Paeoniflorin

Xuebijing injection (catalog number: z20040033, batch number: 1905061) was manufactured by Tianjin Chase Sun Pharmaceutical Co., Ltd (Tianjin, China). Paeoniflorin (CAS #:23180-57-6), Hydroxysafflor yellow A (CAS #: 78281-02-4), Ferulic acid (CAS # 537- 98-4), and protocatechuic aldehyde (CAS #: 139-85-5) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Mouse NT-proBNP ELISA (Cat#: ZC-37812-96T) kit, DCFH-DA (Cat#:CA1410-100T), RIPA tissue/cell lysate (Cat#: R0020), SDS-PAGE gel kit, and Rainbow 245 Spectrum Marker (Cat#:PR 1920), Mouse IL-6 ELISA kit (Cat#:ZC-37988-96T), Mouse IL-1β ELISA kit (Cat#:ZC-37974-96T), and EasySee Western Blot Kit (Lot#:O30807) were purchased from Solarbio (Beijing, China). The primary antibody of rabbit anti-CXCL2 (bs-1162R) was purchased from Bioss Inc., (Beijing, China) and GAPDH, 1:4,000 (14C10) was from Cell Signaling Technology (Beverly, MA, United States). The secondary antibody Goat anti-Rabbit IgG (1:4,000) (ZB-2301) was purchased from ZS bio (Beijing, China). 2, 2, 2-Tribromoalcohol (Cat#:T48402) and Bacterial lipopolysaccharide (Cat#: L2880-10 MG) were purchased from Sigma Aldrich (St. Louis, MO, United States). Rhod2-AM (Cat#: zy0129) and Anti-GRP78 (BiP) antibody (ab21685) were purchased from Abcam (Cambridge, MA, United States).

The reference standards of chlorogenic acid, caffeic acid, ethyl gallate, 3-butyl-phthalide, benzoyloxypaeoniflorin, senkyunolide I, gallic acid, oxypaeoniflorin, vanillic acid, albiflorin, paeoniflorin, ferulic acid, 1,2,3,4,6-pentagalloylglucose, benzoylpaeoniflorin, paeonol, and ligustilide (the purities of all standards were above 98.0%) were supplied by Shanghai Yuanye Bio-Technology Co., Ltd. Appropriate amounts of the reference standards were mixed and dissolved in methanol in a 10-mL measuring flask, then filtered and set aside.

LC-10 ATVP high performance liquid chromatography equipped with a C18 chromatographic column and SLD-10AVP UV detector (Shimadzu, Japan), DGG-9620A thermostatic blast oven (Shanghai, China), Re-52AA rotary evaporator (Shanghai, China).
Chinese materia medica were all provided by Baibao Pharmaceutical company (Gansu, China). They were authenticated by Dr. Jianying Li, School of Pharmacy Lanzhou University. Hydroxy-propyl methyl cellulose (HPMC) and pharmaceutical starch were supplied by Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Standard samples of paeoniflorin and Ferulic acid were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Calycosin-7-glucoside was obtained from China’s Food and Drug Verification Research Institute (Beijing, China). Chromatographic grade methanol was purchased from Fisher Scientific (Shanghai, China). Chromatographic grade acetonitrile was purchased from MREDA Technology Inc. (Beijing, China). All other reagents were analytical grade.

Incomplete Freund's adjuvant (IFA) and Bacillus Calmette-Guérin (BCG) were purchased from Rebio Scientific (Shanghai, China). TNF-α, IL-1β, IL-10, and IL-6 ELISA kits were the products of Multi Science (Hangzhou, China). Biochemical quantification kits for the determination of malondialdehyde (MDA), reduced glutathione (GSH), total superoxide dismutase activity (SOD), total antioxidant capacity (T-AOC), nitric oxide (NO), nitric oxide synthase (inducer) (iNOS), and carbonic oxide (CO) were brought from Jiancheng Bioengineering Institute (Jiangsu, China). The primary antibodies used in immunoblotting and immunohistochemical experiments including anti-MMP3, COX-2, TLR4, SIRT1, PPAR-γ, p65, p-p65, and β-actin antibodies together with secondary antibodies were provided by ABclonal Technology (Wuhan, China). Fluorescein-tagged anti-CD3, CD4, and IL-17α antibodies for the use of flow cytometry analysis were supplied by Beyotime Biotech (Nantong, Jiangsu, China). Rat peripheral blood mononuclear cell (PBMC) isolation kit, TaqMan RT-qPCR kit, and cDNA synthesis kit were purchased from Solarbio Technologies (Beijing, China). All the solvents were of analytical grade and supplied by Merck Chemicals (Shanghai, China). High-purity compounds (>98%) kaempferol, paeoniflorin, matrine, sophocarpine, and sinomenine were all obtained from Yuanye Bio-Technology (Shanghai, China).

All the six herbal medicines (HMR: RM130121-08; DH: 130524; BS: RM130128-11; KXR: RM121026-04; HP: RM121026-05; ZS: RM121210-11) contained in MZRW were supported and authenticated by PuraPharm Company, Nanning department (Nanning, China). The reference standards of gallic acid, amygdalin, paeoniflorin, hesperidin, neohesperidin, naringin, magnolol, honokiol, aloe emodin, physcion, chrysophanol were purchased from Shanghai Yuan-Ye Bio-technology company (Shanghai, China). Emodin, rhein, albiflorin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of all these reference standards was ≥98.0% (HPLC). HPLC grade of acetonitrile, methanol and formic acid were purchased from Merck (Darmstadt, Germany). Deionized water was purified by the Millipore water purification system (Millipore, Milford, MA, United States). All other reagents used were of analytical grade. Twelve male Sprague-Dawley rats (220–250 g) were kindly provided by the Experimental Animal Center of Chinese University of Hong Kong (Hong Kong). The rats were bred in an environmentally controlled room (22 ± 2°C, relative humidity 50 ± 20%) with a natural light-dark cycle for 7 days before the experiment carried out. The animal study was carried out in accordance with the Guideline for Animal Experimentation of Hong Kong.

The standard compounds paeoniflorin, albiflorin, catechin, lactiflorin, benzoylpaeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, ethyl gallate, 3,4-dihydroxybenzaldehyde and 3,4-dihydroxybenzoic acid were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Gallic acid, oxypaeoniflorin, methyl gallate, benzoic acid, and apiopaeonoside were obtained from Chengdu Push Bio-Technology Co., Ltd. (Chengdu, China). The purity of each standard was >98%. The chemical structures of the 14 reference compounds were presented in Supplementary Figure 1. A Milli-Q system (Millipore, MA, United States) was used to prepare ultra-pure water. HPLC-grade acetonitrile and methanol were procured from Tedia (Fairfield, OH, United States), and formic acid (LC-MS grade) was purchased from Aladdin (California, United States). All other reagents were of analytical grade.