Chemical Constituents of the Flowers of Pueraria lobata and Their Cytotoxic Properties

The flower of Pueraria lobata (Puerariae Flos) is a reddish-purple to violet-purple flower that blooms between July and September. In our preliminary study, Puerariae Flos extract exhibited significant activity against a human ovarian cancer cell line. This research aims to identify the active compounds in Pueraria Flos. By repeated chromatography, one new tryptophan derivative (1), two new flavanones (4 and 5), and 19 known compounds, including tryptophan derivatives (2 and 3), flavonoids (6–9), isoflavonoids (10–20), a flavonolignan (21), and a phenolic compound (22), were isolated from a methanol extract of Puerariae Flos. The structures of new compounds were elucidated as 13-N-benzoyl-l-tryptophan-1-N-β-d-glucopyranoside (1), 2-hydroxy-5-methoxy-naringenin (4), and 2-hydroxy-5-methoxy-naringenin 7-O-β-d-glucopyranoside (5). Among the isolates, afromosin (17), tectorigenin (11), apigenin (8), glycitein (16), (-)-hydnocarpin (21), irilin D (12), irisolidone 7-O-glucoside (14), and genistein (10) showed cytotoxicity against human ovarian cancer cell line A2780. Apigenin (8) and (-)-hydnocarpin (21) were the most active (IC50 values of 9.99 and 7.36 μM, respectively).


Introduction
Pueraria lobata (Willd.) Ohwi (Leguminosae) is one of the most important Chinese traditional medicines native to East Asia, Central and South America, and Europe [1]. The flower of P. lobata (Puerariae Flos) is a reddish-purple to violet-purple flower that blooms between late July and September [2]. It has been commonly used as a hangover treatment in traditional oriental medicine due to its enhancing activity of acetaldehyde removal [3,4]. Puerariae Flos mainly contains isoflavonoids, flavonoids, saponins, tryptophan derivatives, and phenolic compounds [5][6][7][8]. Extracts and secondary metabolites from Puerariae Flos have shown potential pharmacological effects, such as an anti-diabetic effect [9,10], an anti-inflammatory effect [11], estrogen-like activity [12], an anti-cancer property [12], an anti-endometriotic effect [13], and a sensitizing effect on paclitaxel-resistant ovarian cancer cells [14].
Ovarian epithelial cancer is one of the most fatal cancers in women [15]. A previous investigation has shown that the 5-year relative survival rate for patients with ovarian epithelial cancer was only 30% [16]. Platinum-taxane chemotherapy after surgical resection is the most usual treatment for ovarian cancer patients. However, tumor cell heterogeneity causes treatment resistance through multiple genetic alterations, leading many patients to relapse or even die [17]. As a result, a novel therapeutic agent for ovarian cancer is urgently needed. Numerous plant-derived medicines and their derivatives induce apoptosis in ovarian carcinoma cell lines [18]. The genus Pueraria has shown cytotoxic and anti-proliferative activity against various cancer cells [11]. In particular, the phytoestrogens extracted from Puerariae rhizome exhibited anti-proliferative activity against ovarian cancer cells [14]. In our preliminary experiment, a methanol extract of Puerariae Flos exhibited a significant cytotoxic effect against the human ovarian cancer cell line A2780. Therefore, the purpose of this study is to find active compounds in Pueraria Flos against human ovarian cancer cells.
Herein, repeated chromatography with the methanol extract of Puerariae Flos was conducted to isolate compounds with cytotoxic activity against human ovarian cancer cell line A2780. The structure of isolated compounds was determined by analyzing 1Dand 2D-nuclear magnetic resonance (NMR) spectroscopic data and high-resolution mass spectroscopy (HRMS). Then, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to assess the cytotoxicity of all isolated compounds (1−22) against human epithelial ovarian cancer cell line A2780 and the immortalized ovarian epithelial cell line IOSE80PC.

Plant Material
The flowers of Pueraria lobata (Willd.) Ohwi (Leguminoseae) were purchased from CK Pharm Co. (Seoul, Korea) in June 2019. The origin of the herbal material was identified by prof. Dae Sik Jang and a voucher specimen (PULO5-2019) has been stored in the Lab. of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Seoul, Korea.

General Experimental Procedures
General experimental procedures are in the Supplementary Materials.   Table 2.

Acidic Hydrolysis of Compounds 1 and 5
Compounds 1 and 5 (each 1.0 mg) were hydrolyzed with 2N HCl at 80 • C for 4 h and with 1N HCl at 100 • C for 3 h, respectively. Each reaction was stopped by the addition of sodium thiosulfate. Additional hydrolysis was proceeded for identifying the absolute configuration of tryptophan moiety of 1. Compound 1 (1.0 mg) was hydrolyzed with 6N HCl at 85 • C for 24 h. Reaction was stopped by the addition of sodium thiosulfate.

Absolute Configuration Analysis of β-Glucoses in Compounds 1 and 5
To determine the absolute configuration of β-glucoses in 1 and 5, the modified analysis method from a reference was conducted [46]. The hydrolysate was dissolved in pyridine (500 µL) and L-cysteine methyl ester hydrochloride (1.2 mg) was added and heated at 60 • C for 1 h. σ-Tolyl isothiocyanate (100 µL) was added and heated again at 60 • C for 1 h. The reaction product was analyzed by HPLC under a gradient system (A: 0.1 % (v/v) formic acid in water, B: 0.1 % (v/v) formic acid in acetonitrile, 10 to 50% B, 45 min). The glucoses in the reaction mixture of 1 and 5 were detected at 28.3 and 28.4 min each. Authentic L-and D-glucose were, respectively, detected at 27.6 and 28.2 min at the same HPLC conditions. Therefore, the absolute configuration of β-glucose in compounds 1 and 5 was identified as the D configuration.

Absolute Configuration Analysis of Tryptophan in Compound 1
The hydrolysate to determine the absolute configuration of tryptophan of 1 was subjected to HPLC by under a gradient system (A: water, B: methanol, 10 to 35% B, 80 min). Tryptophan (0.2 mg) was obtained from the hydrolysate. Analyzing the optical rotation dispersion of the obtained tryptophan revealed that it has L-configuration (

Cell Viability Assay
Human ovarian endometrioid adenocarcinoma cell line A2780 and immortalized ovarian surface epithelial cell line IOSE80PC were provided by Dr. Ie-Ming Shih (Johns Hopkins School of Medicine, Baltimore, MD, USA) and Dr. N. Auersperg (University of British Columbia, Vancouver, British Columbia, Canada), respectively. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 5% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin sulfate (100 µg/mL) in a 5% CO 2 and 95% air humidified atmosphere at 37 • C. RPMI 1640, FBS, streptomycin sulfate, and penicillin were procured from Life Technologies Inc. (Grand Island, NY, USA). The cells were seeded at a density of 1.0 × 10 5 cells/mL in a 96-well plate containing 50 µL of RPMI medium in each well and incubated for 24 h. Various concentrations of compounds dissolved in dimethyl sulfoxide (DMSO) were mixed with RPMI 1640 medium and added into cells in each well. The final concentration of DMSO in the medium did not exceed 0.1%. Following 48 h incubation, 50 µL of MTT (Molecular Probes Inc., Eugene, OR, USA) solution was added into each well to achieve a final concentration of 0.5 mg/mL and then incubated for an additional 4 h. The medium was discarded, and the formazan blue that formed in the cells was dissolved in 50 µL of DMSO. The optical density was measured at 540 nm by microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). Three independent experiments with at least three replicates have been performed for all the tested compounds except quercetin, which has been tested once. IC 50 is defined as the concentration that reduces cell number by 50% compared to control cultures. Results shown in Table 3 and Figure 3 are the representative of the independent experiments.