Blackcurrant ( Ribes nigrum L.) and Raspberry ( Rubus idaeus L.) Ethanolic Extracts: Inhibitory Effects on Pancreatic Lipase and Antioxidant Activity †

: Blackcurrant ( Ribes nigrum L.) and raspberry ( Rubus idaeus L.) fruits, rich sources of phyto-chemicals such as polyphenols, have been shown to possess promising biological properties. Herein, both fruits from southern Italy have been extracted by maceration by using ethanol as the solvent and investigated for their potential pancreatic lipase inhibitory activity. The radical scavenging effects were also analysed by using the 2,2 (cid:48) -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests. Our study showed that the extracts of both blackcurrant and raspberry have signiﬁcant inhibitory activity against lipase with IC 50 of 5.1 and 30.2 µ g/mL, respectively, better than the positive control orlistat (IC 50 of 37.1 µ g/mL).


Introduction
In recent years, several studies analyzing the composition, the bioactivity of red fruits, and the relationship between fruit intake and reduced risk of several chronic diseases, including obesity, have been published.Fruits are sources of not only vitamins, minerals, and dietary fiber but also several healthy phytochemicals.Blackcurrant (Ribes nigrum L.) and raspberry (Rubus idaeus L.) fruits are rich sources of polyphenols that have been shown to possess antioxidant, anti-inflammatory, antimicrobial, and hypoglycaemic properties [1,2].In this study, in order to investigate how environmental factors may influence types and contents of active substances and to prospect a potential new use as functional foods and/or nutraceuticals, we investigated the radical scavenging effects and lipase inhibitory activity of the ethanolic extract of blackcurrant (Ribes nigrum L.) and raspberry (Rubus idaeus L.) fruits collected in southern Italy.Several previous studies have demonstrated that plants that grow in various environments produce different active substance contents because of their wide distribution in different geological zones.This will result in variations in their internal qualities, as well as in the same species from different growing regions [3].

Materials and Methods
Plant materials and extraction procedure.Ripe fruits were collected in southern Italy (WGS84: 39 • 87 13 N, 16 • 06 53 E).Fresh fruits (350 g) were exhaustively extracted by maceration using ethanol as a solvent (4 × 1.2 L).Dry extracts were stored in brown glass bottles and kept at 4 • C before analysis.
Total polyphenols content (TPC).The TPC was determined using the Folin-Ciocalteu method in which extracts (concentration of 1.5 mg/mL) were mixed with water, sodium carbonate 15% (w/v), and Folin-Ciocalteu reagent [4].After an incubation of 2 h at room temperature, the absorbance was read at 765 nm using a UV-vis Jenway 6003 spectrophotometer (Milan, Italy).
Total flavonoids content (TFC).For the TFC determination, extracts (concentration of 1.5 mg/mL) were added to water and sodium nitrite 5% (w/v).After 5 min, aluminium chloride 10% (w/v) was added.After another 6 min, sodium hydroxide 1 M and water were added.Then, the absorbance was read at 510 nm [4].
Radical scavenging activity.The radical scavenging activity was investigated using the 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests [4].In the ABTS test, an ABTS radical cation solution was prepared and mixed with a solution of potassium persulfate and left before use for 12 h in the dark.The ABTS solution was diluted with ethanol to an absorbance of 0.70 at 734 nm.After the addition of the lavender extract to the ABTS solution, the absorbance was read after 6 min at 734 nm.In the DPPH test, a mixture of the DPPH ethanol solution (1.0 × 10 −4 M) and extracts at different concentrations were prepared and kept for 30 min in the dark.The bleaching of the DPPH was determined by reading the absorbance at 517 nm.Ascorbic acid was used as positive control in both assays.
Pancreatic lipase inhibitory activity test.To investigate the lipase inhibitory activity, a previously reported protocol was adopted [4].Concisely, extracts at different concentrations were mixed with the enzyme, Tris-HCl buffer (pH 8.5), and 4-nitrophenyl octanoate, and the mixture was incubated for 30 min at 37 • C.Then, the absorbance was measured at 405 nm.Orlistat was used as a positive control.
Statistical analysis.Experiments were performed in triplicate.The Prism GraphPad Prism Software (San Diego, CA, USA) was used to calculate the concentration causing 50% inhibition (IC 50 ).Data were analyzed using the one-way analysis of variance (ANOVA) and significant differences were calculated using Tukey's test.

Results and Discussion
The fresh fruits of R. nigrum and R. idaeus from Calabria (southern Italy) have been subjected to maceration by using ethanol as the solvent for obtaining extraction.Yields of 11.8 and 11.2% for the blackcurrant and raspberry, respectively, were observed.A TPC value of 501.1 and 483.7 mg chlorogenic acid equivalents/100 g plant materials for raspberry and blackcurrant, respectively, were determined.The blackcurrant extract was characterized by the highest TFC with a value of 35.1 mg quercetin equivalents/100 g plant materials compared to the raspberry (26.3 mg quercetin equivalents/100 g plant materials).
The raspberry extract was the most active in the ABTS test with an IC 50 of 1.6 µg/mL, a value comparable to that of the positive control (Table 1).Data are expressed as media ± standard deviation (n = 3).Differences within and between groups were evaluated by One-way (ANOVA) followed by Tukey's multiple range test.Results followed by different letters in a same column are significantly different at p < 0.01.
In recent years, inhibitors of pancreatic lipase have received great attention from researchers, and inhibitors from natural sources have still attracted much attention due to their wide range of sources, structural diversity, and low toxicity and side effects.The results of our study showed that the ethanolic extracts of both blackcurrant and raspberry have significant inhibitory activity against this enzyme, better than the positive control orlistat.In fact, the R. idaeus exhibited an IC 50 value of 5.1 µg/mL.An IC 50 of 30.2 mg/mL was found for the blackcurrant (Table 2).Both the extracts were more active than the orlistat (IC 50 of 37.1 µg/mL).Data are expressed as media ± standard deviation (n = 3).Differences within and between groups were evaluated by One-way (ANOVA) followed by Tukey's multiple range test.Results followed by different letters in a same column are significantly different at p < 0.01.