Triterpene Acid and Phenolics from Ancient Apples of Friuli Venezia Giulia as Nutraceutical Ingredients: LC-MS Study and In Vitro Activities

Triterpene acid and phenolic constituents from nine ancient varieties of apple (Malus domestica) fruits cultivated in Fanna, Friuli Venezia Giulia region, northeast Italy, were analyzed and compared with four commercial apples (‘Golden Delicious’, ‘Red Delicious’, ‘Granny Smith’ and ‘Royal Gala’). Total phenolic and flavonoid contents were measured by spectrophotometric assays. The quali-quantitative fingerprint of secondary metabolites including triterpene acid was obtained by LC-DAD-(ESI)-MS and LC-(APCI)-MS, respectively. Based on the two LC-MS datasets, multivariate analysis was used to compare the composition of ancient fruit varieties with those of four commercial apples. Significant differences related mainly to the pattern of triterpene acids were found. Pomolic, euscaphyc, maslinic and ursolic acids are the most abundant triterpene in ancient varieties pulps and peels, while ursolic and oleanolic acids were prevalent in the commercial fruits. Also, the content of the phenolic compounds phloretin-2-O-xyloglucoside and quercetin-3-O-arabinoside was greater in ancient apple varieties. The antioxidant (radical scavenging, reducing power, metal chelating and phosphomolybdenum assays) and enzyme inhibitory effects (against cholinesterase, tyrosinase, amylase and glucosidase) of the samples were investigated in vitro. Antioxidant assays showed that the peels were more active than pulps. However, all the samples exhibited similar enzyme inhibitory effects. Ancient Friuli Venezia Giulia apple cultivars can be a source of chlorogenic acid and various triterpene acids, which are known for their potential anti-inflammatory activity and beneficial effects on lipid and glucose metabolism. Our results make these ancient varieties suitable for the development of new nutraceutical ingredients.


Introduction
The consumption of fruits and vegetables is very important in human nutrition. They provide nutrients and non-nutritive constituents with significant biological activities, thus contributing to a healthy diet, with reduction of disease risk. Based on scientific data, nutritionists suggest that increasing fruit and vegetable consumption is one of the best strategies to decrease the burden of several chronic source of health-promoting constituents and for nutraceutical applications offering a new market to these overlooked productions.
In this work we considered nine ancient apple varieties preserved and cultivated in Fanna, Friuli Venezia Giulia (FVG) region, Northeast Italy. Literature data about these apple varieties are scarce. Recently, the volatile constituents of some ancient FVG apples were studied in comparison with extensively-cultivated varieties [32], but to our knowledge information on polyphenols and triterpenes content is still limited as well as their bioactivities such as antioxidant and enzyme inhibitory. Here we reported the chemical characterization of triterpene and phenolic of these old apple cultivars by performing Liquid Chromatography Diode Array Electro Spray Mass Spectrometry n-fragmented, LC-DAD-(ESI)-MSn and Liquid Chromatography Atmospheric Chemical Ionization Mass Spectrometry n-fragmented LC-APCI-MSn analyses. Results revealed the secondary metabolites fingerprint of such ancient apples, furthermore in vitro assays evaluating the antioxidant and enzyme-inhibitory activities were performed thus showing their potential application as a source of novel nutraceutical ingredients.

Secondary Metabolite Fingerprinting of Ancient Apples
Fingerprinting of the secondary metabolites of ancient apple samples resulted in the identification of triterpene acids and polyphenols. In Figure 1 a representative LC-ESI chromatogram (total reconstructed current) showing the fingerprint of the analyzed apple peel sample (H1B) with the main classes of constituents, is reported. Compounds were identified on the basis of their retention times, m/z and collision-induced dissociation spectra plus comparison with authentic standards when available. such fruits as a source of health-promoting constituents and for nutraceutical applications offering a new market to these overlooked productions.
In this work we considered nine ancient apple varieties preserved and cultivated in Fanna, Friuli Venezia Giulia (FVG) region, Northeast Italy. Literature data about these apple varieties are scarce. Recently, the volatile constituents of some ancient FVG apples were studied in comparison with extensively-cultivated varieties [32], but to our knowledge information on polyphenols and triterpenes content is still limited as well as their bioactivities such as antioxidant and enzyme inhibitory. Here we reported the chemical characterization of triterpene and phenolic of these old apple cultivars by performing Liquid Chromatography Diode Array Electro Spray Mass Spectrometry n-fragmented, LC-DAD-(ESI)-MSn and Liquid Chromatography Atmospheric Chemical Ionization Mass Spectrometry n-fragmented LC-APCI-MSn analyses. Results revealed the secondary metabolites fingerprint of such ancient apples, furthermore in vitro assays evaluating the antioxidant and enzyme-inhibitory activities were performed thus showing their potential application as a source of novel nutraceutical ingredients.

Secondary Metabolite Fingerprinting of Ancient Apples
Fingerprinting of the secondary metabolites of ancient apple samples resulted in the identification of triterpene acids and polyphenols. In Figure 1 a representative LC-ESI chromatogram (total reconstructed current) showing the fingerprint of the analyzed apple peel sample (H1B) with the main classes of constituents, is reported. Compounds were identified on the basis of their retention times, m/z and collision-induced dissociation spectra plus comparison with authentic standards when available.  Table 1. As reported in Table 1 the identified apple constituents were summarized according to their main m/z values and fragments. The main classes of analyzed compounds were triterpene acids, flavan-3-ols and proanthocyanidins (PAC), glycosylated flavonoids, dihydrochalcones, and hydroxycinnamic acids that were detectable in all the samples. Signals ascribable to lipid portions were also detected especially in peel samples. Apple triterpene acids present significant bioactivities and have been up to now less considered compared to polyphenols due to the poor UV absorbtion that in many cases prevent the use of LC-DAD or LC-UV methods. The possibility to rapidly identify the triterpene acids in samples can be useful for assessing differences of the various apples related to these compounds. In a previous paper we reported an LC-APCI method was developed and used to characterize apple triterpene acids. [34] As a continuation of that work, here we present the fragmentation pattern of the main triterpenes detected in FVG ancient apples. Typical collision-induced dissociation spectra are reported in supplementary figures showing the different behavior of structurally related derivatives. The apple triterpene acid namely, pomaceic, annurcoic, euscaphic, pomolic, maslinic, oleanolic, betulinic and ursolic acids presents hydroxyl groups in the ring A and E. Tentatively assigned structures of main fragment ions of pomaceic, annurcoic, euscaphic and pomolic acids are reported in Figure 2 and can offer a starting point for the structure elucidation of such triterpene acids by MS/MS fragmentation. The observed fragments losses are ascribable to water, CO 2 and methyl groups. At the same time, we observed that up to MS 3 the pentacyclic triterpene moiety is intact, while several different ion species showed m/z values that suggest water elimination and formation of isolated or conjugated double bonds. Spectra presented in Supplementary Materials of the various derivatives clearly show the possibility to discriminate several different compounds on the basis of MS n behavior. Derivatives differing only for the methyl groups in position 19 and 20, as oleanolic, ursolic, maslinic and corosolic acids were not discriminated enough up to MS 3 . groups in position 19 and 20, as oleanolic, ursolic, maslinic and corosolic acids were not discriminated enough up to MS 3 . The LC-APCI-MS n analysis of apple triterpenes offer the unique opportunity to have structural information on such compounds and to identify the pattern of constituents using low resolution MS n spectrometers. The selectivity of the MS detector represents an improvement in the detection of triterpene acids compared to the proposed methods using (Evaporative Light Scattering Detector) ELSD, UV or diode array detectors, allowing phytochemical screening of different fruits for this class of compounds. The method was also used for quantitative purposes, offering a valuable approach in the quali-quantitative analysis of different apple varieties. In fact, each different derivative was identified on the basis of its [M − H] − and fragmentation pathway, furthermore the triterpene acids amount was measured using oleanolic acid as reference, generally done, for example, for flavonoids or other classes of constituents.  The LC-APCI-MS n analysis of apple triterpenes offer the unique opportunity to have structural information on such compounds and to identify the pattern of constituents using low resolution MS n spectrometers. The selectivity of the MS detector represents an improvement in the detection of triterpene acids compared to the proposed methods using (Evaporative Light Scattering Detector) ELSD, UV or diode array detectors, allowing phytochemical screening of different fruits for this class of compounds. The method was also used for quantitative purposes, offering a valuable approach in the quali-quantitative analysis of different apple varieties. In fact, each different derivative was identified on the basis of its [M − H] − and fragmentation pathway, furthermore the triterpene acids amount was measured using oleanolic acid as reference, generally done, for example, for flavonoids or other classes of constituents. Pulps of all the samples revealed the presence of procyanidins and the analysis of cumulative MS spectra revealed in the first part of the chromatogram (3-10 min) the presence of oligomeric PAC (dimers up to pentamers) while multiple charged ion species ascribable to polymeric PAC (hexamers to decamers) were detectable in the time range from 18-22 min, representative spectra referring to sample E1P ( Figure 3).
Pulps of all the samples revealed the presence of procyanidins and the analysis of cumulative MS spectra revealed in the first part of the chromatogram (3-10 min) the presence of oligomeric PAC (dimers up to pentamers) while multiple charged ion species ascribable to polymeric PAC (hexamers to decamers) were detectable in the time range from 18-22 min, representative spectra referring to sample E1P ( Figure 3).

Multivariate Analysis
The datasets obtained from the LC-ESI and LC-APCI methods, used for the analysis of polyphenols and triterpene acid respectively, were merged together and targeted multivariate data analysis was performed considering triterpene and phenolic derivatives. Figure 4 summarized the results obtained for four commercial and nine ancient apple varieties. PCA clearly shows differences (R2X 0.321, Q2X 0.2285 for pulps and R2X 0.399, Q2X 0.349 for peels), between the two apple groups indicating that the chemical profiles of peels and pulps of the ancient and commercial apples are different. (A)

Multivariate Analysis
The datasets obtained from the LC-ESI and LC-APCI methods, used for the analysis of polyphenols and triterpene acid respectively, were merged together and targeted multivariate data analysis was performed considering triterpene and phenolic derivatives. Figure 4 summarized the results obtained for four commercial and nine ancient apple varieties. PCA clearly shows differences (R2X 0.321, Q2X 0.2285 for pulps and R2X 0.399, Q2X 0.349 for peels), between the two apple groups indicating that the chemical profiles of peels and pulps of the ancient and commercial apples are different. Pulps of all the samples revealed the presence of procyanidins and the analysis of cumulative MS spectra revealed in the first part of the chromatogram (3-10 min) the presence of oligomeric PAC (dimers up to pentamers) while multiple charged ion species ascribable to polymeric PAC (hexamers to decamers) were detectable in the time range from 18-22 min, representative spectra referring to sample E1P ( Figure 3).

Multivariate Analysis
The datasets obtained from the LC-ESI and LC-APCI methods, used for the analysis of polyphenols and triterpene acid respectively, were merged together and targeted multivariate data analysis was performed considering triterpene and phenolic derivatives. Figure 4 summarized the results obtained for four commercial and nine ancient apple varieties. PCA clearly shows differences (R2X 0.321, Q2X 0.2285 for pulps and R2X 0.399, Q2X 0.349 for peels), between the two apple groups indicating that the chemical profiles of peels and pulps of the ancient and commercial apples are different.
(A)   Most of the descriptors are significantly discriminant for the ancient fruit pulps and peels. In addition, a different fingerprint of ancient apples was evidenced with respect to commercial ones. Hydroxycinnamic acids and phloretin-2-O-xyloglucoside were the discriminant phenolic compounds of fruit pulps, while procyanidins with a larger degree of polymerization characterized the commercial samples.
Triterpene acids had a significant role in discriminating peels of the ancient apples with respect to those of commercial ones. Specific compounds can be observed as discriminant of the two groups in particular for pulps, annurcoic, maslinic, pomolic, pomaceic, corosolic acid and oleanolic are present in the ancient apple while they are not detectable in the commercial pulp samples. Phloretin-2-O-xylogucoside peak area is increased in ancient varieties (+35%) while rutin is higher in commercial fruits pulps (+112%) compared to the average area values of each compound in total samples. Related to peels, ancient varieties are characterized by maslinic acid (+44 %), annurcoic acid (+34%), pomaceic acid (not detectable in commercial samples) and quercetin-3-O-arabinoside (+24%) while commercial apple present incremented amounts of oleanolic acid (+174%), and ursolic acid (+175%) compared to ancient varieties.

Spectrophotometric Assays to Assess Total Phenolic and Flavonoid Contents
The total phenolic and flavonoid contents of pulps and peels samples were detected by spectrophotometric assays. Generally, peels had higher concentration of these compounds as compared to pulps ( Figure 5). The highest levels of phenolic and flavonoids were detected in F1B (15.83 mgGAE g −1 ) and I2B (8.05 mgRE g −1 ), respectively. Such preliminary data are useful to have a rapid comparison of all the samples and to assess any possible relation also with antioxidant results. To assess a more accurate variation in the quali-quantitative profile of the apple samples, quantitative data on phenolics were also obtained using LC-DAD-ESI to avoid any interference from other apple constituents. Most of the descriptors are significantly discriminant for the ancient fruit pulps and peels. In addition, a different fingerprint of ancient apples was evidenced with respect to commercial ones. Hydroxycinnamic acids and phloretin-2-O-xyloglucoside were the discriminant phenolic compounds of fruit pulps, while procyanidins with a larger degree of polymerization characterized the commercial samples.
Triterpene acids had a significant role in discriminating peels of the ancient apples with respect to those of commercial ones. Specific compounds can be observed as discriminant of the two groups in particular for pulps, annurcoic, maslinic, pomolic, pomaceic, corosolic acid and oleanolic are present in the ancient apple while they are not detectable in the commercial pulp samples. Phloretin-2-O-xylogucoside peak area is increased in ancient varieties (+35%) while rutin is higher in commercial fruits pulps (+112%) compared to the average area values of each compound in total samples. Related to peels, ancient varieties are characterized by maslinic acid (+44 %), annurcoic acid (+34%), pomaceic acid (not detectable in commercial samples) and quercetin-3-O-arabinoside (+24%) while commercial apple present incremented amounts of oleanolic acid (+174%), and ursolic acid (+175%) compared to ancient varieties.

Spectrophotometric Assays to Assess Total Phenolic and Flavonoid Contents
The total phenolic and flavonoid contents of pulps and peels samples were detected by spectrophotometric assays. Generally, peels had higher concentration of these compounds as compared to pulps ( Figure 5). The highest levels of phenolic and flavonoids were detected in F1B (15.83 mgGAE g −1 ) and I2B (8.05 mgRE g −1 ), respectively. Such preliminary data are useful to have a rapid comparison of all the samples and to assess any possible relation also with antioxidant results. To assess a more accurate variation in the quali-quantitative profile of the apple samples, quantitative data on phenolics were also obtained using LC-DAD-ESI to avoid any interference from other apple constituents.

Pulp Composition
Triterpene acid was present in low amounts compared to peels (p < 0.05) for both groups. The average values were 0.0723 and 0.0363 mg/g for ancient and commercial samples, respectively. Significant differences (p < 0.05) were observed with the ancient varieties being richer in pomolic, pomaceic, maslinic and annurcoic acids than commercials. Higher amounts of ursolic and oleanolic acids were found in commercial apples, representing the 79-95% of total triterpene content. Average PAC and catechin derivatives values in pulps were significantly different (p < 0.05) between ancient (1.06 ± 0.64 mg g −1 ) and commercial (32 ± 0.19 mg g −1 ) varieties ( Figure 6). Total PAC in E1P was of 2.44 mg/g, two-and three-folds higher than the average of ancient and commercial samples. In all the ancient apple pulps, PAC dimers (m/z 577) were the most abundant compounds of PAC ranging from 24-38% of total PAC content. In the commercial samples they ranged from 3-24% of total PAC content. Alike, the concentration of caffeic acid derivatives in ancient apples were significantly (p < 0.05) higher (2.70 mg g −1 ) than in commercial samples (0.41 mg g −1 ). Sample H1P containing 5.4 mg/g of caffeic acid derivatives was thirteen-fold higher than the commercial samples. Triterpene acid was present in low amounts compared to peels (p < 0.05) for both groups. The average values were 0.0723 and 0.0363 mg/g for ancient and commercial samples, respectively. Significant differences (p < 0.05) were observed with the ancient varieties being richer in pomolic, pomaceic, maslinic and annurcoic acids than commercials. Higher amounts of ursolic and oleanolic acids were found in commercial apples, representing the 79-95% of total triterpene content. Average PAC and catechin derivatives values in pulps were significantly different (p < 0.05) between ancient (1.06 ± 0.64 mg g −1 ) and commercial (32 ± 0.19 mg g −1 ) varieties ( Figure 6). Total PAC in E1P was of 2.44 mg/g, two-and three-folds higher than the average of ancient and commercial samples. In all the ancient apple pulps, PAC dimers (m/z 577) were the most abundant compounds of PAC ranging from 24-38% of total PAC content. In the commercial samples they ranged from 3-24% of total PAC content. Alike, the concentration of caffeic acid derivatives in ancient apples were significantly (p < 0.05) higher (2.70 mg g −1 ) than in commercial samples (0.41 mg g −1 ). Sample H1P containing 5.4 mg/g of caffeic acid derivatives was thirteen-fold higher than the commercial samples.

Peels Composition
The triterpene content in ancient apple peels (45.9 mg g −1 ) doubled that of commercial ones (21.5 mg g −1 ) being significantly higher (p < 0.05) for all the ancient varieties compared to the four commercial ones. As for pulp samples, a different pattern of constituents was observed. Cuneataol, pomaceic, annurcoic, maslinic, pomolic and corosolic acids represented the most abundant derivatives in ancient apples, whereas ursolic and oleanolic acids were the predominant compounds in commercial samples. This suggests the possibility to consider triterpene acids as marker compounds to check the authenticity of such ancient varieties.
Catechin, PAC and flavonoid contents of peels of ancient and commercial samples were in the same concentration range both from a qualitative and quantitative point of view. Procyanidin dimer (m/z 577) represented the 18-40% of total PAC content in ancient varieties (19-26% in commercial ones). Average values of PAC derivatives in ancient apple peels were higher than those of commercial apples, being 1.51 and 1.09 mg g −1 , respectively (Figure 7). Average values of caffeic acid derivatives were 1.90 and 0.18 mg/g for ancient and commercial samples, respectively. However, a high variation was observed in the various ancient varieties, with concentrations ranging from 0.19 (E1B) to 4.97 mg/g (I1B). The average amount of flavonoids was similar in the two apple groups being 3.86 and 2.16 mg/g for ancient and commercial samples, respectively. Quercetin

Peels Composition
The triterpene content in ancient apple peels (45.9 mg g −1 ) doubled that of commercial ones (21.5 mg g −1 ) being significantly higher (p < 0.05) for all the ancient varieties compared to the four commercial ones. As for pulp samples, a different pattern of constituents was observed. Cuneataol, pomaceic, annurcoic, maslinic, pomolic and corosolic acids represented the most abundant derivatives in ancient apples, whereas ursolic and oleanolic acids were the predominant compounds in commercial samples. This suggests the possibility to consider triterpene acids as marker compounds to check the authenticity of such ancient varieties.
Catechin, PAC and flavonoid contents of peels of ancient and commercial samples were in the same concentration range both from a qualitative and quantitative point of view. Procyanidin dimer (m/z 577) represented the 18-40% of total PAC content in ancient varieties (19-26% in commercial ones). Average values of PAC derivatives in ancient apple peels were higher than those of commercial apples, being 1.51 and 1.09 mg g −1 , respectively (Figure 7). Average values of caffeic acid derivatives were 1.90 and 0.18 mg/g for ancient and commercial samples, respectively. However, a high variation was observed in the various ancient varieties, with concentrations ranging from 0.19 (E1B) to 4.97 mg/g (I1B). The average amount of flavonoids was similar in the two apple groups being 3.86 and 2.16 mg/g for ancient and commercial samples, respectively. Quercetin

Biological Activities
To provide a full picture for antioxidant properties of the apple samples, different in vitro assays (phosphomolybdenum, metal chelating, reducing power (CUPRAC and FRAP) and radical quenching (DPPH and ABTS)) were performed, data are reported in Figure S5 and Tables S1-S3 in Supplementary Materials. In phosphomolybdenum assay, the samples exhibited similar activities and the best activity was observed in peels of 'Royal Gala' (0.74 mmolTE/g), followed by peels of 'Red delicious' (0.72 mmolTE/g) and 'Granny Smith' (0.66 mmolTE/g) ( Figure 5). In previous papers [35][36][37], the assay was used to assess total antioxidant capacity of extracts. In reducing power assays, F1B exhibited the strongest ability (62.72 mgTE/g for CUPRAC and 37.66 mgTE/g for FRAP), followed by E2B, H1B and B1B ( Figure 5). These results seem to be linked to the fact that these extracts had a higher concentration of total phenolics and flavonoids. This approach was also confirmed by several researchers, who reported a linear correlation between total phenolic and reducing abilities. [38,39]. Regarding radical quenching assays (ABTS and DPPH), B1B and F1B had significant higher activity than other samples ( Figure 5). Similar to total bioactive compounds, peel samples were more active on these radicals than pulp samples. In this sense, observed activity could be attributed to levels of total bioactive compounds and this is accordance with earlier reports [40,41].

Biological Activities
To provide a full picture for antioxidant properties of the apple samples, different in vitro assays (phosphomolybdenum, metal chelating, reducing power (CUPRAC and FRAP) and radical quenching (DPPH and ABTS)) were performed, data are reported in Figure S5 and Tables S1-S3 in Supplementary Materials. In phosphomolybdenum assay, the samples exhibited similar activities and the best activity was observed in peels of 'Royal Gala' (0.74 mmolTE/g), followed by peels of 'Red delicious' (0.72 mmolTE/g) and 'Granny Smith' (0.66 mmolTE/g) ( Figure 5). In previous papers [35][36][37], the assay was used to assess total antioxidant capacity of extracts. In reducing power assays, F1B exhibited the strongest ability (62.72 mgTE/g for CUPRAC and 37.66 mgTE/g for FRAP), followed by E2B, H1B and B1B ( Figure 5). These results seem to be linked to the fact that these extracts had a higher concentration of total phenolics and flavonoids. This approach was also confirmed by several researchers, who reported a linear correlation between total phenolic and reducing abilities. [38,39]. Regarding radical quenching assays (ABTS and DPPH), B1B and F1B had significant higher activity than other samples ( Figure 5). Similar to total bioactive compounds, peel samples were more active on these radicals than pulp samples. In this sense, observed activity could be attributed to levels of total bioactive compounds and this is accordance with earlier reports [40,41].
Chelating ability of transition metal is another important mechanism and this is closely related to production of hydroxyl radical, which is the most dangerous radical. The potent metal chelating abilities were found to be in the peel of C1 (6.76 mgEDTAE/g), 'Royal Gala' (6.53 mgEDTAE/g) and H1 (6.34 mgEDTAE/g). These observation differed for radical scavenging and reducing power assays. This fact could be explained with the presence of non-phenolic chelators such as polysaccharides, or peptides in the extracts [42,43].
The enzyme inhibitory capacity of the apple samples was investigated towards cholinesterases, tyrosinase, amylase and glucosidase due to the very well-known importance of such enzymes in some pathological diseases (Figure 8). The pulps and peels had similar AChE and BChE inhibitory effects. The strongest abilities was observed in the pulp of 'Granny smith' (1.29 mgGALAE/g for AChE) and E2P (0.68 mgGALAE/g for BChE), respectively. Additionally, G1B (29.35 mgKAE/g) and F1B (29.27 mgKAE/g) showed very close tyrosinase inhibitory effects. A similar case was found for A2B (6.99 mgACAE/g) and the pulp of 'Golden delicious' (6.94 mgACAE/g) against amylase. The most potent glucosidase inhibitory effects were found in E2P, D2B and I1B and their results were similar. Generally, the peel samples had higher inhibitory effects when compared to pulp samples against the tested enzymes. From this point, the presence of proanthocyanidins, triterpenoids or hydroxycinnamic acids might be attributed to observed enzyme inhibitory effects. These compounds have been already reported as anti-Alzheimer and anti-diabetic agents [44][45][46][47][48]. Chelating ability of transition metal is another important mechanism and this is closely related to production of hydroxyl radical, which is the most dangerous radical. The potent metal chelating abilities were found to be in the peel of C1 (6.76 mgEDTAE/g), 'Royal Gala' (6.53 mgEDTAE/g) and H1 (6.34 mgEDTAE/g). These observation differed for radical scavenging and reducing power assays. This fact could be explained with the presence of non-phenolic chelators such as polysaccharides, or peptides in the extracts [42,43].
The enzyme inhibitory capacity of the apple samples was investigated towards cholinesterases, tyrosinase, amylase and glucosidase due to the very well-known importance of such enzymes in some pathological diseases (Figure 8). The pulps and peels had similar AChE and BChE inhibitory effects. The strongest abilities was observed in the pulp of 'Granny smith' (1.29 mgGALAE/g for AChE) and E2P (0.68 mgGALAE/g for BChE), respectively. Additionally, G1B (29.35 mgKAE/g) and F1B (29.27 mgKAE/g) showed very close tyrosinase inhibitory effects. A similar case was found for A2B (6.99 mgACAE/g) and the pulp of 'Golden delicious' (6.94 mgACAE/g) against amylase. The most potent glucosidase inhibitory effects were found in E2P, D2B and I1B and their results were similar. Generally, the peel samples had higher inhibitory effects when compared to pulp samples against the tested enzymes. From this point, the presence of proanthocyanidins, triterpenoids or hydroxycinnamic acids might be attributed to observed enzyme inhibitory effects. These compounds have been already reported as anti-Alzheimer and anti-diabetic agents [44][45][46][47][48].

Discussion
Apples are one of the most important fruits in the human diet and in the last thirty years the market has moved towards modern cultivar due to organoleptic and yield issues. For these reasons, in many countries, the ancient cultivars have been considered obsolete and replaced, leading to a loss of fruit biodiversity. Nevertheless in the context of renewed consumer interest in locally produced foods, the ancient varieties are now being re-considered. Another aspect is related to environmentally

Discussion
Apples are one of the most important fruits in the human diet and in the last thirty years the market has moved towards modern cultivar due to organoleptic and yield issues. For these reasons, in many countries, the ancient cultivars have been considered obsolete and replaced, leading to a loss of fruit biodiversity. Nevertheless in the context of renewed consumer interest in locally produced foods, the ancient varieties are now being re-considered. Another aspect is related to environmentally acceptable cultivar practice, and these ancient varieties posses also particular traits of resistance to climate and pathogens. The safeguard of ancient apple varieties appears relevant for the possible exploitation in the food market given the specific organoleptic qualities. But an attractive opportunity to valorize such ancient fruits is to develop their use for nutraceutical purposes, thanks to the high content in health promoting secondary metabolites.
In this context the results obtained in the present paper report for the first time the characterization of nine ancient apple varieties of Friuli Venezia Giulia region. The triterpene acid content result was different than the compared conventional varieties and should be taken into account for the possible use as nutraceutical. In fact, different papers reported the beneficial effects of triterpene acids on glucose metabolism with possible application in glycemic control [28,29]. Oleanolic acid has a potential role in treating drug-induced hepatic steatosis due to its interaction with liver X receptor alpha and pregnane X receptor reducing ligand-induced lipogenesis [49]. In addition, it is well known that different triterpene acids from various natural sources present anti-inflammatory effects. For instance, recent papers showed significant effects of maslinic acid in inflammation [50] and explained part of its activity by NF-κB inactivation [51]. Anti-inflammatory and anti-arthritic effects through NF-κB inactivation were reported for maslinic and pomolic acids [50]. Ancient FVG apple varieties are significant sources of such compounds and further investigations are needed to assess potential anti-inflammatories and glycemic control effects of their constituents.
Significantly higher levels of dihydrochalcones (p < 0.05) and hydroxycinnamic acids (p < 0.05) were measured in peels and pulps of the ancient varieties compared to the commercial ones.
The phenolic composition of the pulp, which represents more than the 95% of the total fruit dry weight, is especially rich in chlorogenic acid and, for the samples H1P, G1P and I1P, in procyanidins. Chlorogenic acid has pronounced effects on glucose and lipid metabolism [49] being considered useful for weight management and obesity. Green coffee beans are in general used for the production of nutraceutical ingredients as they contain chlorogenic acid ranging from 3.5-14% (w/w dry matter) [52]. Among the ancient cultivars, the highest chlorogenic acid contents (0.4-0.5% w/w dry matter) were found in the pulp of I1 and H1 fruits. Apples of the ancient cultivars can be thus considered as a good source of chlorogenic acid offering a different source than green coffee with the advantage of the absence of caffeine and trigonelline.
Preliminary in vitro assays revealed significant antioxidant and inhibitory enzyme activities showing potential usefulness. These data showed the effects on enzymes that are involved in metabolic and degenerative diseases and indicate the need for further investigations into the possible usefulness of such compounds related to Alzheimer's disease, hyperpigmentation and type 2 diabetes. The observed biological effects could be attributed to the phytochemicals in these extracts, such as triterpenoids, proanthocyanidins and hydroxycinnamic acids. Thus, the ancient apples could be regarded as a valuable source for biologically-active compounds in view of nutraceuticals. LC-MS n analysis with different ion sources allowed the identification of secondary metabolites yielding an accurate fingerprinting of these fruits. Ancient FVG apple cultivars can be a source of chlorogenic acid and different triterpene acids, which have been studied in literature for their potential anti-inflammatory activity and beneficial effects on lipid and glucose metabolism.

Plant Materials and Sampling
Apples of nine ancient varieties (10 kg each) were collected with the assistance of Associazione Amatori Mele Antiche (Pordenone, Fanna, Friuli Venezia Giulia, Italy, N 46 • 11 ; E 12 • 45 , 274 m a.s.l.). Fruits were washed and peels and pulps separately cut in thin slices. Samples indicated with "B" and "P" letters refer to peels and pulps, respectively. Sliced peels and pulps were put into a ventilated oven at 55 • C and dried up to constant weight. Samples were then grinded, transferred into Eppendorf tubes (50 mL) and stored at −20 • C until sample preparation and analysis. The nine varieties were indicated with letters as follows: Di Corone A2; Rosso Invernale B1; Limoncello antiche C1; Pomacia D2; De la rosa E1; Belladonna F1; Regina G1; Del Mieli H1; Di Giulio I1. Commercial samples of 'Golden Delicious', 'Red Delicious', 'Granny Smith' and 'Royal Gala' varieties were bought in local stores of Padova, Italy. Samples of peels and pulps were indicated for all the apples varieties with letter P and B respectively. A voucher sample of each apple dried material (peels and pulps) is stored at the Department of Pharmaceutical and Pharmacological Sciences of University of Padova.

Chemicals and Instruments
Methanol, acetonitrile, formic acid, rutin, phloridzin and catechin were obtained from Sigma-Aldrich (Milan, Italy). Procyanidin B2, oleanolic acid, asiatic acid were obtained from Extrasynthese (Genay Cedex, France). An Agilent Eclipse XDB C-18 3.0 × 150 mm (3.5 µm) was used as stationary phase for analytical procedures. For semipreparative HPLC separation of triterpene acids, an Agilent Eclipse C-18 30 × 210 mm (5 µm) stationary phase was used. An Agilent 1100 system equipped with 1100 series diode array, Sedex 60 Evaporative Light Scattering Detector (ELSD) and Agilent 1260 series fraction collector was used for purification of triterpene acids. For LC-DAD-MS analysis an Agilent 1260 chromatograph equipped with 1260 series diode array was used (Agilent, Santa Clara, CA, USA). After the chromatography column a "T" junction split the flow to DAD and Varian MS 500 mass spectrometer (Varian, Santa Clara, CA, USA). MS spectra were acquired using two different approaches, one using Electrospray (ESI) and one Atmospheric Pressure Chemical Ionization (APCI) sources for polyphenols and triterpenes, respectively. For both analyses the spectrometer operated in negative ion mode acquiring spectra in the range m/z 50-2000.

Sample Preparation
Dried pulp and peel samples were weighed in triplicate (500 mg ± 0.1 mg) and transferred in flasks. Fifteen mL of methanol-water (50%) solution was added and ultrasound assisted extraction was performed at room temperature for 15 min. Liquid was decanted and filtered; further 5 mL of solvent was added to solid material and sonicated for further 15 min. The supernatants were combined and volume adjusted to 25 mL with the same solvent. Samples were then filtered through 0.45 µm membrane filters and used for analysis. After the chromatography column a "T" junction split the flow to DAD and Varian MS 500 mass spectrometer with ESI as ion source. Flow rate was set at 500 µL/min. For qualitative purposes, MS n spectra was used for identification of the compound, with the use of reference when available. For quantification, DAD detector was set at 280, 330 and 350 nm. Compounds were identified on the basis of MS data and UV spectra and classified in four classes namely proanthocyanidins, dihydrochalcones, hydroxycinnamic acids and flavonols. For quantification of these classes of compounds the following reference compounds were used namely catechin, phloridzin, chlorogeinc acid and rutin. Standard solutions were prepared for the calibration curves in the concentration range of 5-100 µg/mL: catechin at 280 nm, y = 20.525x + 3.2962 (r 2 = 0.999); phloridzin at 280 nm, y = 87.029x -1.832 (r 2 = 0.9998); chlorogenic acid at 330 nm, y = 47.359x + 439.99 (r 2 = 0.9951); rutin at 350 nm, y = 27.788x + 330.7 (r 2 = 0.9981).

HPLC-(APCI)-MS Analysis
Triterpenes were separated on an Agilent Eclipse XDB C-18 (3.0 × 150 mm) 3.  55). Flow rate was 500 µL/min. After the chromatography column, a Varian MS 500 mass spectrometer was used using APCI as ion source. Identification of triterpene was done on the base of MS n spectra and NMR spectra of isolated compounds as reported in our previous work. [34]. For quantification purposes we adopted oleanolic acid as the reference standard and the calibration curve was obtained in the range 5-150 µg/mL, y = 18755x + 961.31 (r 2 = 0.9992). Quantification was obtained using [M − H] − of each triterpene acid referring to oleanolic acid calibration.

Multivariate Analysis
Data modelling was performed by applying the multivariate technique based on projection. Specifically, principal component analysis (PCA) was used for exploratory data analysis and for highlighting differences of composition between the various grouped samples. Matrix was created merging data obtained by LC-DAD-ESI-MS for the phenolic constituents as well as the LC-APCI-MS for the triterpene acids. Twenty-eight different compounds were used as descriptors of the ancient and commercial apple samples. The data matrix was imported in SIMCA 12 (Umetrics, Sweden) and used to perform PCA analysis. Significant variation of the area of the considered compounds in ancient versus commercial samples were calculated using ANOVA (p < 0.05).

Total Bioactive Compounds
With reference to our previous studies [35], the total amount of phenolics (by standard Folin-Ciocalteu method) and flavonoids (by AlCl 3 method) were determined. Standard compounds (gallic acid (mg GAE/g) for TPC and rutin (mg RE/g) for TFC, respectively) were used to express the obtained results.

Assays for Enzyme Inhibition and Antioxidant Capacity
Tyrosinase, α-amylase, α-glucosidase and cholinesterases (acetylcholinesterase (AChE) and butyrlcholinesterase (BChE)) were selected as the target enzyme and the procedures of these assays were described in our earlier work [35]. Standard inhibitors (acarbose (for α-amylase and α-glucosidase), galantamine (for AChE and BChE), and kojic acid (for tyrosinase)) were used to express the enzyme inhibitor properties.
The antioxidant capacity of the extracts was spectrophotometrically screened by different experiments, namely the ferrozine assay (for chelating abilities), phosphomolybdenum, reduction potentials (by FRAP and CUPRAC assays) and radical scavenging activity (using DPPH and ABTS radicals). Standard compounds (TE/g and EDTAE/g) were used to express the antioxidant properties. The procedures of assays were given reported in our earlier work [35].

Statistical Evaluation for Total Bioactive Compounds and Biological Activity Assays
All tests were carried out in triplicates and findings are expressed as mean value ± SD. One-way ANOVA followed by Tukey's multiple range was done to investigate significant differences (p < 0.05) between the tested samples. SPSS v. 17.0 (SPSS Institute Inc., Cary, NC, USA) was employed for statistical analysis.

Conclusions
The results presented related to the ancient apple fruits of FVG show their phytochemical composition and in vitro bioactivities offering a starting point for these fruits to be exploited as vegetal material for the production of new nutraceutical ingredients. The valorization of this varietal patrimony by nutraceutical application can offer new profitable opportunities for their cultivation and strengthen the preservation of local biodiversity.