Ferulic acid

The root and rhizome of Angelica sinensis (Oliv.) Diels and stem bark of Cinnamomum cassia (L.) J. Presl, were purchased from Guangzhou Zisun Pharmaceutical Co., Ltd., China, which are accordant with the standards of Chinese Pharmacopoeia (2015 edition) by confirmation of Professor Quan Zhu. We performed the method of steam distillation for the extracts of AC (1 : 1 of two herbal medicine weight ratio), and detailed procedures refer to our previous article [21 (link)]. The extracts (yield: 25%) were obtained by lyophilizing the concentrated sample with a Virtis Freeze Dryer (The Virtis Company, New York, USA). 5 mg of AC extract was dissolved in 5 ml of 30% methanol and filtered, which was directly subjected to ACQUITY UPLC system on PAD λe detector (λ = 200–400 nm), autosampler, in-line degasser, Waters ACQUITY-UPLC CLASS system (Waters Corp., Milford, USA) on an ACQUITY UPLC HSS T3 column (150 mm × 2.1 mm, 1.8 μm). Reference compounds are ferulic acid (t R = 2.59 min, purity HPLC > 98%), Senkyunolide I (t R = 4.42 min, purity HPLC > 98%), and Trans-Cinnamic acid (t R = 6.32 min, purity HPLC > 98%), which were purchased from Chengdu Chroma-Biotechnology Co., Ltd., China.

Terminalia chebula Retz., Terminalia bellirica (Gaertn.) Roxb. and Phyllanthus emblica Linn. were purchased from Zhongyong Pharmaceutical Co., Ltd. (Sichuan, China). All herbs were identified by Professor Jin Pei, deposited at the Chengdu University of TCM, and met Chinese Pharmacopoeia requirements (2015 Edition). Standards of Chebulic acid (CHB180831), Gallic acid (CHB171107), Punicalin (CHB190211), Catechin (CHB170301), EpigalloCatechin gallate (CHB180307), EpiCatechin (CHB180831), Corilagin (CHB190106), GalloCatechin gallate (CHB180327), 1,3,6-tri-O-galloylglucose (CHB191021), EpiCatechin gallate (CHB170317), Ferulic acid (CHB 180201), Chebulagic acid (CHB190109), 1,2,3,4,6-O-penta-galloyl glucose (CHB190125), Chebulinic acid (CHB190124), and Ellagic acid (CHB170303) were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (Chengdu, China), the purity of all standard products is ≥ 98%. CCl₄ (20181010) was purchased from Tianjin Bodi Chemical Co., Ltd. (Tianjin, China), Dimethyl diphenyl bicarboxylate (DDB) (200603) was purchased from Bond Pharmaceuticals Group Co., Ltd. (Wenling, China). Test kits for ALT (20191003), AST (20191005), SOD (20191101), MDA (20191028), and GSH-Px (20191029) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α (A28291045) and IL-6 (A20691132) were obtained by Multi Sciences Biotech Co., Ltd. (Hangzhou, China).

To evaluate the quality and stability of the extracts of JHD, high-performance liquid chromatography (HPLC) analysis was performed by Agilent 1260 HPLC system (Agilent Technologies). The mobile phase system was composed of water with 0.05% phosphoric acid (A) and methanol (B). The gradient elution profile was: 0∼20min, 5%∼21% B; 20∼55 min, 21%∼88% B; 55∼65 min; 88% B. The extracts of JHD were separated by Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 µm) at 30°C, with 1.0 ml/min of mobile phase flow rate. The UV spectrum was set at 283nm. Hesperidin (HPLC, > 98% purity, CAS:520-26-3), p-coumaric acid (HPLC, > 98% purity, CAS: 501-98-4), ferulic acid (HPLC, > 98% purity, CAS: 1135-24-6), 5,7-dimethoxycoumarin (HPLC, > 98% purity, CAS: 487-06-9) and bergapten (HPLC, > 98% purity, CAS: 484-20-8) were purchased from Chengdu Chroma-Biotechnology Co., Ltd (Chengdu, China). The reference substances were detected under the same condition of JHD. HPLC chromatograms of JHD is shown at Figure 1. Besides, the methodology analysis of JHD, which was included linear relation, stability, repeatability, precision and recovery test, was also performed, and the results were shown at Supplementary Material 3. Concentration of the identified monomer was the weight in the lyophilized powder per gram.