The Electrophoretical Spectra of Kiwi Fruits Proteins
The investigated varieties of kiwi fruits are presented on Fig. 1a–c. The SDS-PAGE spectrum from fruit juices of six hardy kiwi varieties (Fig. 2a) consists of six to seven bands and only one of them (‘M1’) was distinguished from other hardy kiwi varieties (Fig. 2a, arrow a). Extracts from fruit lyophilizates revealed significant differences in the protein patterns of three kiwi fruit species. The electrophoretic spectrum of hardy kiwis consisted of 13–14 bands of different intensities with molecular weights of 14–52 kDa, while A. deliciosa and A. eriantha were composed only of some bands (Fig. 2b, arrows a–c and h showed differences between kiwi fruit species). The major protein bands of hardy kiwi cultivars were in the ranges from 17 to 22 kDa and 28–36 kDa. In ‘Hayward,’ the major bands were at 22, 24, and 32 kDa. It was possible to obtain only six sharp bands in the protein pattern of ‘Bidan,’ using urea buffer. One of the bands, characterized by molecular weight of 23 kDa, was more intensive by the amount of protein stained on the gel and comparable to bands intensity in patterns of other cultivars. Hardy kiwi fruits varieties were nearly monomorphic by protein patterns, and the intervarietal differences were small or any differences were not detected (Fig. 2b, arrows d–g, j, and k showed differences among four of six varieties examined). ‘M1’ cultivar differs from others by the presence of bands that are shown as d, e, j, k, but in ‘Ananasnaya,’ ‘Weiki,’ and ‘Jumbo’ the number of differentiating bands is limited to one (shown as f and g bands). Four groups of hardy kiwi fruit cultivars can be distinguished on the basis of protein spectrum: the first and the second were two pairs of identical cultivars: ‘Bingo’ and ‘Geneva,’ and ‘Ananasnaya’ and ‘Weiki’. ‘M1’ and ‘Jumbo’ clearly differ from other cultivars. Especially, the protein spectrum of ‘M1’ was distinct from other patterns due to the bands: 17 kDa (Fig. 2b, arrow d) and 52 kDa (Fig. 2b, arrow k), while no band of 31 kDa common for other hardy kiwi varieties was detected (Fig. 2b, arrow e). In the case of A. deliciosa and A. eriantha cultivars (‘Hayward’ and ‘Bidan’), three distinct interspecies differences were found (Fig. 2b, arrows b, c and h).
Protein Patterns of ‘Bingo’ and ‘Ananasnaya’ Cultivars
In protein patterns of two cultivars, collected in 2011 and 2013, no changes were determined (Fig. 2c). Storage had no effect on fruit protein patterns.
Electrophoresis of Hardy Kiwi Proteins Extracted from 100 to 200 mg Seed Samples
Electrophoresis of each of examined cultivars revealed the same patterns (Fig. 3a). The protein spectrum of bulk samples consisted of 24–26 bands with three zones of very intensive stained bands: 16–22, 29–33, and about 50 kDa. Two varieties ‘Jumbo’ and ‘Anananasnaya’ slightly differ from other cultivars (Fig. 3a, arrows a and b). Electrophoretic spectra of proteins extracted from singular seeds of six hardy kiwi cultivars consisted of 18–20 bands, but revealed slightly higher polymorphism of patterns than those obtained from “bulk samples.” Slight intervarietal differences were detected in patterns of ‘M1,’ ‘Jumbo,’ and ‘Bingo’ cultivars (Fig. 3a, arrows c–e, respectively).
Electrophoretic Spectra of Proteins Extracted From Singular Seeds of Hardy Kiwis
‘Ananasnaya’ cultivar was identical (Fig. 3b).
A low level of polymorphism between protein patterns of hardy kiwi cultivars has been confirmed. A low level of polymorphism determined high level of genetic similarity between patterns-lanes of particular cultivars. Value of 100 % similarity was found for two pairs of cultivars: ‘Bingo’ and ‘Geneva,’ and ‘Anananasnya’ and ‘Weiki’ (similarity 100 % was shown on matrix as a coefficient 1.00, Fig. 4a). These cultivars pairs represented identical lanes. It means that from this point of identity, such genotypes were the same. Coefficient similarities of other pairs of hardy kiwi fruit cultivars were lower, but not lower than 0.67 (Fig. 4a). Coefficient similarities between ‘Hayward’ and ‘Bidan’ cultivars were lower and estimated as 0.57. These kiwi fruit genotypes differed significantly from hardy cultivars, showing the level of similarity varying from 0.29 to 0.67 (Fig. 4a). Dendrogram analysis of cultivars was not able to group them distinct clusters, except a cluster of two pairs of identical cultivars and, partially, cluster was generated of ‘Hayward’ and ‘Bidan’ (Fig. 4b). The densitometry analysis of protein profiles has generated curves-lanes partially “covered” each other (Fig. 4c). It means that in the course of densitometry lanes that were composed of peaks, quanititative differences by height of peaks were weak or no differences were shown, especially those that represented A. arguta cultivars. Any significance in a feature of peaks height which caused by lack of the differences in the presence of bands or their intensities was not estimated. It was impossible to distinguish A. arguta lanes on the basis of the densitometry lanes of particular cultivars, except ‘M1’. This corresponded with an evidence of low protein polymorphism of A. arguta cultivars. Such phenomenon was in other parts of curves in major peaks. Maximum height was reached for lane 2 (that represented ‘M1’ cultivar of A. arguta)—12,278,143. Heights of other cultivars were slightly lower. The densitometry curve of ‘Bidan’ cultivar was nearly “flat” and differed from other curves. As it was shown above, two protocols were used for extraction of total proteins (or urea-soluble proteins), of whole fruits to find a rapid, reproducible method of protein electrophoresis that could be used initially for A. arguta cultivars and then for other kiwi fruit species. Proteins were extracted in the presence of high level of DTT to ensure complete reduction of proteins. The use of buffer sample solution for extraction of A. arguta kiwi juice proteins was not promising, until the new extraction buffer for fruit lyophilizate was applied and a small progress was achieved. We have achieved a progress in ability of protein extraction from kiwi tissues (fruit lyophylizate, seeds) by the use of urea buffer. Our results are in line with Vergara-Barberan et al. (2015), who improved the protein extraction from olive leaves using an enzyme-assisted protocol with a cellulase enzyme. Different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH, and extraction temperature and time, were optimised. The improvement of electrophoregrams for the practice purposes was an increase of the number of bands detected on the electrophoregrams (up to 22–24 bands for seeds proteins). Other researchers have obtained a seed protein pattern for kiwi fruit which consisted only of 11 bands (Miraghaee at al. 2011; Miraghaee at al. 2012). Common opinion among seed scientists is that seed proteins are especially suitable for the aim of characterization and distinguishing of crop cultivars (Cooke 1989). We were interested whether this opinion can be considered as common one for seeds of fruit plants too. Unfortunately in the case of hardy kiwis, the level of the seed protein intervarietal polymorphism has remained relatively low, and also of limited suitability for this purpose. According to molecular taxonomy of genus Actinidia, molecular similarity between kiwi fruit species was approximately 0.28. Three examined in this study kiwi fruit species were classified to different clusters of the dendrogram (Huang at al. 2002). Our results have confirmed significant differences in the biochemical identity between three kiwi fruit species. We have confirmed data provided by others (Tamburrini et al. 2005; Miraghaee et al. 2011) that the most distinct bands were in the range from 20 to about 30 kDa, and particularly protein band of 30 kDa was identified by N-terminal amino acid sequencing as actinidin. Western blotting analysis allowed to find actinidin band at ~24 kDa in protein spectrum of A. deliciosa (among them ‘Hayward’ cv.), A. chinensis, and A. arguta cultivars. Band of 24 kDa was not detected in A. eriantha cultivars. This supported our data that protein pattern of A. eriantha cultivars was composed of lower number of bands than were detected in protein patterns of ‘Hayward’ and A. arguta cultivars (Maddumage et al. 2013). As it was shown previously, actinidin was the most abundant protein in fruit and leaves of kiwi fruit (Afshar-Mohammadian et al. 2011; Testolin and Ferguson 1997; Miraghaee et al. 2011), but three of A. arguta cultivars contained higher levels of actinidin than ‘Hayward’ (Afshar-Mohammadian et al. 2011; Nishyama 2007). Only trace amount of actinidin (25 kDa band) was found in juice of some kiwi fruit varieties, and intensity of actinidin band was the same as other bands of protein patterns. In the case of other varieties, intensity of actinidin band was higher (Nishyama 2007). For kiwellin, a band at ~20 kDa was observed in A. deliciosa, A. arguta, A. chinensis, and A. eriantha cultivars. TLP proteins (band at ~22 kDa) in A. eriantha occurred only in traces or completely in no detectable amount of protein (Maddumage et al. 2013). Our results showed that the coefficient intervarietal (A. arguta) similarity was higher than 0.67. This confirmed the conclusions of other researches that genetic similarity between botanical varieties of particular species of Actinidia was relatively high (0.54 in average) and probably was lower than those of cultivated ones. Molecular markers analysis also supported our data that kiwi fruit cultivars, including botanical varieties, were genetically similar. For example, RAPD markers allowed grouping of botanical varieties into a singular cluster. The forms of A. deliciosa var. chlorocarpa, A. deliciosa var. deliciosa, and A. deliciosa with A. chinensis var. chinensis were classified into two clusters, but nearby on the dendrogram (Huang et al. 2002). Similarly, clustering based on SGE method allowed to group A. arguta cultivars in one cluster, with the level of similarity between cultivars higher than 0.7 (Testolin and Ferguson 1997). And indeed, a factor of high genetic similarity between cultivars examined by us has not allowed to distinguish all A. arguta varieties on the basis of proteins patterns obtained from fruit and seeds tissues. Although, all examined varieties of A. arguta origin from different countries [‘Jumbo,’ ‘Weiki,’ and ‘Geneva’ were selected in Italy, Germany, and USA, respectively; ‘M1’ and ‘Bingo’ are hybrid varieties selected in Poland], the protein polymorphism detected on gels was low. We have obtained interesting data that ‘Weiki’ and ‘Ananasnaya’ (unknown origin) plants, according to the morphological description, were similar varieties (Latocha and Krupa 2007). Protein patterns isolated from juice and seeds became undistinguished. It should be emphasized that distinguishing of A. deliciosa varieties on the basis of isoenzyme features was difficult (Testolin and Ferguson 1997). Only a small improvement in cultivars discrimination was achieved by high-resolution polyacrylamide gradient gels. One of four examined hardy kiwi cultivars was clearly distinct on gel from others (Maddumage et al. 2013). This supported our suggestions of low genetic variability among hardy kiwi genotypes. ‘Hayward’ and ‘Bidan’ cultivars can be easily distinguished by protein patterns, although the number of bands detected on gels was low, especially in ‘Bidan.’ The explanation of these results can be that both genotypes are classified to different genetic pools of materials. Nevertheless, an increase of detected distinct bands on gels was obtained, but the number of bands that characterize A. eriantha cultivars remained lower than other examined kiwi fruits (Maddumage et al. 2013). Densitometry analysis showed a flat curve of densitometry peaks, which was typical for ‘Bidan.’ According to this feature, ‘Bidan’ differed from other examined cultivars. On the other hand, peaks of ‘M1’ cv. were higher than others and it can be connected with the highest antioxidant activity that this cultivar exhibits. It should be emphasized that the most wanted biochemical, e.g., electrophoretical feature of identity, is the presence of lack of particular band/s on gel, in comparison of genotypes in detecting of polymorphism. Peaks height cannot be considered as identity feature: it indicates only quantitative, variable differences between cultivars. Similarly, Nishyama (2007) and other researches (Grozdanovic et al. 2014; Maddumage et al. 2013) have found intervarietal differences in actinidin activity on SDS-PAGE gel shown as differences of intensity of actinidin band. The difficulties in distinguishing A. arguta varieties on the basis of biochemical features of proteins can be explained by low range of genetic variability of materials on the market. We suppose that selection of new varieties was performed with a material of relatively limited pool of genetic variability. Very important factor which we could explain was the phenomena of low genetic variability. It is a common fact for this plant group where the used method of propagation is vegetative (non sexual procedure). It is known that the level of seed protein polymorphism, detected on gels, is usually lower than those of molecular markers, but it is suitable for distinguishing varieties, for the purposes of genotype identification. It aims for describing varietal identity and evaluation of genetic purity of seed samples and lots of agricultural crops, as cereals, grasses, and legume plants. We have demonstrated a possibility of successful extraction of proteins from singular seeds of kiwis A. arguta, followed by SDS-PAGE electrophoresis. Although obtained low intervarietal polymorphism of proteins was low and not allowed to distinguish all varieties of hardy kiwis, based on juice and seeds patterns, we supposed that proposed method can be suitable for distinguishing kiwi cultivars that were derived from more wide range of genetic pool. Our results are in accordance with others (Cooke 1989; Hameed et al. 2014; Shivashankar et al. 2010; Singh et al. 2011; Tamkoc and Arslan 2011) that SDS-PAGE method is the most suitable method of electrophoresis for the purpose of describing and distinguishing species and varieties of dicotyledonous plants-legumes, some vegetables-varieties such Chilli (Capsicum annuum L.), Brassica juncea genotypes, Poa L. (Poaceae) species, sunflower, tomato germplasm, and other dicots as pseudocereal plants. We had no problems with recognizing genetically different varieties as ‘Hayward’ and ‘Bidan’ cultivars, on the basis of method proposed by us. The problems with discrimination of more related varieties as A. arguta kiwis appeared. There is well documented data that seed protein markers generated by SDS-PAGE are suitable for discrimination of crop varieties (Cooke 1989), but some problems with distinguishing were described in the case of white and yellow lupines cultivars. Explanation was proposed that it may be caused by a limited genetic pool of genotypes for selected cultivars, and the similar problem has occurred in the case of examination of hardy kiwi cultivars. On the example of ‘Ananasnaya’ cultivar, we have confirmed genetic homogeneity of plants that are propagated by the way of vegetative process. It is known that on the market, new kiwi fruit genotypes and the number of seeds for sale increased. This factor implicates necessity of developing methods that can be suitable for laboratory check of identity material and evaluation of its genetic purity. Biochemical marker-protein pattern derived from single seeds can be used for plant breeder’s protection, before application of a new variety registration, also in the cases about doubts in samples identity on the market. The checking of crop seeds identity is based on the well-documented statement that biochemical and electrophoretical markers are independent of any environment changes, including year of harvest, and they are constant (Cooke 1989). Also others concluded that seed storage protein profiles could be useful markers in the studies of genetic diversity and genetic relationships of Poa species. SDS-PAGE method was preferred to inter- and intraspecific determinination of the genetic relationships of Poa species in order to facilitate genotype selection in breeding programs (Tamkoc and Arslan 2011). Our aim was to check whether this principle is obligated for kiwis too on the example of fruit proteins that were collected in 2 years. The answer for such question is positive. Any changes were not found in the electrophoregrams after the period of 3 weeks of fruits storage. Previously, other researchers have been interested in the problem whether any protein changes occur during maturing with ethylene treatment. After 2 days of maturing in 20 °C, only some minor changes in the intensity of 32 kDa band (by 1-D SDS-PAGE) were found (Park et al. 2008). One-week fruit maturing period caused about 1300 spots, which were recognized and corresponded to particular peptides. Nevertheless, the changes (presence of particular spots or lack of spots) were noticed only in the case of 32 spots (Li et al. 2014).
During the process of 3-week period of storage at 1 °C and 90–95 % of RH in kiwi fruit harvested in commercial maturity, any possible changes of protein activity were not detected.
The significant progress in detecting kiwi fruit proteins was achieved by 2-D electrophoresis, followed by proteomics analysis. On the other hand, it is known that the use of 2-D electrophoresis, followed by proteomic analysis, was successful for solving problem of detecting biochemical changes during the storage of A. arguta fruits (Li et al. 2014). We supposed that the method used in our investigation (SDS-PAGE, followed by Coomassie staining) was not suitable for detecting minor changes due to low sensitivity of the stain used. On the other hand, the use of 2-D electrophoresis for kiwi genotypes, followed by proteome analysis, can be performed only for scientific work purposes, but not for routine tests—seed lots evaluation, tests performed by plant breeders, and seed testing laboratories. The present analysis of plants by protein markers as 1-D SDS-PAGE, IF, or A-PAGE markers represented the most effective testing of initial and breeding material (Cooke 1989). Our results are in line with Singh et al. (2011), where genetic variability among 59 B. juncea genotypes was assessed, based on morphological and biochemical markers. Based on the SDS-PAGE patterns, all genotypes were classified into five clusters.
Fourier Transform Infrared Spectra of Polyphenols
For illustration of the distinct patterns of the kiwi fruit cultivars, averaged spectra of each cultivar are given in Fig. 5. The IR spectra of kiwi A. arguta methanol extracts were compared between them (Fig. 5b, c) and also with standard kiwi fruit such as ‘Hayward’ and ‘Bidan’ in the range of common peaks (Fig. 5a). In our previous study, the IR spectra data showed that the main bands in the kiwi fruit samples slightly shifted (Park et al. 2015c). IR spectrum of polyphenols showed common peaks for all kiwi fruit samples at 3299–3300 cm−1, 2924–2930 cm−1, 1232–1235 cm−1, and 919–924 cm−1, except cultivar ‘M1’ (Fig. 5). A. arguta cultivars had bands of 3011–3007, 2851, 2353, and 2357 cm−1 (Fig. 5b, c). Then A. arguta cultivars showed peaks at 1743, 1737, 1651, 1603, 1605, 1317–1319, 1233, 1182, 1106–1140, 1042, 989, 924, 817, and 779 cm−1. ‘Hayward’ and ‘Bidan’ showed limited number of bands as A. arguta cultivars at 1716, 1603, 1593, 1402, 1340, 1232, 1027, 919, 866, 816, and 777 cm−1. A predominant band in the region of 1042–1140 cm−1 was assigned to the C-O stretching vibration of the sugars (Fig. 5). In all samples, the region at 3300–2900 cm−1 was dominated by one broad band, which represented the H-bonded O-H stretching of carbohydrate, carboxylic acids, and residual water. In the spectrum of the methanol soluble fraction of poly(rutin), a broad peak centered at 3300 cm−1 due to the vibration of O–H linkage of phenolic and hydroxyl groups and peaks at ca. 1 570 cm−1 ascribed to the C=C vibration of aromatic group were observed, the methanol-soluble and insoluble polymers. The doublets near 2925–2930 cm−1 were caused by the C-H asymmetric and symmetric stretching of the methyl C-H group. This may have been due to the presence of a phenolic hydroxyl group in the kiwi fruit polyphenol molecules. The characteristic functional groups stretches indicate the presence of alkane (3011 and 2930 cm−1), N-methyl (2851 cm−1), and C-O stretch (1182 cm−1) in the samples. The three bands between 1402 and 1603 cm−1 were attributed to aromatic ring vibrations. Multiple bands between 1140 and 1319 cm−1 comprised intricate absorption of the C-O stretch and C-O-H bending of phenols, carboxylic acids, and carbohydrates (Silverstein et al. 1981; Coates 2000; He et al. 2007; Lopez-Sánchez et al. 2010; Bureau et al. 2012). The spectral region giving the best results for phenolic compound prediction was 1010–1700 cm−1. This area corresponds to the spectral data of catechin obtained with the same ATR technique and showing important peaks between 1700 and 1000 cm−1. According to Coates (2000), this area shows phenolic compounds bands (C=C-C aromatic ring stretch, 1603 cm-1 in both A. arguta and A. deliciosa cultivars), to phenol OH bend (1402–1318 cm−1), to aromatic C-H in-plane bend (1232–966 cm−1), and to CO stretch of phenol (1232 cm−1). The total phenolic content showed numerous characteristic peaks in the spectral region (1010–1700 cm−1). Bands at around 1605, 1593, 1402, 1340, 1233, 1145, 1182, and 1042 cm−1 are found in all spectra with various relative intensities and minor, but varietal, characteristic shifting in absolute band positions. ‘M1’ differed from other A. arguta cultivars in the range of 1042–990 cm−1 and showed only one peak at 1015 cm−1. Our results are similar to others, showing the polyphenols differences in the spectral region giving the best results for phenolic compound prediction of 1010–1700 cm−1 (Bureau et al. 2012). In the region of 865–775 cm−1, ‘M1’ is similar to ‘Hayward’ and ‘Bidan.’ Thus, for each cultivar, a unique phenolic fingerprint in the IR spectral region could be obtained. Most remarkable are the differences from 1232 till 777 cm−1 (He et al. 2007; Lopez-Sánchez 2010; Bureau et al. 2012). A shift in the difference between the standard and the investigated samples can be explained by the extraction procedures of the total phenols. The matching (%) of the peaks in the region from 4000 to 700 cm−1 (short regions: 3400–2900 cm−1, 1800–900 cm−1, and 1100–700 cm−1) for Hayward/Bidan, Weiki/Bingo, and Jumbo/Ananasnaya was the highest (96–100 %). Cultivar ‘M1’ which showed differences in the electrophoretic separation as well showed lower matching in the same spectral region as: Hayward/M1, Ananasnaya/M1, Bidan/M1, Bingo/M1, and Geneva/M1 from 72 to 99 % (Fig. 5, Table 1). As it was discussed previously, by Bureau et al. (2012), the best matching region for polyphenols is 1100–700 cm−1 and the following values of matching were obtained for Hayward/M1 (90 %) and Bidan/M1 (93 %), and lower for Ananasnaya/M1 (84 %) and Geneva/M1 (76 %).
NMR Spectra of Polyphenols
NMR spectra of some cultivars are shown on Fig. 6. The assignment of 1H spectrum of kiwi fruit organic extract (DMSO) was obtained, where the main peaks appeared between 2 and 6 ppm. The spectra were similar for three cultivars with slight differences between 4–5 ppm for ‘Ananasnaya’ (Fig. 6c). In fact, the difference between the concentrations of the major and minor components of a metabolite mixture can be beyond the limited dynamic range of a NMR spectrometer. In this case, the limit of detection for the minor component in the mixture is higher than the limit of detection for the same compound isolated from the mixture. In the case of DMSO, mostly the sugars (Fig. 6a–c) were the main compounds and the aromatic part (Fig. 6d–f) was the minor (ppm): ‘Bidan’ (8.16, 7.22, 6.60, 6.23), ‘Hayward’ (8.17, 7.22, 6.64, 6.23), ‘Ananasnaya’ (8.15, 7.23, 6.58, 6.21). Using the NMR approach, it has been possible to identify primary as well as secondary metabolites of different fruits such as grape, orange, apple juice, mandarin, orange, kiwi fruits, mango, black raspberry, melon, watermelon, blueberry, and peaches (Sobolev et al. 2015; Capitani et al. 2010; Capitani et al. 2013a and 2013b). As it can be seen from the obtained data, all peaks in aromatic region in the cultivars were completely similar in DMSO solvent. It is known that phenolic compounds are poorly soluble in water. The mixture of water and methanol is usually used for extraction and subsequent NMR analysis (Fig. 6g, h): for ‘Hayward’ (9.37, 8.53, 8.34, 8.25, 8.16, 7.87, 7.80, 7.72, 7.46, 7.29, 7.23, 7.22, 7.16, 7.10, 6.98, 6.82, 6.78, 6.60, 6.46 ppm) and for ‘Ananasnaya’ (9.19, 8.05, 7.96, 7.95, 7.30, 7.10, 7.05, 6.92, 6.86, 6.73, 6.61, 6.42, 6.32 ppm). ‘Bidan’ showed exactly the same peaks as ‘Hayward.’ In ‘Ananasnaya,’ the number of peaks was sligthly lower that in the two varieties. In NMR-based analyses, the extraction procedure is probably the most critical step aimed to the quantitative transfer of the metabolites from the solid matrix into the solution. Metabolites identified in 1H-NMR spectra of kiwi fruit water extracts showed the following compounds: acids—ascorbic, citric, lactic, malic, quinic; sugars and sugar alcohols—β-Arabinose, α-d-fructofuranose, β-d-fructofuranose, β-d-fructopyranose, α-galactose, β-galactose, α-glucose, β-glucose, α-glucose-6P, β-glucose-6P, β-mannose, Myo-inositol, raffinose, sucrose, α-xylose, β-xylose; amino acids, peptides and derivatives—alanine, γ-amino-butyrate, arginine, asparagine, aspartate, glutamate, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophane, valine; alcohols, polyols, amines, aldehydes, ketones, esters—choline; nucleic acid derivatives—adenosine-triphosphate, uridine; aromatic compounds—(−)-epicatechin, neochlorogenic acid, quercetin-3-O-rhamnoside, O3-β-d-glucopyranosyltrans caffeic acid; O3-β-d-glucopyranosyl-cis-caffeic acid (Sobolev et al. 2015; Capitani et al. 2010; Capitani et al. 2013b). Sugars such as glucose, sucrose, and fructose have been detected in the investigated kiwi fruit cultivars. The observation of secondary metabolites in the 1H-NMR spectra of crude extracts can be hindered by the presence of major components (Sobolev et al. 2015; Capitani et al. 2010; Capitani et al. 2013b).