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SUPPLEMENTARY MATERIAL
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Quantification and antioxidant and anti-HCV activities of the constituents from
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the inflorescences of Scabiosa comosa and S. tschilliensis
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Jian-Nan Ma, Sambuu Bolraa, Min Ji, Qian-Qian He, Chao-Mei Ma *
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College of Life Sciences, Inner Mongolia University, 235 Daxuexilu, Huhhot 010021,
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China
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To investigate the bioactive constituents of the inflorescences of Scabiosa
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comosa and S. tschilliensis which are used traditionally for liver diseases, we tested
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the antioxidant activity using ABTS, FRAP, DPPH-UPLC-MS and DPPH assay. In
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addition, cell based anti-HCV acitivity of the major compounds were evaluated. The
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plant
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3,4-dicaffeoylquinic acids (DCQA), 3,5-DCQA and 4,5-DCQA were identified from
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genus Scabiosa. An UPLC-MS method in MRM mode was established to quantify 18
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constituents in the inflorescences of Scabiosa. The 3,5-DCQA, chlorogenic acid and
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some glycosides of luteolin or apigenin were found to be the most abundant
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constituents. Chlorogenic acid and 3,5-DCQA showed excellent radical scavenging
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activity and demonstrated anti-HCV activity. These findings provided scientific
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evidences for the clinic use of this herbal medicine for liver diseases.
extracts
showed
strong
antioxidant
activity.
For
the
first
time,
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Keywords: Scabiosa; dicaffeoylquinic acid; antioxidant; anti-HCV; quantification
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.
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*Corresponding author. Email: cmma@imu.edu.cn
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3. Experimental
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3.1. Chemicals and instruments
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The 2, 2′ - Diphenyl - 1 - picrylhydrazyl (DPPH•) was purchased from
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Sigma-Aldrich, Co., Germany), while 2,2′- azinobis - (3- ethylbenzthiazoline - 6 -
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sulphonic acid) (ABTS•+) and the ferric reducing antioxidant potential (FRAP) assay
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kit were purchased from Beyotime Institute of biotechnology (S0119 and S0116,
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Nantong, China). Maltase assay kit was purchased from Nanjing Jiancheng Bio
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Company (A082-3, Nan Jing, China). Authentic 3,4-DCQA, 4,5-DCQA and
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protocatechuic acid were purchased from Beijing Century Aoke Biotechnology Co.
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Ltd (Beijing, China); Quinic acid was from Sigma-Aldrich Co. (Shanghai, China);
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Caffeic acid and p-coumaric acid were from Alfa Aesar Chemical Co. Ltd. (Shanghai,
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China). Other compounds were purified in our laboratory in the present experiment or
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in a previous research (Jin et al. 2014). Varioskan Flash spectral scanning multimode
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reader was from Thermo Fisher Scientific Oy Microplate Instrumentation Co. Ltd.
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(Vantaa, Finland). Ultra high performance liquid chromatography/Mass spectrometer
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(UPLC-MS) experiments were performed on an Agilent 1290 infinity UPLC and
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Agilent 6430 triple Quad MS system (Agilent, USA) with an auto-sampler and a
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photo-diode array detector (DAD). Analytical grade solvents were used for the
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extraction and isolation. HPLC grade solvents used for UPLC-MS were purchased
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from Fisher scientific Co., China.
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3.2. Plant materials
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The inflorescence of S. comosa was purchased from KuLun Mongolia
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Pharmaceutical Factory (Lot NO. 0203) in Tongliao, Inner Mongolia, China, and that
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of S. tschilliensis from KangMeng Medicine Co. Ltd., Huhhot, Inner Mongolia,
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China. Information about the plant species was provided by the suppliers and the
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plants were further identified by the authors. Voucher specimens (NPFFS-1 for S.
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comosa and NPFFS-2 for S. tschilliensis) were stored in the Lab of Natural Product &
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Functional Foods, College of Life Sciences, Inner Mongolia University.
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3.3. Determination of the free radical scavenging and antioxidant activity
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3.3.1. Sample preparation for bio-activity assessment
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The dried inflorescences of SC or ST were pulverized in a metal blender (QE-100 g,
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650W, 25000 rpm. Yi Li Industry and Trade Co., Ltd. Zhejiang, China). The resulted
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powder was weighed (40 mg) in a centrifugal tube and subsequently extracted with 2
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mL 70% ethanol under sonication (Power: 500 W, Frequency: 40 KHz) for 3 times
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(10 min/time). After 5 min of centrifugation at 13500 rpm (12225 × g), the
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supernatant was filtered with a 0.22 µm microfilter and stored at 4 °C for the
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following bio-activity analysis.
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3.3.2. DPPH• scavenging activity
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DPPH• scavenging activity was determined as described by Ma (Ma et al. 2012) in
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96-well micro-plates. The test sample (10 μL) with a concentration range of 0.625-10
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mg/mL, was added to 190 μL DPPH solution (0.1 mM in 70% ethanol). An equal
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amount of 70% ethanol in place of sample solution was used as a control. The mixture
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was incubated for 20 min in the dark at room temperature. The absorbance (A) of the
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reaction solution was measured at 520 nm. Activity represented as scavenging (%)
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was calculated by the following formula:
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DPPH• scavenging activity (%) = (Acontrol – Asample) / Acontrol × 100
The concentration of each sample to cause a 50% decrease of the initial DPPH• was
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defined as IC50 value.
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3.3.3. ABTS•+ scavenging activity
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ABTS•+ scavenging activity was assayed according to instruction by the
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manufacturer. The working solution was prepared by mixing ABTS and oxidant
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solutions in equal quantities and allowing them to react for 12 h at room temperature
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in dark. The solution was then diluted by mixing 1 mL working solution with 59 mL
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70% ethanol in order to obtain an absorbance of 0.68±0.03 at 734 nm. Sample
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solution (10 μL) with a concentration range of 0.625-10 mg/mL was mixed with 190
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μL of freshly prepared ABTS solution and the mixture was left at room temperature
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for 8 min. The absorbance at 734 nm was the measured and the ABTS•+ scavenging
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activity was calculated as follows:
ABTS•+ scavenging activity (%) = (Acontrol – Asample) / Acontrol × 100
The IC50 value was determined to be an effective concentration at which the
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ABTS•+ was scavenged by 50%.
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3.3.4. Ferric reducing antioxidant power (FRAP)
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FRAP experiment was performed according to instruction of Beyotime Institute of
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Biotechnology. Stock solutions included TPTZ (2,4,6-tripyridyl-s-triazine) solution,
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TPTZ dilution and detective buffer. The working FRAP reagent was freshly prepared
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by mixing TPTZ dilution, detective buffer and TPTZ solution in the ratio of 10:1:1
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(v/v/v). A sample solution (5 μL) at different concentrations ranging from 0.625 to 10
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mg/mL was mixed with 180 μL of FRAP reagent and kept at 37 ℃ for 5 min. The
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absorbance of the reaction mixture was then recorded at 593 nm. Various
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concentrations (0.31-5.00 mM) of FeSO4 were used to establish the standard curve.
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The assay result was expressed by FeSO4 values, which were calculated using
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standard curves.
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3.4. Analysis of caffeoylquinic acids and flavonoids in SC and ST
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3.4.1. Identification of 18 major constituents
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The structures of compounds 1-18 are shown in Figure 1: 3,5-DCQA (1),
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4,5-DCQA (2), 3,4-DCQA (3), chlorogenic acid (4), quinic acid (5), caffeic acid (6),
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protocatechuic acid (7), p-coumaric acid (8), apigenin (9), apigenin-4'-glucoside (10),
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apigenin-7-glucoside (11), apigenin-7-arabino(1~6)-glucoside (12), luteolin (13),
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luteolin-4'-glucoside (14), luteolin-7-glucoside (15), luteolin-6-C-glucoside (16),
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quercetin-3-glucoside (17), quercetin-3-rutinoside (rutin) (18).
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Compounds 1-3, 5-8, 11, 16-18 were identified using UPLC-MS by comparing with
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authentic reference standards. Compounds 4, 9, 10, 12-15 were isolated from SC in
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our laboratory in a previous work (Ji et al. 2014). Related compounds:
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apigenin-6-C-glucoside (isovitexin), apigenin-8-C-glucoside (vitexin), quercetin,
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quercetin-3-rhamnoside (quercitrin), and quercetin-3-galactoside (hyperoside) were
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screened by UPLC-MS to see if they existed in SC or ST, and the result indicated that
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these 5 compounds were below detection limits in the herb extracts.
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3.4.2. Sample preparation for UPLC-MS analysis
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The plant powder was extracted with 70% ethanol containing two internal standards
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(I.S.s, 10 μg/mL), which were abrusin 2′′-O-β-apioside (Ma et al. 1998) for flavonoids,
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and 1,7-(4-heptanone) ketal of chlorogenic acid (Ma et al. 2008) for caffeoylquinic
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acids. Other steps were the same as for sample preparation for determination of
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bio-activity. After microfiltration, the sample solution was transferred to
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automsampler vials for UPLC-MS analysis. The calibration standards were prepared
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at 10 concentration levels.
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3.4.3. UPLC-MS conditions.
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An Agilent ZORBAX SB-C18 column (50 mm×2.1 mm i.d.; particle size 1.8 µm)
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was used for the separation. The mobile phase composed of water /formic acid
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(100:0.1, v/v) (solvent A) and 100% methanol (solvent B). The elution program was:
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0-12 min, 6-12% B; 12-13 min, 12-21% B; 13-43 min, 21-25% B; 43-44 min, 25-32%
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B; 44-54 min, 32-45% B; 54-55 min, 45-100% B; 55-57 min, 100% B. The flow rate
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was 400 µL/min and injection volume was 2 µL. MS analysis was performed in
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multiple reaction monitoring (MRM) mode with capillary 4 kV, gas flow 11 L/min,
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nebulizer 45 psi, source temperature 350 °C. The optimized analysis conditions are
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listed in Table S1 and the representative chromatograms are shown in Figure S2.
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3.4.4. Quantification analysis
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The calibration formulas is expressed in Y=kX+b (R2 > 0.9995), where X stands for
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the concentration of the analyte and Y is the response factor (peak area of the
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analyte/peak area of the I.S). The quantification limit was defined as the concentration
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at which the signal-to-noise ratio (S/N) was ≥ 10. Limits of quantification ranged from
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0.21 to 5.12 µg/mL and limits of determination ranged from 12.3 to 72.1 ng/mL for the
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18 major compounds.
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3.5 . Screening and determination of antioxidants in the SC and ST
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3.5.1. DPPH-UPLC-MS method
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For characterization and identification of antioxidants in SC or ST, the method
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based on DPPH spiking test combined with UPLC-MS was carried out according to
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the literature (Li et al. 2011) with some modifications. Briefly, after the samples and
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DPPH solution were filtered with 0.22 µm microfilters, 50 µL of SC or ST (20 mg/mL)
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solution was mixed with 150 µL of DPPH (2.8 mM). As a control, 150 µL of 70%
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ethanol instead of the DPPH solution, was mixed with 50 µL SC or ST. The mixtures
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were kept in dark for 20 min at room temperature. The resultant solutions were
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analyzed by UPLC-MS. The UPLC-MS conditions were the same as for constituent
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quantification.
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Percentage of the reduced peak area of compounds in each sample was determined
with the following formula:
Reduced ratio (%) = 100 × (Area before reaction - Area after reaction)/Area before
reaction
The antioxidant ability of the compounds was expressed by the reduced ratio, that
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is, higher reduced ratio representing stronger oxidation resistance.
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3.5.2. DPPH radical scavenging method
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According to the DPPH-UPLC-MS screening results, 11 compounds were
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identified as antioxidants in SC or ST. Various concentrations of the 11 compounds
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were used instead of the extracts to measure their IC50 values in 96-well micro-plates
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and a plate reader using the procedure described in 3.4.2.
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3.6. Determination of anti-HCV activity
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Anti-HCV activities were evaluated in vitro in the virus infection human hepatoma
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cell lines (Huh7) with a procedure described in the literature (Liu et al. 2012).
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3.7. Statistical analysis
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Date was expressed as mean ± standard deviation (SD) of triplicate experiments.
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IC50 values were obtained from concentration-dependent inhibition curves for
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bio-activity tests. Linear regression and calibration were used in all quantification
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analysis.
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References
6
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Ji M, Li SJ, Ma CM. 2014. Chemical Constituents of the Inflorescence of Scabiosa
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comosa Fisch and Their Antioxide and α-Glucosidase Inhibitory Activities.
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Journal of Inner Mongolia University (Natural Science Edition). 4: 398 – 403.
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Li YJ, Chen J, Li Y, Li Q, Zheng YF, Fu Y, Li P. 2011. Screening and characterization
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of nature antioxidants in four Glycyrrhiza species by liquid chromatography
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coupled with electrospray ionization quadrupole time-of-flight tandem mass
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spectrometry. J Chromatogr A. 1218: 8181 – 8192.
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Ma CM, Hattori M, Daneshtalab M, Wang LL. 2008. Chlorogenic Acid Derivatives
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with Alkyl Chains of Different Lengths and Orientations: Potent #-Glucosidase
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Inhibitors. J Med Chem. 51: 6188 – 6194.
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Ma CM, Nakamura N, Hattori M. 1998. Saponins and C-glycosyl flavones from the
seeds of Abrus precatorius. Chem Pharml Bull. 46: 982 – 987.
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Components
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Apple-Shaped Pear (Fruit of Pyrus pyrifolia cv. pingguoli). J Food Sci. 77: 1097
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– 1102.
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and Antioxidant Activity of
the Peels
of Commercial
Meng HC, Ma CM. 2013. Flavan-3-ol-cysteine and acetylcysteine conjugates from
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edible reagents and the stems of Cynomorium songaricum as potent antioxidants.
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Food Chem. 141: 2691 – 2696.
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Song WH, Liu MM, Zhong DW, Zhu YL, Bosscher M, Zhou L, Ye DY, Yuan ZH.
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2013. Tetrazole and triazole as bioisosteres of carboxylic acid: Discovery of
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diketo tetrazoles and diketo triazoles as anti-HCV agents. Bioorg Med Chem
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Lett. 23: 4528 – 4531.
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Table S1. Analytical parameters of interest from UPLC-MS-MRM chromatograms
a
Product ion
Compound name
tR
(min)
Parent ion
－
[M-H]
Base
Secondary
3,5-DCQA (1)
4,5-DCQA (2)
3,4-DCQA (3)
Chlorogenic acid (4)
Quinic Acid (5)
Caffeic acid (6)
Protocatechuic acid (7)
p-Coumaric acid (8)
Apigenin (9)
Apigenin-4'-glucosied (10)
Apigenin-7-glucosied (11)
Apigenin-7-arabino(1~6)-glucoside (12)
Luteolin (13)
Luteolin-4'-glucoside (14)
Luteolin-7-glucoside (15)
Luteolin-6-C-glucoside (16)
Quercetin-3-glucoside (17)
Quercetin-3-rutinoside (18)
I.S.-1 a,b
I.S.-2 a,b
28.00
26.37
40.74
7.42
0.40
6.94
2.06
11.55
52.04
32.64
31.16
30.64
48.20
35.05
24.37
19.74
25.48
26.92
34.43
55.208
515.0
515.0
515.0
353.0
191.1
179.0
153.1
163.0
269.1
431.0
431.0
563.0
285.0
447.0
447.0
447.0
463.0
609.0
607.0
449.0
353.0
352.9
352.9
191.0
85.1
135.1
109.0
119.0
117.0
268.0
268.0
269.0
133.0
285.0
285.0
357.0
300.0
300.0
354.0
179.0
191.0
178.9
173.0
93.1
107.0
107.0
327.0
325.0
135.0
I.S. is the internal standard.
2′′-O-β-apioside; I.S.-2: 1,7-(4-Heptanone) ketal of chlorogenic acid
bI.S.-1: Abrusin
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203
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Table S2. The reduced ratios of screened antioxidants in 70% ethanol extracts of ST and SC by
DPPH-UPLC-MS method
Compound name
3,5-DCQ (1)
4,5-DCQ (2)
3,4-DCQ (3)
Chlorogenic acid (4)
Caffeic acid (6)
Protocatechuic acid (7)
Luteolin (13)
Luteolin-7-glucoside (15)
Luteolin-6-C-glucoside (16)
Quercetin-3-glucoside (17)
Quercetin-3-rutinoside (18)
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aThe
Reduced ratio (%)
ST a
SC a
75.38
81.34
80.52
78.44
41.09
44.89
82.66
89.76
86.51
84.90
86.43
81.65
88.65
83.70
81.48
43.65
46.53
87.29
95.22
89.97
86.45
89.58
final concentrations of ST and SC were 5 mg/mL
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Figure captions
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Figure S1. Antioxidant activity of SC and ST at various concentrations in FRAP
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assay
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Figure S2. Multiple reaction monitoring (MRM) chromatograms in UPLC-MS of (top)
authentic compounds, (middle) ST and (bottom) SC
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10
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Figure S1. Antioxidant activity of SC and ST at various concentrations in FRAP assay
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223
224
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226
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Figure S2. Multiple reaction monitoring (MRM) chromatograms in UPLC-MS of (top) authentic
compounds, (middle) ST and (bottom) SC
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