Potassium persulfate

The 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assay was performed by an adaptation of the method described by Nenadis et al. [59 (link)]. The ABTS radicals were obtained by the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Alfa Aesar, Tewksbury, MA, USA) with potassium persulfate (Alfa Aesar, Tewksbury, MA, USA). A quantity of 47 mg of ABTS and 12.8 mg of potassium persulfate were added to 250 mL of distilled water and the reaction was placed in the dark at room temperature for 16 h. A volume of 2 mL of the ABTS radical solution was added to 2 mL of aqueous extract sample (0.6 mg ML⁻¹ in distilled water) and 2 mL of distilled water. Each sample was prepared individually, stirred for 1 min and absorbance was measured at 734 nm by an UV-Visible Spectrophotometer (Evolution201, Waltham, MA, USA). The ABTS radical scavenging activity was calculated using the following formula: $$\mathrm{<a\; class="product"\; href="/product/7SriCZIBPBHhf-iFT6QF/"\; target="\_blank">ABTS</a>}\mathrm{radical}\mathrm{scavenging}\mathrm{activity}(\%)=\frac{A_B-A_S}{A_B}\times 100$$
where A_B is the absorbance of the blank and A_S is the absorbance of the sample, both at 734 nm.

Red clover samples were collected in red clover fields in Laičiai, Kupiškis district, Lithuania (latitude 55°53024.2″ N; longitude 25°19036.0″ E). The collections of flower buds and flowers were made on the 26 September 2020. Samples were dried and stored at room temperature. Before use, clover flowers were ground to a fine powder using an Ultra Centrifugal Mill ZM 200 (Retsch, Haan, Germany). Grinding was performed at 4025 g using a 0.5 mm trapezoid hole sieve.
Purified water was prepared with GFL2004 (GFL, Burgwedelis, Germany). Deionized water was prepared with Milipore, SimPak 1 (Merck, Darmstadt, Germany). The following reagents were used: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), aluminium chloride, hexaethylenetetraamine, acetic acid obtained from Sigma-Aldrich (Buchs, Switzerland); potassium persulfate from Alfa Aesar (Karlsruhe, Germany); monosodium phosphate, ferrous sulfate heptahydrate, saline phosphate buffer and hydrogen peroxide from Sigma Aldrich (Schnelldorf, Germany); disodium hydrogen phosphate obtained from Merck (Darmstadt, Germany); 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and α-, β-, and γ-CDs purchased from Sigma Aldrich (Hamburg, Germany); ethanol (96%) (Vilniaus Degtinė, Vilniaus, Lithuania).

Analytical- and chromatographic-grade chemicals and solvents were used for this study: acetonitrile, methanol, glacial acetic acid, erythrodiol, maslinic, oleanolic acids from Sigma-Aldrich (GmbH, Karlsruhe, Germany); uvaol, betulin, corosolic acids, ursolic acid from Carl Roth (Karlsruhe, Germany); polyphenols (gallic acid, ellagic acid, neochlorogenic acid, chlorogenic acid, oenothein B, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, guaijaverin, afzelin) were purchased from ChromaDex (Santa Ana, CA, USA), Sigma-Aldrich (Saint Louis, MO, USA), and Hwi Analytik (Rülzheim, Germany); all solvents used were of HPLC grade. Water was obtained using a Mili-Q purification system (Millipore, Burlington, MA, USA). Also, other reagents were used, such as sodium carbonate (Sigma-Aldrich, Scnelldorf, Germany), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), aluminium chloride, hexaethylenetetraamine, acetic acid obtained from Sigma-Aldrich (Buchs, Switzerland); potassium persulfate from Alfa Aesar (Karlsruhe, Germany); Trolox (98%) was received from Fluka Chemika (Buchs, Switzerland).

Graphite (Grafguard 160–50N) was obtained from GrafTech. Sulfuric acid (95.0–98.0%), phosphorus pentoxide (>98%), potassium persulfate (>99.0%), potassium permanganate (>99.0%), titanium n-butoxide (>99%), hydrofluoric acid (48–51%), and potassium dichromate (>99.0%) were purchased from Alfa Aesar and used as received without further purification.

I. indigotica leaf was purchased from an online market (http://hanyakjae.net/, accessed on 5 January 2020) and produced in July 2019 from China. Potassium persulfate, 3,4,5-trimethoxycinnamic acid (TMCA, CAS 90-50-6), 2,2-diphenyl-1-1picrylhydrazyl (DPPH, CAS 1898-66-4), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, CAS 30931-67-0) were purchased from Alfa Aesar (Haverhill, MA, USA). Distilled water was freshly produced using a Milli-Q system (Shinhan Company, Seoul, Korea). All reagents used in this study were of analytical grade. Acetonitrile, methanol, ethyl acetate, ethanol, 1,3-butylene glycol, and dimethyl sulfoxide (DMSO) were purchased from Burdick & Jackson (Muskegon, MI, USA). The primers were obtained from Bioneer Inc. (Daejeon, Korea). The absorbance values for antioxidant activity assays were measured using an infinite F200 microplate reader from Tecan (Mannedorf, Switzerland).