Phenolic Characterization, Antioxidant Activity, and Enzyme Inhibitory Properties of Berberis thunbergii DC. Leaves: A Valuable Source of Phenolic Acids

Berberis species are known for their use in traditional medicine. Here, we report the phenolic composition and bioactivity of methanolic and aqueous extracts of Berberis thunbergii DC. leaves. The phenolic profiling and the quantitation of the main compounds were performed by high-performance liquid chromatography with diode array and mass spectrometry detections. The most abundant compounds in both extracts were caffeoylquinic acids (chlorogenic acid, particularly, with a concentration of 90.1–101.3 mg g−1 dried extract), followed by caffeoylglucaric acids and quercetin glycosides. Antioxidant and radical scavenging assays (phosphomolybdenum, DPPH, ABTS, CUPRAC, FRAP, metal chelating activity), as well as enzyme inhibitory assays (acetylcholinesterase, butyrylcholinesterase, tyrosinase, amylase, glucosidase, and lipase), were carried out to evaluate the potential bioactivity of B. thunbergii. The methanolic extract presented the highest antioxidant and radical scavenging values, in agreement with its higher phenolic content. Regarding enzyme inhibitory potential, the methanolic extract was also more potent than the aqueous one. Hence, B. thunbergii leaves represent a suitable candidate for the preparation of pharmaceutical or nutraceutical products.

voltage, 4500 V; capillary exit voltage, -117.3 V. We used the auto MS n mode for the acquisition of MS n data, with isolation width of 4.0 m/z, and fragmentation amplitude of 0.6 V (MS n up to MS 4 ).

Assays for Total Phenolic and Flavonoid Contents
The total phenolic content was determined by employing the methods given in the literature with some modification. Sample solution (0.25 mL) was mixed with diluted Folin-Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vigorously. After 3 min, Na2CO3 solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after a 2 h incubation at room temperature. The total phenolic content was expressed as milligrams of gallic acid equivalents (mg GAE/g extract) .
The total flavonoid content was determined using the AlCl3 method. Briefly, sample solution (1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol.
Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl3. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) .
For the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay: Sample solution was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after a 30 min incubation at room temperature in the dark. DPPH radical scavenging activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid) radical scavenging assay: Briefly, ABTS + was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12-16 in the dark at room temperature. Prior to beginning the assay, ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after a 30 min incubation at room temperature. The ABTS radical scavenging activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For CUPRAC (cupric ion reducing activity) activity assay: Sample solution was added to premixed reaction mixture containing CuCl2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH4Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl2 . Then, the sample and blank absorbances were read at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For phosphomolybdenum method: Sample solution was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95 °C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract).
For metal chelating activity assay: Briefly, sample solution was added to FeCl2 solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL).
Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl2 solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was sub-tracted from that of the sample. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract).
The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI, Sigma) or butyrylthiocholine chloride (BTCl, Sigma) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as galanthamine equivalents (mg GALAE/g extract).
For Tyrosinase inhibitory activity assay: Sample solution was mixed with tyrosinase solution (40 μL, Sigma) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of L-DOPA (40 μL, Sigma). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mg KAE/g extract).
Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated 10 min at 37 °C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACAE/g extract).
For α-glucosidase inhibitory activity assay: Sample solution was mixed with glutathione (50 µL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 µL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside, Sigma) (50 µL) in a 96-well microplate and incubated for 15 min at 37 °C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 µL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalents (mmol ACAE/g extract).
Porcine pancreatic lipase (type-II) activity was performed using p-nitrophenyl butyrate (p-NPB) as substrate (Roh and Jung, 2012). Enzyme solution (1 mg/mL) was prepared in 50 mM Tris-HCl (pH 8.0). Fraction solution (25 µL) was mixed with a lipase solution (50 µL) in a 96-well microplate and incubated for 20 minutes at 25°C. The reaction was initiated with the addition of p-NPB (5 mM, 50 µL). Similarly, a blank sample (prepared in the same manner but without the extract) was prepared for each of the samples and analysed according to this procedure. Results were expressed as orlistat equivalents (mg OE/g extract).
Chromatograms. Figure S1. HPLC-ESI/MS n base peak chromatograms (BPC) of the methanolic and aqueous extracts of Berberis thunbergii leaves. Figure S2. UV chromatograms of the methanolic and aqueous extracts of Berberis thunbergii leaves at 320 and 350 nm.
UV-chromatograms at 320 nm; used for the quantification of phenolic acids. Time windows have been selected for a better comparison of the chromatograms.