Comparison of bioactive compounds and health promoting properties of fruits and leaves of apple, pear and quince

This paper presents characterization of healthy potential new sources of functional constituents with reference to basic plant sources. In this study, the phenolics, triterpene, isoprenoids (chlorophylls and carotenoids), amino acids, minerals, sugars and organic acids of different cultivars of pome species—apple, pear, quince—leaves vs. fruits and their enzymatic in vitro enzyme inhibition of hyperglycemic (α-glucosidase, α-amylase), obesity (pancreatic lipase), cholinesterase (acetylcholinesterase, butylcholinesterase), inflammatory (15-LOX, COX-1 and -2) and antioxidant capacity (ORAC, FRAP, ABTS) were evaluated. Leaves of pome species as a new plant sources were characterized by higher content of bioactive and nutritional compounds than basic fruits. The dominant fraction for quince, pear, and apple fruits was polymeric procyanidins. In quince and pear leaves flavan-3-ols, and in apple dihydrochalcones dominated. Triterpene was present in equal content in leaves and fruits. Leaves are excellent sources of amino acids and minerals (especially Ca, Mg, Fe, and K), with high content of organic acids and low content of sugars compared to fruits of pome species. Leaves of apples and pears most effectively inhibited COX-1, COX-2, α-amylase, and α-glucosidase enzyme but quince leaves showed the most effective inhibition of pancreatic lipase, AChE and BuChE, 15-LOX, and antioxidant capacity, which particularly correlated with bioactive compounds. Present study shows that leaves are promising sources of valuable compounds and may be used to produce functional foods as well as for medical purposes.

www.nature.com/scientificreports/ Mg was the most abundant macro-element for quince leaves and fruit cultivars, 457.3-534.0 mg and 20.3-41.9 mg/100 g dw, respectively. Only quince fruits and leaves showed high content of Na, contrary to apple and pears fruits and leaves (< 7 mg/100 g dw). This fact could be of interest to people with special low sodium diets because nowadays salt is available everywhere. Among the foods evaluated by Leterme et al. 25 , leaves are unconventional sources of minerals and appear as outstanding mineral sources, which have the highest contents of Ca, Mg, S, Fe, Mn and Cu. The leaves of Trichanthera have average Ca content of 6.2 g/100 g dw (Leterme et al., 2006). Other unconventional sources of minerals are peels of fruits (pomelo, orange, lemon, mandarin), i.e. K, Ca and Mg 24 .     www.nature.com/scientificreports/ It is known that fruits of pome species have high nutritional properties but leaves should be classified as a 'source of minerals' . The differences in content of minerals may also be ascribed to different species, fruit maturity, agricultural practice, and ecological conditions, such as climate, altitude, soil fertility and seasonal variations 22 . Micronutrients are involved in numerous biochemical processes and an adequate intake of certain micronutrients relates to the prevention of deficiency diseases. Phosphorus, together with Ca, participates in the formation of strong bones and teeth 24,25 . The daily requirements of an adult man are as follows (mg/d): [10][11][12][13][14][15] Fe, 300-400 Mg, 700-800 P, 800-1200 Ca, 500 Na, 12-15 Zn, 2-3 Cu (FAO).
Amino acids. The concentrations of free amino acids present in apple, pears, and quince fruits vs. leaves are shown in Fig. 1. Apple leaves and fruits are characterized by significantly higher content of amino acids than pears and quince samples. All pome fruits have significantly higher content of amino acids than leaves, and cultivars were also differentiated by their content. As previously mentioned by Mykhailenko et al. 26 the above ground organs (i.e. leaves, flowers) have a higher content and more diverse composition of amino acids than their underground organs (i.e. corms and rhizomes).
Nineteen amino acids were determined in pome species and they were categorized into essential (histidine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan), conditionally essential (arginine, glutamine, glycine, proline, tyrosine), and non-essential (alanine, asparagine, aspartic acid, cysteine, glutamic acid, serine, alanine) amino acids 27,28 . Methionine and valine were absent. As a general rule, the non-essential fraction was significantly dominant in all fruits and leaves, besides apple leaves where essential and non-essential fractions were equal. Leaves of apple, pear, and quince had higher content of essential amino acids while for fruits this fraction was marginal. The content of essential amino acids for people is important because they are synthesized only by plants and people are still looking for new rich sources of these type amino acids.
The pome leaves had a more diverse amino acid composition compared to pome fruits ( Fig. 1). In apple and pear fruits and leaves aspartic acid was dominant but additionally in fruits O-phospho-l-serine, and in pear leaves additionally glutamic acid and cysteine content was observed. Meanwhile in the fruit of quince aspartic acid dominated, but in leaves cysteine, aspartic and glutamic acid dominated. Generally, the amino acid profile is similar as previously reported in the literature 27 . For comparison, the sum of amino acids ranged from approx. 0.03 to 0.14 and 0.05 to 0.18 mg/100 g for quince pulps and peels, respectively 27 . It seems that apple has some similarities with quince and pear; asparagine and aspartic acid are usually two of the major free amino acids in these pome species 27 . Compared to results present by Turkiewicz et al. 29 apple, pear and quince fruits and leaves are characterized by lower content of amino acids than some cultivars of flowering quince fruits (between 15.87 and 2326.33 mg/100 g dw), but they present similar content to a different species of rosehip 28 or sprouts and microgreens 30 .
Aspartic acid is synthesized by direct amination but alanine and glutamic acid are formed as a result of reductive amination. All other amino acids are secondary ones, because they are formed as a result of transamination of the amino acids listed above with the corresponding keto acids that arise during the metabolism, as well as by the conversion of some acids to others 26,28 . Changes in amino acid contents in different plants depend on numerous factors such as source of nutrition, nutritional requirement, especially nitrogen fertilization, biotic and abiotic factors. Apart from nutritional properties, amino acids also affect taste and flavor, as a number of them have a distinctively bitter taste (e.g. tyrosine, arginine, leucine, valine, methionine, and histidine) [26][27][28] Additionally amino acids exhibit different actions benefiting human health such as preventing cardiovascular disease, improving digestion, protection from arteriosclerosis and diabetes mellitus, etc. 28 . Table 1, among the investigated sample, species, cultivar, and fruits vs. leaves had a significant influence of the content of polyphenols. Significantly higher concentration of polyphenols was noted for leaves (approx. 10,825.9 mg/100 g dw) than in fruits (approx. 3275.0 mg/100 g dw). Higher content of polyphenols was noted for quince fruits and leaves (approx. 9128.2 mg/100 g dw) than for apple (approx. 7289.0 mg/100 g dw) and pears (approx. 5904.8 mg/100 g dw). Cultivar had a significant influence on content of polyphenols except monomers of flavan-3-ols and phenolic acids. Leaves turned out to be richer in phenolics than fruits due to the complexity of the biosynthesis process in plants, which is dependent on various factors, including place of cultivation, environmental conditions, drought resistance, frost hardiness, biotic and abiotic stress 31,32 . As reported by Jaakola et al. 31 , a higher content of polyphenolic compounds was identified in leaves of plants growing under intensive sunlight, which results in enhanced gene expression coupled with phenolic biosynthesis.

Polyphenols. As shown in
The profile and content of polyphenols were quite diverse and strongly dependent on the presence in different fruits and leaves according to the morphological part tested. In quince, pear and apple fruits procyanidins were the dominant fraction of polyphenols. In quince leaves were richer in flavan-3-ols than flavonols, in pear there was dominance of flavan-3-ols > > flavonols > phenolic acids, and finally in apple the major content was dihydrochalcones > flavonols ~ flavan-3-ols. These results are similar to data previously mentioned in the literature 20,21,33 .
Flavan-3-ols were quantified as one of the predominant phenolics in the analyzed samples. These compounds present different properties, and beside them, they have antioxidative and antiproliferative properties. In addition, they also play a crucial role in shaping the astringent taste of foods. The highest content of these compounds was recorded in all quince and pear cultivars of fruits and leaves. A lower content was noted for apple fruits and leaves. Additionally, the analyzed cultivars of fruits and leaves exhibited large differences in content of flavan-3-ols, especially the cultivar Szampion, where fruits were noted to be richer than leaves. Szampion is a scab-resistant cultivar and, as previously reported, it accumulates significantly higher amounts of flavonoids, mainly flavan-3-ol compounds 34  www.nature.com/scientificreports/ Flavonols were the next most abundant phenolic group evaluated in leaves but not in fruits. Pear leaves were characterized by significantly higher content of flavonols than apple and quince, but the largest differences between fruits and leaves were noted for pear and apple. Leaves of pome species are interesting sources of flavonol compounds. This difference in content results from the fact that these compounds are mainly located in the top layer of plants, protecting them from harmful UV-A and UV-B radiation. Previous studies reported that flavonols (especially moieties of quercetin or kaempferol) accumulate in higher amounts in response to increased UV-B radiation 32 . Shading of the fruits (flavonols in fruits mainly located in skin) during development has been found to reduce the accumulation of flavonols and to inhibit the transcription of the corresponding flavonoid pathway genes 31,32 . Jaakola et al. 31 discovered that the content of flavonol derivatives in sun-exposed leaves was three times higher than in shaded leaves. The content of flavonols in the daily diet, which can vary between 5 and 40 mg/day 35 , is important because they reduce the incidence of cardiovascular diseases and inhibit cancer cell Table 1. Comparison of polyphenols [mg/100 g dw] content in fruits and leaves of some selected species and cultivars of apple, pear, quince fruits and leaves. Mean of 3 replications ± standard deviation followed by the same letter, within the same column were significantly different (p < 0.05) according to Tukey's least significant differences test. Letters a,b,c,d, represent significance in content (p < 0.05). PP polymeric procyanidins, nd not detected.

Species Cultivars
Flavan-3-ols  35 . High concentration of flavonol glycosides in apple leaves was noted earlier 20 . As previously reported 35 , among the richest sources of flavonols are onions (2.5-6.5 g/g), sea buckthorn (0.9 g/100 g dw 36 ; and cranberry (1.2 g/100 g dm) 10 . Phenolic acids were the next abundant group evaluated in leaves and fruit. Only pear leaves were characterized by a higher content of phenolic acid than fruits. Similar results were presented previously by Kolniak et al. 37 . Differences between leaves and fruits for quince were marginal, but for apple, the trend was not obvious because the content of phenolic acid was strongly dependent on cultivar (Table 1).
Another important class of naturally occurring flavonoids is dihydrochalcones, found only in apple leaves and fruits. These observations have also been confirmed previously 20 . Their content ranged from 3155.7 to 3552.7 mg/100 g dw for leaves and 10.9 to 12.0 mg/100 g dw for fruits. Florina cv. had the lowest content of dihydrochalcones compared to Szampion and Empire cultivars Mikulic-Petkovsek et al. 34 reported that apple leaves exhibited low content of chlorogenic acid (0.0-1.0 mg/g dw) and procyanidin (0.5-0.9 mg/g dw) while the concentration of phloridzin ranged between 70 and 115 mg/g dw. Plants containing chalcones have been employed in traditional herbal medicine for centuries 35 . For that reason, the recent results of research present a wide spectrum of biological activities including antioxidative, antibacterial, anti-inflammatory by inhibiting COX-1 and COX-2 activity, anticancer against prostate cancer cells, and immunosuppressive potentials 35 .
Anthocyanin contents in fruits are marginal and for apple fruits the level was > 2 mg/100 g dw. In leaves, the composition of anthocyanins was more diverse and the concentrations were considerably higher, even tenfold, than in the fruits (Table 1). These compounds are strongly UV-absorbing, and accumulate in leaves mainly in the epidermal cells of the plant tissues 31,32 . Higher accumulation of anthocyanins and their photoprotective role have been observed in various species after UV-B-or UV-A-induced lights 32 , e.g. for cranberry leaves, where leaves are red 31 . Additionally, anthocyanins are compounds which show a number of health-promoting properties. In vitro and in vivo research trials have demonstrated anthocyanidins as compounds with biological activity, but this effectiveness also depends on the amount in a given plant 35 .
Isoprenoid. Carotenoids are associated with chlorophylls in the photosynthetic apparatuses of plants, and play a crucial role protect against photooxidation. The carotenoid and chlorophyll contents, presented in Table 2, significantly (p < 0.05) varied depending on the (i) species, (ii) morphological part (leaves vs. fruits) and (iii) cultivars.
Regarding the carotenoid profile, 9-cis or 9-cis'-lutein and Σ of β-carotene with 9-cis-β-carotene were significantly higher for quince than for apple and pears. Among the carotenoids, 9-cis or 9-cis'-lutein predominated in both fruits and leaves but the content was differentiated by cultivar. In turn, leaves were up to 4 times richer in carotenoids content than fruits. As previously reported by Pop et al. 38 , content of carotenoids in sea buckthorn leaves depends on cultivar and equals 3.8-4.2 mg/100 g dw, and lutein was found in the highest concentration followed by β-carotene (0.9 and 0.7 mg/100 g dw, respectively). Lakshminarayana et al. 39 reported the carotenoid content in some green leafy vegetables; it was 166.36 mg/100 g dw in dill and 238.62 mg/100 g dw in spinach.
The levels of chlorophylls were considerably higher relative to those of carotenoids, especially in leaves. The chlorophyll fraction was represented in leaves by chlorophylls and pheophytin as a/b and a'/b'. The content of chlorophylls in fruits was marginal, especially in apple and pear cultivars (> 10 mg/100 g dw). Regarding the chlorophyll profile, chlorophylls and pheophytin a were predominant because type a is a precursor for b type components. Pheophytin is synthesized from chlorophylls when naturally chelated magnesium in the chlorophyll macrocycle is readily substituted by hydrogen. Total chlorophyll concentration of fresh sea buckthorn leaves was 98.8 mg/100g 38 , which is lower than or comparable with our results. As reported by Ponder et al. 14 , raspberry leaves also contain chlorophylls in the range 6.7-9.6 mg/100 g fw, which significantly depends on cultivar.
Many epidemiological studies suggest that increased daily consumption of isoprenoid compounds decreases the risk of several degenerative and other diseases such as cardiovascular disease and skin cancer 14,38,39 .
Triterpenoids. The content of eleven triterpenoid compounds in the analyzed morphological parts of leaves and fruits of apple, pear, quince is shown in Table 3. Total triterpene for apple, pears and quince fruits and leaves was: 136.3-214.4 and 118.9-219.9 mg/100 g dw, 70.2-117.1 and 35.1-235.0 mg/100 g dw, and 65.2-152.3 and 91.3-226.4 mg/100 g dw, respectively. Overall, it has been observed that the leaves are an equal source of triterpenes as the analyzed fruits.
Generally, pear and apple had significantly (p < 0.05) higher content of triterpene compounds than quince. Oleanolic, corosolic, ursolic acids and erythrodiol prevailed in the apple fruits but ursolic and oleanolic acids were characteristic compounds for apple leaves. Corosolic and oleanolic acids and erythrodiol were predominant triterpenoid compounds for pear fruits and leaves. Ursolic and corosolic acids were major triterpenes for quince leaves and fruits, but in addition quince fruits were rich in erythrodiol and leaves were rich in uvaol. As previously mentioned, ursolic, oleanolic acids and uvaol are the main compounds identified in apple and cherry, oleanolic acid in grape berry and bilberry, olive, maslinic acid in olive but α-,β-,δ-amyrins was characterized for tomato 40 . There was no significant (p > 0.05) difference in total content of triterpene between leaves and fruits but leaves were significantly (p < 0.05) higher content of uvaol, α-boswellic, betulin, betulinic, maslinic and tormentic acids than fruits, where the major (p < 0.05) triterpenes were erythrodiol and corosolic acids. Szakiel et al. 41 reported that mainly triterpene compound levels of bilberry leaves (oleanolic and ursolic acids) were significantly lower than those of berries.
A diet rich in triterpenoid compounds has been associated with some beneficial effects such as reduced incidence of many chronic diseases including cardiovascular, ischemic stroke, neurodegenerative disorders and aging and with numerous biological activities such as cytostatic, anti-inflammatory (COX inhibition), antibacterial and antiviral (including anti-HIV), anticarcinogenic or hypolipidemic and cholesterol-lowering 40  Anti-oxidant, inhibitory of diabetic, obesity, cholinergic, lipo-and cyclooxygenase activity of apple, pear and quince fruits and leaves. Anti-oxidant capacity. The antioxidant capacity assayed in fruits vs. leaves of different cultivars of apple, pear and quince is presented in Table 4. For ABTS, FRAP and ORAC assay obtained results clearly indicate that all pome leaves showed significant differences between fruits and leaves. These values were 3.8, 3.5 and 3.0 and 1.2, 1.8 and 3.2 times for ABTS and FRAP higher than in the apple, pear and quince fruits of the respective cultivars, respectively. For ORAC assay these values were 10.1, 3.3 and 14.5 times higher than in the apple, pear and quince fruits of the respective cultivars, respectively. Higher Table 2. Comparison of isoprenoids (carotenoid and chlorophylls; mg/100 g dw) content in fruits and leaves of some selected species and cultivars of apple, pear, quince fruits and leaves. Mean of 3 replications ± standard deviation followed by the same letter, within the same column were significantly different (p < 0.05) according to Tukey's least significant differences test. Letters a,b,c,d, represent significance in content (p < 0.05). nd not detected.
Species Cultivars 9-cis or 9-cis'lutein Σ β-and 9-cisβ-carotene   www.nature.com/scientificreports/ antioxidant potential was determined in quince than in pears and apple, whether fruit or leaves were analyzed. Additionally, it was mentioned that cultivars were one factor influencing antioxidant capacity. It is known that the antioxidative potential depends on the content of bioactive components such as polyphenols, isoprenoid, vitamins or other molecules present in plants. As presented in Fig. 2, there was a significantly high correlation between chlorophylls, carotenoids, flavonols and ABTS, FRAP, ORAC (r 2 > 0.808). Additionally, antioxidant capacity had an influence on polymeric procyanidins (FRAP as r 2 = 0.789). Similar results were previously presented in the literature 10,14 .
Inhibitory of hyperglycemic and obesity activity. Pome fruits and leaves were also analyzed as sources of bioactive substances with hyperglycemic and obesity activities. The α-amylase activity was significantly dependent on type of pome species where quince and apple presented greater activity than pear. Quince leaves are more active Table 3. Comparison of triterpene [mg/100 g dw] content in fruits and leaves of some selected species and cultivars of apple, pear, quince fruits and leaves. Mean of 3 replications ± standard deviation followed by the same letter, within the same column were significantly different (p < 0.05) according to Tukey's least significant differences test. Letters a,b,c,d, represent significance in content (p < 0.05). www.nature.com/scientificreports/ inhibitors of α-amylase than other samples, especially pears. Contrary to α-amylase activity leaves of pome species showed significantly higher α-glucosidase and lipase activity than fruits. Higher potential of α-glucosidase inhibitory was shown by quince leaves and fruits than apple and pears. Higher lipase inhibitory potential was presented by pear and apple leaves and fruits than quince. Cultivar had no significant influence on α-amylase, α-glucosidase or lipase activity. Figure 2A presents a high, significant correlation between α-amylase and phenolic acid (r 2 = 0.827) where other compounds play a minor impact. There was a correlation between α-glucosidase and non-essential amino acids (r = 0.902) and between lipase and polyphenols or sugars (r 2 = 0.617 and 0.532, respectively). The enzymes α-amylase and α-glucosidase are responsible for breakdown of complex saccharides before absorbates during Table 4. Anti-oxidant capacity and inhibition of hyperglycemic, obesity, inflammatory and cholinesterase enzyme, 15-lipooxygenase activity of apple, pear, quince fruits and leaves. Mean of 3 replications ± standard deviation followed by the same letter, within the same column were significantly different (p < 0.05) according to Tukey's least significant differences test. Letters a,b,c,d, represent significance in content (p < 0.05).

Species Cultivars
Anti www.nature.com/scientificreports/ digestion and their active inhibition is important for people with type 2 diabetes mellitus. Pancreatic lipase enzyme is responsible for the breakdown of dietary fats to become absorbable in the intestinal lumen as monoacylglycerols and free fatty acids and their inhibition is important for people for obesity prevention and weight management 23,30,42,43 . Similar results were previously presented by Spinola et al. 42 , whereas leaves of Elaeagnus umbellata and Sambucus lanceolata present significantly higher inhibition than their berries against α-glucosidase and pancreatic lipase enzyme but opposite for α-amylase. www.nature.com/scientificreports/ Cholinergic enzyme activity. Significant differences concerning the inhibitory activity towards AChE and BuChE activity were noted between all the pome fruits and leaves (Table 4). Our findings showed higher inhibition of leaves against AChE and BuChE activity than fruits. Inhibitory effects in the tested leaves were highest (p < 0.05) for quince > pear > apple and were 71.7, 56.9, 51.3% for AChE and 72.9, 66.4 78.2% for BuChE, respectively. Cultivar as a factor had no significant influence on these activities. BuChE and AChE are key enzymes in the breakdown of an important neurotransmitter, acetylcholine, playing a key role in pathogeneses of Alzheimer's disease, currently one of the most prevalent neurodegenerative disorders. Therefore, these results present new promising sources of cholinergic inhibitory. AChE activity was only highly correlated with polymeric procyanidins and organic acids (r 2 = 0.719 and 0.622, respectively). The other compounds present weak influences on AChE and BuChE activity (r 2 < 0.650-0.413).
Anti-inflammatory activity. The anti-inflammatory effects of pome fruits and leaves were evaluated based on their ability to inhibit the activities of 15-LOX (as %), COX-1 and COX-2 (as IC 50 ), as presented in Table 4. LOX and COX are important enzymes in lipid metabolism and oxidation of some linoleic and arachidonic acids for their corresponding metabolites, cis-, trans-conjugated hydroperoxides and prostaglandin, respectively 44 45 . These results indicated that leaves have high anti-inflammatory effects, which can be presumably related to bioactive compounds present in leaves and fruits. Flavonoids inhibit biosynthesis of prostaglandins (the end products of the COX and lipoxygenase pathways), which act as secondary messengers and are involved in various immunologic activities 44 . LOX and COX play an important role in several inflammatory diseases such as cancer, bronchial asthma, osteoporosis, allergic, atherosclerosis, and arthritis 44 .

Principal component analysis (PCA) and hierarchical clustering analysis (HCA).
To investigate and better understand their variation and the relationship between biological activity, nutritional and bioactive compounds of apple, pear and quince fruits vs. leaves, PCA and HCA were performed ( Fig. 2A, B). The first two PCA account for about 65.37% of the total variance, which showed that nearly all the data variation can be explained by PC1-PC2. As can be seen, pome leaves samples are found on the right half of the PCA graphic, whereas pome fruits appear mainly on the negative half of the graphic. Pome leaves were associated with some nutritional compounds (minerals, amino acids (conditional and nonconditional), organic acids) and bioactive compounds (polyphenols, carotenoids, chlorophylls, triterpenic) whereas fruits were associated only with high content of sugars and non-essential amino acids. Additionally, the PCA relationship indicates that leaves were associated with all biologically activity. Quince leaves are rich in polymeric procyanidins, organic acids, chlorophylls, carotenoids and they primarily associated with AChE and pancreatic lipase. Apple and pear leaves are rich in triterpenic, phenolics (phenolic acids, flavonols, anthocyanins, dihydrochalcones, flavan-3-ols), essential and non-essential amino acids, and minerals primarily associated with α-amylase, α-glucosidase, COX-1 and COX-2. All investigated leaves showed high antioxidant potential as FRAP, ABTS, ORAC and 15-LOX activity.
Similar to PCA, HCA (Fig. 2B) presented two separate clusters for leaves vs. fruits. Inspection of the groups showed that the quince clustered separately from apple and pears, being characterized by high concentrations of polymeric procyanidins, organic acids, chlorophylls and carotenoids.

Conclusions
Various plant materials and their morphological parts are currently being investigated as potential sources of bioactive compounds. This study demonstrated significant differences between leaves and fruits of pome species-apple, pear, and quince-in the content of both bioactive and some nutritional compounds and biological activity. Leaves of pome species were richer in isoprenoid such as chlorophylls and polyphenolics. The dominant fraction for quince, pear, and apple fruits comprised polymeric procyanidins. In quince and pear leaves flavan-3-ols and flavonols dominated, while in apple the polyphenols were represented by dihydrochalcones > flavonols ~ flavan-3-ols. The differences in the content of triterpene in the analyzed morphological parts occur since these compounds are mainly accumulated in the waxy layer of the plants and therefore fruits are richer in triterpene than leaves. Leaves are excellent sources of amino acids and mineral compounds, especially Ca, Mg, Fe, and K. They are characterized by a high content of organic acids and low content of sugars compared to fruits of pome species. Leaves of apples and pears showed the most effective inhibition of COX-1, COX-2, α-amylase, and α-glucosidase but quince leaves showed the most effective inhibition of pancreatic lipase, AChE and BuChE, 15-LOX, and antioxidant capacity, which were particularly correlated with bioactive compounds.
Thus, these results indicate that leaves of pome species are attractive, unconventional sources of bioactive compounds for preparing some nutraceutical foods for use in the prevention of selected disease entities, if they will be acceptance by the consumers. Additionally, leaves of pome species can also be used as valuable sources for the pharmaceutical and cosmetic industries. Inhibitory of hyperglycemic (α-glucosidase and α-amylase) and obesity (pancreatic lipase) enzyme. Analysis of: α-amylase, α-glucosidase and pancreatic lipase were determined using the method proposed previously by Wojdyło et al. 30 .
The α-amylase inhibitory activity is based on a result of a reaction of iodine in potassium iodide with the remaining starch after enzymatic hydrolysis after incubation at 37 °C and absorbance was measured at 600 nm. The analysis of α-glucosidase inhibitory activity consists of the reaction of the enzyme with a β-D-glucosidase substrate measured at 405 nm. As in the above analysis, the reference samples contained buffer instead of enzymes and for above analysis the acarbose was included as a positive control.
The analysis of pancreatic lipase inhibitory activity is based on the amount of p-nitrophenol formed from p-nitrophenyl acetate. Basic samples with enzyme and substrate incubated at 37 °C and absorbance was measured at 400 nm. The reference samples contained buffer instead of enzymes and for above analysis the orlistat was used as a positive control.
The results of α-amylase, α-glucosidase, and pancreatic lipase activity are presented as the amount of the sample that is able to reduce enzyme activity by 50% as IC 50 in mg/mL. Inhibitory of 15-lipoxygenase assay. The 15-lipooxygenase inhibitory assay were determined using the method proposed previously by 23 as on a results of a reaction of extract, enzyme and linoleic acid incubated at 37 °C for 20 min. This method defined the increase on absorbance at 210 nm as a result of the formation of conjugate double bonds in the linoleic acid hydroperoxide. Reference samples contained Tris-HCl buffer instead of the enzyme. The results were expressed as a % of inhibition.
Anti-inflammatory activity as cyclooxygenase (COX-1 and COX-2) assay. Anti-inflammatory activity as COX-1 and COX-2 inhibition enzyme was determined according using a protocol described in COX Inhibitor Screening Assay Kit (Cayman, No. 560131). The results of COX-1 and COX-2 are presented as the amount of the sample that is able to reduce enzyme activity by 50% as IC 50 in mg/mL.
Antioxidant capacity by FRAP, ABTS ·+ and ORAC assay. The FRAP (involves determining the ability to reduce Fe +3 ions), ABTS (based on measuring the decrease in the color intensity inversely proportional to the antioxidant content) and ORAC assays (decrease in fluorescence caused by oxidation of a fluorescent substance under the influence of free radicals) were determined using the method proposed previously by 48,49 and 50 , respectively. The 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) diluted in HCl and FeCl 3 × 6H 2 O were mixed with sample extract and after 10 min of reaction the absorption at the 593 nm was measured.
The 2,2′-azine-bis-(3-ethylene-benzothiazoline-6-sulfonic acid (ABTS) were mixed with sample extract and after 6 min of reaction the absorption at the 734 nm was measured. www.nature.com/scientificreports/ The 2,2'-azobis(2-amidinopropane)dihydrochloride was added to sample extract, phosphate buffer, and fluorescein (incubated at 37 °C) were mixed and measured performed every 5 min at an excitation and an emission wavelength 493 and 515 nm, respectively. The blank was a phosphate buffer.
The results for FRAP and ABTS were calculated based on the calibration curve (R 2 = 0.9950) for Trolox concentrations 0.050 to 0.900 mM and 0.100 to 0.900 mM, respectively. The results of ORAC assay were obtained by comparing the surface under the fluorescence decrease curves over time with the surface for pure Trolox solutions (12.5, 25.0, 50.0, and 75.0 μM). The FRAP, ABTS, and ORAC results were expressed in mmol TE (Trolox)/100 g sample.
Statistical analysis. Results are presented as mean values of n = 3 for each cultivar analysis ± standard deviation. Principal Components Analysis (PCA) and Hierarchical Clustering Analysis (HCA) were performed on XLSTAT 2017 (Addinsoft, New York, NY, USA). One-way analysis of variance (ANOVA) by Tukey's test were performed.
Ethic statement. Research did not include any human subjects and animal experiments. All the authors declare that plants were used in accordance with relevant guidelines and regulations. The authors had permission to collect all plant samples used in the study.