090 ± 0.001 0.097 ± 0.000YP.141 dry fruit 50% methanol 0.097 ± 0.001 0.088 ± 0.001 0.095 ± 0.000YP.141 dry fruit pure water 0.094 ± 0.001 0.087 ± 0.000 0.098 ± 0.000YP.141 leaf pure methanol 0.105 ± 0.000 0.155 ± 0.000 0.241 ± 0.001YP.141 leaf 80% methanol 0.108 ± 0.000 0.165 ± 0.001 0.254 ± 0.000YP.141 leaf 60% methanol 0.100 ± 0.000 0.154 ± 0.001 0.250 ± 0.000YP.141 leaf 50% methanol 0.106 ± 0.001 0.162 ± 0.000 0.252 ± 0.002YP.141 leaf pure water 0.101 ± 0.000 0.141 ± 0.000 0.247 ± 0.001YP.141 leaf 0.5% acidified ethanol 0.088 ± 0.000 0.123 ± 0.001 0.186 ± 0.001YP.141 leaf 1% acidified ethanol 0.082 ± 0.000 0.108 ± 0.000 0.148 ± 0.000YP.141 leaf hexane 0.070 ± 0.001 0.102 ± 0.000 0.162 ± 0.000YP.188 fresh fruit pure methanol 0.092 ± 0.001 0.111 ± 0.000 0.146 ± 0.000YP.188 fresh fruit 80% methanol 0.094 ± 0.000 0.107 ± 0.001 0.136 ± 0.001YP.188 fresh fruit 60% methanol 0.090 ± 0.000 0.104 ± 0.001 0.123 ± 0.000YP.188 fresh fruit 50% methanol 0.095 ± 0.000 0.096 ± 0.001 0.112 ± 0.000YP.188 fresh fruit pure water 0.099 ± 0.000 0.103 ± 0.000 0.126 ± 0.000YP.188 dry fruit pure methanol 0.090 ± 0.001 0.086 ± 0.000 0.110 ± 0.000Continuation of the Table 360Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Extracts and Standards 5.88* 14.7* 29.41*YP.188 dry fruit 80% methanol 0.091 ± 0.000 0.088 ± 0.000 0.094 ± 0.001YP.188 dry fruit 60% methanol 0.089 ± 0.001 0.087 ± 0.000 0.098 ± 0.000YP.188 dry fruit 50% methanol 0.092 ± 0.000 0.094 ± 0.001 0.099 ± 0.000YP.188 dry fruit pure water 0.093 ± 0.000 0.087 ± 0.001 0.100 ± 0.000YP.188 leaf pure methanol 0.102 ± 0.000 0.182 ± 0.001 0.252 ± 0.001YP.188 leaf 80% methanol 0.115 ± 0.001 0.164 ± 0.000 0.263 ± 0.000YP.188 leaf 60% methanol 0.116 ± 0.000 0.176 ± 0.000 0.279 ± 0.000YP.188 leaf 50% methanol 0.109 ± 0.001 0.159 ± 0.000 0.253 ± 0.001YP.188 leaf pure water 0.111 ± 0.000 0.169 ± 0.000 0.271 ± 0.000YP.188 leaf 0.5% acidified ethanol 0.098 ± 0.000 0.157 ± 0.001 0.218 ± 0.0001YP.188 leaf 1% acidified ethanol 0.088 ± 0.000 0.110 ± 0.001 0.147 ± 0.000YP.188 leaf hexane 0.076 ± 0.001 0.102 ± 0.001 0.167 ± 0.001BHA 0.690 ± 0.001 1.346 ± 0.000 1.984 ± 0.000BHT 0.504 ± 0.000 0.939 ± 0.000 1.290 ± 0.002α-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 as1 mg/mL and completing the total volume of 3.750 μmLthe EP.4 hybrid wet fruit extract obtained with puremethanol. The reducing capacities of the standards were1.984 ± 0.001, 1.290 ± 0.002, 0.872 ± 0.001 for BHA,BHT, and α-toceferol, respectively, at the highestconcentration of 29.41 μg/mL.No significant difference was observed between therootstock kumquat plant and its mutants. Although thereducing power is an important factor of antioxidantactivity, in our study, the reducing power was lower inthe extracts with high antioxidant activity. Other studiesalso show that extracts with high antioxidant activitymay have low reducing power [33, 34]. This is becausein the systems where free iron ions are present in traceamounts, the net oxidation rate increases with theFenton reaction. Substances with high reducing powermay cause further acceleration of oxidation by reducingFe(III) to Fe(II). The presence of trace levels of iron ionsin kumquat materials may have caused its low reducingpower and ncreased antioxidant activity [35].Phenolic and flavonoid content. Since phenolicand flavonoid compounds contain hydroxyl groups intheir structures and can easily give a hydrogen radicalin hydroxyl groups, they have free radical quenchingproperties. Therefore, it is important to know thetotal phenolic and flavonoid contents of the samplesto determine their contribution to the antioxidantactivity, including radical scavenging activity tests. Forthis, we used the Folin-Ciocalteu method, a standardmethod in antioxidant studies. The basis of the methodis that phenolic compounds dissolved in water andother organic solvents form a colored complex witha Folin reagent in an alkaline medium. The totalphenolic content of the extracts obtained by Soxhletextraction with different solvents was calculated usingthe regression equation (y = 0.0292x + 0.0749 andR² = 0.9994) of the calibration line of the standard gallicacid solution prepared in the concentration range of5–50 μg/mL and expressed as gallic acid equivalent (mgGAE/g extract). The gallic acid standard curve is shownFigure 1 Standard calibration curve of gallic acid to determinetotal phenolic contentFigure 2 Calibration curve of standard quercetin to determinetotal flavonoid content% free-radical scavenging activity = 𝐴𝐴C− 𝐴𝐴S/S𝐴𝐴C× 100 (1)y = 0.0292x + 0.0749R² = 0.99940.00.20.40.60.81.01.21.41.60 10 20 30 40 50AbsorbanceConcentration, μg/mLy = 0.045x + 0.0305R² = 0.99130.000.020.040.060.080.100.12Absorbance% free-radical scavenging activity = 𝐴𝐴C− 𝐴𝐴S/S𝐴𝐴C× 100 (1)y = 0.0292x + 0.0749R² = 0.99940.00.20.40.60.81.01.21.41.60 10 20 30 40 50AbsorbanceConcentration, μg/mLy = 0.045x + 0.0305R² = 0.99130.000.020.040.060.080.100.120 5 10 15 20 25AbsorbanceConcentration, μg/mLContinuation of the Table 361Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Table 4 Total phenolic and total flavonoid contents in kumquat fruit and leaf extractsExtracts Total Phenolic Substance,mg GAE/g extractTotal Flavonoid Substance,mg QUE/g extractRootstock fresh fruit pure methanol 16.096 ± 0.045 42.222 ± 0.018Rootstock fresh fruit 80% methanol 8.432 ± 0.024 24.444 ± 0.014Rootstock fresh fruit 60% methanol 5.808 ± 0.012 22.222 ± 0.012Rootstock fresh fruit 50% methanol 7.089 ± 0.018 26.667 ± 0.018Rootstock fresh fruit pure water 13.747 ± 0.011 41.111 ± 0.020Rootstock dry fruit pure methanol 8.959 ± 0.038 46.667 ± 0.016Rootstock dry fruit 80% methanol 9.856 ± 0.033 10.022 ± 0.010Rootstock dry fruit 60% methanol 5.829 ± 0.011 10.100 ± 0.012Rootstock dry fruit 50% methanol 5.425 ± 0.010 5.556 ± 0.011Rootstock dry fruit pure water 3.705 ± 0.011 14.444 ± 0.016Rootstock leaf pure methanol 66.356 ± 0.034 454.444 ± 0.046Rootstock leaf 80% methanol 72.548 ± 0.021 258.889 ± 0.024Rootstock leaf 60% methanol 68.979 ± 0.023 213.333 ± 0.034Rootstock leaf 50% methanol 67.096 ± 0.018 248.889 ± 0.032Rootstock leaf pure water 54.062 ± 0.023 174.444 ± 0.024Rootstock leaf 0.5% acidified ethanol 31.925 ± 0.030 314.444 ± 0.042Rootstock leaf 1% acidified ethanol 31.062 ± 0.018 308.889 ± 0.014Rootstock leaf hexane n.d. n.d.EP.4 fresh fruit pure methanol 20.281 ± 0.013 67.778 ± 0.026EP.4 fresh fruit 80% methanol 8.678 ± 0.025 32.222 ± 0.024EP.4 fresh fruit 60% methanol 5.479 ± 0.012 26.667 ± 0.018EP.4 fresh fruit 50% methanol 7.760 ± 0.021 35.556 ± 0.012EP.4 fresh fruit pure water 7.534 ± 0.011 25.556 ± 0.010EP.4 dry fruit pure methanol 11.247 ± 0.013 25.556 ± 0.014EP.4 dry fruit 80% methanol 11.315 ± 0.022 16.667 ± 0.016EP.4 dry fruit 60% methanol 14.288 ± 0.023 27.778 ± 0.022EP.4 dry fruit 50% methanol 9.137 ± 0.014 30.000 ± 0.023EP.4 dry fruit pure water 7.521 ± 0.021 23.333 ± 0.024EP.4 leaf pure methanol 63.438 ± 0.015 410.000 ± 0.032EP.4 leaf 80% methanol 64.797 ± 0.017 271.111 ± 0.023EP.4 leaf 60% methanol 64.685 ± 0.010 231.111 ± 0.023EP.4 leaf 50% methanol 65.568 ± 0.022 248.889 ± 0.023EP.4 leaf pure water 73.034 ± 0.015 255.556 ± 0.023EP.4 leaf 0.5% acidified ethanol 33.068 ± 0.032 315.556 ± 0.023EP.4 leaf 1% acidified ethanol 33.952 ± 0.014 355.556 ± 0.023EP.4 leaf hexane n.d. n.d.EP.29 fresh fruit pure methanol 14.596 ± 0.011 42.222 ± 0.023EP.29 fresh fruit 80% methanol 8.884 ± 0.021 31.111 ± 0.023EP.29 fresh fruit 60% methanol 8.842 ± 0.021 20.000 ± 0.023EP.29 fresh fruit 50% methanol 11.534 ± 0.018 30.000 ± 0.023EP.29 fresh fruit pure water 13.404 ± 0.016 21.111 ± 0.023EP.29 dry fruit pure methanol 12.404 ± 0.012 65.556 ± 0.023EP.29 dry fruit 80% methanol 12.918 ± 0.012 16.667 ± 0.023EP.29 dry fruit 60% methanol 9.623 ± 0.018 26.667 ± 0.023EP.29 dry fruit 50% methanol 9.747 ± 0.017 21.111 ± 0.023EP.29 dry fruit pure water 7.205 ± 0.013 13.333 ± 0.023EP.29 leaf pure methanol 60.836 ± 0.022 438.889 ± 0.023EP.29 leaf 80% methanol 67.589 ± 0.032 223.333 ± 0.023EP.29 leaf 60% methanol 70.226 ± 0.043 256.667 ± 0.023EP.29 leaf 50% methanol 64.822 ± 0.023 268.889 ± 0.023EP.29 leaf pure water 50.390 ± 0.013 184.444 ± 0.023EP.29 leaf 0.5% acidified ethanol 41.158 ± 0.011 486.667 ± 0.023EP.29 leaf 1% acidified ethanol 25.856 ± 0.033 242.222 ± 0.023EP.29 leaf hexane n.d. n.d.EP.31 fresh fruit pure methanol 6.384 ± 0.014 38.889 ± 0.023EP.31 fresh fruit 80% methanol 9.952 ± 0.012 20.000 ± 0.02362Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66Extracts Total Phenolic Substance,mg GAE/g extractTotal Flavonoid Substance,mg QUE/g extractEP.31 fresh fruit 60% methanol 17.500 ± 0.023 42.222 ± 0.023EP.31 fresh fruit 50% methanol 5.822 ± 0.023 14.444 ± 0.023EP.31 fresh fruit pure water 8.164 ± 0.013 27.778 ± 0.023EP.31 dry fruit pure methanol 12.212 ± 0.015 105.556 ± 0.023EP.31 dry fruit 80% methanol 7.452 ± 0.028 25.556 ± 0.023EP.31 dry fruit 60% methanol 7.767 ± 0.026 23.333 ± 0.023EP.31 dry fruit 50% methanol 7.486 ± 0.024 26.667 ± 0.023EP.31 dry fruit pure water 6.568 ± 0.022 13.333 ± 0.023EP.31 leaf pure methanol 61.973 ± 0.022 450.000 ± 0.023EP.31 leaf 80% methanol 64.739 ± 0.018 284.444 ± 0.023EP.31 leaf 60% methanol 74.082 ± 0.020 260.000 ± 0.023EP.31 leaf 50% methanol 72.363 ± 0.014 281.111 ± 0.023EP.31 leaf pure water 50.274 ± 0.024 180.000 ± 0.023EP.31 leaf 0.5% acidified ethanol 47.699 ± 0.010 454.444 ± 0.023EP.31 leaf 1% acidified ethanol 43.603 ± 0.018 632.222 ± 0.033EP.31 leaf hexane n.d. n.d.YP.117 fresh fruit pure methanol 13.322 ± 0.022 36.667 ± 0.023YP.117 fresh fruit 80% methanol 8.527 ± 0.012 16.667 ± 0.023YP.117 fresh fruit 60% methanol 8.486 ± 0.014 17.778 ± 0.023YP.117 fresh fruit 50% methanol 7.349 ± 0.022 158.889 ± 0.023YP.117 fresh fruit pure water 8.308 ± 0.018 112.222 ± 0.023YP.117 dry fruit pure methanol 9.445 ± 0.012 36.667 ± 0.023YP.117 dry fruit 80% methanol 8.822 ± 0.010 16.667 ± 0.023YP.117 dry fruit 60% methanol 7.705 ± 0.016 17.778 ± 0.023YP.117 dry fruit 50% methanol 6.986 ± 0.020 158.889 ± 0.023YP.117 dry fruit pure water 5.740 ± 0.018 112.222 ± 0.023YP.117 leaf pure methanol 65.356 ± 0.016 458.889 ± 0.023YP.117 leaf 80% methanol 70.205 ± 0.014 194.444 ± 0.023YP.117 leaf 60% methanol 68.514 ± 0.023 298.889 ± 0.023YP.117 leaf 50% methanol 65.616 ± 0.022 285.556 ± 0.023YP.117 leaf pure water 55.425 ± 0.020 248.889 ± 0.023YP.117 leaf 0.5% acidified ethanol 43.603 ± 0.016 312.222 ± 0.023YP.117 leaf 1% acidified ethanol 41.205 ± 0.022 381.111 ± 0.023YP.117 leaf hexane n.d. n.d.YP.141 fresh fruit pure methanol 9.342 ± 0.022 313.333 ± 0.023YP.141 fresh fruit 80% methanol 7.630 ± 0.020 40.000 ± 0.023YP.141 fresh fruit 60% methanol 10.740 ± 0.014 40.000 ± 0.023YP.141 fresh fruit 50% methanol 9.164 ± 0.018 31.111 ± 0.023YP.141 fresh fruit pure water 8.432 ± 0.012 27.778 ± 0.023YP.141 dry fruit pure methanol 15.637 ± 0.020 97.778 ± 0.023YP.141 dry fruit 80% methanol 9.089 ± 0.022 26.667 ± 0.023YP.141 dry fruit 60% methanol 10.918 ± 0.018 50.000 ± 0.023YP.141 dry fruit 50% methanol 8.295 ± 0.014 55.556 ± 0.023YP.141 dry fruit pure water 6.144 ± 0.022 26.667 ± 0.023YP.141 leaf pure methanol 72.342 ± 0.023 564.444 ± 0.023YP.141 leaf 80% methanol 76.658 ± 0.010 387.778 ± 0.023YP.141 leaf 60% methanol 64.322 ± 0.022 354.444 ± 0.023YP.141 leaf 50% methanol 63.767 ± 0.016 357.778 ± 0.023YP.141 leaf pure water 60.082 ± 0.014 305.556 ± 0.023YP.141 leaf 0.5% acidified ethanol 51.048 ± 0.012 470.000 ± 0.023YP.141 leaf 1% acidified ethanol 32.329 ± 0.012 300.000 ± 0.023YP.141 leaf hexane n.d. n.d.YP.188 fresh fruit pure methanol 11.336 ± 0.010 111.111 ± 0.023YP.188 fresh fruit 80% methanol 8.993 ± 0.012 87.778 ± 0.023YP.188 fresh fruit 60% methanol 9.986 ± 0.008 86.667 ± 0.023YP.188 fresh fruit 50% methanol 8.979 ± 0.016 104.444 ± 0.023YP.188 fresh fruit pure water 20.144 ± 0.022 102.222 ± 0.023Continuation of the Table 463Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66in Fig. 1. We found that the kumquat leaf extracts hadthe highest total phenolic content (Table 4). In particular,the highest total phenolic content (86.329 ± 0.022 mgGAE/g extract) was in the YP.188 mutant extractobtained with 60% methanol. In the fruit samples, thehighest total phenolic content (20.281 mg GAE/g extract)was found in the EP.4 mutant extract obtained with puremethanol. There was no significant difference in totalphenolic contents between the fresh and dried fruitsamples.Lou et al. compared total phenolic contents infresh and dried kumquat fruits [36]. The scientistsinvestigated changes in total phenolic matter bychanging the drying degree and time. They found thatthe total amount of phenolic substances increased withdrying, 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 fruitin hot air, under vacuum, and in a microwave oven [27].The authors found that the total phenolic content of hotair-dried fruit was approximately 5 mg GAE/g extract,but with other drying methods, it varied in the range of25–30 mg GAE/g extract [37].Yıldız Turgut et al. studied the functional qualityparameters of the powder obtained from Fortunellamargarita kumquat varieties grown in Turkey. Theyreported the total phenolic content of kumquat between2.62 ± 0.051 – 6.97 ± 0.053 mg GAE/g depending on thetype of drying method [38].Having determined the total phenolic content, wemeasured the total flavonoid content of the samples.Total flavonoid concentration was determined colorimetricallyusing a UV spectrophotometer according tothe method applied by Zhishen et al. [27].In our study, quercetin was used as a standard andthe results were calculated as quercetin equivalent (mgQUE/g extract) from the quercetin standard calibrationchart (y = 0.0185x – 0.0019 and R² = 0.9666) (Fig. 2).The highest amount of total flavonoid substance wasExtracts Total Phenolic Substance,mg GAE/g extractTotal Flavonoid Substance,mg QUE/g extractYP.188 dry fruit pure methanol 9.151 ± 0.014 15.556 ± 0.023YP.188 dry fruit 80% methanol 8.212 ± 0.028 16.667 ± 0.023YP.188 dry fruit 60% methanol 7.048 ± 0.014 21.111 ± 0.023YP.188 dry fruit 50% methanol 7.021 ± 0.012 38.889 ± 0.023YP.188 dry fruit pure water 5.418 ± 0.008 26.667 ± 0.023YP.188 leaf pure methanol 72.637 ± 0.010 446.667 ± 0.023YP.188 leaf 80% methanol 85.651 ± 0.030 330.000 ± 0.023YP.188 leaf 60% methanol 86.329 ± 0.022 345.556 ± 0.023YP.188 leaf 50% methanol 75.418 ± 0.022 300.000 ± 0.023YP.188 leaf pure water 70.849 ± 0.018 313.333 ± 0.023YP.188 leaf 0.5% acidified ethanol 62.890 ± 0.020 582.222 ± 0.023YP.188 leaf 1% acidified ethanol 33.226 ± 0.018 275.556 ± 0.023YP.188 leaf hexane n.d. n.d.n.d.: not detectedseen in kumquat leaves (Table 4). In particular, thehighest flavonoid content was found in the EP.31 mutantextract (632.222 ± 0.033 mg QUE/g extract) obtainedwith 1% acidified ethanol.Among the fruit samples, the highest amount(313.333 ± 0.023 mg QUE/g extract) was found in theYP.141 mutant extract obtained with pure methanol.There were no significant differences between the totalflavonoid amounts in the fresh and dried fruits.Lou et al. reported that the total amount of flavonoidsubstance in kumquat varied between 58.23–91.42 mg/gdepending on the drying temperature [36]. In anotherstudy, Lou et al. found that the total phenolic andflavonoid contents were higher in the extracts fromkumquat and calamondin peel compared to fruit pulp,and that they were higher in the extracts from unripekumquat compared to those from ripe kumquat [39, 40].CONCLUSIONIn antioxidant activity studies, it is common to use adifferent polarity solvent system in order to determinewhich compound types have the highest activity. Theremay be a relationship between phenolic or flavonoidamounts and antioxidant capacity determinationmethods. In particular, a relationship between methodssuch as the DPPH, which is based on radical capture,and total phenolic and flavonoid amounts may beimportant in some plant structures. Phenolic acids andflavonoids are soluble in polar solvents and show strongactivity in polar systems.In this study, we investigated the effect of differentsolvents and their concentrations on the bioactivityof kumquat fruit and leaf extracts. We found that thesolvent type was extremely important for the extracts’bioactivity. In particular, the extraction performed withpure methanol in the fruits and 60 or 80% methanolin the leaves showed the highest total phenolic andflavonoid contents, the highest extraction efficiency(50.18–59.95%), and the highest antioxidant capacity.Continuation of the Table 464Büyükkormaz Ç. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 51–66We found no statistically significant differencebetween the total amount of phenolic/flavonoidsubstances and % inhibition value in the extractionperformed with 60 and 80% methanol solutions. Thisshows that the amount of phenolic substances wasaffected by the polarity of the solvent, depending on thedifference in phenolic compounds found in kumquatfruit and leaves. We concluded that phenolic componentsin the structure of a kumquat fruit could be extractedwith a single solvent type, whereas those in the structureof a kumquat leaf could be extracted better with anaqueous solution of the relevant solvent, rather than asingle solvent type.We also observed that the aqueous solutions gavebetter results than the pure solutions in the productionof phenolics from kumquat leaves, maximizing atcertain water ratios and showing different distributionsaccording to the solvents. These results can be explainedby the fact that water increases diffusion by causingswelling in the leaf structure. In this context, methanolwas the most effective solvent for bioactive componentextraction 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 solventensured the highest total phenolic content, extractionefficiency, and antioxidant capacity. In addition, sincemethanol is a toxic solvent, it must be removed so thatthe obtained extract can be used in foods or consumed asa food supplement.Plants are complex systems by nature and havemultiple reaction characteristics and dissolutionproperties in different phases. Thus, it is not possiblefor a single method to reveal all of their radical sourcesor antioxidants [41–43]. For these reasons, we useda combination of methods, namely the DPPH, metalchelation, and iron reduction. In addition, we used theFolin-Ciocalteu method and the aluminum chloridemethod to determine the total phenol and flavonoidcontents, respectively. The results clearly showed thatthe differences in the phenolic contents affected theplants’ antioxidant properties.We found that having a high phenolic content orhigh radical scavenging activity did not yield highresults in all antioxidant activity studies. Thus, weconcluded that determining the antioxidant activity witha single method was not the right approach and that itwould be more accurate to simulate biochemical eventsin living systems by using a variety of methods. Insummary, antioxidant structures can demonstrate theirantioxidant activities by different mechanisms such asbinding transition metal ions, breaking down peroxides,preventing hydrogen absorption, and removing radicals.Our study revealed that the kumquat leaf extractshad a higher DPPH radical scavenging power thanthe fruit extracts. However, both the fruit and leafextracts showed high levels of free radical scavengingactivity with high antioxidant activity at a 125 μg/mLconcentration. Due to high antioxidant activity,kumquat leaves can be recommended to be used asfood, just as kumquat fruit, against many diseases –from gastrointestinal to infertility, from cardiovascularto respiratory and excretory disorders, especially toprevent cell damage caused by free radicals in humanand animal bodies.CONTRIBUTIONThe authors were equally involved in writing themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors have declared no conflicts of interest forthis manuscript.ACKNOWLEDGMENTSThe authors are thankful to Mr. M. Murat HOCAGİLfor his helpful contribution with the plant materialcollection.