Chlorogenic acid

Acetonitrile and methanol were HPLC-grade from Fisher Scientific (Pittsburg, PA, United States). All other reagents were of analytical grade and obtained by Anaqua™ Chemicals Supply (Wilmington, DE, United States). Ultrapure water is prepared by the Millipore Ultra-Pure Water System. Harpagide, protocatechuic acid, atractyloside A, verbenalin, paeoniflorin, p-Coumaric acid, sinapic acid, vitexin, liquiritin, liquiritin apioside, isoliquiritin apioside, cimifugin, scutellarin, apigenin-7-O-glucuronide, ononin, isoliquiritin, naringenin, glycyrrhizic acid, baicalein, icariin (IS, internal standards), isopimpinellin (IS), astragaloside II (IS) were purchased from Chengdu Desite Bio-Technology Co., Ltd (Chengdu, China). Quercitrin were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Catechin, cryptochlorogenin acid, hyperoside, wogonoside, chlorogenic acid, ginsenosides Rf, ginsenosides Rh1 were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). Isoferulic acid, hesperidin, baicalin, harpagoside, wogonin, ferulic acid were purchased from National Institutes for Food and Drug Control (Beijing, China). Their purity was higher than 98%. QJYQ were made by the laboratory of Tianjin University of Traditional Chinese Medicine, and the batch numbers in this study was 210601.

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.

We obtained the nine batches of TSMT samples (S1-S9, No. 210401 ~ 210409) from Jiangsu Kanion Pharmaceutical Co., Ltd (Jiangsu, China). These were prepared from eight herbal pieces, i.e., Angelica sinensis (Oliv.) Diels (DG, danggui), Lonicera japonica Thunb. (JYH, jinyinhua), Codonopsis pilosula (Franch.) Nannf. (DS, dangshen), Scrophularia aestivalis Griseb (XS, xuanshen), Astragalus membranaceus (Fisch.) Bunge (HQ, huangqi), Achyranthes bidentata Bl. (NX, niuxi), Dendrobium nobile Lindl. (SH, shihu), and Glycyrrhiza uralensis Fisch. (GC, gancao) which were also checked with http://www.theplantlist.org on February 14, 2022. Next, we purchased 17 reference standards (RS) from Chengdu MUST Bio-Technology Co, Ltd (Chengdu, China), which included Astragaloside IV, Calycosin-7-glucoside, Atractylenolide II, Atractylenolide III, Ferulic acid, Senkyunolide I, Cynaroside, Chlorogenic acid, Luteolin, Harpagoside, Harpgide, Acteoside, β-Ecdysone, Ginsenoside Ro, Glycyrrhizic acid, Liquiritin, and Gallic acid. The purity of all the reference standards was over 98%. Mobile phases including the HPLC-grade acetonitrile, phosphoric acid, and formic acid were purchased from Thermo Fisher Scientific (Thermo scientific, USA).

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).

HPLC grade methanol and acetonitrile were obtained from Fisher Scientific (USA). HPLC grade formic acid was purchased from Dikma Co. (USA), and Wahaha purified water was obtained from Hangzhou Wahaha Group (China). Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B (3,4-diCQA), isochlorogenic acid A (3,5-diCQA), isochlorogenic acid C (4,5-diCQA), bergapten, columbianetin acetate, isoimperatorin, osthol, and columbianadin were obtained from Chengdu Must Biotechnology Co., LTD (China), and columbianetin, xanthotoxin, psoralen, and chloramphenicol were obtained from Shanghai Nature Standard Biotechnology Co., LTD (China), and daidzein was obtained from Chengdu Purechem-Standard Biotechnology Co., LTD (China). The purity of the involved standards and reagents were over 98%, with their chemical structures exhibited in Figure 1.
The APR samples was obtained from Hubei Province (30°24N, 115°30E) picked in April 2019, and identified by Professor Lianjie Su from Heilongjiang University of Chinese Medicine, as the dry root of Angelica pubescens Maxim. f. biserrata Shan et Yuan and a voucher specimen were deposited at department of Chinese herbal medicine processing of Heilongjiang University of Chinese Medicine, Harbin, China (voucher numbers: APQH201904-7).

Yuanye Biotechnology Co., Ltd. (Shanghai, China) was paid for bovine lactoferrin (BLF, HPLC ≥ 95%). Chlorogenic acid (CA, HPLC ≥ 98%) came from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China) Kangwei Century Biotechnology (Beijing, China) Co., Ltd. and Beijing Solaibao Technology (Beijing, China) Co., Ltd. both sold BCA protein quantification kits and SDS-PAGE gel preparation kits. Coomas Bright Blue G-250 was purchased from Tianjin Comeiou Chemical Reagent (Tianjin, China) Co., Ltd. Rainbow 130 Broad-spectrum Protein marker (15–180 kDa) was purchased from Beijing Soleibao Technology Co., Ltd. It was decided to buy sodium dodecyl sulfate (SDS) from Tianjin Fuchen Chemical Reagent Factory (Tianjin, China) Co., Ltd. Protein was dissolved in phosphate-buffered saline (PBS, 0.1 mol L⁻¹, pH 7.4). Analytical-grade reagents were also employed in the experiment, and ultra-pure water was used. In further studies, none of the samples underwent more purification.

Acetonitrile and formic acid were of HPLC grade (Burdick and Jackson, Honeywell International Inc., Muskegon, MI, USA). Water used in HPLC analysis was prepared using a Milli-Q Water purification system (Millipore, MA, USA). Reference standards, chlorogenicacid, mangiferin, isomangiferin, and astragalin were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), while trifolin was from Shanghai Tanto Biotech Co., Ltd. (Shanghai, China). Their purity was verified to be more than 98% by HPLC analysis. The samples of Pyrrosiae Folium were collected from different geographic areas and were authenticated as the leaves of Pyrrosia sheareri (Bak.) Ching, P. lingua (Thunb.) Farwell, and P. petiolosa (Christ) Ching (Figure 1), respectively, by Dr. Sibao Chen based on herbarium specimen (deposited in the Herbarium of State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen, China).