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12 protocols using caffeic acid

1

Antioxidant Capacity Evaluation of Phenolic Compounds

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Six reference compounds, including caftaric acid (S1), chlorogenic acid (S2), caffeic acid (S3), cichoric acid (S4), 3,5-di-O-caffeoylquinic acid (S5), and luteolin (S6) were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). The structures of the six reference compounds are shown in Figure 5. The purities of these compounds were determined to be higher than 98% by high performance liquid chromatography diode array detection analysis. Total antioxidant capacity assay kits (ABTS and FRAP method) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu reagent were purchased from Macklin (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China). (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was purchased from Aladdin (Shanghai Aladdin Bio-Chem Technology Co., LTD, Shanghai, China). Gallic acid and rutin were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China).
Methanol, acetonitrile and formic acid of HPLC grade were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Deionized water was prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other reagents and chemicals used in the experiment were of analytical grade.
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2

Isolation and Identification of Phytochemicals from Forsythia suspensa

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Lianqiaoxinside A was isolated by the author from the fruits of F. suspensa. Chlorogenicacid, caffeic acid, forsythiaside B, rutin, hyperoside, forsythiaside A, cynaroside, scutellarin, (+)-pinoresinol-β-d-glucoside, isochlorogenic acid A, baicalein, baicalin, phillyrin, phillygenin, arctiin, quercetin, luteolin (≥98.0%)were purchased from the Chengdu MUST Biotechnology Co., Ltd. (Chengdu, China). HPLC grade acetonitrile (ACN) and formic acid were obtained from Fisher Scientific (Waltham, MA, USA). Purified water was used from a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents were of analytical grade.
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3

Synthesis of Phenolic Metabolites

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P. appendiculatum plants were maintained in a greenhouse under a 12 h photoperiod and a temperature of ~25°C. Flavonoids and their methylated products, caffeic acid, SAM, and other reagents were purchased from Chengdu Must Bio-technology (Chengdu, China), Alfa Aesar (Heysham, UK), and Sigma-Aldrich (St. Louis, MO, USA), respectively. The synthesis of caffeoyl aldehyde, caffeoyl alcohol, 5-hydroxy coniferyl aldehyde, and 5-hydroxy coniferyl alcohol was performed following procedures published elsewhere [23 (link),24 (link)]. CCoA was synthesized from caffeic acid using liverwort 4-coumarate CoA ligase [25 (link)].
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4

Quantitative Analysis of Bioactive Compounds

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Reference standards including chlorogenic acid, caftaric acid, caffeic acid, cichoric acid, luteolin, luteolin-7-O-β-d-glucoside, isochlorogenic acid A, isochlorogenic acid C, apigenin and quercetin were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). DPPH (1,1-Diphenyl-2-picrylhydrazyl), copper (II) chloride, ascorbic acid, sodium acetate trihydrate, iron (III) chloride hexahydrate, TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), copper (III) chloride, neocuproine, Folin-Ciocalteu reagent, gallic acid, sodium nitrite, and catechin were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents used in this study were of analytical grade.
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5

Quantification of Phenolic Compounds in Semen Cuscutae

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Methanol and acetonitrile (HPLC pure grade) were purchased from Fisher Co., Ltd. Formic acid was of chromatographic purity obtained from ROE Co., Ltd. Ultrapure water for the HPLC-MS/MS analysis was purified by Milli-Q water purification system (Millipore, Milford, MA, USA). Hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercitrin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, astragalin, and liquiritin (internal standards, IS) were obtained from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China). Quercitrin and quercetin were purchased from National Institutes for Food and Drug Control. Semen Cuscutae was purchased from Anguo, Hebei province. The structures of 13 compounds are displayed in Figure 1.
Sprague–Dawley rats (SPF, 200 ± 10 g, male) were purchased from HFK Laboratory Animal Technology Co., Ltd (license number: SCXK 2014-0004).
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6

Polyphenol Extraction and Characterization

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Castalin, vescalagin, castalagin were obtained from Shanghai ZZBIO Co., Ltd (Shanghai, China). Gallic acid, ellagic acid, vanillic acid, caffeic acid, polydatin (+)-catechin, (−)-epicatechin were purchased from Chengdu Must Bio Technology Co., Ltd. (Chengdu, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Proanthocyanidin dimer B1, B2 and B2-3′-O-gallate (B2-3′-O-G), trimer C1 and anthocyanin malvidin-3-O-glucoside were extracted and purified (with purity > 95%) as our previous methods.18–20 (link)Chemicals used for wine total polyphenols, flavones, proanthocyanidins and anthocyanins analyses, and all organic solvents used for sample preparation (analytical grade) and HPLC analysis (chromatographic grade) were purchased from Chemical Branch of Shandong Yuwang industrial Co., Ltd. (Shandong, China).
One hydrolysable tannin sample was provided from Proenol Industria Biotecnologica Lda., another condensed tannin sample was provided from Biocrático Lda. (Portugal). Two Central Valley red wines from Chile (RWA, 2015; RWB, 2016) were selected for this study.
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7

Neuraminidase Inhibition Assay Protocol

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LC-MS grade acetonitrile was purchased from Fisher Scientific (Beijing, China). De-ionized water was purified through a Milli-Q system (Millipore, Bedford, MA, USA). The analytical grade reagents used for extraction were obtained from Beijing Chemical Plant Co. Ltd. (Beijing, China). Gallic acid, ellagic acid, ethyl gallate, methyl-gallate, rutin, epicatechin, procyanidin B2, protocatechuic acid, quercitrin, hesperidin, eriodictyol, and caffeic acid were purchased from Chengdu Must Biotechnology Co. Ltd. (Chengdu, China). Oseltamivir acid was purchased from Medchem Express, LLC (Monmouth Junction, NJ, USA). Neuraminidase Inhibitors Screen Kit (NO. P0309) was purchased from Beyotime Institute of Biotechnology Co. Ltd. (Shanghai, China) and includes 10 mL buffer, 1 mL NA, 1 mL fluorescent substrate, and 1.2 mL Milli-Q water. Ninety six-well microplate reader (3925, Costar Company, Bethesda, MD, USA).
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8

Quantification of Bioactive Compounds in Lanqin Oral Liquid

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Lanqin oral liquid (lot no. 19122821) was purchased from Yangtze River Pharmaceutical Group (Jiangsu, China). Reference compounds (purity > 98%) of chlorogenic acid, caffeic acid, scutellarin, baicalin, palmatine, wogonoside, apigenin, baicalein, wogonin, indirubin, and indigo were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). Acetonitrile and formic acid of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Purified water was purchased from Hangzhou Wahaha Corporation (Hangzhou, China). All other chemical reagents used in this study were of analytical grade.
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9

Phytochemical Analysis of Lianhua Qingwen

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Lianhua Qingwen capsule (Lot No. B2008172) were obtained from Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China). Reference compounds (purity>98 %) of chlorogenic acid, esculetin, caffeic acid, luteolin, apigenin, liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, glycyrrhizic acid and glycyrrhetinic acid were purchased from Chengdu Must Bio-Technology Co., Ltd (Chengdu, China). Acetonitrile and formic acid of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Purified water was provided from Hangzhou Wahaha Corporation (Hangzhou, China).
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10

Analytical Characterization of Herbal Medicines

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Methanol, acetonitrile, and formic acid were of LC–MS grade, and 2-propanol was high-performance liquid chromatography (HPLC) grade. These chemicals were all purchased from Thermo Fisher Scientific Inc (Waltham, Massachusetts, United States). The ferulic acid (MW: 194.19, LOT: MUST-17010908, purity ≥98% HPLC),caffeic acid (MW: 180.15, LOT: MUST-17010908, purity ≥98% HPLC), calycosin (MW: 284.26, LOT: MUST-14070923, purity ≥98% HPLC), and formononetin (MW: 268.26, LOT: MUST-14091205, purity ≥98% HPLC) were provided by Chengdu Must Bio-Technology Co. Ltd., Ligustilide (MW: 284.26, LOT: wkq-00246, purity ≥98% HPLC LOT: wkq-00246) was obtained from Sichuan Weikeqi Biological Technology Co., Ltd. Rat SOD, MDA, TNF-α, IL-1β, IL-18, IL-6 and insulin ELISA Kits and ROS kit were all purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.
Danggui (the root of A. sinensis (Oliv.) Diels, Place of Origin: Gansu Province, Batch number: 259200103) and Huangqi (the root of Astragalus mongholicus Bge, Place of Origin: Inner Mongolia, Batch number: 190919001) were purchased from Beijing Tong Ren Tang Nanjing Pharmacy (Han Zhong Road, Nanjing, China). After purchase, the herbs were maintained in closed, lucifugal, and dry conditions at 23 ± 2°C.
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