Journal of Functional Foods 25 (2016) 477–485
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Poncirus trifoliata (L.) Raf.: Chemical composition, antioxidant properties and hypoglycaemic activity via the inhibition of α-amylase and α-glucosidase enzymes R. Tundis a,*, M. Bonesi a, V. Sicari b, T.M. Pellicanò b, M.C. Tenuta a, M. Leporini a, F. Menichini a, M.R. Loizzo a a
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Via P. Bucci – Edificio Polifunzionale, 87036 Rende, Cosenza, Italy b Department of Agricultural Science, Mediterranean University of Reggio Calabria, Via Graziella, Feo di Vito, 89123 Reggio, Calabria, Italy
A R T I C L E
I N F O
A B S T R A C T
The aim of this study was to investigate the phytochemical content of Poncirus trifoliata (L.)
Received 2 May 2016
Raf. (Rutaceae) and to assess its hypoglycaemic and antioxidant effects. Hesperidin, naringin
Received in revised form 22 June
and chlorogenic acid were the most abundant compounds of the juice. Peel essential oil
showed that limonene, myrcene, p-cimene and β-pinene were its main components. The
Accepted 27 June 2016
antioxidant activity was studied through different in vitro tests. The hypoglycaemic effects
were analysed via inhibition of carbohydrate-hydrolysing enzymes. IC50 values in the range 30.38–39.25 µg/mL were found respectively for juice and peel oil in the DPPH test. Juice in-
hibited α-amylase and α-glucosidase with IC50 values respectively of 138.14 and 81.27 µg/
Poncirus trifoliata (L.) Raf
mL. Polyphenolic compounds identified in the juice were investigated. While neoeriocitrin
displayed the strongest antioxidant activity and inhibited α-amylase (IC50 of 4.69 vs 77.45 µM
for acarbose), didymin was found to be the most bioactive flavonoid against alpha-
glucosidase (IC50 of 4.20 vs 54.99 µM for acarbose).
© 2016 Elsevier Ltd. All rights reserved.
The metabolic disease diabetes mellitus is rapidly expanding to the extent that it is expected to affect about 439 million people by 2030 (Shaw, Sicree, & Zimmet, 2010). Type 2 diabe-
tes is a progressive condition, whereby the human body eventually resists the effects of insulin and/or progressively loses the capacity to produce sufficient insulin from pancreatic β-cells (WHO, 2014). Pancreatic β-cells become dysfunctional due to persistent high glucose or lipid levels, the release of inflammatory
* Corresponding author. Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, CS, Italy. Fax: +39984493107. E-mail address: [email protected]
(R. Tundis). Chemical compounds: Didymin (PubChem CID: 16760075); Hesperetin (PubChem CID: 72281); Hesperidin (PubChem CID: 10621); Isorhamnetin (PubChem CID: 5281654); Naringin (PubChem CID: 442428); Narirutin (PubChem CID: 442431); Neoeriocitrin (PubChem CID: 114627); Neohesperidin (PubChem CID: 442439); Poncirin (PubChem CID: 442456); Rhamnetin (PubChem CID: 5281691). http://dx.doi.org/10.1016/j.jff.2016.06.034 1756-4646/© 2016 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 25 (2016) 477–485
mediators and/or because of oxidative stress, thus producing an increased amount of reactive oxygen species (ROS) (Giacco & Brownlee, 2010). Failure of insulin-stimulated glucose uptake by fat and muscle, in fact, produces high glucose levels in the blood. An increased glucose flux subsequently reduces antioxidant defences while enhancing oxidant production through several pathways. Oxidative stress plays a key role in the development of diabetes complications. Many studies have demonstrated that fruit consumption is associated with disease prevention and/or protection, emphasizing the potential of using natural compounds in the treatment of type 2 diabetes and in managing its complications (Kyriakis et al., 2015; Ríos, Francini, & Schinella, 2015; Tundis, Loizzo, & Menichini, 2010). The Rutaceae family, consisting of several genera including Citrus and Poncirus, is rich in flavonoids, limonoids, and vitamin C, which have proven to have health-promoting properties (Hollman & Katan, 1999; Tundis, Loizzo, & Menichini, 2014). Poncirus trifoliata (L.) Raf., a deciduous or semi-deciduous shrub native to China and Korea, also known as “trifoliate orange”, is closely related to the genus Citrus. Its bitter fruits are commonly used in dried and powdered marmalades (Reuther, Batchelor, & Webber, 1989). P. trifoliata has been widely used in traditional medicine to treat gastro-intestinal disorders, including digestive ulcers, gastritis, dysentery, and inflammation (Lee, Lee, Kim, & Jeong, 2009; Zhou et al., 2007). Several works have highlighted the antiinflammatory, antibacterial, anti-anaphylactic effects, as well as the antitumour activity of P. trifoliata (Kim, Kim, & Park, 1999; Rahman, Siddiqui, Jakhar, & Kang, 2015; Yi et al., 2004). Phenolic acids (mainly hydroxycinnamic acids), flavonoids (mainly glycosides such as hesperidin and narirutin), coumarins, alkaloids, triterpenoids and sterols are the main classes of phytochemicals identified in P. trifoliata fruits, roots, bark and seeds (Chung et al., 2011; Eom et al., 2011; Feng, Wang, Cai, Zheng, & Luo, 2010; Jayaprakasha et al., 2007; Kim et al., 2011; Starkenmann, Niclass, & Escher, 2007; Wu, Ru, Huang, & Furukawa, 1986). In particular, P. trifoliata seeds are also a source of 7α-oxygenated limonoids (Bennet & Hasegawa, 1982). A literature survey revealed that scant studies have been conducted on the characterization of volatiles. Limonene, myrcene and β-phellandrene have been identified as the most abundant constituents of peel essential oil (Heinrich, Schultze, & Wegener, 1980). The volatile compounds of flavedo, pulp and seeds from two cultivars, namely, P. trifoliata var. trifoliata and var. monstrosa, grown in Italy, were analysed in a recent study (Papa et al., 2014). Peel essential oils were mainly characterized by monoterpene hydrocarbons, with limonene and myrcene as the most representative components. The present study was designed to investigate the chemical profile, and the antioxidant and hypoglycaemic properties of P. trifoliata juice, seed extracts and peel essential oil. For this purpose, high performance liquid chromatography-diode array detection (HPLC-DAD), gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) analyses were performed. The antioxidant activity was investigated through three different in vitro assays, namely, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability power (FRAP), and β-carotene
bleaching tests. Hypoglycaemic properties were investigated via the inhibition of carbohydrate-hydrolysing enzymes, α-amylase and α-glucosidase. The inhibition of these enzymes, together with the consequent decrease of post-prandial hyperglycaemia, is of particular interest as it represents one of the therapeutic approaches to managing diabetes type 2.
Materials and methods
Chemicals and reagents
Caffeic acid and chlorogenic acid were purchased from SigmaAldrich Chem. Co. (Milwaukee, WI, USA). Hesperetin, narirutin, naringin, hesperidin, neohesperidin, quercetin, rhamnetin, isorhamnetin, rutin, neoeriocitrin, didymin and poncirin were supplied by Extrasynthese (Genay, France). Acetonitrile, formic acid and water were obtained from Carlo Erba Reagents (Milan, Italy). Ethanol, NaOH, chloroform, sodium acetate buffer, and phosphate buffer were obtained from VWR International s.r.l. (Milan, Italy). β-Carotene, tripyridyltriazine (TPTZ), butylated hydroxytoluene (BHT), propyl gallate, 2,2diphenyl-1-picrylhydrazyl (DPPH), linoleic acid, Tween 20, ascorbic acid, α-amylase from porcine pancreas (EC 184.108.40.206), and α-glucosidase from Saccharomyces cerevisiae (EC 220.127.116.11) were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). Acarbose from Actinoplanes sp. was purchased from Serva (Heidelberg, Germany).
P. trifoliata fruits were collected in October 2014 in the Botanic Garden of the University of Calabria (Italy), and were identified by Dr. N.G. Passalacqua, Natural History Museum of Calabria and Botanic Garden at the same University. Fruits were examined for integrity and the absence of dust and insect contamination.
Eighty-seven fruits (3.16 kg) were squeezed and the juice (300 mL) was centrifuged and filtered until analysis. Foreign materials, including peels etc. were removed in order to obtain P. trifoliata seeds. A quantity of seeds (657 g) was exhaustively extracted by methanol at room temperature (5 × 700 mL). Immediately after collection, the peels (1.37 kg) were subjected to hydro-distillation in a Clevenger type apparatus for 3 h. The yield (w/w) was 0.14%, and the oil was collected in dark-brown sealed glass vials and stored at −20 °C until analysis.
2.4. High performance liquid chromatography-diode array detection (HPLC-DAD) analyses P. trifoliata juice and seeds extract were analysed using a Knauer (Asi Advanced Scientific Instruments, Berlin) system equipped with two pumps (Smartiline Pump 1000), a Rheodyne injection valve (20 µL), and a photodiode array detector UV/VIS
Journal of Functional Foods 25 (2016) 477–485
equipped with a semi micro-cell. Data processing was carried out with the support of Clarity Software (Chromatography Station for MS Windows). Compounds were separated on a Knauer RP C18 column (250 mm × 4.6 mm, 5 µm). The mobile phase was water/formic acid (99.9:0.1, v/v; solvent A) and acetonitrile/formic acid (99.9:0.1, v/v; solvent B). The gradient profile was 0.01–20.00 min 5% B isocratic; 20.01–50.00 min, 5–40% B; 50.01–55.00 min, 40–95% B; 55.01–60.00 min 95% B isocratic (La Torre, Saitta, Vilasi, Pellicanò, & Dugo, 2006). The column temperature recorded was 30 °C and the flow rate 1.0 mL/ min. Samples were filtered through a 0.45 µm millipore filter (GMF Whatman) before injection took place. The injection volume was 20 µL. Peaks were monitored at 280 and 350 nm. Fourteen selected compounds belonging to different phenolic classes (caffeic acid, chlorogenic acid, didymin, hesperidin, hesperetin, naringenin, naringin, narirutin, neohesperedin, neoeriocitrin, poncirin, quercetin, rhamnetin, and rutin) were quantified. A standard mixture was prepared by adding an accurately weighed amount of each compound (100 mg) to a 100 mL volumetric flask and was brought to the mark with methanol (90:10). A calibration straight for each standard was obtained by analysing the standard solution diluted at different concentrations. All solutions were filtered through a 0.45 µm millipore filter (GMF Whatman) and injected into the HPLC system to determine retention times. Identification and quantification were carried out based on recorded retention times in comparison with authentic standards. Analyses were performed in triplicate.
2.5. Gas chromatography (GC) and gas chromatography– mass spectrometry (GC–MS) analyses P. trifoliata essential oil was analysed by means of gas chromatography (GC), using Shimadzu GC17A gas chromatograph (Shimadzu, Milan, Italy) fitted with a HP-5 MS capillary column (30 m × 0.25 mm i.d.; 0.25 µm film thickness) (Agilent, Milan, Italy) and controlled by Borwin Software. Flame ionization detection (FID) was performed at 280 °C. Nitrogen was the carrier gas (1 mL/min). Column temperature was initially kept at 50 °C for 5 min, and then gradually increased to 280 °C at 13 °C/ min, held for 10 min at 280 °C. Diluted P. trifoliata essential oil (1/100 v/v, in n-hexane) was injected (1.0 µL). Gas chromatography–mass spectrometry (GC–MS) analyses were performed on a Hewlett-Packard 6890 gas chromatograph fitted with a fused silica HP-5 capillary column (30 m length, 0.25 mm i.d., 0.25 µm film thickness). Ionization energy voltage 70 eV was used. The carrier gas was helium and the gas chromatographic conditions were as given above. Constituents were tentatively identified through gas chromatography, comparing their retention indices either with those in the literature or with those of authentic compounds available in our laboratories. Retention indices were determined in relation to a homologous series of n-alkanes (C9–C31) under the same operating conditions. Further identification was made by comparing their mass spectra on both columns with either those stored in Wiley 275 and NIST 98 libraries, or with mass spectra from the literature and from our in-house library (Adams, 2007; Davies, 1990; Jennings & Shibamoto, 1980). Component relative concentrations were calculated based on GCFID peak areas without using correction factors.
Physico-chemical parameters of P. trifoliata juice
The pH was measured in sample juices by pH meter (Basic Model 20, Crison) and the total titratable acidity (TA) was assessed by titration with NaOH (0.1 N) to pH 8.1, and expressed as citric acid %. The ascorbic acid content was evaluated using iodometric titration with an iodine 0.01 N solution. Results are expressed in mg/100 mL of juice. The total soluble solids were estimated by a digital refractometer PR-201α (Atago, Tokyo, Japan) and expressed as °Brix (sucrose percentage) at 20 °C. Fresh-juice colour was measured at 25 °C using a Konica Minolta CM-700/600d spectrophotometer (Konica Minolta Sensing, Japan). Data were expressed as L* (lightness/darkness in a range 0–100), a* (greenness/redness in a range between −60 and +60) and b* (blueness/yellowness in a range between −60 and +60).
2.7.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay Measurement of the radical scavenging activity was performed by DPPH assay according to the procedure afore described (Loizzo et al., 2010). The DPPH assay is based on the measurement of the scavenging ability of antioxidants towards DPPH radicals. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine. When DPPH reacts with an antioxidant, which can donate hydrogen, it is reduced.The change in colour from violet to yellow was read at 517 nm (Perkin Elmer Lambda 40 UV/VIS spectrophotometer). A decrease in the absorbance of the DPPH solution indicates an increase of DPPH radical scavenging activity. The positive control was ascorbic acid.
The ferric reducing ability power (FRAP) assay
The reduction of TPTZ (2,4,6-tripyridyl-S-triazine)-Fe3+ to the TPTZ–Fe2+ in the presence of antioxidants is measured in the FRAP assay (Loizzo et al., 2010). Concisely, 0.2 mL of sample solutions (concentration of 1 µg/mL) were mixed with 1.8 mL of freshly prepared FRAP reagent, and the absorption of the reaction mixture was measured at 595 nm. FRAP value represents the ratio between the slope of the linear plot for reducing Fe3+– TPTZ reagent by P. trifoliata samples compared to the slope of the plot for FeSO4.
β-Carotene bleaching test
The β-carotene bleaching assay is based on the oxidation of linoleic acid. Linoleic acid hydroperoxides react with β-carotene resulting in the rapid disappearance of colour. The presence of an antioxidant can obstruct the extent of β-carotene by acting on linoleate free radicals and other free radicals formed in the system. Therefore, the absorbance rapidly decreased in samples without antioxidants, whereas they maintained their absorbance and colour for a longer period in the presence of an antioxidant. Propyl gallate was used as positive control. In this assay, 1 mL of β-carotene (0.2 mg/mL) was added to 0.02 mL of linoleic acid and 0.2 mL of 100% Tween 20.29. After evaporation of the solvent and dilution with water, 5 mL of the emulsion was transferred into different tubes containing 0.2 mL of P. trifoliata samples at different concentrations. The absorbance was measured at 470 nm.
Journal of Functional Foods 25 (2016) 477–485
Inhibition of carbohydrate-hydrolysing enzymes
The α-amylase inhibition assay
The α-amylase inhibition assay was performed as previously described (Iauk et al., 2015). The enzyme solution was prepared by adding 0.0253 g of enzyme in 100 mL of cold distilled water. The starch solution was prepared by stirring 0.125 g of potato starch in 25 mL of sodium phosphate buffer 20 mM and sodium chloride 6.7 mM (65 °C for 15 min). The colorimetric reagent was prepared by mixing a sodium potassium tartrate solution and 96 mM 3,5-dinitrosalicylic acid solution. P. trifoliata juice, seed extract, peel essential oil and control were added to the starch solution and left to react with α-amylase solution at 25 °C for 5 min. Acarbose was used as positive control. The percentage of enzyme inhibition (% I) was calculated spectrophotometrically at 540 nm by using the following equation:
⎛ [maltose] test ⎞ ⋅ 100 %I = 100 − ⎜ ⎝ [maltose] control ⎟⎠
The α-glucosidase inhibition assay
In α-glucosidase inhibition assay a maltose solution was prepared by dissolving 12 g of maltose in 300 mL of 50 mM sodium acetate buffer (Iauk et al., 2015). The enzyme solution was prepared by adding 1 mg of enzyme (10 units/mg) in 10 mL of icecold distilled water. DIAN solution was prepared by dissolving 1 tablet in 25 mL of distilled water. PGO system–colour reagent solution was obtained by dissolving 1 capsule in 100 mL of icecold distilled water. Samples and control were added to maltose solution and left to equilibrate for 5 minutes at 37 °C. The addition of α-glucosidase solution started the reaction. After 30 minutes of incubation at 37 °C, the reaction was stopped by adding a solution of perchloric acid. The supernatant of tube of step one was mixed with DIAN and PGO and was left to incubate at 37 °C for 30 min. Acarbose was used as positive control. The percentage of enzyme inhibition (% I) was calculated by using spectrophotometric data at 500 nm and by the equation:
⎛ [glucose] test ⎞ ⋅ 100 %I = 100 − ⎜ ⎝ [glucose] control ⎟⎠
Statistical analysis of data
The half maximal inhibitory concentration (IC50) was calculated by nonlinear regression with the use of GraphPad Prism version 6 for MS Windows (GraphPad Software, San Diego, CA, USA). The concentration–response curve was obtained by plotting the percentage inhibition vs. concentration. Differences concerning parameters were analysed using the one-way ANOVA test and multi-comparison Dunnett’s test.
Results and discussion
Chemical profile of P. trifoliata
P. trifoliata juice and seeds were investigated for their polar constituents by HPLC-DAD. Peels were subjected to hydrodistillation and analysed by GC-FID and GC–MS. Flavonoids constitute one
of the most important groups of naturally occurring phenols that are beneficial as protective agents for plants and for human health (Arena, Fallico, & Maccarone, 2001; Ferlazzo et al., 2015; Rotelli, Guardia, Juárez, De La Rocha, & Pelzer, 2003; Shen, Ko, Tseng, Tsai, & Chen, 2004). P. trifoliata juice was characterized by having a large amount of flavonoids, in particular flavanones (Deterre et al., 2013). Herein, fourteen compounds were chosen as markers and quantified in P. trifoliata juice and seeds extract. Data are reported in Table 1. Nine flavanones, namely, narirutin, naringin, hesperidin, neohesperidin, neoeriocitrin, didymin, poncirin, hesperetin and naringenin, were identified and quantified in the juice. Consistently with previous studies (Deterre et al., 2013; Nogata et al., 2006), hesperidin was the dominant flavanone glycoside (129.33 µg/mL), while hesperetin was the principal flavanone aglycones (55.13 µg/mL). Naringin was the second most abundant flavanone in P. trifoliata juice (115.79 µg/mL), followed by didymin (78.83 µg/mL) and narirutin (75.73 µg/mL). Lower values of naringin have been previously found (Rajkumar & Jebanesan, 2008). Neohesperidin and naringenin were the least abundant flavanones in the samples (respectively 32.75 and 28.65 µg/mL). Three flavones, namely rutin, quercetin and rhamnetin, were identified. Rutin was the most abundant with a value of 1.85 µg/mL of juice, followed by quercetin (0.76 µg/ mL). Unlike the flavanones, their content in the juice was lower. The two phenolic acids, caffeic acid and chlorogenic acid, were quantified, and the latter was found to be the most abundant (112.54 µg/mL). Amongst the selected standards, four compounds were detected in the methanol extract of seeds. Naringin was the most abundant (156.42 µg/g ex), followed by neohesperedin (80.12 µg/g ex), narirutin (37.62 µg/g ex) and caffeic acid (32.85 µg/g ex). The quality parameters, including pH, total soluble solids, total acidity, ascorbic acid content, and colour of P. trifoliata juice,
Table 1 – Identified constituents of P. trifoliata juice and seed extracts. Compound Flavanone-O-glycosides Narirutin Naringin Neohesperedin Hesperidin Neoeriocitrin Didymin Poncirin Flavanone aglycones Hesperetin Naringenin Flavone-O-glycosides Rutin Flavone aglycones Quercetin Rhamnetin Phenolic acids Caffeic acid Chlorogenic acid
Seed extracts (µg/g ex)
75.73 ± 0.03 115.79 ± 0.06 32.75 ± 0.04 129.33 ± 0.12 12.44 ± 0.14 78.83 ± 0.12 49.37 ± 0.07
37.62 ± 0.53 156.42 ± 0.35 80.12 ± 0.25 nd nd nd nd
55.13 ± 0.10 28.65 ± 0.08
1.85 ± 0.06
0.76 ± 0.07 0.37 ± 0.06
18.46 ± 0.20 112.54 ± 0.22
Data are expressed as mean ± S.D (n = 3). nd: not detected.
32.85 ± 0.11 nd
Journal of Functional Foods 25 (2016) 477–485
were investigated. These parameters are important for the sensory characteristics of fruits (Humayun, Gautam, Madhav, Sourav, & Ramalingam, 2014). The values of total acidity (1.9 g citric acid/100 mL) and total soluble solids (14.0 ° Brix) were in agreement with those found in the literature (Deterre et al., 2013). P. trifoliata juice has a pH of 3.17 and it is a rich source of ascorbic acid (352.5 mg/L). Hunter parameters (L*, a* and b*) of juice were measured. P. trifoliata juice showed the brightness colour coordinate L* of 9.57, the chromaticity coordinate a* of 2.31, and b* of 9.96 (greenness/redness in a range between −60 and +60). A Chroma value (C*) of 10.22 was calculated. The chemical composition of the essential oil of P. trifoliata peel was analysed by GC-FID and GC–MS (Table 2). Fortyeight constituents, representing 98.62% of the total oil composition, were identified. The essential oil was mainly composed of monoterpene hydrocarbons (76.26%). Limonene (41.73%) was the main component, followed by myrcene (15.68%), (E)-β-ocimene (5.05%), α-phellandrene (4.11%) and β-pinene (3.95%). Sesquiterpene hydrocarbons were also present in good amount (8.18%) with trans-caryophyllene (3.59%) and β-farnesene (1.16%) as the principal ones. Oxygenated monoterpenes accounted for 3.06%, where linalool (0.69%) and nerol (0.65%) were the main compounds. Oxygenated sesquiterpenes constituted a minor quantity (1.48%) with farnesol being the most representative. The abundance of esters, such as ethyl octanoate (2.12%) and ethyl hexanoate (2.22%), was noteworthy. Esters are, in fact, significant aroma constituents of many fruits. Monoterpene hydrocarbons have been reported as the main class of volatile constituents of P. trifoliata var. trifoliata and var. monstrosa peel essential oils (Papa et al., 2014). Limonene (41.3– 54.1%) and myrcene (18.2–23.2%) were the main representative compounds of this fraction, followed by β-pinene (2.2–4.8%) and (E)-β-ocimene (0.7–4.2%).
Several methods have been developed to determine antioxidant capacity; the most frequently used are in vitro methods based on capturing or scavenging free radicals generated in the reaction or in the reduction of metal ions. Antioxidant activity is a complex process, which includes the decomposition of peroxides, as well as free radical scavenging activity, reducing ability, prevention of hydrogen abstraction, and binding of transition metal ion catalysts. For this reason, P. trifoliata samples were investigated in this work for their potential antioxidant capacity by using three in vitro methods (DPPH, β-carotene bleaching and FRAP) that measure different types of antioxidant functions (Table 3). P. trifoliata juice, seeds extract and peel essential oil exerted DPPH radical scavenging activity in a concentration-dependent manner. IC50 values in the range 30.38–39.25 µg/mL for juice and peel essential oil were respectively found. Significant differences were recorded in the β-carotene bleaching test, in which seed extract showed the highest protection of lipid peroxidation with an IC50 value of 46.13 µg/mL after 60 minutes of incubation. A twice as lower activity was found for peel essential oil and juice (IC50 value of 84.89 and 86.77 µg/mL, respectively). Mitochondrial dysfunction and ROS are often implicated in diseases involving oxidative stress and elevated iron. The latter is a com-
Table 2 – The main chemical components of P. trifoliata essential oil. Componentsa
Relative amount (%)
α-Pinene Sabinene β-Pinene Myrcene α-Phellandrene α-Terpinene p-Cimene Limonene (E)-β-Ocimene γ-Terpinene Terpinolene p-Mentha-1,3,8-triene Linalool Terpinen-4-ol α-Terpineol Nerol Geraniol Neryl acetate Geranyl acetate α-Cubebene trans-Caryophyllene β-Farnesene α-Humulene Germacrene B β-Elemene γ-Cadinene δ-Cadinene (E)-Nerolidol Spathulenol (E,Z)-Farnesol Ethyl hexanoate Hexyl butanoate Nonanal Ethyl octanoate Decanal Ethyl decanoate Dodecanal Ethyl laurate Myristic acid Ethyl myristate Octadecane Nonadecane Eicosane Ethyl linoleate Ethyl stearate Pentacosane Heptacosane Nonacosane
938 973 980 993 1005 1012 1025 1030 1052 1057 1086 1108 1098 1176 1189 1236 1240 1370 1388 1352 1415 1441 1455 1554 1387 1515 1526 1564 1578 1742 1000 1065 1102 1198 1205 1396 1417 1601 1780 1793 1800 1900 2000 2160 2193 2500 2700 2900
1.19 1.17 3.95 15.68 4.11 tr 1.50 41.73 5.05 0.70 0.54 0.46 0.69 tr 0.30 0.65 0.52 0.34 0.56 0.28 3.59 1.16 0.72 0.51 0.59 0.20 0.73 0.42 0.44 1.02 2.22 0.98 tr 2.12 0.42 0.72 0.22 0.46 0.26 0.31 0.14 0.12 0.35 0.23 0.35 0.42 0.32 tr
1,2,3 1,2,3 1,2,3 1,2,3 1,2 1,2,3 1,2 1,2,3 1,2 1,2,3 1,2,3 1,2 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2 1,2 1,2 1,2,3 1,2 1,2 1,2 1,2 1,2,3 1,2,3 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2 1,2,3 1,2,3 1,2,3 1,2 1,2 1,2,3 1,2,3 1,2,3
Components are listed in order of their elution from an HP-5MS column. Retention Index (RI) on HP-5 MS column. b M: Identification Methods: 1, comparison of retention times; 2, comparison of mass spectra with MS libraries, 3, comparison with authentic compounds. tr: trace (<0.1%).
ponent of numerous oxidases and oxygenases and an essential element in the utilization of oxygen. Data obtained from the FRAP test demonstrated that P. trifoliata samples are not very active as antioxidant by reduction of iron. Seed extracts and juice respectively showed values of 3.42 and 0.64 µM Fe(II)/g. Peel essential oil was not active at the tested concentration.
Journal of Functional Foods 25 (2016) 477–485
Table 3 – The antioxidant activity of P. trifoliata. P. trifoliata
Juice Peel oil Seed extracts Positive control Ascorbic acid Propyl gallate BHT
β-carotene bleaching test
30.38 ± 2.5*** 39.25 ± 2.3*** 33.34 ± 2.1***
86.77 ± 3.7*** 84.89 ± 3.7*** 46.13 ± 2.4***
0.64 ± 0.08*** NA 3.42 ± 0.009***
5.0 ± 0.8 1.0 ± 0.04 63.2 ± 4.3
Data are expressed as mean ± S.D (n = 3). NA: not active. One-way ANOVA ***p < 0.0001 followed by a multicomparison Dunnett’s test: ***p < 0.01 compared with positive control.
Eom et al. (2011) have investigated dried powdered P. trifoliata extracts and fractions for its antioxidant potential, using several assays, amongst which DPPH, hydroxyl, alkyl radicals, and superoxide. Water extract and dichloromethane fraction showed the most promising scavenging activity against reactive radicals. In addition, P. trifoliata water extract reduced the hydrogen peroxide-induced intracellular reactive oxygen species on CCL 13 cell line and improved cell viability against hydrogen peroxide-induced oxidative damage. Natural products like phenols and flavonoids proved to be efficient antioxidant agents. Given that several synthetic antioxidants have demonstrated to be toxic and/or mutation inducers, many researchers have directed their studies in search of natural antioxidants. P. trifoliata juice has proven to be a rich source of flavonoids and ascorbic acid. Table 4 reported the antioxidant properties of narirutin, poncirin, didymin, naringin, hesperidin and neoeriocitrin. Tested flavonoids demonstrated a DPPH radical scavenging activity in a concentration-dependent manner. The most active was neoeriocitrin with an IC50 value of 2.85 µM, followed by hesperidin and naringin (IC50 values respectively of 16.54 and 21.53 µM). Interesting results were obtained from the β-carotene bleaching test in comparison to the positive control propyl gallate (IC50 value of 4.71 µM) with neoeriocitrin (IC50 value of 3.18 µM) and narirutin (IC50 value of 6.72 µM). Flavonoids were
Table 4 – The antioxidant activity (IC50 µM) of identified flavonoids. Flavonoids
β-Carotene bleaching test
Narirutin Poncirin Didymin Naringin Hesperidin Neoeriocitrin Positive control Ascorbic acid Propyl gallate
45.30 ± 1.4 44.23 ± 1.6 36.16 ± 1.5 21.53 ± 2.0 16.54 ± 1.3 2.85 ± 0.04
6.72 ± 0.09 47.40%a 35.42%a 13.44 ± 1.1 10.81 ± 1.0 3.18 ± 0.08
28.40 ± 2.1 4.71 ± 0.8
Data are expressed as mean ± S.D. (n = 3). a At a concentration of 200 µg/mL. One-way ANOVA ***p < 0.0001 followed by a multicomparison Dunnett’s test: ***p < 0.01 compared with positive control.
Table 5 – In vitro α-amylase and α-glucosidase inhibitory activity [IC50 (µg/mL)] of P. trifoliata samples. P. trifoliata
Juice Seed extracts Peel essential oil Acarbose
138.14 ± 3.1a 459.58 ± 4.8a 664.54 ± 4.7a 50.0 ± 0.9
81.27 ± 3.5a 170.54 ± 4.4a 300.17 ± 4.4a 35.5 ± 1.2
Data are expressed as media ± SD (n = 3). Acarbose was used as positive control. α-Amylase: One-way ANOVA ***p < 0.0001 followed by a multicomparison Dunnett’s test: α = 0.01 (F = 22120, R2 = 0.999). α-Glucosidase: One-way ANOVA ***p < 0.0001 followed by a multicomparison Dunnett’s test: α = 0.01 (F = 3280, R2 = 0.999); ap < 0.01 compared with acarbose.
also investigated by using FRAP assay, but inactivity was evidenced at the test concentration of 1 mg/mL for all samples except for seeds (0.40 µM Fe(II)/g).
Hypoglycaemic potential effects of P. trifoliata were analysed by means of the inhibition of α-amylase and α-glucosidase assays as reported in Table 5. Although all samples were able to inhibit both enzymes in a concentration-dependent manner, the most interesting activity was found against α-glucosidase enzyme. In particular, juice exhibited an IC50 value of 81.27 µg/ mL, followed by seed extracts (IC50 value of 170.54 µg/mL). The same trend was observed against α-amylase with IC50 values of 138.14 and 459.58 µg/mL, respectively, for juice and seed extract. As flavonoids have revealed positive effects in the treatment of hyperglycaemia (Stuart, Gulve, & Wang, 2004), the main flavonoids identified in P. trifoliata juice were investigated for their α-amylase and α-glucosidase inhibitory activity as reported in Table 6. All tested compounds demonstrated α-amylase and α-glucosidase inhibitory properties in a concentration-dependent manner, and were more active than the positive control acarbose (IC50 of 4.69–70.80 µM vs IC50 of 77.45 µM). The most active against α-amylase was neoeriocitrin with an IC50 value of 4.69 µM, while didymin demonstrated the most promising activity against α-glucosidase (IC50 of 4.20 µM), followed by naringin (IC50 of 10.33 µM) and narirutin (IC50 of
Table 6 – α-Amylase and α-glucosidase inhibitory activity (IC50 µM) of identified P. trifoliata constituents. Flavonoids
Narirutin Poncirin Didymin Naringin Hesperidin Neoeriocitrin Positive control Acarbose
70.80 ± 2.5 39.19 ± 1.3 31.62 ± 2.8 36.35 ± 1.9 26.04 ± 1.7 4.69 ± 0.9
14.30 ± 3.5 64.58 ± 2.6 4.20 ± 0.6 10.33 ± 1.1 15.89 ± 1.8 25.31 ± 1.2
77.45 ± 1.8
54.99 ± 1.3
Data are expressed as mean ± S.D (n = 3). One-way ANOVA ***p < 0.0001 followed by a multicomparison Dunnett’s test: ***p < 0.01 compared with positive control.
Journal of Functional Foods 25 (2016) 477–485
14.30 µM). All tested flavonoids were more active than acarbose (IC50 of 4.20–25.31 µM vs IC50 of 54.99 µM), but an exception is made for poncirin. Significant results were also obtained for hesperidin, the most abundant flavonoid identified in the juice, which inhibited α-amylase and α-glucosidase with IC50 values of respectively 26.04 and 15.89 µM. Several Citrus juices are able to exert hypoglycaemic effect. For example, the administration of Citrus paradisi juice was found to significantly reduce rapid blood glucose levels without any effect on 1.5-h plasma insulin levels (Owira & Ojewole, 2009). More recently, Menichini et al. (2015) demonstrated that Citrus medica cv Diamante peel extract administered in Zucker diabetic fatty (ZDF) rats exerts a dose-dependent effect on serum glucose levels. The leaves, mesocarp and endocarp extracts of C. medica cv Diamante exerted a moderate carbohydrate hydrolysing enzyme inhibition (Menichini et al., 2011). In the same year, another research group demonstrated the effect of bergamot juice extract on diet-induced hyperlipaemia in Wistar rats and in 237 patients either with hyperlipaemia associated or not with hyperglycaemia. After 30 days, a significant decrease in blood glucose was observed in both rats and patients (Mollace et al., 2011). All authors speculate that Citrus flavonoids are responsible for this bioactivity. Sixteen flavonoids, divided into six groups, have been previously investigated for their ability to inhibit yeast and rat small intestinal α-glucosidases and porcine pancreatic α-amylase (Tadera, Minami, Takamatsu, & Matsuoka, 2006). Anthocyanin, flavonol and isoflavone groups strongly inhibited yeast α-glucosidase with IC50 values less than 15 µM. The analysis of structure–activity relationships revealed that the unsaturated C ring, a carbonyl group at 4 position, a hydroxyl group at the 3 position or the linkage of the B ring at the 3 position, and the presence of hydroxyl substituents on the B ring enhanced the inhibitory activity. Luteolin, myricetin and quercetin were the most active flavonoids against α-amylase. The presence of a 2,3 double bond, a hydroxyl group at C5 position, the linkage of the B ring at the C3 position and the presence of hydroxyl substituents on the B ring enhanced the inhibitory activity. Moreover, the inhibitory activity against α-amylase was reduced by the presence of a hydroxyl group at C3 position. Shen, Xu, and Lu (2012) evaluated the effect of Citrus flavonoids (i.e., hesperidin, naringin, neohesperidin, and nobiletin) on amylase-catalysed starch digestion, pancreatic α-amylase and α-glucosidase, and glucose use in HepG2 cells. All phytochemicals significantly inhibited amylase-catalysed starch digestion. In particular, naringin and neohesperidin principally inhibited amylose digestion, whereas hesperidin, the main abundant flavanone of P. trifoliata juice, inhibited both amylose and amylopectin digestion. Additionally, glucose consumption, glycogen concentration, and glucokinase activity were significantly elevated, and glucose6-phosphatase activity was markedly decreased by flavonoids. More recently, Jia et al. (2015) have demonstrated that neohesperidin significantly decreased serum glucose and glycosylated serum protein in diabetic mice. Collectively, these results demonstrate that these flavonoids prevent the progression of hyperglycaemia, partly by binding to starch, increasing hepatic glycolysis and glycogen concentration, lowering hepatic gluconeogenesis, elevating oral glucose tolerance and insulin sensitivity, and decreasing insulin resistance.
The ability of antioxidants to protect against the effects of hyperglycaemia and to improve glucose metabolism and intake must be considered as leads of choice for the management of diabetes. In addition to their antioxidant activity, many phytochemicals, and in particular flavonoids, proved to act on biological targets involved in type 2 diabetes mellitus such as α-amylase and α-glycosidase inhibition. In this context, flavonoids behaving as antioxidants were studied as potential drugs by acting as biological targets involved in the development of diabetes. Of particular interest is P. trifoliata juice, which demonstrated a significant inhibition of carbohydrate hydrolysing enzymes. Juice is featured by a high content of flavonoids, in particular flavanones like hesperidin, naringin, didymin and narirutin that inhibited α-amylase and α-glucosidase more than the commercial drug acarbose. Compared to a common glass of juice, which is about 150–200 mL, the content in flavonoids is remarkable. Nonetheless, the health effects of flavonoids and more in general of polyphenols, depend on their bioavailability. Flavanones are the flavonoids with the best profile of bioavailability, and plasma concentrations may reach 5 µmol/ L. For this reason, further studies will be conducted to monitor the bioaccessibility and bioavailability of these particular phytochemicals. In sum, the present study suggests that P. trifoliata fruits are a promising source for the development of key ingredients, which contribute to creating functional foods or supplements for treating and/or preventing several diseases associated with oxidative stress, such as type 2 diabetes.
Acknowledgements The authors wish to thank Dr. N.G. Passalacqua, Natural History Museum of Calabria and Botanic Garden, University of Calabria (Italy), for supplying the P. trifoliata fruits used in this study. The authors also thank Dr. Anna Franca Plastina, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria (Italy), for proofreading a previous version of the current manuscript.
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