Isoquercitrin

Acetonitrile was of HPLC grade from Tedia (Fairfield, OH, USA); deionized water was further purified by a Milli-Q purification system (Millipore, Millford, MA, USA); HPLC-grade formic acid was purchased from Aladdin Co., Ltd. (Shanghai, China), and other reagents were all of analytical reagent grade. The reference standards of 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, quercetin-3-O-glucuronide, quercetin-3-O-galactoside, isoquercitrin, kaempferol-3-O-glucuronide, kaempferol-3-O-glucoside, quercetin-3-O-rhamnoside, hederagenin and oleanolic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), and arjunolic acid was purchased from BioBioPha Co., Ltd. (Kunming, China), whereas kaempferol-3-O-rhamnoside, cyclocaric acid B, pterocaryoside A and pterocaryoside B were isolated and purified previously from the leaves of C. paliurus in the laboratory of China Pharmaceutical University (Nanjing, China) and were elucidated by comparison of spectral data (UV, MS, ¹H-NMR, ¹³C-NMR) with those of published references [16 (link),17 (link)]. The purity of each compound was determined to be more than 98% by normalization of the peak area detected by HPLC–UV.

The following standards with purity higher than 98% were purchased from Yuanye Biotechnology Co., Ltd.: isoquercitrin, isorhamnetin‐3‐O‐neohesperidoside, narcissin, quercitrin, ellagic acid, gallocatechin, apigenin, rutin, and kaempferol. Epicatechin was obtained from Chengdu Pulse Biotechnology Co., Ltd. Citric acid, sucrose, tannic acid, sodium glutamate, and quinine hydrochloride was acquired from Adamas Beta Chemical Reagents Co., Ltd. 2,2′‐Azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulphonate acid; ABTS) and ferric ion‐reducing antioxidant power (FRAP) kits were provided by Shanghai Biyuntian Biotechnology Co., Ltd. Diphenyl‐1‐picrylhydrazyls (DPPH) was supplied by Sigma‐Aldrich. Chromatography‐grade formic acid was obtained from Shanghai Aladdin Reagent Co., Ltd. Methanol was acquired from Tianjin Beilian Fine Chemical Depot. Experimental ultrapure water was produced with a Mili‐Q system (Millipore Corp.).

The coding sequence of FeUGT1, FeUGT2, FtUGT1, and FtUGT2 were cloned and inserted into the PET30b( +) expression vector, and then recombinant plasmid of these four genes were transformed into E. coli Rosetta (DE3) (Tsingke Biotechnology Co., Ltd., Beijing). pET30b( +)-transformed E. coli Rosetta (DE3) cells were treated in parallel as a control. Four recombinant proteins were extracted by ultrasonic cell breaker (on/off: 3 s/7 s, power: 90%), purified using nickel-nitrilotriacetic acid (Ni–NTA) agarose, and eluted with 250 mM imidazole. After concentrating, each 5 ug of the four purified proteins were incubated at 30 °C with 100 mM Tris–HCl (pH 8.0), 100 mM Tris–HCl (pH 7.5), 14 mM β-mercatoethanol, 4 mM UDP-rhamnose or UDP-glucose, and 0.1 mM substrate for 30 min and reaction was stopped by adding methanol. Glycosylated products were detected using a LC–ESI–MS/MS system (LC, Shimadzu LC30AD; MS, QTRAP 6500 +) with a Thermo Hypersil Gold analytical column (100 × 2.1 mm, 1.9 μm). Data analysis was performed using Analyst 1.7.0. Standards of quercetin, isoquercitrin, and rutin were purchased from Yuanye Bio-Technology (Shanghai, China).

Hibiscus manihot L. was provided and identified by Yulin Xinbang Pharmaceutical Co., Ltd. (Yulin, China). It was dried in a tray at 65 °C, destemmed and crushed (0.42 mm), and stored at 4 °C for further use.
The fermentation strain was Rhizopus arrhizus JHK31. We obtained it through pressure screening. It is stored in the China Center for Type Culture Collection under the deposit number CCTCC M 2023722.
Reagents including rutin (P98%), vitamin C (P99%), aluminum nitrate nonahydrate (P99%), DPPH (P96%), iron trichloride hexahydrate (P99%), trichloroacetic acid (P99%), dibasic sodium phosphate (P99%), and sodium dihydrogen phosphate (P99%) were from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) provided other chemicals such as ferrous sulfate (P98%), green vitriol (P99%), sodium hydroxide (P96%), Tris (P99.8%), and salicylic acid (P99%). Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) provided hyperoside (P98%), isoquercitrin (P98%), myricetin (P98%), and quercetin (P98%). Other chemicals such as sodium nitrite (P99%), potassium ferricyanide (P99.5%), and pyrogallol (P99%) were analytically pure. Acetonitrile and methanol were of chromatographic grades. The resin used was LX-83 macroporous resin from Sunresin New Materials Co., Ltd. (Xi’an, China). We used ultrapure water in all experiments.

The mulberry leaves in this experiment were collected from Deqing (119°97′ E, 30°53′ N), Huzhou City, Zhejiang Province, and were authenticated by Dr. Chu Chu (Zhejiang University of Technology, Hangzhou, China). The harvest time is shown in Table 3. After being freeze-dried, the mulberry leaves were stored in a refrigerator at −20 °C before analysis. Chlorogenic acid (≥98%), rutin (≥98%), and isoquercitrin (≥98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Gallic acid (≥99%) and Folin–Ciocalteu reagent were purchased from Beijing Zhongkezhijian Biotechnology Co., Ltd. (Beijing, China).
HPLC-grade acetonitrile and methanol were supplied by Merck (Darmstadt, Germany). HPLC-grade formic acid was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MΩ) was purified using the Milli-Q^® IQ 7000 Purification System (Molsheim, France). Other reagents used in this experiment were all of analytical grade and were obtained from Yongda Chemical Reagent Company (Tianjin, China).