The contents of DBT, Astragali Radix (AR) and Angelicae sinensis Radix (ASR) in the ratio of 5:1, were kindly provided and authenticated by Chinese medicine manufacturer Sun Ten Pharmaceutical Co., Ltd, in Taiwan. Before extraction, Angelicae sinensis Radix was stir fried in rice wine (alcohol content, 75% v/v) and the pedicles were removed as per the traditional preparation method. AR and ASR were boiled in water for 1 h, and then separated into polysaccharide-enriched (DBT-PE) and polysaccharide-depleted (DBT-PD) fractions via precipitation in 50% ethanol. Both fractions were concentrated by freeze-drying. The purity of the indicator components of AR and ASR respectively, ferulic acid and astragaloside IV, were determined separately by HPLC-UV and HPLC-MS analysis and compared with the standard compounds of ferulic acid (ChromaDex, Irvine, CA, USA) and astragaloside IV (Tauto biotech, Shanghai, China) [21 ].
Ferulic acid
Manufactured by ChromaDex
Sourced in United States
Ferulic acid is a naturally occurring organic compound. It is a white to pale yellow crystalline solid. Ferulic acid has the chemical formula C₁₀H₁₀O₄.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (4 mentions)
Caffeic acid, by ChromaDex (4 mentions)
Chlorogenic acid, by ChromaDex (4 mentions)
Gallic acid, by ChromaDex (4 mentions)
Gallic acid, by Merck Group (3 mentions)
Caffeic acid, by Merck Group (3 mentions)
5 4 hydroxyphenyl valeric acid 4 hpva, by Thermo Fisher Scientific (3 mentions)
Pierce lc ms water, by Thermo Fisher Scientific (3 mentions)
3 4 dihydroxyphenylacetic acid 3 4 dihpaa, by ChromaDex (3 mentions)
Catechin, by Merck Group (2 mentions)
Quercetin, by Merck Group (2 mentions)
Vanillic acid, by ChromaDex (2 mentions)
Glacial acetic acid, by Merck Group (2 mentions)
Protocatechuic acid, by ChromaDex (2 mentions)
Cinnamic acid, by ChromaDex (2 mentions)
P coumaric acid, by ChromaDex (2 mentions)
Methanol, by Carl Roth (2 mentions)
Acetonitrile, by Carl Roth (2 mentions)
Ethyl acetate, by Thermo Fisher Scientific (2 mentions)
Homovanillic acid, by Merck Group (2 mentions)
13 protocols using ferulic acid
1
Extraction and Characterization of DBT Polysaccharides
2
HPLC Analysis of Phenolic and Flavonoid Compounds
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The phenolic and flavonoid compounds in both extracts were determined by HPLC according to the method of Pintha et al. [24 (link)]. Briefly, the solution was filtered through 0.2 μm and then subjected to separation by the ODS-3-C18 column (4.6 mm × 250 mm, 5 μm particle diameter). The 0.1% trifluoroacetic acid (TFA) in methanol and methanol was used as mobile phases A and B, respectively. The gradient was performed as follows: 0–35 min, 90–10% mobile phase A and 10–90% mobile phase B; 35–40 min, 10–90% mobile phase A and 90–10% mobile phase B with a flow rate of 1.0 ml/min and injection volume of 10 μl. The phenolic and flavonoid absorption peaks were measured by a UV detector with wavelengths at 280 and 320 nm, respectively. Peak area and retention time of the compounds in the extract were compared to phenolic and flavonoid standards including chlorogenic acid, gallic acid, catechin, and quercetin (Sigma-Aldrich, USA); caffeic acid, rosmarinic acid, luteolin, rutin, and apigenin (Chengdu biopurify phytochemicals, China); vanillic acid, and ferulic acid (ChromaDex, USA).
3
Analytical LC-MS Phenolic Standards
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Water, methanol, acetonitrile, 2-propanol and formic acid were of Optima® LC/MS grade (Fisher Scientific, Fair Lawn, NJ, USA). Glacial acetic acid provided by Merck (Darmstadt, Germany) was of Suprapur® quality. Sodium fluoride (Fluka Chemie, Buchs, Switzerland) and ascorbic and formic acid (Panreac, Barcelona, Spain) were of ACS grade.
The phenolic standards (+)-catechin, (-)-epicatechin, procyanidin B1, procyanidin B2, ferulic acid, quercetin-3-O-galactoside, isorhamnetin, isorhamnetin-3-O-glucoside and isorhamnetin-3-O-rutinoside were supplied by Extrasynthèse (Genay, France); procyanidin C1, quercetin-3-O-glucoside, quercetin-3-O-rhamnoside and quercetin-3-Oarabinofuranoside, by Chromadex (Santa Ana, CA, USA); caffeic acid, 5 ´-O-caffeoylquinic acid (5-CQA), p-coumaric acid, phloretin, phloretin-2′-O-glucoside and quercetin, by Sigma-Aldrich Chemie (Steinheim, Germany); phloretin-2′-O-xylosylglucoside, by Polyphenols Biotech (Bordeaux, France); and quercetin-3-O-rutinoside, by PhytoLab (Vestenbergsgreuth, Germany).
The phenolic standards (+)-catechin, (-)-epicatechin, procyanidin B1, procyanidin B2, ferulic acid, quercetin-3-O-galactoside, isorhamnetin, isorhamnetin-3-O-glucoside and isorhamnetin-3-O-rutinoside were supplied by Extrasynthèse (Genay, France); procyanidin C1, quercetin-3-O-glucoside, quercetin-3-O-rhamnoside and quercetin-3-Oarabinofuranoside, by Chromadex (Santa Ana, CA, USA); caffeic acid, 5 ´-O-caffeoylquinic acid (5-CQA), p-coumaric acid, phloretin, phloretin-2′-O-glucoside and quercetin, by Sigma-Aldrich Chemie (Steinheim, Germany); phloretin-2′-O-xylosylglucoside, by Polyphenols Biotech (Bordeaux, France); and quercetin-3-O-rutinoside, by PhytoLab (Vestenbergsgreuth, Germany).
4
Quantitative Analysis of Phytochemicals
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Chemicals and reagents: Syringin, eleutheroside E, isofraxidin, hyperoside, rutin, kaempferol, oleanolic acid, and L-phenylalanine were purchased from the Chinese National Institute of Control of Pharmaceutical and Biological Products (Beijing, China). Syringic acid, vanillic acid, p-hydroxybenzoic, protocatechuic acid, cinnamic acid, gallic acid, ferulic acid, chlorogenic acid, caffeic acid, p-coumaric acid, luteolin, genistein, and quercetin were purchased from ChromaDex Inc. (Santa Ana, CA, USA). The water used for UPLC-MS/MS was prepared by a Milli-Q (Millipore, Bedford, MA, USA). Acetonitrile (J&K Scientific Ltd., Beijing, China) was HPLC grade. All other chemicals used in the research were of analytical grade.
5
Analytical Standards Acquisition Protocol
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Analytical standards, such as: gallic acid, p-coumaric acid, ferulic acid were purchased in ChromaDex (Irvine, CA, USA) and syringic acid, sinapinic acid and dihydroxybenzoic acid in Sigma Aldrich.
6
Analytical Standards for Phytochemicals
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Acetonitrile and methanol were of HPLC grade and purchased from Roth GmbH (Karlsruhe, Germany). Reference compounds (crocin, safranal, apigenin, rutin, caffeic acid, ferulic acid, chlorogenic acid, gallic acid) were purchased from ChromaDex (Santa Ana, CA), Sigma-Aldrich (Saint Louis, MO), HWI Analytik GmbH, and Roth GmbH (Karlsruhe, Germany).
7
HPLC Analysis of Bioactive Compounds
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In order to determine the bioactive compound present in the plant extract, a standardized HPLC method was utilized, which has been described in the USP 30-NF25 Pharmacopoeia [49 ]. The HPLC system used consisted of an Agilent 1200 chromatogram equipped with a quaternary pump, DAD, thermostat, degas system, and autosampler (Agilent Tehnologies, Boblingen. Germany). A C18 Zorbax XDB column (250 mm × 4.6 mm; 5 µm) was employed, and the eluents were composed of 0.1% phosphoric acid (A) and acetonitrile (B), with a linear gradient of 10% B for 13 min, 22% B for 1 min, 40% B for 3 min, and 10% B for 1 min. The flow rate was set to 1.5 mL/min and the column temperature was maintained at 35 °C. The DAD system was set to detect wavelengths of 310 nm, 335 nm, and 360 nm simultaneously. The standards used in the study were purchased from Chromadex (Wesel, Germany) and included E-resveratrol, Z-resveratrol, caffeic acid, chlorogenic acid, cinnamic acid, ellagic acid, vanillin, gallic acid, ferulic acid, astragalin, isorhamnetin, kaempferol, scutellarin, rutoside, and quercetin. All assays were run in triplicate, and the results were expressed as mean ± SD.
8
HPLC Analysis of Phenolic Compounds
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The acetonitrile and methanol were of HPLC grade and were purchased from Roth GmbH (Karlsruhe, Germany). The reference compounds (chlorogenic acid, caffeic acid, ferulic acid, t-cinnamic acid, mangiferin, kaempferol, tectoridin, iristectorigenin B, nigricin, irigenin, isoorientin, genistein-7-glucoside, and biochanin A) were purchased from ChromaDex (Santa Ana, CA, USA), Sigma-Aldrich (Saint Louis, MO, USA), HWI ANALYTIK GmbH, and Roth GmbH (Karlsruhe, Germany). All other chemicals were of analytical grade.
9
Characterization of Chlorogenic Acid Derivatives
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Chlorogenic acid and caffeic acid were purchased from Acros Organics (Pittsburgh, PA, USA). 4,5-Dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 1,3-dicaffeoylquinic acid, and 1,5-dicaffeoylquinic acid were obtained from Chengdu Biopurify Phytochemicals Ltd., (Chengdu, China). Protocatechuic acid, ferulic acid, and 1,4-dicaffeoylquinic acid were obtained from ChromaDex (Irvine, CA, USA), Wako Chemicals (Osaka, Japan), and ChemFaces (Wuhan, China), respectively. The purity of these reference standards was >98.0% by HPLC analysis. The chemical structures of these components are shown in Figure 1 . The HPLC-grade solvents methanol, acetonitrile, and water were obtained from JT Baker (Phillipsburg, NJ, USA). Glacial acetic acid (analytical grade) was purchased from Merck KGaA (Darmstadt, Germany).
10
Phenolic Standards Characterization Protocol
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