Naringin

The α-L-rhamnosidase gene of T. petrophila DSM 13995 (TpeRha, GenBank accession: ABQ47687.1) was codon-optimized, synthesized and cloned into the vector pET-28a by General Biosystems Co., Ltd. (Anhui, China). Details of strains and plasmids used in this study are listed in Table 1. Primers are listed in the Supplementary Table S1. Competent cells of E. coli BL31(DE3) and E. coli DH5α were purchased from Weidi Biotechnology Co., Ltd. (Shanghai, China). Plasmid Mini Kit I was purchased from Omega Bio-tek Inc. (Georgia, United States). The PrimeSTAR^® Max DNA Polymerase, used for site-directed mutagenesis, was purchased from Takara Biomedical Technology Co., Ltd. (Dalian, China). Restriction enzymes were purchased from Thermo Fisher Scientific Co., Ltd. (Beijing, China). The HiPure Gel Pure DNA Micro Kit was acquired from Magen Co., Ltd. (Guangzhou, China). The standards epimedin C, naringin, rutin, hesperidin, naringin dihydrochalcone (NDHC), icariin, and icariside I were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). The epimedin C and icariin was purchased from Tianben Bio-Engineering Co., Ltd. (Xian, China). Other general chemical reagents used in this study were obtained from standard suppliers.

All the Chinese herbs were provided by Tongde Hospital of Zhejiang Province (Hangzhou, China). All the chemical standards, including l (+)-arginine, proline, adenine, succinic acid, isoleucine, leucine, gallic acid, phenylalanine, protocatechuic acid, l-tryptophan, protocatechuic aldehyde, neo-mangiferin, chlorogenic acid, caffeic acid, amygdalin, mangiferin, rutin, liquiritin, luteoloside, naringin, berberrubine, baicalin, quercetin, epimedin A, epimedin B, epimedin C, icarrin, isorhamnetin, glycyrrhizic acid, limonin, aaikosaponin A, and 3-N-butyl-4,4-dihydrophthalide, were purchased from Shanghai Yuanye Bio-Technology Co. Ltd (Shanghai, China). Azoxymethane (AOM) was obtained from Sigma-Aldrich (St. Louis, MO, United States) and dextran sodium sulfate (DSS) from MP Biomedicals (Santa Ana, CA United States). TUNEL apoptosis assay kits, radioimmunoprecipitation assay (RIPA) buffer, BCA protein assay kits, and anti-MMP9 antibody (AF5234) were purchased from Beyotime (Beijing, China). Mouse IL-6 and IL-17A ELISA kits were from LiankeBio (Hangzhou, China), and antibodies to p-STAT3 (AF3293), STAT3 (AF6294), NF-κB-p65 (BF8005), Bcl-2 (BF9103), Lamin B (AF5161), and β-actin (T0022) were purchased from Affinity Biosciences (Changzhou, China). Antibodies to IL-17RA (sc-376374) and Bax (sc-20067) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

SMD, used for gastrointestinal dysmotility in China (approval number: guo-yao-zhun-zi Z20025044; specification: 10 mL/division), was obtained from Hunan Hansen Pharmaceutical Company, Ltd. (Yi Yang, China). HSA was acquired from Sigma Chemical Co. (St. Louis, MO, USA), standards, including arecaidine, arecoline, norisoboldine, linderane, costunolide, dehydrocostus lactone, synephrine, rutin, limonin, eriocitrin, narirutin, naringin, hesperidin, heohesperidin, poncirin, naringenin, hesperetin, nobiletin, tangeretin, and sorbic acid with a purity of over 98%, were purchased from Yuan-ye Bio-Technology Co., Ltd. (Shanghai, China). The formic acid, acetonitrile, and methanol used for HPLC analysis were chromatographic grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Reference standards of neochlorogenic acid (1, Purity ≥ 98%), chlorogenic acid (2, Purity ≥ 98%), quercetin-3-O-glucuronide (3, Purity ≥ 98%), kaempferol-3-O-rhamnoside (4, Purity ≥ 98%), quercetin (5, Purity ≥ 98%), and kaempferol (6, Purity ≥ 98%) were supplied by Baoji Herbest Bio-Tech Co., Ltd. (Shanxi, China). Naringin (7, internal standard (IS)) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Their chemical structures are illustrated in Fig. 1. Acetonitrile, methanol, and formic acid (HPLC grade) were purchased from Merck (Darmstadt, Germany). Ultra-pure water was purified by the Millipore water purification system (Millipore, Milford, MA, United States).Fig. 1

Chemical structure of six components. Neochlorogenic acid (1), chlorogenic acid (2), quercetin-3-O-glucuronide (3), kaempferol-3-O-rhamnoside (4), quercetin (5), kaempferol (6), and Naringin (IS)

Eleven samples of Aurantii fructus (AF) samples from different habitats were collected and identified by Associate Professor Tang Qi. Reference standards included synephrine, eriocitrin, neoeriocitrin, rutin, narirutin, naringin, hesperidin, neohesperidin, limonin, xanthotol, poncirin, naringenin, hesperetin, nobiletin, tangeretin and auraptene with a purity of over 98% were purchased from Yuan-ye Bio-Technology Co., Ltd. (Shanghai, China). Chromatographic grade formic acid, methanol, ethanol and acetonitrile were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used for extractive and analytical procedures.

Naringenin, eriodictyol, apigenin, luteolin, liquiritigenin, butin, sakuranetin, kaempferol, quercetin, dihydrokaempferol, dihydroquercetin, eriocitrin, pinocembrin, narirutin, naringin, chrysin, genkwanin, baicalein, scutellarein, norwogonin, wogonin, and neoeriocitrin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Hesperidin, neoHesperidin, sinensetin, isosinensetin, and nobiletin were obtained from Sigma-Aldrich (St Louis, MO, USA). 7-O-methyleriodictyol, isosakuranetin, and 5-HPMF were purchased from BioBioPha Co., Ltd (Kunming, China).

Citri Reticulatae Pericarpium was supplied by Scientific Research Institute of Beijing Tongrentang Co., Ltd. Total 21 reference standards, including vicenin-2 (6), vicenin-3 (25), eriocitrin (40), isoquercitroside (45), troxerutin (49), rhoifolin (66), naringin (67), naringenin (68), naringenin-7-O-glucoside (71), hesperidin (78), neohesperidin (82), hesperetin (108), isosinensetin (122), sinensetin (133), quercetagetin-3,5,6,7,3′,4′-hexamethyl ether (144), nobiletin (151), 4´,5,6,7-tetramethoxyflavone (153), tangeretin (168), 5-demethylnobiletin (174), 8-hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone (180), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (181), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The purity of all the reference standards was > 98%. The distilled water was obtained from Watsons, whereas LC–MS grade formic acid was obtained from Fisher-Scientific (Fair Lawn, USA). The LC–MS-grade acetonitrile and methanol were purchased from Merck (Germany).