Journal of Applied Botany and Food Quality 91, 39 - 46 (2018), DOI:10.5073/JABFQ.2018.091.006 1Department of AGRARIA, University of Reggio Calabria, Italy 2 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Italy Portulaca oleracea L. (Purslane) extracts display antioxidant and hypoglycaemic effects Vincenzo Sicari1*, Monica Rosa Loizzo2, Rosa Tundis2, Antonio Mincione1, Teresa Maria Pellicanò1 (Submitted: November 3, 2017; Accepted: February 7, 2018) * Corresponding author Summary Purslane (Portulaca oleracea L.) is a member of the family Por- tulacaceae. Due to its many health benefits, it is listed in a World Health Organization database. The aim of this work is to investigate the purslane extracts for their chemical profile and bioactivity. In this study, two different solvents (MeOH/H2O and EtOH) were applied to fresh and dried leaves. The extracts were analysed using HPLC-DAD. Phenolic acids (caffeic acid, p-coumaric acid and fe- rulic acid) and flavonoids (apigenin, kaempferol, luteolin, quercetin, isorhamnetin, kaempferol-3-O-glucoside and rutin) were identified in all samples. Quercetin and p-coumaric acid were the most abun- dant compounds. Total antioxidant activity was measured by using the ABTS and DPPH tests, and the ferric reducing antioxidant power (FRAP) assay. Hypoglycaemic properties were investigated via the inhibition of carbohydrate-hydrolysing enzymes, α-amylase and α- glucosidase. Fresh hydroalcoholic purslane extract exhibited the highest radical scavenging potential in both ABTS and DPPH test (IC50 values of 52.86 and 66.98 μg/mL, respectively), whereas dried hydroalcoholic purslane extract showed the highest α-glucosidase inhibitory poten- tial (IC50 value of 45.05 μg/mL). Collectively these data show the health properties of this widely consumed salad plant. Keywords: Portulaca oleracea, Phenols, HPLC-DAD, Antioxidant potential, Carbohydrate hydrolysing enzymes. Introduction Purslane (Portulaca oleracea) is an annual succulent plant of the family Portulacaceae. Originally, from India, today it has spread throughout the world’s temperate zones, including Italy. The plant, which can grow to a height of 35 cm, has reddish-brown, prostrate, fleshy, branching stems, and light-green, fleshy, oval leaves. Although often considered a weed, all the aerial parts can be eaten, especially as a salad plant. Purslane is rich in vitamins (Uddin et al., 2014), protein, carbohy- drates and minerals (Uddin et al., 2014; MohaMed and hUssein, 1994) such as calcium, iron, magnesium, potassium, zinc and sodium (aberoUMand, 2009). Apart from is alimentary use, purslane has been traditionally used as a medicinal plant. It has anti-inflamma- tory and analgesic properties (Lee et al., 2012; ZhoU et al., 2015; rafieian-Kopaei and aLesaeidi, 2016; Meng et al., 2016), anti- cancer activity (ZhoU et al., 2015; Lee et al., 2014; ahangarpoUr et al., 2016) and antioxidant activity (LiM and QUah, 2007; siriaMornpUn and sUttajit, 2010; erKan, 2012). This plant can be used externally for various skin complaints, such as eczema, ulcers and acne, and to give relief from insect bites. It can also be used for coughs, bronchitis and fever (Zhang et al., 2002). Due to its many health benefits, it is listed in a World Health Organization database. Moreover, purslane is an excellent vegetable source of omega-3 fatty acids (Uddin et al., 2014; LiU et al., 2002; siMopoULos et al., 2005) since 100 g of purslane leaves contain around 350 mg of α-linolenic acid. Several works reported the presence of flavonoids as main bioactive purslane constituents (erKan, 2012; XU et al., 2006; ZhU et al., 2010). In recent decades, the role of foods in disease prevention and treat- ment has been increasingly recognised. The role played by reac- tive oxygen species (ROS) in the pathogenesis of several diseases including diabetes mellitus (DM) has been clarified (aLfadda and saLLaM, 2012; tUndis et al., 2016; LoiZZo et al., 2017). To pre- vent complications, the stabilization of blood glucose levels in DM patients is crucial (Mai and ChUyen, 2007). Several therapeutic approaches may be used to achieve this objective: stimulating insulin release, increasing the amount of glucose transporters, inhibiting gluconeogenesis, reducing the absorption of glucose or decreasing the post-prandial hyperglycaemia (KiM et al., 2005). This last ap- proach could be obtained by inhibiting carbohydrate hydrolysing en- zymes α-amylase and α-glucosidase, using acarbose, voglibose and miglitol (rios et al., 2005; LoiZZo et al., 2016; LoiZZo et al., 2017). However, these drugs are characterized by several gastrointestinal side effects including abdominal discomfort, flatulence, bloating, and diarrhoea. For these reasons natural sources are being investigated to provide new hypoglycaemic drugs (rios et al., 2005; tUndis et al., 2010). Both fresh and dried samples are used in medicinal plants studies. In most cases, dried samples are preferred considering the time needed for experimental design. Purslane is highly perishable in the fresh state; it has the shortest shelf life among fruits and vegetables due to its high metabolic reactions, which lead to loss quality. Therefore, the aim of this work is to investigate and compare the chemical com- position, antioxidant and hypoglycaemic properties of dried and fresh purslane leaves. Materials and methods Chemicals and reagents All reagents were of analytical grade and were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). Acarbose from Actinoplanes spp. was obtained from Serva (Heidelberg, Germany). HPLC sol- vents were obtained from VWR International S.r.l. (Milan, Italy). Sample, extraction and analysis procedure Purslane plants were bought from a supermarket in Reggio Calabria (Italy) in November 2016. Leaves were manually separated from the stems and divided into two groups: fresh and dried (35 °C for 48 h). Samples were subsequently homogenized in a commercial blender and subjected to different extraction procedures: a) MeOH:H2O (80:20 v/v) and b) 100% EtOH, in IKA Ultra-Turrax T25 and cen- trifuged for 10 min at 5000 rpm, after which the supernatant was filtered through a 0.45 mm Millipore filter (GMF Whatman) before analysis. For fresh leaves, yields (%) of 15.3 and 8.2 were obtained for hydroalcoholic and EtOH extracts, respectively, while for dried leaves, yields (%) of 10.7 and 6.6 were obtained for hydroalcoholic and EtOH extracts, respectively. 40 V. Sicari, M.R. Loizzo, R. Tundis, A. Mincione, T.M. Pellicanò Extraction of bioactive compounds and RP-HPLC/DAD analysis RP-HPLC/DAD analyses of all samples were obtained as reported by siCari et al. (2017) using a Knauer (Asi Advanced Scientific Instruments, Berlin) system equipped with two pumps Smartline Pump 1000, a Rheodyne injection valve (20 μL) and a photodiode array detector UV/VIS equipped with a semi micro-cell. Compounds were separated on a Knauer RP-C18 (250 mm × 4.6 mm, 5 μm). The chromatographic method used was a gradient elution of solvent A (water/formic acid, 99.9:0.1 v/v) and B (acetonitrile/formic acid 99.9:0.1 v/v). The gradient was used as follows: 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. The column temperature was 30 °C and the flow rate was 1.0 mL/min. The injection volume was 20 μL. Peaks were monitored at 254, 330 and 305 nm. The identification and quantification of antioxidant compounds were carried out from the retention times in comparison with authentic standards. Data process- ing data were carried out using Clarity Software (Chromatography Station for Windows). All analyses were performed in triplicate and the results were expressed as mg/Kg of leaves. Total phenolic content (TPC) The total phenolic content of the extracts was determined as de- scribed by singLeton et al. (1999). An aliquot of 350 μL of ex- tract was mixed with 1 mL of Folin-Ciocalteau reagent and 10 mL of 20% Na2CO3 solution. Absorbance was measured at λ = 760 nm using a UV-Vis Agilent 8453 spectrophotometer (Agilent Tech- nologies, Italy) after 2 h in the dark. The results were expressed in milligram gallic acid equivalents per 100 g (mg/100 g) weight of the sample. All samples were analysed in triplicate. DPPH Radical Scavenging Activity Assay DPPH radical scavenging activity of P. oleracea was determined according to the technique previously described (LoiZZo et al., 2016) at 517 nm using a UV-Vis Jenway 6003 Spectrophotometer. The DPPH radical scavenging activity was calculated as follows: [(A0-A1/A0) × 100], where A0 is the absorbance of the control and A1 is the absorbance in the presence of the sample. Ascorbic acid was used as positive control. ABTS Radical Scavenging Activity Assay As reported by LoiZZo et al. (2016) the ABTS radical cation solution was mixed with potassium persulphate and left in the dark for 12 h. The ABTS solution was diluted with methanol to an absorbance of 0.70 ± 0.05 at 734 nm. After addition of sample (1-1000 mg/mL in methanol) to the ABTS solution, absorbance was measured after 6 min. Ascorbic acid was used as positive control. Ferric Reducing Activity Power (FRAP) Assay The FRAP assay is based on the redox reaction that involves TPTZ (2,4, 6-tripyridyl-s-triazine)-Fe3+ complex. FRAP reagent was pre- pared as previously described (LoiZZo et al., 2015). Extracts were dissolved in methanol and tested at 2.5 mg/mL. BHT was used as control. Carbohydrate hydrolysing enzymes inhibition study A starch solution, α-amylase (EC 3.2.1.1) solution and colorimetric reagent were prepared. Both control and juice were added to starch solution and left to react with enzyme at room temperature for 5 min (LoiZZo et al., 2014). The absorbance was read at 540 nm. The enzyme inhibition (%) was obtained by the following equation: In the α-glucosidase inhibition test a maltose solution, α-glucosidase solution (EC 3.2.1.20) and o-dianisidine (DIAN) solution were pre- pared (LoiZZo et al., 2014). A mixture of juice maltose solution and enzyme were left to incubate at 37 °C for 30 min. Then perchloric acid was added and mixture was centrifuge. The supernatant was collected and mixed with DIAN and PGO and left to incubate at 37 °C for 30 minutes. The absorbance was read at 500 nm. The α-glucosidase inhibition (%) was calculated by using the following equation: Relative antioxidant capacity index (RACI) calculation Relative antioxidant capacity index (RACI) is an integrated approach to evaluate the antioxidant capacity generated from different in vitro tests (sUn and tanUMihardjo, 2007). For the calculation, standard scores were used with no unit limitation and no variance among methods. Data obtained from TPC, ABTS, DPPH and FRAP tests were used to calculate RACI values for purslane samples. Statistical analyses Excel software (Office 2007) was used to calculate the means and the standard deviation. Statistical analysis was carried out using SPSS software for Windows (SPSS Inc., Elgin, IL, U.S.A.) 15.0 version. The means of all parameters were examined for significance using ANOVA with Tukey test to determine any significant difference be- tween the treatments at P < 0.05. Further multivariate analysis was performed using Principal Component Analysis (PCA). All samples were analysed in triplicate. Results and Discussion Identification and quantification of the phenolic compounds present in P. oleracea Both fresh and dried purslane leaves were analysed by HPLC-DAD to determine their bioactive compounds and the effect of drying on selected phenolic compounds. Tab. 1 reported the chemical profile of both fresh and dried purslane leaves extracts. Quercetin, apigenin, luteolin, kaempferol, isorhamnetin, kaempferol-3-O-glucoside and rutin were the flavonoids (erKan, 2012; ZhU et al., 2012), and caffe- ic, p-coumaric, and ferulic acids were the phenolic acids found in all extracts (siriaMornpUn and sUttajit, 2010; erKan, 2012; yang et al., 2009). Among identified flavonoids, quercetin was the most abundant compound with values in the range 16.01-6.02 mg/kg, fol- lowed by rutin (6.17-4.12 mg/kg) and kaempferol (3.25-1.85 mg/kg). Apigenin, luteolin, isorhamnetin, kaempferol-3-O-glucoside were found in smaller quantities (Tab. 1). The level of flavonoids depends on the portion of the plant, and is normally higher in the root, fol- lowed by the stem and leaves (XU et al., 2006). p-Coumaric acid was the main phenolic acid with the highest value in hydroalcoholic fresh leaf extract (20.53 mg/kg) followed by EtOH dried sample (18.77 mg/ kg). The same trend was found for ferulic acid. Wide investigations have shown that p-coumaric acid and quercetin exhibit various bioactivities, including antioxidant, anti-inflamma- tory and anti-cancer activities, in addition to mitigating atheroscle- rosis, oxidative cardiac damage, diabetes and many other biological actions (LiM, 2007; dKhiL et al., 2011; pei et al., 2015). In addition, quercetin is a versatile antioxidant known to possess protective abili- % Inhibition = 100 - [Maltose] test [Maltose] control x 100 ± S.D. % Inhibition = 100 - [Glucose] test [Glucose] control x 100 ± S.D. Bioactivity of Portulaca oleraceae extract 41 ties against tissue injury induced by various drug toxicities (anand david, 2016). The efficiency of extraction varies according to the polarity of the solvent, pH, temperature, extraction time, and composition of the sample. When extraction time and temperature are the same, the sol- vent used and the composition of sample were shown to be the most important parameters. In the present study, extraction was performed on samples of purslane leaves using two different solvents: methanol/ water (80/20 v/v) and ethanol (100%). Ethanol is known as a good solvent to extract phenol, as well as being safe for human consump- tion. Methanol is considered to be more efficient when extracting phenols of lower molecular weight (dai and MUMper, 2010). hUang et al. (2007) identified isorhamnetin, quercetin and kaempferol with values of 2.8, 1.3 and 1.1 mg/100 g, respectively. More recently, apigenin, bergapten, caffeic acid, p-coumaric acid, ferulic acid, scopoletin, quercetin, and quercetin-3-O-rhamnoside, were quantified in P. oleracea from different localities (ai et al., 2015). XU et al. (2006) identified mirycetin and luteolin. Antioxidant potential In this study, two in vitro tests, DPPH and ABTS, were used to screen the radical scavenging activity of P. oleracea extract. The total phenolic content and ferric reducing power was also tested. The different methods for measuring the radical scavenging potential can give different results according to which specific free radical is being used as a reactant. DPPH is often used to test how far com- pounds can act as free radical scavengers or hydrogen donors, and to quantify antioxidants in complex systems. The procedure which inhibits the production of the ABTS radical cation did not involve a substrate (antoLoviCh et al., 2002). The ferric thiocyanate method determines the quantity of peroxide, the main product of lipid oxi- dation, which is produced in the initial stages of oxidation. In this test, hydroperoxides formed from linoleic acid added to the reaction mixture, oxidized in air during the experiment, were indirectly mea- sured. Antioxidants may be reductants and inactivation of oxidants by reductants are redox reactions in which one reaction species is reduced when the other is oxidized (apaK et al., 2004). Total phe- nols were calculated and expressed as gallic acid/100 g of extract. The highest value was found in fresh leaves hydroalcoholic extract (565 mg/100 g) followed by fresh leaves ethanol extract (488.04 mg/ 100 g) (Tab. 2). A concentration-dependent activity was observed for all tested purs- lane extracts. Fresh purslane leaf ethanolic extract exerted the great- est DPPH radical scavenging activity with IC50 value of 52.86 mg/ mL. This extract was followed by fresh leaf hydroalcoholic extract (IC50 value of 53.92 mg/mL) (Tab. 2). A similar trend was also seen in ABTS·+ radical scavenging ability where the IC50 values of 66.98 and 72.60 mg/mL were found for F and F1 samples, respectively. Generally, a promising ferric reducing ability was observed. In par- ticular, fresh leaf hydroalcoholic extract showed a FRAP value com- parable to the positive control BHT (56.11 vs 63.2 mM Fe (II)/g). The RACI calculation was used to integrate the antioxidant capacity generated from different in vitro methods of each samples. For this calculation, the mean of standard scores was used, taken from the raw data of the different antioxidant tests. Any differences between units and in variances in the raw data did not influence the RACI. Stepwise regression between RACI and different tests demonstrated the following: 1) each test was selected as a significant variable with no one applied method being removed, 2) each method had the same weight in building RACI, and 3) the regression was highly significant (r = 1, p < 0.001). Based on RACI the following order of antioxidant ability was found: dried leaves (EtOH) > dried leaves (MeOH:H2O) > fresh leaves (MeOH:H2O) (Fig. 1). This trend plainly showed that dried leaves, independently of which solvent was used for extraction, had the highest antioxidant potential. Recently, yoUssef et al. (2014) reported the radical scavenging po- tential of purslane leaves fresh and under different drying procedures (hot-air drying, microwave drying and freeze-drying). Fresh purslane leave extracts showed values of 53.23% for DPPH and 147.78 μmol Trolox for ABTS per 100 g dry weight. All the examined methods of drying significantly lowered the antioxidant capacity of the sample. Analysis of data demonstrated that among investigated dried sample, hot air dried and freeze-dried purslane leaves retained a better an- tioxidant capacity independently by the temperature applied while microwave procedure drastically reduced the antioxidant potential of Tab. 1: Quantification (mg/kg) of selected phenols of purslane leaves extracts. MeOH:H2O EtOH (80:20 v/v) Fresh leaves Dried leaves Fresh leaves Dried leaves Flavonoids Apigenin 0.29 ± 0.05a 0.11 ± 0.03c 0.17 ± 0.05b 0.09 ± 0.01c Kaempferol 3.25 ± 0.15a 2.03 ± 0.12c 2.75 ± 0.15b 1.85 ± 0.14d Luteolin 0.55 ± 0.02a 0.03 ± 0.08d 0.32 ± 0.02b 0.23 ± 0.03c Quercetin 16.01 ± 0.33a 6.02 ± 0.03d 11.11 ± 0.33c 14.14 ± 0.30b Isorhamnetin 0.36 ± 0.01a 0.08 ± 0.03c 0.27 ± 0.01b 0.25 ± 0.07b Kaempferol-3-O-glucoside 0.63 ± 0.05a 0.23 ± 0.08c 0.55 ± 0.01b 0.53 ± 0.08b Rutin 6.10 ± 0.12b 4.12 ± 0.04d 5.11 ± 0.10c 6.17 ± 0.12a Phenolic acids Caffeic acid 7.35 ± 0.08a 3.48 ± 0.08d 6.33 ± 0.24b 5.58 ± 0.88c p-Coumaric acid 20.53 ± 0.46a 11.03 ± 0.15d 16.44 ± 1.18c 18.77 ± 1.2b Ferulic acid 9.62 ± 0.41a 4.12 ± 0.28d 7.53 ± 0.88c 9.27 ± 1.01b Sign. ** ** ** ** Values are mean ± SD of three sample seed oils, analyzed in triplicate. Different letters indicate significant differences. Differences were evaluated by one-way analysis of variance (ANOVA) test completed with a multicomparison Tukey’s test. **P<0.01 compared with the positive control. 42 V. Sicari, M.R. Loizzo, R. Tundis, A. Mincione, T.M. Pellicanò the matrix. These modifications in terms of bioactivity could be re- lated to the different phytochemical content in investigated samples. However, the generation and accumulation of antioxidants during food dehydration may cause antagonistic or synergistic effects with each other or with other compounds present in the sample. The com- plex interactions influencing the functional properties of food during drying require further research (hsU et al., 2003; di sCaLa et al., 2011; LoiZZo et al., 2013; LòpeZ et al., 2013). The methanol extracts edible fresh parts of thirteen P. oleracea from Malaysia were examined for their phytochemical content and anti- oxidant activity by using the DPPH radical scavenging method and FRAP assay (aLaM et al., 2014). The IC50 values ranged from 2.52 to 3.29 mg/mL for DPPH test, and for 7.39 to 104.2 μmol TE/g DW for FRAP assay. These results are better than our data in terms of both radical scavenging activity and ferric reducing power. Differently, similar DPPH radical scavenging results were obtained with air- dried powered of Iranian P. oleracea. In fact, the n-hexane, dichlo- romethane, chloroform, ethyl acetate and methanol extracts showed IC50 values in the range from 62.9 to 91.0.8 mg/ml for ethyl acetate and dichloromethane extracts, respectively (saLehi et al., 2013). The influence of area of collection on the phytochemical content and antioxidant potential of this plant species was confirmed also by siLva et al. (2014) that investigated leaves, flowers and stems of P. oleracea from different area of Portugal. Results revealed that in the DPPH assay, samples from Vendas Novasr reached the 50% inhibition rate in lower concentrations than plants from Tavira. Recently, hydroalcoholic extracts of the aerial parts of P. oleracea from Bulgaria (POB) and Greece (POG) were studied for their radi- cal scavenging activity and ferric reducing power (gevrenova et al., 2016). Both purslane extracts revealed a similar radical scavenging potential with IC50 values of 1.98 and 2.00 mg/mL, and 0.88 and 0.92 mg/mL in DPPH and ABTS test for POB and POG, respec- Tab. 2: Total phenols content, radical scavenging activity and ferric reducing potential of purslane leaves extracts. Total Phenols DPPH ABTS FRAP (mg GAE/100 g) (IC50 mg/mL) (IC50 mg/mL) (mM Fe(II)/g) MeOH:H2O (80:20 v/v) Fresh leaves 565.07 ± 3.23 52.86 ± 0.8 66.98 ± 1.9 54.35 ± 0.5 Dried leaves 244.17 ± 4.04 53.92 ± 1.3 72.60 ± 1.7 56.11 ± 3.9 EtOH Fresh leaves 488.04 ± 1.54 55.92 ± 1.1 85.91 ± 1.9 45.14 ± 3.3 Dried leaves 260.19 ± 2.07 56.87 ± 1.3 89.46 ± 2.3 36.22 ± 3.2 Positive control Ascorbic acid 2.0 ± 0.9 1.7 ± 0.8 BHT 63.2 ± 2.8 Data are expressed as means ± S.D. (n = 3). DPPH Radical Scavenging Activity Assay: One-way ANOVA ***p<0.0001 followed by a multicomparison Dunnett’s test: ***p<0.01 compared with ascorbic acid. Antioxidant Capacity Determined by Radical Cation (ABTS+): One-way ANOVA ***p<0.0001 fol- lowed by a multicomparison Dunnett’s test ***p<0.01, *p<0.05 compared with ascorbic acid. Ferric Reducing Antioxidant Power (FRAP): One-way ANOVA ***p<0.0001 followed by a multicomparison Dunnett’s test **p<0.01 compared with positive control. Fig. 1: Relative antioxidant capacity index of purslane leaves samples. 0,47 -0,40 0,54 0,67 Fresh leaves (MeOH/water) 12*3456/7*)8 Dry leaves (MeOH/water) 12*3456/7*)8 Fresh leaves (EtOH) 1;7348 Dry leaves (EtOH) 1;7348 R A C l v al u es Bioactivity of Portulaca oleraceae extract 43 tively. However, considering that all measured values are greater than those are found for the Calabrian extracts, our sample showed a most promising antioxidant potential. The sample from Bulgaria showed two-time highest ferric reducing ability respect POG with value of 0.16 mM Trolox equivalent. Carbohydrate hydrolysing enzymes inhibition Following our research interest in starch hydrolase inhibitors from edible plants we have investigate purslane extracts against α-amylase and α-glucosidase enzymes. These extracts inhibited carbohydrate- hydrolysing enzymes depending upon their concentration. Generally, α-glucosidase enzyme was most sensible since the IC50 values are in the range from 45.05 to 195.01 mg/mL for fresh leaf hydroalcoholic extract and fresh leaf ethanol extract, respectively. On α-amylase dried leaves, hydroalcoholic extract showed the highest inhibitory activity (IC50 value of 488.49 mg/mL) (Tab. 3). All these values are greater than those for the positive control acarbose. Our values are in agreement with those reported by saLehi et al. (2013), which found an IC50 value of 93.2 μg/mL for P. oleracea methanol extract against α-glucosidase. Previously, the effect of P. oleracea were screened in vivo in rats with type 2 DM. Results clearly evidenced that the extract reduced body weight, improved impaired glucose tolerance, attenuated hyperinsu- linemia and elevated insulin sensitivity. The mechanism might be related to improved lipid metabolism and decreased free fatty acids (Lan and fUer, 2003). Successively, eL-sayed (2011) studied the effect of P. oleracea seeds in thirty type-2 DM patients. Patients were split into two groups, one received 5 g of seeds two times a day, while the other received 1.5 mg of metformin daily. The treatment caused a significant drop in total cholesterol, low-density lipoprotein and serum levels of triglycerides and a rise in high-density lipopro- tein. Other effects included modifications of liver transaminase, total and direct bilirubin, body weight and body mass index, fasting and post-prandial blood glucose, and insulin. In the metformin group similar effects were obtained. Tab. 3: Carbohydrate hydrolysing enzymes of purslane extracts. Assay α-Amylase α-Glucosidase (IC50 mg/mL) (IC50 mg/mL) MeOH:H2O (80:20 v/v) Fresh leaves 902.74 ± 3.8 195.01 ± 1.7 Dried leaves 640.01 ± 3.5 45.05 ± 1.1 EtOH Fresh leaves 774.02 ± 3.7 132.88 ± 2.1 Dried leaves 488.49 ± 3.5 138.51 ± 2.3 Positive control Acarbose 50.0 ± 0.9 35.5 ± 1.2 Data are expressed as means ± S.D. (n = 3). α-Amylase: One-way ANOVA ***p<0.0001 followed by a multicomparison Dunnett’s test: ***p<0.01 com- pared with acarbose. α-Glucosidase: One-way ANOVA ***p<0.0001 fol- lowed by a multicomparison Dunnett’s test: ***p<0.01 compared with acar- bose. 6 Fig. 2: PCA loading plot (PC1 vs PC2) for the first and second principal components. Dry leaves (EtOH) Fresh leaves (EtOH) Fresh leaves (MeOH/H2O) Dry leaves (MeOH/H2O) PC2: 33,8 % PC1: 61,24 % Fig. 2: PCA loading plot (PC1 vs PC2) for the first and second principal components. Principal component analysis PCA showed that the two principal components accounted for 95.12% of total variance, with PC1 for 61.24% and PC2 for 33.88% of total variance. The first principal component (Fig. 2) shows strong correlation with apigenin, kaempferol, luteolin quercetin isorhamne- tin, kaempferol-3-O-glucoside, rutin, caffeic acid, p-coumaric acid, ferulic acid, total phenols, α-glucosidase and a lower correlation with α-amylase, DPPH, and ABTS test. This suggests that these thirteen variables are grouped together. In addition, from the analysis of vari- able loads, it was seen that the PC1 has a negative correlation with FRAP. The second principal component is strongly correlated with apigenin, kaempferol, FRAP and α-amylase, while it is strongly negatively correlated with DPPH and ABTS. Total phenols (TPC) 44 V. Sicari, M.R. Loizzo, R. Tundis, A. Mincione, T.M. Pellicanò is positively correlated with both PC1 and PC2. The significant cor- relations obtained support the hypothesis that phenolic compounds contribute significantly to the total antioxidant capacity (Cai et al., 2004; djeridane et al., 2006; siCari et al., 2016; siCari et al., 2016a). Fig. 3 shows that PC1 positive correlation with hydroalcoholic ex- tracts obtained from the fresh leaves and was characterized by the presence of total phenols, flavonoid compounds, phenolic acids, α-amylase, α-glucoside and a low value of ABTS and DPPH. More- over, values of the original variables are greater than those of the ethanolic extracts obtained from fresh and dried leaves, respectively. Methanol is the most suitable solvent in the extraction in polypheno- lic compounds from plant tissue, due to its ability to inhibit the action of polyphenol oxidase that causes the oxidation of polyphenols (yao et al., 2004; LiM and QUah, 2007). In addition, the different relation- ships between the antioxidant activity and the total phenolic content can be due to many factors; in fact the total phenolic content does not incorporate all the antioxidants. Also, it must be taken into account the synergism between the antioxidants in the extracts that makes the antioxidant activity not only dependent on the concentration, but also on the structure and the interaction between the antioxidants (piLUZZa and bULLitta, 2011). The ethanolic extract obtained from fresh leaves has a positive cor- relation with PC1, while the same obtained from the dried leaves shows low positive correlation and was characterized by assay of antioxidant activity (DPPH and ABTS). Hydroalcoholic extracts (dried leaves) were grouped at the negative side of PC1 (showing low flavonoid compounds, phenolic acids, total phenols and high FRAP values). Conclusions In this work, extracts from fresh and dried leaves of P. oleracea were tested to evaluate the most efficient process in terms of extracting bioactive molecules. Was carried out a qualitative analysis of phenolic compounds present in the leaves of purslane examined by using LC-DAD by comparison with standard and literature data. Analysis of results revealed that fresh hydroalcoholic purslane extract exhibited a promising radical scavenging activity. A great difference was observed in hypogly- caemic whereas dried hydroalcoholic purslane extract exhibited the highest α-glucosidase inhibitory potential. Therefore, the results of this paper, with the addition of further stu- dies, will allow this plant with a large amount of biomolecules use- ful for beneficial effects on humans to be reevaluated. 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