1SUPPLEMENTARY MATERIAL

2 Quantification and antioxidant and anti-HCV activities of the constituents from 3 the inflorescences of Scabiosa comosa and S. tschilliensis 4 5 6 Jian-Nan Ma, Sambuu Bolraa, Min Ji, Qian-Qian He, Chao-Mei Ma * 7 8College of Life Sciences, Inner Mongolia University, 235 Daxuexilu, Huhhot 010021, 9China 10 11 To investigate the bioactive constituents of the inflorescences of Scabiosa 12comosa and S. tschilliensis which are used traditionally for liver diseases, we tested 13the antioxidant activity using ABTS, FRAP, DPPH-UPLC-MS and DPPH assay. In 14addition, cell based anti-HCV acitivity of the major compounds were evaluated. The 15plant extracts showed strong antioxidant activity. For the first time, 3,4- 16dicaffeoylquinic acids (DCQA), 3,5-DCQA and 4,5-DCQA were identified from 17genus Scabiosa. An UPLC-MS method in MRM mode was established to quantify 18 18constituents in the inflorescences of Scabiosa. The 3,5-DCQA, chlorogenic acid and 19some glycosides of luteolin or apigenin were found to be the most abundant 20constituents. Chlorogenic acid and 3,5-DCQA showed excellent radical scavenging 21activity and demonstrated anti-HCV activity. These findings provided scientific 22evidences for the clinic use of this herbal medicine for liver diseases. 23 24Keywords: Scabiosa; dicaffeoylquinic acid; antioxidant; anti-HCV; quantification 25 26 . 27*Corresponding author. Email: [email protected] 28 29 30 313. Experimental

1 1 323.1. Chemicals and instruments

33 The 2, 2′ - Diphenyl - 1 - picrylhydrazyl (DPPH•) was purchased from Sigma- 34Aldrich, Co., Germany), while 2,2′- azinobis - (3- ethylbenzthiazoline - 6 - sulphonic 35acid) (ABTS•+) and the ferric reducing antioxidant potential (FRAP) assay kit were 36purchased from Beyotime Institute of biotechnology (S0119 and S0116, Nantong, 37China). Maltase assay kit was purchased from Nanjing Jiancheng Bio Company 38(A082-3, Nan Jing, China). Authentic 3,4-DCQA, 4,5-DCQA and protocatechuic acid 39were purchased from Beijing Century Aoke Biotechnology Co. Ltd (Beijing, China); 40Quinic acid was from Sigma-Aldrich Co. (Shanghai, China); Caffeic acid and p- 41coumaric acid were from Alfa Aesar Chemical Co. Ltd. (Shanghai, China). Other 42compounds were purified in our laboratory in the present experiment or in a previous 43research (Jin et al. 2014). Varioskan Flash spectral scanning multimode reader was 44from Thermo Fisher Scientific Oy Microplate Instrumentation Co. Ltd. (Vantaa, 45Finland). Ultra high performance liquid chromatography/Mass spectrometer (UPLC- 46MS) experiments were performed on an Agilent 1290 infinity UPLC and Agilent 6430 47triple Quad MS system (Agilent, USA) with an auto-sampler and a photo-diode array 48detector (DAD). Analytical grade solvents were used for the extraction and isolation. 49HPLC grade solvents used for UPLC-MS were purchased from Fisher scientific Co., 50China. 513.2. Plant materials

52 The inflorescence of S. comosa was purchased from KuLun Mongolia 53Pharmaceutical Factory (Lot NO. 0203) in Tongliao, Inner Mongolia, China, and that 54of S. tschilliensis from KangMeng Medicine Co. Ltd., Huhhot, Inner Mongolia, 55China. Information about the plant species was provided by the suppliers and the 56plants were further identified by the authors. Voucher specimens (NPFFS-1 for S. 57comosa and NPFFS-2 for S. tschilliensis) were stored in the Lab of Natural Product & 58Functional Foods, College of Life Sciences, Inner Mongolia University. 593.3. Determination of the free radical scavenging and antioxidant activity

603.3.1. Sample preparation for bio-activity assessment

2 2 61 The dried inflorescences of SC or ST were pulverized in a metal blender (QE-100 62g, 650W, 25000 rpm. Yi Li Industry and Trade Co., Ltd. Zhejiang, China). The 63resulted powder was weighed (40 mg) in a centrifugal tube and subsequently 64extracted with 2 mL 70% ethanol under sonication (Power: 500 W, Frequency: 40

65KHz) for 3 times (10 min/time). After 5 min of centrifugation at 13500 rpm (12225 × 66g), the supernatant was filtered with a 0.22 µm microfilter and stored at 4 °C for the 67following bio-activity analysis. 683.3.2. DPPH• scavenging activity

69 DPPH• scavenging activity was determined as described by Ma (Ma et al. 2012) in 7096-well micro-plates. The test sample (10 μL) with a concentration range of 0.625-10 71mg/mL, was added to 190 μL DPPH solution (0.1 mM in 70% ethanol). An equal 72amount of 70% ethanol in place of sample solution was used as a control. The mixture 73was incubated for 20 min in the dark at room temperature. The absorbance (A) of the 74reaction solution was measured at 520 nm. Activity represented as scavenging (%) 75was calculated by the following formula:

76 DPPH• scavenging activity (%) = (Acontrol – Asample) / Acontrol × 100

77 The concentration of each sample to cause a 50% decrease of the initial DPPH• was

78defined as IC50 value. 793.3.3. ABTS•+ scavenging activity

80 ABTS•+ scavenging activity was assayed according to instruction by the 81manufacturer. The working solution was prepared by mixing ABTS and oxidant 82solutions in equal quantities and allowing them to react for 12 h at room temperature 83in dark. The solution was then diluted by mixing 1 mL working solution with 59 mL 8470% ethanol in order to obtain an absorbance of 0.68±0.03 at 734 nm. Sample 85solution (10 μL) with a concentration range of 0.625-10 mg/mL was mixed with 190 86μL of freshly prepared ABTS solution and the mixture was left at room temperature 87for 8 min. The absorbance at 734 nm was the measured and the ABTS•+ scavenging 88activity was calculated as follows:

+ 89 ABTS• scavenging activity (%) = (Acontrol – Asample) / Acontrol × 100

3 3 90 The IC50 value was determined to be an effective concentration at which the 91ABTS•+ was scavenged by 50%. 923.3.4. Ferric reducing antioxidant power (FRAP)

93 FRAP experiment was performed according to instruction of Beyotime Institute of 94Biotechnology. Stock solutions included TPTZ (2,4,6-tripyridyl-s-triazine) solution, 95TPTZ dilution and detective buffer. The working FRAP reagent was freshly prepared 96by mixing TPTZ dilution, detective buffer and TPTZ solution in the ratio of 10:1:1 97(v/v/v). A sample solution (5 μL) at different concentrations ranging from 0.625 to 10

98mg/mL was mixed with 180 μL of FRAP reagent and kept at 37 ℃ for 5 min. The 99absorbance of the reaction mixture was then recorded at 593 nm. Various

100concentrations (0.31-5.00 mM) of FeSO4 were used to establish the standard curve.

101The assay result was expressed by FeSO4 values, which were calculated using 102standard curves. 1033.4. Analysis of caffeoylquinic acids and flavonoids in SC and ST

1043.4.1. Identification of 18 major constituents

105 The structures of compounds 1-18 are shown in Figure 1: 3,5-DCQA (1), 4,5- 106DCQA (2), 3,4-DCQA (3), chlorogenic acid (4), quinic acid (5), caffeic acid (6), 107protocatechuic acid (7), p-coumaric acid (8), apigenin (9), apigenin-4'-glucoside (10), 108apigenin-7-glucoside (11), apigenin-7-arabino(1~6)-glucoside (12), luteolin (13), 109luteolin-4'-glucoside (14), luteolin-7-glucoside (15), luteolin-6-C-glucoside (16), 110quercetin-3-glucoside (17), quercetin-3-rutinoside (rutin) (18). 111 Compounds 1-3, 5-8, 11, 16-18 were identified using UPLC-MS by comparing with 112authentic reference standards. Compounds 4, 9, 10, 12-15 were isolated from SC in 113our laboratory in a previous work (Ji et al. 2014). Related compounds: apigenin-6-C- 114glucoside (isovitexin), apigenin-8-C-glucoside (vitexin), quercetin, quercetin-3- 115rhamnoside (quercitrin), and quercetin-3-galactoside (hyperoside) were screened by 116UPLC-MS to see if they existed in SC or ST, and the result indicated that these 5 117compounds were below detection limits in the herb extracts. 1183.4.2. Sample preparation for UPLC-MS analysis

4 4 119 The plant powder was extracted with 70% ethanol containing two internal standards 120(I.S.s, 10 μg/mL), which were abrusin 2′′-O-β-apioside (Ma et al. 1998) for 121flavonoids, and 1,7-(4-heptanone) ketal of chlorogenic acid (Ma et al. 2008) for 122caffeoylquinic acids. Other steps were the same as for sample preparation for 123determination of bio-activity. After microfiltration, the sample solution was 124transferred to automsampler vials for UPLC-MS analysis. The calibration standards 125were prepared at 10 concentration levels. 1263.4.3. UPLC-MS conditions.

127 An Agilent ZORBAX SB-C18 column (50 mm×2.1 mm i.d.; particle size 1.8 µm) 128was used for the separation. The mobile phase composed of water /formic acid 129(100:0.1, v/v) (solvent A) and 100% methanol (solvent B). The elution program was: 1300-12 min, 6-12% B; 12-13 min, 12-21% B; 13-43 min, 21-25% B; 43-44 min, 25-32% 131B; 44-54 min, 32-45% B; 54-55 min, 45-100% B; 55-57 min, 100% B. The flow rate 132was 400 µL/min and injection volume was 2 µL. MS analysis was performed in 133multiple reaction monitoring (MRM) mode with capillary 4 kV, gas flow 11 L/min, 134nebulizer 45 psi, source temperature 350 °C. The optimized analysis conditions are 135listed in Table S1 and the representative chromatograms are shown in Figure S2. 1363.4.4. Quantification analysis

137 The calibration formulas is expressed in Y=kX+b (R2 > 0.9995), where X stands for 138the concentration of the analyte and Y is the response factor (peak area of the 139analyte/peak area of the I.S). The quantification limit was defined as the concentration 140at which the signal-to-noise ratio (S/N) was ≥ 10. Limits of quantification ranged 141from 0.21 to 5.12 µg/mL and limits of determination ranged from 12.3 to 72.1 ng/mL 142for the 18 major compounds. 1433.5 . Screening and determination of antioxidants in the SC and ST 1443.5.1. DPPH-UPLC-MS method

145 For characterization and identification of antioxidants in SC or ST, the method 146based on DPPH spiking test combined with UPLC-MS was carried out according to 147the literature (Li et al. 2011) with some modifications. Briefly, after the samples and 148DPPH solution were filtered with 0.22 µm microfilters, 50 µL of SC or ST (20

5 5 149mg/mL) solution was mixed with 150 µL of DPPH (2.8 mM). As a control, 150 µL of 15070% ethanol instead of the DPPH solution, was mixed with 50 µL SC or ST. The 151mixtures were kept in dark for 20 min at room temperature. The resultant solutions 152were analyzed by UPLC-MS. The UPLC-MS conditions were the same as for 153constituent quantification. 154 Percentage of the reduced peak area of compounds in each sample was determined 155with the following formula:

156 Reduced ratio (%) = 100 × (Area before reaction - Area after reaction)/Area before 157reaction

158 The antioxidant ability of the compounds was expressed by the reduced ratio, that 159is, higher reduced ratio representing stronger oxidation resistance. 1603.5.2. DPPH radical scavenging method

161 According to the DPPH-UPLC-MS screening results, 11 compounds were 162identified as antioxidants in SC or ST. Various concentrations of the 11 compounds

163were used instead of the extracts to measure their IC50 values in 96-well micro-plates 164and a plate reader using the procedure described in 3.4.2. 1653.6. Determination of anti-HCV activity 166 Anti-HCV activities were evaluated in vitro in the virus infection human hepatoma 167cell lines (Huh7) with a procedure described in the literature (Liu et al. 2012). 1683.7. Statistical analysis 169 Date was expressed as mean ± standard deviation (SD) of triplicate experiments.

170IC50 values were obtained from concentration-dependent inhibition curves for bio- 171activity tests. Linear regression and calibration were used in all quantification 172analysis. 173 174References 175Ji M, Li SJ, Ma CM. 2014. Chemical Constituents of the Inflorescence of Scabiosa 176 comosa Fisch and Their Antioxide and α-Glucosidase Inhibitory Activities.

177 Journal of Inner Mongolia University (Natural Science Edition). 4: 398 – 403.

6 6 178Li YJ, Chen J, Li Y, Li Q, Zheng YF, Fu Y, Li P. 2011. Screening and characterization 179 of nature antioxidants in four Glycyrrhiza species by liquid chromatography 180 coupled with electrospray ionization quadrupole time-of-flight tandem mass

181 spectrometry. J Chromatogr A. 1218: 8181 – 8192. 182Ma CM, Hattori M, Daneshtalab M, Wang LL. 2008. Chlorogenic Acid Derivatives 183 with Alkyl Chains of Different Lengths and Orientations: Potent #-Glucosidase

184 Inhibitors. J Med Chem. 51: 6188 – 6194. 185Ma CM, Nakamura N, Hattori M. 1998. Saponins and C-glycosyl flavones from the

186 seeds of Abrus precatorius. Chem Pharml Bull. 46: 982 – 987. 187Ma JN, Wang SL, Zhang K, Wu ZG, Hattori M, Chen GL, Ma CM. 2012. Chemical 188 Components and Antioxidant Activity of the Peels of Commercial Apple-Shaped

189 Pear (Fruit of Pyrus pyrifolia cv. pingguoli). J Food Sci. 77: 1097 – 1102. 190Meng HC, Ma CM. 2013. Flavan-3-ol-cysteine and acetylcysteine conjugates from 191 edible reagents and the stems of Cynomorium songaricum as potent antioxidants.

192 Food Chem. 141: 2691 – 2696. 193Song WH, Liu MM, Zhong DW, Zhu YL, Bosscher M, Zhou L, Ye DY, Yuan ZH. 194 2013. Tetrazole and triazole as bioisosteres of carboxylic acid: Discovery of 195 diketo tetrazoles and diketo triazoles as anti-HCV agents. Bioorg Med Chem

196 Lett. 23: 4528 – 4531.

7 7 197 Table S1. Analytical parameters of interest from UPLC-MS-MRM chromatograms

Compound name tR Parent ion Product ion (min) [M-H]－ Base Secondary 3,5-DCQA (1) 28.00 515.0 353.0 191.0 4,5-DCQA (2) 26.37 515.0 352.9 178.9 3,4-DCQA (3) 40.74 515.0 352.9 173.0 Chlorogenic acid (4) 7.42 353.0 191.0 - Quinic Acid (5) 0.40 191.1 85.1 93.1 Caffeic acid (6) 6.94 179.0 135.1 - Protocatechuic acid (7) 2.06 153.1 109.0 - p-Coumaric acid (8) 11.55 163.0 119.0 - Apigenin (9) 52.04 269.1 117.0 107.0 Apigenin-4'-glucosied (10) 32.64 431.0 268.0 - Apigenin-7-glucosied (11) 31.16 431.0 268.0 - Apigenin-7-arabino(1~6)-glucoside (12) 30.64 563.0 269.0 - Luteolin (13) 48.20 285.0 133.0 107.0 Luteolin-4'-glucoside (14) 35.05 447.0 285.0 - Luteolin-7-glucoside (15) 24.37 447.0 285.0 - Luteolin-6-C-glucoside (16) 19.74 447.0 357.0 327.0 Quercetin-3-glucoside (17) 25.48 463.0 300.0 - Quercetin-3-rutinoside (18) 26.92 609.0 300.0 - I.S.-1 a,b 34.43 607.0 354.0 325.0 I.S.-2 a,b 55.208 449.0 179.0 135.0 198a I.S. is the internal standard. 199bI.S.-1: Abrusin 2′′-O-β-apioside; I.S.-2: 1,7-(4-Heptanone) ketal of chlorogenic acid 200 201

8 8 202 203Table S2. The reduced ratios of screened antioxidants in 70% ethanol extracts of ST and SC by 204 DPPH-UPLC-MS method Compound name Reduced ratio (%) ST a SC a 3,5-DCQ (1) 75.38 81.65 4,5-DCQ (2) 81.34 88.65 3,4-DCQ (3) 80.52 83.70 Chlorogenic acid (4) 78.44 81.48 Caffeic acid (6) 41.09 43.65 Protocatechuic acid (7) 44.89 46.53 Luteolin (13) 82.66 87.29 Luteolin-7-glucoside (15) 89.76 95.22 Luteolin-6-C-glucoside (16) 86.51 89.97 Quercetin-3-glucoside (17) 84.90 86.45 Quercetin-3-rutinoside (18) 86.43 89.58 205aThe final concentrations of ST and SC were 5 mg/mL

9 9 206Figure captions 207 208Figure S1. Antioxidant activity of SC and ST at various concentrations in FRAP 209assay 210Figure S2. Multiple reaction monitoring (MRM) chromatograms in UPLC-MS of 211(top) authentic compounds, (middle) ST and (bottom) SC 212

10 10 213

214 215 Figure S1. Antioxidant activity of SC and ST at various concentrations in FRAP assay

11 11 216 217

218 219Figure S2. Multiple reaction monitoring (MRM) chromatograms in UPLC-MS of (top) authentic 220compounds, (middle) ST and (bottom) SC 221

12 12