Apigenin

PPHRCs were initiated by culturing a high-productive hairy root line (PPHRL II) under the optimal conditions reported previously [10]. Apart from cajaninstilbene acid (separation and purification by our laboratory), all flavonoid standards were provided by Weikeqi Biological Technology Co., Ltd. (Chengdu, China). MiniBEST Plant RNA Extraction, PrimeScript™ RT reagent, and SYBR Premix Ex Taq™ II Kits were purchased from TaKaRa (Dalian, China).

Pigeon pea seeds were provided

by Honghe Taifu Agriculture and Forestry Technology Development

Co., Ltd. (Honghe, China). Cajaninstilbene acid was separated and

purified in our laboratory with a purity higher than 98%. Other

standards were bought from Weikeqi Biological Technology Co., Ltd.

(Chengdu, China).

The standard compound 2 was a gift from associate professor Peng Wu and the standard compound 4 was obtained from Jie Zhang, Guangzhou University of Chinese Medicine. The other standards were purchased from Sichuan Weikeqi Bio-Technology CO. LTD. (Chengdu, China) and Chengdu Must Bio-Technology Co. Ltd (Chengdu, China). Chemical reagents were obtained from Thermo Fisher Scientific Co. Ltd. (Shanghai, China), and Sigma-Aldrich (St. Louis, MO, USA).

Orientin, vitexin, isovitexin, genistin, luteolin, genistein, apigenin, isorhamnetin, biochanin A, and pinostrobin were purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Cajaninstilbene acid was obtained by the separation and purification from pigeon pea leaves by column chromatography in our laboratory. All standards were identified with the purity higher than 98%.

Orientin (CAS Registry Number 28608-75-5), vitexin (CAS Registry Number 3681-93-4), isovitexin (CAS Registry Number 38953-85-4), genistin (CAS Registry Number 529-59-9), luteolin (CAS Registry Number 491-70-3), genistein (CAS Registry Number 446-72-0), apigenin (CAS Registry Number 520-36-5), isorhamnetin (CAS Registry Number 480-19-3), and biochanin A (CAS Registry Number 491-80-5) were purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Cajaninstilbene acid (CAS Registry Number 87402-84-4) was obtained by the separation and purification from pigeon pea leaves by column chromatography in our laboratory.

The reference standards of isorhmnetin (3), diosmetin (6), isochlorogenic acid A (7), kaempferol (10), quercetin (14), isochlorogenic acid C (15), apigenin (19), forsythin (20), chlorogenic acid (23), liquiritin (25), luteolin-7-O-glucoside (26), astragalin (28), luteolin (29), buddleoside (31), quercetin-3-O-glucoside (33), amygdalin (37), caffeic acid (38), forsythoside A (39), glycyrrhizic acid (40), ferulic acid (42), rutin (44), hyperoside (50), and platycodin D (51) were purchased from Sichuan Weikeqi Biological Technology, Co., Ltd. (Chengdu, China). Mulberry leaf, chrysanthemum, Fructus Forsythiae, licorice, Semen Armeniacae Amarum, Platycodi Radix and Phragmitis Rhizoma

The reference standards ginkgolide M (3), apigenin-7-O-glucoside (7), bilobalide (8), ginkgolide C (9), quercetin (14), luteolin (15), apigenin (16), kaempferol (17), isorhamnetin (18), ferulic acid (19), ginkgolide A (20), myricetin (22), genistein (23), chlorogenic acid (24), caffeic acid (26), luteolin-7-O-glucoside (33), kaempferol-3-O-glucoside (34), kaempferol-7-O-glucoside (35), quercetin-7-O-rhamnoside (40), isoquercitrin (45) and rutin (65) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China), and their structures are shown in Fig. 1. Purities of all the reference standards were over 98% according to HPLC-DAD analysis.

Substrates including tectorigenin (1), formononetin (2), genistein (3), daidzein (4), apigenin (5), luteolin (6), naringenin (7), eriodictyol (8), hesperetin (9), kaempferol (10), isorhamnetin (11), 3,4-dichloroaniline (13), 3,4-dichlorobenzenethiol (14), emodin (15), tectoridin (16), puerarin (17), vitexin (18), orientin (19), and mangiferin (20) were purchased from Sichuan Weikeqi Bio-Technology CO., LTD. (Chengdu, China) and Chengdu Must Bio-Technology Co., Ltd (Chengdu, China) while the compound phloretin (12) as a gift was obtained from Dr. Peng Wu, Guangzhou University of Chinese Medicine.

Voucher specimens (No. 20140312) were deposited at School of Pharmacy, Liaoning University of Traditional Chinese Medicine. Oleracone C (purity >99%) was isolated from P. oleracea in our laboratory (Fig. 1). The IS, apigenin (purity ≥98%), was provided by Sichuan Weikeqi Biological Technology Co. (Sichuan, China; Fig. 1). HPLC-grade methanol and acetonitrile were provided by Damao Chemical Reagent Co.

were supplied from Chengdu Pufeide Biological Technology Co., Ltd. (Sichuan, China). The standards of scutellarin, isochlorogenic acid A, isochlorogenic acid C, and apigenin were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, China). The internal standard gallic acid (IS1) was purchased from the National Institute for Control of Pharmaceutical

The standards of puerarin, daidzin, p-hydroxycinnamic acid, daidzein, apigenin, biochanin A, ononin, naringenin, genistein, formoononetin, β-sitosterol, umbelliferone (internal standard 1, IS1), and rutin (internal standard 2, IS2) were purchased from Sichuan Weikeqi Biological Technology (Sichuan, China). The purity of all standards was over 98%. The structure of analytes and ISs are demonstrated in Figure 1.

The roots of Daphne genkwa were collected from Yueyang City, Hunan Province (lot: 170,525), which was authenticated by Prof. Wu Qinan. Five flavonoids, including genkwanin, 3′-hydroxygenkwanin, apigenin, luteolin and liquiritin were purchased from Sichuan Weikeqi Biotechnology Co. Yuanhuacine and genkwadaphnin were obtained from Nanjing Jin Yibai Biological Technology Co.

The reference standards of apigenin, luteolin, quercetin, isorhamnetin,ILG, ILQ, and liquiritigenin (purity >98.5%, HPLC) were purchasedfrom Sichuan Weikeqi Biological Technology, Co., Ltd. (Chengdu,China). Acetonitrile and methanol of HPLC grade were purchasedfrom Fisher Scientific (Waltham, MA, USA).

Reference compounds, comprising specnuezhenide, oleuropein, nuezhenidic acid, ligustroflavone, G13, oleoside 11-methyl ester, tyrosol, hydroxytyrosol, salidroside, acteoside, protocatechuic acid, galuteolin, apigenin, quercitrin, rutin, luteolin, quercetin, oleanolic acid, ursolic acid, betulin, emodinmonomethyl ether, 3β-O-acetylpomolic acid, oleonuezhenide, 6′-O-trans-cinnamoyl-8-epikingisidic acid, ligulucidumoside A, ligulucidumoside C, oleuropeinic acid, echinacoside, 3β-O-acetylpomolic acid, oleonuezhenide, 6′-O-trans-cinnamoyl-8-epikingisidic acid, ligulucidumoside A, ligulucidumoside C and geniposide (purity ≥98%), were provided by Yifang S&T Co. Ltd (Tianjin, China), the National Institutes for Food and Drug Control and Sichuan Weikeqi Pharmaceutical Technology Co. Ltd. Methanol and acetonitrile (chromatographic purity) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). The Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to produce Ultrapure-water. All reagents were analytical grade.