The high consumption of fruits and vegetables has been associated to a good health state. Indeed, it is reported that the low mortality rate due to fruit and vegetable consumption is due to the phytochemicals they contain [1
]. Apple fruit (Malus domenica
Borkh) have shown to be one of the most important dietary sources of polyphenols, whose consumption has been associated with human wellness [2
]. It has also been reported that traditional and overlooked apples were proved to be higher sources of phytonutrients than the commercial ones [3
]. Despite their particularity (shape, taste, nutritional values), these old apples are on the verge of extinction because of globalization. Hence, the characterization of old apple cultivars is important to enhance their value and increase their production [4
Polyphenols and triterpenes are secondary metabolites usually found in the apple varieties. Five classes of polyphenols are reported for this fruit: flavan-3-ols/proanthocyanidins (catechin, epicatechin, procyanidin of the group A and B), flavonols (quercetin, rutin, kaempferol), dihydrochalcones (phloretin and phloridzin), hydroxycinnamic acids (chlorogenic acid, p
-coumaric acid, caffeic acid, ferulic acid) and hydroxybenzoic acids (gallic acid) [6
]. The triterpene acids, generally found in the peel, are mainly represented by ursolic acid, oleanolic acid, betulinic acid, and annurcoic acid [7
Many pharmaceuticals of current use with therapeutic applications act mechanistically as enzyme inhibitors. The consumption of dietary polyphenols is linked to the reduction of the incidence of neurodegenerative diseases such as Alzheimer’s disease (AD) [9
]. Some plant extracts rich in polyphenols as those from green tea and blueberry have demonstrated some inhibitory activities against acetylcholinesterase (AChE) exhibiting thus neuroprotective capacity [9
]. α-Glucosidase inhibitors are currently used in the management of type 2 diabetes and hyperglycemia [10
]. Polyphenols from plant extracts have shown to be efficient α-glucosidase and amylase inhibitors and also to block glucose absorption by the inhibition of the Na+
- dependent glucose transporters, SGLT1 and SGLT2; contributing thus to the prevention of hyperglycemia [10
]. One of the strategies of the prevention of obesity, which is becoming a global health issue, is the reduction of the activity of pancreatic lipase, which is involved in the digestion of triglycerides into monoglycerides and free fatty acids readily absorbable [11
]. It is reported that many extracts from foods potentially inhibit lipase enzyme [11
]. Many plant polyphenols have also shown inhibitory properties against monoamine oxidase A (MAO-A), an enzyme involved in depression [12
]. It is suggested that the skin-lightening effect of the crude polyphenolic extract of the acerola fruit and other natural products may be due the tyrosinase activity inhibition in melanocytes, suppressing thus melanogenesis [14
]. Other enzymes such as 5α-reductase and carbonic anhydrase can also be inhibited by some polyphenols [16
]. The study of the inhibitory effect of polyphenols on the biological enzymes can be an important way to counteract and manage many diseases.
The Mela Rosa dei Monti Sibillini (MR) is an ancient apple cultivated since the age of the Roman empire in central Italy at an altitude between 400 and 900 m [4
]. Due to its uniqueness given by its particular shape, smell, taste and shelf life, this apple is regaining attention. To recover and preserve the germplasm of this overlooked old variety, many actions such as the increase of its cultivation and research of the health benefits have been put in place by the local authorities. Our previous studies also showed that samples of this cultivar have a high content of polyphenols and triterpenes such as annurcoic acid which was firstly found in the Annurca cultivar [4
The consumption of the hydroalcoholic extracts of this cultivar has shown protective effects against renal ischemia/reperfusion injury and CCl4
-induced hepatotoxicity in rats [18
The development of nutraceuticals from fruits and vegetables to be employed in the prevention and treatment of different diseases is a great challenge and can lead to huge benefits for the health and the environment [11
]. On this basis, the aim of this work was to assess the inhibitory effects of the peel and pulp methanolic extracts of the Mela Rosa dei Monti Sibillini obtained from dried (DEPe and DEPu respectively) and lyophilised (LEPe and LEPu, respectively) material on different enzymes, namely α-glucosidase, lipase, MAO-A, AChE and tyrosinase (TYR).
2.1. Polyphenols and Triterpenes Composition
The HPLC-DAD-MS analysis of polyphenolic and triterpenic compounds was carried out on the extracts obtained through two different dehydration methods, i.e., drying at 45 ± 5 °C and freeze-lyophilization. The monitored compounds were flavan-3-ols (catechin, epicatechin, procyanidin A2 and procyanidin B2), flavonols (rutin, quercetin, quercetin-3-D-galactoside, kaempferol and kaempferol-3-glucoside), anthocyanins (cyanidin-3-glucoside), hydroxycinnamic acids (p
-coumaric acid, neochlorogenic acid, chlorogenic acid, caffeic acid and trans
-ferulic acid), hydroxybenzoic acid (gallic acid), dihydrocalchones (phloretin and phloridzin) and triterpenes (oleanolic and ursolic acids) as reported in Table 1
and Table 2
HPLC analysis showed that the LEPe (8160.7–12687.5 mg/kg) were richer in polyphenols than DEPe (4520.3–6868.8 mg/kg). Whereas, triterpenes seem to be higher in DEPe (1960.2–21390.4 mg/kg) than in LEPe (903.9–18736.0 mg/kg). The most represented class was triterpenes, followed by flavan-3-ols and dihydrochalcones. In the samples, the most abundant compounds were epicatechin (1740.2–3999.0 and 938.1–2083.5 mg/kg for LEPe and DEPe, respectively), procyanidin B2 (1655.9–2732.7 and 855.0–2041.4 mg/kg for LEPe and DEPe, respectively) and phloridzin (730.2–1809.5 and 387.7–1315.0 mg/kg for LEPe and DEPe, respectively) with an exception for some samples which exhibited a high content of chlorogenic acid (sample 2: 1726.3 and 1551.7 mg/kg for LEPe and DEPe, respectively), quercetin-3-D-galactoside (sample 5: 1313.8 mg/kg for LEPe) and rutin (sample 4:656.7 mg/kg for DEPe; sample 7: 1422.4 and 553.2 mg/kg for LEPe and DEPe, respectively). Ursolic acid was the most abundant phytoconstituent in all samples (583.4–12541.8 mg/kg for LEPe and 1361.7–15088.8 mg/kg for DEPe) apart from sample 2 of LEPe and sample 5 of DEPe where oleanolic acid was present at higher concentrations (10835.5 and 2340.1 mg/kg respectively). The samples with the highest content of the phytochemicals analyzed were samples 6 (31423.5 mg/kg), 2 (26890.9 mg/kg) and 7 (23274.6 mg/kg) for the LEPe, and samples 1 (28250.3 mg/kg), 7 (20256.2 mg/kg) and 2 (20056.9 mg/kg) for DEPe.
In pulp extract analyses, as in the case of peel, the LEPu had a higher content of polyphenol compounds than the DEPu. The most abundant classes present in both DEPu and LEPu were flavan-3-ols and hydroxycinnamic acids. Epicatechin (494.5–1725.2 mg/kg for LEPu and 586.9–1410.9 mg/kg for DEPu), chlorogenic acid (167.9–1946.8 mg/kg for LEPu and 136.0–1438.7 mg/kg for DEPu) and procyanidin B2 (292.1–1262.2 mg/kg for LEPu and 325.6–1015.6 mg/kg for DEPu) were more concentrated in almost all the samples with an exception of the LEPu samples 5, 7 and 8, and DEPu samples 4 and 5 which also showed a high content of catechin (340.1, 195.1 and 416.9 mg/kg for LEPu; 144.9 and 285.3 mg/kg for DEPu). The samples with the highest content of all bioactive compounds analyzed were samples 8 (4242.2 mg/kg), 2 (3408.4 mg/kg) and 5 (3317.6 mg/kg) for LEPu, and samples 8 (3749.6 mg/kg), 6 (3246.6 mg/kg) and 5 (2902.7 mg/kg) for DEPu.
This analysis also showed variability between the samples studied. In the DEPe, a variation of 15.2% of the total polyphenol was noticed and was similar to that of the LEPe. Whereas in the LEPe, the triterpenes content varied highly in respect to that of the DEPe (83.7 and 56.7% respectively). In the pulp samples, the variability was quite similar for both total polyphenols (29.4 and 31.1% for DEPu and LEPu respectively) and triterpenes concentration (41.2 and 41.8% for DEPu and LEPu respectively).
2.2. Enzymatic Inhibitory Effects
2.2.1. α-Glucosidase Inhibition
From the results, both DEPu and LEPu at concentrations of 1 mg/mL inhibited the enzyme at a percentage less than 50% (Figure 1
a). Similarly, almost all LEPe (exception with sample 5) and samples 1, 2, 3 and 4 of DEPe have shown an inhibitory effect less than 50%. While at the same concentration (1 mg/mL), DEPe from samples 5 and 6 inhibited α-GLU with a percentage higher than that of acarbose (90% and 80%, respectively) (Figure 1
b). DEPe and LEPe inhibited α-GLU in a concentration dependent manner (Figure 1
values calculated by nonlinear regression are reported in Table 3
2.2.2. Lipase Inhibition
The extracts showed lipase inhibitory activity (Figure 2
c). Indeed, at concentration of 1 mg/mL lipase was inhibited at a percentage lower than 50% by all the samples even if the pulp extracts in this case seemed more active than the peel ones (Figure 2
a,b). In contrast, at 10 mg/mL of DEPe, the inhibition obtained was 100%. IC50
values were calculated by nonlinear regression (Table 3
2.2.3. MAO-A Inhibition
The extracts were able to inhibit MAO-A in a dose-dependent manner with a similar profile of clorgyline (Figure 3
c). The percentages of inhibition were between 67% and 88% with all the samples studied at 1 mg/mL of extract concentration (Figure 3
a,b). However, there were significant differences between the IC50
of the extracts (DEPe: 533 μg/mL; LEPe: 473 μg/mL; DEPu: 1793 μg/mL and LEPu: 846 μg/mL) and the reference (0.2 μg/mL), calculated by nonlinear regression (Table 3
2.2.4. AChE Inhibition
The extracts had a lower inhibitory effect against AChE than the positive control, galantamine (Figure 4
a–c). At 1 mg/mL, the DEPu seems to be more active (inhibition less than 52%) than the DEPe and LEPe (inhibition less than 27%). On the other hand, LEPu showed no activity against the enzyme. Furthermore, the values of IC50
obtained from extracts (DEPe: 1889 μg/mL; LEPe: 2261 μg/mL and DEPu: 3963 μg/mL) were significantly different from those achieved by galantamine (Table 3
2.2.5. TYR Inhibition
As can be noted from the following figures (Figure 5
a,b), the extracts were not able to inhibit significantly TYR at concentration of 1 mg/mL (inhibition less than 50%), and increasing the concentration to 10 mg/mL, the percentage remains almost the same (between 49% and 60%). IC50
values were calculated by nonlinear regression (Table 3
2.3. Correlation Analysis between Phytochemical Composition and Bioactivity
In order to establish a relationship between phytochemical content and the enzymatic inhibitory capacity of the extracts, correlation analyses were performed for each type of extract. Results showed a strong negative correlation between the content of triterpenes and MAO-A inhibition (Pearson r
Value = −0.8241) in LEPe, without a good correlation between polyphenols and the MAO-A bioassay (Pearson r
Value = −0.2331). A strong positive correlation was found in the DEPu sample between the content of phenolic compounds and tyrosinase inhibition (Pearson r
Value = 0.7924). Additionally, a very strong positive correlation in the analysis was found for the triterpene content versus lipase inhibition (Pearson r
Value = 0.9568) in the same sample (Table 4
Two methods were used in this study for the dehydration of the fresh fruit: drying at 45 ± 5 °C and freeze-lyophilization. HPLC-DAD analysis revealed a higher content of phytochemicals in the LEPe and LEPu than in the DEPe and DEPu that can be due to the neutralization of the degradative enzymes by liquid nitrogen (−195.8 °C) used during the crushing of the fresh samples [2
]. The peel extracts showed the highest content in polyphenols than the pulp ones because of the accumulation of these compounds in the peel according to the ecological role of this part such as protection against ultraviolet radiations, attraction for fruit dispersion and defense against pathogens [20
]. Indeed, it is also known that triterpenes are concentrated on the surface of fruit peel [22
]. This confirms the high concentrations detected in our samples. The variability of the total polyphenols and triterpenes content in extracts obtained from dried and freeze-lyophilized materials showed that the dehydration method used can influence the phytochemical content of a sample and is thus an important parameter to consider during the preparation of samples.
An increasing interest in the utilization of natural products as candidates for drug discovery, coadjuvant or alternative to drugs (food supplements, nutraceuticals) in the treatment of different pathologies has been demonstrated in the last years [23
]. Thus, the present study was conducted in order to evaluate the nutraceutical or pharmaceutical potential of an old Italian apple variety. In particular, it was assayed the possible inhibitory effect of the apple extracts towards the enzymes α-glucosidase (α-GLU), lipase, monoamine oxidase A (MAO-A), tyrosinase (TYR) and acetylcholinesterase (AChE). These enzymes are related to pathologies such as diabetes, obesity, neurodegenerative disorders and melanogenesis. The phytochemical composition, in terms of concentration of phenolics and triterpenes [24
], is strictly related to the inhibition of these enzymes.
In our investigation, the extracts from dried peels inhibited better α-GLU than the ones from freeze-lyophilized peels, especially the samples 5 and 6 that showed a greater activity than the reference compound although without significant differences. This may be the result of the formation of some bioactive compounds during apple drying as reported by Birtic et al. [25
As known α-GLU and lipase are digestive tract enzymes, involved in the metabolism of carbohydrates and fats, respectively. The inhibition of α-GLU can thus have an impact on diabetes treatment due to the reduction of intestinal absorption and decrease of post-lunch insulin values, maintaining the glycemic variations under control [26
]. It is reported that the preventive effects of polyphenols against diseases such as diabetes and obesity may be the results of the modulation of receptors and enzymes such as α-GLU and lipase [10
]. Some flavan-3-ols (e.g., catechin) showed an inhibitory effect against the enzymes α-GLU and lipase [27
]. However, according to our results, we did not find a positive correlation for polyphenols and α-GLU inhibition.
It is reported that procyanidins inhibit the gastrointestinal lipase, thus decreasing the plasma triglycerides [28
]. Triterpenes such as ursolic acid are known to significantly inhibit the pancreatic lipase [29
]. In fact, a very strong positive correlation was found in this study between triterpenes and lipase inhibition in the DEPu samples, which reveals that these compounds are responsible for this activity at least in that extract. Quercetin and other flavonoids contributed significantly to the inhibition of the MAO-A, as well their antioxidant activity is related to the central protective action [30
]. The polyphenols contained in the apple are able to cross the brain-blood barrier and for this reason showed antidepressant activity [31
]. It has previously been reported that the flavonol quercetin and some polyphenol-rich extracts are able to inhibit the enzymes anticholinesterase (AChE) and butyrylcholinesterase (BChE), demonstrating thereby neuroprotective effect against pathologies such as Alzheimer’s disease [9
]. It is noteworthy to mention that in spite of the use of the pulp for food and nutritive purposes, the peel contains a higher proportion of phytochemicals exerting better bioactive potential. There are no significant differences in triterpenes composition between the crude methanolic extracts prepared from dry material and the ones prepared from the freeze-lyophilized material, although the latter showed higher concentrations of polyphenols. Thus, the complex phytochemical composition and the synergism of each phytochemical in the extracts can be the result of the activity seen in the study [32
]. The high phytochemical content of the peel extracts might justify their effectiveness with respect to pulp samples and their use for pharmaceutical applications.
The maximum activities achieved by the different extracts were at high concentrations. Although these doses are not physiological, these are in vitro studies, where the ability of the extracts to interact with the different enzymes is confirmed. In order to corroborate this action at a physiological level, subsequent studies should be considered.
This study investigated the phytochemical composition as well as the enzyme inhibitory properties of the extracts of an overlooked traditional apple, the Mela Rosa dei Monti Sibillini. Its polar extracts, especially those obtained from the peel have demonstrated a rich content of bioactive compounds such as flavan-3-ols, flavonols, dihydrocalchones, and triterpenes. The freeze-lyophilization dehydration method was more effective in maintaining the phenolic constituents than the drying method. Some extracts have demonstrated inhibitory properties against α-GLU, lipase and MAO-A. The extracts obtained from the peel dried material exert better bioactivities than those obtained from lyophilized material. These results thereby demonstrated that this variety is a potential source of bioactive compounds for the production of pharmaceuticals or nutraceuticals to be used for the prevention and co-treatment of pathologies such as diabetes, obesity, Alzheimer’s disease, depression, and hypermelanosis.