Formic acid

The roots of Puraria labata were purchased from Beijing Tongrentang Pharmaceutical Company, (Beijing, China). The authentic standards of PU (98.40%), MPU (98.70%), 3′-hydroxypuerarin (HPU) (99.40%), daidzein (DAZ) (99.30%), and daidzein-8-C-apiosyl-(1-6)-glucoside (DAC) (92.1%) were prepared in our previous studies. The chemical structures of detected isoflavonoids are presented in Figure 1. Icarisid II (ICA) used as internal standard was purchased from Shanghai Winherb Medical Technology Company (ShangHai, China). The standards of DA (dopamine) and GLu (glutamic acid) were provided by the National Institutes for Food and Drug control (Beijing, China); The standards of 5-HT (5-hydroxytryptamine), HVA (High vanillin aldehyde acid), 5-HIAA (5-hydroxy indole acetic acid), Ach (acetylcholine), GABA (Gamma Amino Acid Butyric Acid) and Vitamin C, as well as DHBA (3, 4-2 hydroxy benzyl amine) used as the internal standard, were obtained from Sigma-Aldrich Co. Ltd. (Saint Louis, MO, USA). Acetonitrile and methanol with HPLC grade were purchased from Fisher Co.Ltd. (Emerson, USA). Formic acid and other reagents of analytical grade were purchased from Beijing Chemical Reagent Company (Beijing, China). Milli-Q water (18.3 MΩ; Milford, MA, USA) was used throughout the study.

Urea (industrial grade, 98%) was purchased from the Henan Zhongyuan Chemical Company, China. Formaldehyde (aqueous solution, industrial grade, 37%) was purchased from the Guangdong Xilong Chemical Factory, China. Sodium hydroxide (solid, analytical grade, 95%), formic acid (aqueous solution, analytical grade, 98%), and ammonium chloride (powder, analytical grade, 99%) were purchased from the Beijing Chemical Factory, China. WF was obtained from the Beijing Guchuan Flour Company, China. Poplar (Populus tomentosa Carr.) veneers having 8% moisture content were purchased from Hebei province, Langfang, China. SEP (moisture content ≤2%, 1 g/cm³, 200–300 mesh) was purchased from the QiLiPing sepiolite factory, Nanyang, China.

Analytical grade methanol and formic acid were purchased from Beijing Chemical Works (China). Acetonitrile, methanol, and formic acid were purchased in chromatographic grade from Fisher Scientific (USA). Trifluoroacetic acid (TFA; ≥99%) was purchased from Zhanyun Chemical Industry (China). Cyanidin 3,5-O-diglucoside was purchased from Extrasynthese (France). For the HPLC analysis, de-ionized water was prepared from a Milli-Q System (Millipore, USA).

The DPPH (2,2-Diphenyl-1-picrylhydrazyl, Sigma Co., Ltd., Croydon, UK) radical scavenging activity was determined using an ELISA microplate reader (Beijing Perlong New Technology Co., Ltd., Beijing, China) with 96-well plates (Nantong lineng Experiment Equipment Co., Ltd., Nantong, China). The T. quinquecostatus plant was obtained from yulin in Shaanxi province and qingyang and yanchi in the Gansu province in China. The samples were identified by Dr Shengjun Ma, department of Chinese Medicinal Resources, Beijing University of Chinese Medicine. Chlorogenic acid (Batch No. 110753), vanillic acid (Batch No. 110776-200402), scutellarin (Batch No. 110842-201207), galuteolin (Batch No. 111720-201307), apigenin (Batch No. 111901-201102), and danshensu (Batch No. 110855-201311) were reference compounds. Acetonitrile of HPLC-grade and LC-MS-grade was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Phosphoric acid (Tianjin Fuchen Chemical Reagents Factory, Tianjin, China) and formic acid (Beijing Chemical works, Beijing, China) were HPLC grade. Ultra-pure waterused throughout the experiment was obtained from Guangzhou Watsons Food & Beverage Co., Ltd. (Guangzhou, China).

ART and DHA were obtained from the Chongqing Wuling Mountain Pharmaceutical, Kunming Pharmaceutical Group (Chongqing, China, the batch number is MFCD00081057, C00220160402, respectively). The compound had purity ≥99%. De-ART was provided by Professor Wang M.Y. (Capital Medical University, Beijing, China). Cunninghamella elegans CICC 40250 was purchased from the China Center of Industrial Culture Collection (Beijing, China). Reagents used were high-performance liquid chromatography (HPLC) grade. Acetonitrile and methanol were purchased from Fisher (Fisher Chemical, Geel, Belgium), while formic acid and ethyl acetate were purchased from Beijing Chemical Works (Beijing, China). Water was prepared using a Milli-Q system operating at 18.2 MΩ (Millipore, Bedford, MA, USA). All other chemicals used were purchased from Fisher Scientific or Beijing Chemical Works, and were of the highest purity available.

ART and DHA were obtained from the Chongqing Wuling Mountain Pharmaceutical, Kunming Pharmaceutical Group (Chongqing, China, the batch numbers are MFCD00081057 and C00220160402, resp.). The compound had a purity of ≥99%. 3α-Hydroxy deoxy DHA was provided by Professor J.Y. Wang (China Academy of Chinese Medical Sciences, Beijing, China). The reagents used were of high-performance liquid chromatography (HPLC) grade. Acetonitrile and methanol were purchased from Fisher (Fisher Chemical, Geel, Belgium); formic acid and ethyl acetate were purchased from Beijing Chemical Works (Beijing, China). Sodium pentobarbital was purchased from Merck (Darmstadt, Germany). Water was prepared using a Milli-Q system operating at 18.2 MΩ (Millipore, Bedford, MA, United States). All other chemicals used were purchased from Fisher Scientific or Beijing Chemical Works and were of the highest purity available. Potassium dichromate was obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). Ethylenediaminetetraacetic acid disodium salt (EDTA-Na₂) was purchased from Beijing Solaria Science & Technology Co., Ltd (Beijing, China).