Polyphenol Profile and Biological Activity Comparisons of Different Parts of Astragalus macrocephalus subsp. finitimus from Turkey

The members of the genus Astragalus have great interest as traditional drugs in several folk systems including Turkey. In this sense, the present paper was aimed to explore the biological properties and chemical profiles of different parts (aerial parts, leaves, flowers, stems, and roots) of A. macrocephalus subsp. finitimus. Antioxidant (radical quenching, reducing power, and metal chelating) and enzyme inhibitory (α-amylase and tyrosinase) effects were investigated for biological properties. Regarding chemical profiles, individual phenolic compounds were detected by LC-MS, as well as total amounts. The leaves extract exhibited the strongest antioxidant abilities when compared with other parts. However, flowers extract had the best metal chelating ability. Hyperoside, apigenin, p-coumaric, and ferulic acids were identified as main compounds in the tested parts. Regarding enzyme inhibitory properties, tyrosinase inhibitory effects varied from IC50: 1.02 to 1.41 mg/mL. In addition, the best amylase inhibition effect was observed by leaves (3.36 mg/mL), followed by aerial parts, roots, stems, and flowers. As a result, from multivariate analysis, the tested parts were classified in three cluster. Summing up the results, it can be concluded that A. macrocephalus subsp. finitimus could be a precious source of natural bioactive agents in pharmaceutical, nutraceutical, and cosmeceutical applications.


Assays for total phenolic and flavonoid content
For total phenolic content, sample solution (0.25 mL) was mixed with diluted Folin-Ciocalteu reagent (1 mL, 1:9) and shaken vigorously. After 3 min, Na2CO3 solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after 2 h incubation at room temperature. Total phenolic content was expressed as equivalents of gallic acid.
For total flavonoid content, sample solution (1 mL) was mixed with the same volume of aluminium 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 absorbance were read at 415 nm after 10 min incubation at room temperature. Absorbance of the blank was subtracted from that of the sample. Total flavonoid content was expressed as equivalents of quercetin.

Biological activity
Total antioxidant activity of the samples was evaluated by phosphomolybdenum method. Sample solution (0.2 mL) was combined with 2 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 90 min incubation at 95 C.
For 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, sample solution (1 mL) was added to a 4 mL of 0.004% methanol solution of DPPH. Sample absorbance was read at 517 nm after 30 min incubation at room temperature in dark.
For ABTS cation radical scavenging activity, briefly, ABTS .+ radical cation was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 1216 h in dark at the room temperature. Prior to beginning the assay, ABTS solution was diluted with methanol to obtain an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (1 mL) was added to ABTS solution (2 mL) and mixed. Sample absorbance was read at 734 nm after 7 min incubation at room temperature.
For metal chelating activity on ferrous ions, briefly, sample solution (2 mL) 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 absorbance were read at 562 nm after 10 min incubation at room temperature.
For cupric ion reducing activity (CUPRAC), sample solution (0.5 mL) was added to a 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 a premixed reaction mixture (3 mL) without CuCl2. Then, the sample and blank absorbance were read at 450 nm after 30 min incubation at room temperature.
Inhibitory activity on α-amylase was performed using Caraway-Somogyi iodine/potassium iodide (IKI) method. Sample solution (25 µL) was mixed with α-amylase solution (50 µL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well micro plate and incubated for 10 min at 37 C. After preincubation, the reaction was initiated by the addition of starch solution (50 µ L, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme solution (α-amylase). 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 the addition of iodine-potassium iodide solution (100 µL). The sample and blank absorbance were read at 630 nm. Absorbance of the blank was subtracted from that of the sample.
Tyrosinase inhibitory activity was measured using a modified dopachrome method with L-DOPA as substrate. Sample solution (25 µL) was mixed with tyrosinase solution (40 µL) 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). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbance were read at 492 nm after 10 min incubation at 25 °C .
The sample concentration, which decreases the initial concentration by 50% for enzyme inhibition, radical scavenging and metal chelation tests, was defined as IC50, while the EC50 values were calculated as sample concentration providing 0.500 absorbance for reducing power and phosphomolybdenum assays. The biological activities of the extracts were also expressed as mg standard equivalent/g extract and compared with those of the standards, including trolox, ethylenediaminetetraacetic acid (disodium salt) (EDTA), kojic acid, and acarbose, used as positive controls.