SUPPLEMENTARY MATERIAL Antiradical and antioxidant activity of flavones from Scutellariae baicalensis radix

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1 SUPPLEMENTARY MATERIAL Antiradical and antioxidant activity of flavones from Scutellariae baicalensis radix Dorota Woźniak A, Andrzej Dryś B, and Adam Matkowski* A A Department of Pharmaceutical Biology and Botany, B Department of Physical Chemistry, Medical University of Wrocław, Borowska 211, Wrocław, Poland *corresponding author: fitoterapia@prokonto.pl Abstract We evaluated antioxidant properties of four main flavones from Scutellaria baicalensis: baicalein, wogonin and their glucuronides - baicalin and wogonoside. We used three in vitro assays: Free radical scavenging with 2,2 -diphenylpicrylhydrazyl radical (DPPH), transition metal ions reducing power by phosphomolybdenum assay, and inhibition of the hydroxyl radical-induced peroxidation of linoleic acid assay. All flavones have antioxidant capacity, which differs depending on the structure and mechanisms of activity. In all tests, only baicalein the aglycone with three adjacent hydroxyl groups exhibited consistent antioxidant effect. Wogonin protected linoleic acid against oxidation. Baicalin displayed less potent antioxidant properties whereas wogonoside did not have significant antioxidant activity. key words: baicalin; wogonin; lipid peroxidation; Scutellaria baicalensis; antioxidant. 1. Experimental 1.1 Materials Plant material The roots of Baikal skullcap (Scutellariae baicalensis radix) were harvested in October from the plants cultivated in a certified collection of the Medicinal Plants Botanic Garden of Wroclaw Medical University, Poland. The voucher specimen is stored in the herbarium of the Medicinal Plants Botanic Garden under the accession No. Lamiaceae-Sbaicalensis2012/1. The crude drug was washed, airdried and ground Chemicals 2,2-diphenyl-1-picrylhydrazyl (DPPH) was from Sigma-Aldrich (USA), trichloroacetic acid from Ubichem (U.K.), linoleic acid from Fluka (Switzerland), all other chemicals from POCh (Gliwice, Poland). 3.1 Methods Sample preparation

2 To obtain the methanolic extract, 1000 g of the dry, ground crude drug was extracted twice under reflux with 1000 ml 50% aqueous MeOH (v/v) at 80ºC for 12 h each time. The crude aqueous MeOH extract was evaporated to dryness using a rotary vacuum evaporator (Laborota, Heidolph, Germany). Then, the dried extract was dissolved in analytical-grade EtOH acidified with 1M HCl to obtain ph 6.0. The acid ethanol solution was left at room temperature for sedimentation, and then it was decanted to obtain crude crystals and a liquor (Figure S1). The sedimented crystals were redissolved in EtOH and crystallized, resulting in crystalline baicalin (F1, 8.24g). The liquor was evaporated as above to yield yellow powder, fractionated by silica gel G60 (Merck) column (40 cm x 2cm) chromatography with n-hexane: EtOAc 2:1, to get F3 and F4 followed by 6% HOAc in MeOH to obtain F5 and F6 fractions. Fractions F4 and F5 contained wogonoside (2.6g) as the main compound. Wogonin (F7, 0.93g) and baicalein (F2, 1.8g) - aglycones of wogonoside and baicalin were obtained after enzymatic hydrolysis. The glycosides were dissolved in 2 ml of buffer ph 5 and about 1 mg of β- glucuronidase (Sigma) was added. The mixture was allowed to stand overnight at 37ºC. All the stages of flavone-enriched fractions isolation were monitored by using TLC and HPLC, the chromatogram peaks were identified by comparison of the retention times and UV spectra to authentic standards as described by Woźniak et al. (2004) Anti-oxidant assays To estimate the direct antioxidant action such as scavenging or reducing power towards transition metal ions of tested flavones we used DPPH free radical assay and phosphomolybdenum assay, respectively. The linoleic acid peroxidation test allows assessment of preventive properties of flavones against oxidation of substrate DPPH free radical scavenging. The samples containing any of the four flavones at concentration range µg/ml in methanol, were mixed with the same volume of methanolic DPPH solution (0.2 mm). The absorbance was measured at 517 nm immediately after mixing and subsequently after 1, 5, 10, 15, 20, and 30 minutes using μquant multiplate reader (Biotek, USA). Additionally 0.1 mm MeOH solution of DPPH was taken as a negative control. Antiradical activity was expressed as the concentration of an antioxidant generating 50% of the maximum scavenging effect (EC 50) calculated from nonlinear regression curve (GraphPad Prism 5.0) Reducing power assay. Phosphomolybdenum assay according to Prieto et al. (1999) was used to estimate the capacity of the flavones samples to reduce transition-metal ions. The reagent mixture was comprised of ammonium molybdate tetrahydrate (4mM), tribasic sodium phosphate dihydrate (28mM), sulfuric acid (600mM, analytical grade) and the methanolic solution of flavones at the concentration range of µg/ml. The samples were incubated at 40 ºC and 90 ºC for 90 minutes. After cooling down to room temperature, the absorbance of the green Mo(V) complex (max. absorption at 695 nm) was measured as a result of the reduction of Mo(VI) to Mo(V) by the flavones. For reference, appropriate solutions of ascorbic acid were used, and the reducing capacity of the analyzed fractions was expressed as the ascorbic acid equivalents (AAE) expressed as K1/K2 ratio

3 where K1 was the slope coefficient of linear dose response function of the samples, and K2 the slope coefficient of ascorbic acid Linoleic acid peroxidation assay. In this experiment, we assessed an indirect antioxidative action in linoleic acid assay to test the ability of antioxidants to prevent hydroxyl radical-induced peroxidation of a polyunsaturated fatty acid. The flavone samples in the concentration range of µg/ml in methanol, were mixed with 300 µl 50mM phosphate buffer, ph 7.5, 500 µl of 20 mm linoleic acid and 100µl of 5 mm ascorbic acid. The peroxidation was started with addition of 100 µl 4 mm FeSO 4 7H 2O. The samples were incubated for 60 min at 37ºC. Thereafter, 2 ml of 10% ice cold trichloroacetic acid was added and 1 ml of the samples was mixed with 1 ml of 1% thiobarbituric acid (TBA). The TBA/sample mixture was heated in a water bath at 95 ºC for another 60 minutes. The absorbance of colored TBARS (ThioBarbituric Acid Reactive Substances) a product of lipid peroxidation was measured at 532 nm. The percent of inhibition was calculated using appropriate controls and the following formula: % inhibition = (ABS control (ABS sample ABS flavone) x 100/ABS control. ABS control includes methanol instead of the flavone fraction. ABS flavone is the absorbance of the sample containing flavones but without linoleic acid solution that was replaced by water. IC 50 was also calculated from nonlinear regression using GraphPad Prism Statistical analysis The comparison of means was performed by use of one-way ANOVA with post hoc evaluation of statistical significance by Tukey s test at p<0.05 (Statistica 8.0, Statsoft, Poland). DPPH and lipid peroxidation assays were performed in six repetitions (n=6). Phosphomolybdenum assay was performed in eight replicates (n=8). The ANOVA was run for DPPH and LPO blanked absorbance results expressed as percent of scavenging/inhibition and for computed ascorbic acid equivalents in phosphomolybdenum test. In the DPPH assay, we performed ANOVA for 10 minutes incubation time as the most differentiating the four tested flavonoids. For comparing slopes in phosphomolybdenum assay we used GraphPad Prism 6 ANCOVA (Analysis of Covariance module) References Prieto P, Pineda M, Aguilar M. 1999, Spectrophotometric quantitation of antioxidant capacity thrugh the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 269: Woźniak D, Lamer-Zarawska E, Matkowski A. 2004, Antimutagenic and antiradical properties of flavones from the roots of Scutellaria baicalensis Georgi. Nahrung/Food 48: 9-12.

4 Supplemental figures Figure S1. The structures of four flavones from Scutellaria baicalensis roots tested in this study. Figure S2. The diagram of isolation of flavone fractions from dried Scutellariae radix

5 Figure S3. Concentration response in DPPH scavenging assay after 10 minutes of incubation. Data points represent means of six measurements. Figure S4. A comparison of the time course of DPPH scavenging by baicalein, baicalin, and wogonoside. The data points are means of six measurements with error bars indicating standard deviation.