Two New Isoprenoid Flavonoids from Sophora flavescens with Antioxidant and Cytotoxic Activities

Sophora flavescens is a regularly used traditional Chinese medicine. In an attempt to discover adequate active agents, the isoprenoid flavonoids from S. flavescens were further investigated. In this work, two new compounds (1–2, kurarinol A-B) together with 26 known ones (3–28) were isolated and elucidated on the basis of extensive NMR, UV and MS analyses. Furthermore, the antioxidant activity of all constituents was assessed through ABTS, PTIO and DPPH methodologies and also were evaluated for cytotoxic activity by three tumor cell lines (HepG2, A549 and MCF7) and one human normal cell line (LO2 cells). As a result, a multitude of components revealed significant inhibitory activity. In particular, compound 1–2 (kurarinol A-B), two new flavanonols derivatives, exhibited the most potent ABTS inhibitory activity with IC50 of 1.21 µg/mL and 1.81 µg/mL, respectively. Meanwhile, the new compound 1 demonstrated remarkable cytotoxicity against three cancer cells lines with IC50 values ranging from 7.50–10.55 μM but showed little effect on the normal cell. The two new isoprenoid flavonoids could be promising antioxidant and anti-tumor nature agents.


Introduction
Sophora flavescens (Ku-Shen in Chinese), derived from the Fabaceae family, is widely distributed in China as a herb or shrub, and has a strong bitter taste ( [1], pp. 202-203). In 200 A.D., S. flavescens was recorded in the traditional Chinese medicine masterpiece Shen Nong's Herbal Classic for the first time for treating inflammation, solid tumors, and other disorders [2]. It was also included in the Pharmacopoeia of the People's Republic of China in 1977 for the treatment of dysentery, hematochezia, jaundice, oliguria, vulvar swelling, and ulcers [3]. In addition, in some other Asian countries, such as Korea and Japan, S. flavescens is known as a commonly herb for antipyretic, analgesic, antihelmintic and stomachic therapies [4][5][6].
According to reports, alkaloids and flavonoids were the main bioactive components of S. flavescens. To date, a total of more than 60 alkaloids and 120 flavonoids have been isolated from S. flavescens [7]. Among them, matrine and oxymatrine are the most frequently employed as quality control markers in China ( [1], pp. [189][190]. Recently, flavonoids from the flowers of S. flavescens, polygonum cuspidatum, radix sophorae tonkinensis, herba ephedrae, salvia miltiorrhiza and astragali radix have drawn worldwide attention for their potential health benefits [8]. The structures of flavonoids with a lavanduly, prenyl or 1,1-dimethylallyl group show noticeable biological activity and 55 isoprenoid flavonoids were reviewed from S. flavescens in our previous work, all of which played an important roles in antimicrobial, anti-inflammatory, antidiabetic and anti-tumor activities [9][10][11]. However, few studies have concentrated on isoprenoid flavonoids for antioxidant capacity and cytotoxicity. Several studies have shown that a mass of free radicals can be generated during metabolic processes in the human body [12]. These free radicals could lead to oxidative stress and homeostatic imbalance without treatment and removal in a timely manner, and could further bring about some chronic diseases such as diabetes, angiocardiopathy and cancer [13]. To address this point, we decided to explore the active substances from S. flavescens. In this work, 2 new and 26 known compounds were identified by combined spectroscopy. These compounds were tested for the inhibitory activities of cytotoxicity on human cancer cells (human liver cancer cell HepG2, human lung cancer cell A549 and human breast cancer MCF7) and human normal liver cells (LO2 cells), together with ABTS (2-2 -azinobis-3-ethylben-zthia zoline-6-sulphonate), PTIO (2-phenyl-4,4,5,5tetramethylimidazoline-1-oxyl 3-oxide) and DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging inhibition activities. To sum up, a new compound named kurarinol A (1) exhibited excellent cytotoxic and antioxidant activities.

Cytotoxic Activities (HepG2, A549 and MCF7 Cancer Cells, LO2 Human Normal Cells)
Natural products with isoprenoid moieties are an important focus of research for scholars. Previous literatures reported that S. flavescens exhibited promising cytotoxicities against cancer cell lines [29,35]. In the work, cytotoxic activities of 28 compounds (10 µM) and the ethyl acetate (EtOAc) extract (25 µg/mL) were evaluated with three human cancer cell lines and one normal cell, with irinotecan as the positive control. IC 50 values of 0.6 µM, 1.0 µM, and 0.9 µM for HepG2, A549, MCF7, respectively, were found ( Figure 6). The results showed the EtOAc extract exhibited the strongest activity (73-96% inhibition against the three cancer cell lines at 25 µg/mL). Thus, we isolated the EtOAc extract to obtain 28 compounds, and tested their bioactivities. and their isoprenylated derivative, 1 (85.0% against MCF7 cells). We also tested the cytotoxicities of the above compounds against LO2 human normal cell lines ( Figure 6). All of them showed weaker cytotoxic activity against the normal cells than the cancer cells.

Reagents and Materials
The extraction and separation of S. flavescens was performed using analytical or chromatographic grade organic solvents from Anhui Tiandi high-purity Solvent Co., Ltd. The ABTS, PTIO, DPPH, MTS and DMSO were obtained from Sigma-Aldrich (St. Louis, MO, USA). DMEM medium, the dual antibiotic mixture (penicillin-streptomycin), 0.25% Trypsin-EDTA, and FBS were purchased from Gibco Company. Phosphate Buffered Saline (PBS) was acquired from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Three human cancer cell lines (HepG2, A549 and MCF7 cells) were obtained from American Type Culture Collection (ATCC, Manassas, MD, USA) and human normal cells were acquired from The Key Laboratory of Optimal Utilization of Natural Medicine Resources (Guiyang, China). The positive control irinotecan was obtained from Shanghai Hongye As a result, kushenol A (13) displayed selectivity against different tumor cell lines and revealed IC 50 values of 6.85 µM for HepG2 cells, could hardly inhibit MCF7 and A549 cells, and was discovered as cytotoxic agent for the first time. Interestingly, a new compound 1 (kurarinol A) demonstrated potent cytotoxicities against all the three human cancer cell lines with IC 50 values in the range of 7.50-10.55 µM. The two lavandulyl groups might remarkably improve the cytotoxicity. Compounds 2, 3 and 4 showed poor inhibitory activity against HepG2 cells, with inhibition rates of 19.9%, 1.9% and 4.6% at 10 µM. However, their isoprenoid derivative 1 inhibited the cells by 84.1%. Similar results were observed for compounds 2, 3, and 4 (10.3%, 4.5% and 0.7% inhibition against MCF7 cells) and their isoprenylated derivative, 1 (85.0% against MCF7 cells). We also tested the cytotoxicities of the above compounds against LO2 human normal cell lines ( Figure 6). All of them showed weaker cytotoxic activity against the normal cells than the cancer cells.

Reagents and Materials
The extraction and separation of S. flavescens was performed using analytical or chromatographic grade organic solvents from Anhui Tiandi high-purity Solvent Co., Ltd.

Plant Material
The roots of S. flavescens were collected in September 2019 in Dafang county, Bijie city, Guizhou province, People's Republic of China. The plant species of S. flavescens was confirmed by DNA barcoding analysis using the ITS sequences (Supplementary Information). Voucher specimen was deposited at the School of Pharmaceutical Sciences, Guizhou Medical University (Guiyang, China).

Extraction and Isolation
The dried power of S. flavescens (25 kg) was fully dipped in 95% and 75% EtOH for 7 days, three times, respectively. After concentrating the filtered liquor in a vacuum we obtained the extract. The extract was then dispersed in water and successively extracted with EtOAc and n-BuOH. The EtOAc extract (420 g) was separated on a silica gel column eluted with petroleum ether/ethyl acetate (1:0, 50:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 0:1, v/v) to obtain fractions A-H. The eight fractions were separated by repeated column chromatography and preparative liquid chromatography to obtain the isoprenoid flavonoids 1-28. The detailed separation procedure is described in Support Information (Support Information). Purities for all the compounds were above 95% by HPLC/UV analysis.

DPPH Antioxidant Activity Assay
The in vitro antioxidant activity tests were conducted according to previous reports [30]. Among them, the DPPH free radical scavenging assay was slightly modified. Briefly, the DPPH material was precisely weighed, ethanol solution was added to obtain a concentration of 0.1 mmol/L (to use immediately, or store in the dark). Then, the compounds (100 µL, 40 µg/mL) with DPPH solution (100 µL) (A i ) were added to 96-well plates and incubated for 30 min at room temperature. Subsequently, the activity was measured using the Thermo Scientific Varioskan LUX (Berthold, VL0L00D0, USA), with an absorbance of 570 nm. Vitamin C (VC) (40 µg/mL) was used as the positive control. The radical scavenging effect was calculated using the this equation: Free radical clearance = [1 − (A i − A j )/A 0 ] × 100% (the value of 100 µL ethanol with 100 µL DPPH solution as A 0 , the value of 100 µL compounds with 100 µL ethanol as A j ).

PTIO Antioxidant Activity Assay
PTIO free radical scavenging was also performed following previous reports with some modification [28]. VC was used as the positive control. PTIO radicals were dissolved with phosphate buffer (PBS) (pH 7.4, 50 mM) with a concentration of 0.05 mg/mL. Then, the samples (40 µL, 40 µg/mL) and PTIO solution (160 µL) were added into 96-well plates (as the value of A i ). After being thoroughly mixed, the reaction solution was incubated at 37 • C in a water-bath for 2 h. Then, the absorbance was measured at 557 nm. PTIO free radical scavenging was calculated using the above equation. (40 µL PBS with 160 µL PTIO as A 0 , 40 µL compounds with 160 µL PBS as A j ).

ABTS Antioxidant Activity Assay
The third assay, the ABTS free radical scavenging screen was performed by previous experiments with slight changes [29]. First, we needed to prepare for the ABTS + solution. The ABTS and potassium persulfate reagents were both dissolved in deionized water, to obtain concentrations of 7 mmol/L and 2.38 mmol/L, respectively. Then, they were mixed with equal volume at room temperature and kept away from light for 12 h, the mixed solution was diluted with ethanol until the absorbance of 0.70 ± 0.20. Thus, the ABTS + solution was obtained. Furthermore, the compounds (100 µL, 40 µg/mL) with ABTS + solution (100 µL) were also added to the 96-well plates (as the value of A i ) and were interacted for 10 min at room temperature in the dark, then the absorbance at 734 nm was measured. VC was used as the positive control. Antioxidant capacity was calculated according to the first formula (the value of 100 µL ethanol with 100 µL ABTS + solution as A 0 , the value of 100 µL compounds with 100 µL ethanol as A j ). All experiments were carried out in triplicate.

Cytotoxic Activity Assay
The tests were conducted according to our previous reports [36,37]. The cells were grown in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) in a 37 • C and 5% CO 2 incubator. The cytotoxic activities were performed by the MTS assay. Briefly, 28 compounds were dissolved with DMSO. The cells were seeded at 1 × 10 5 cells/mL in 96-well plates (100 µL/well) and cultured for 18 h when the cells filled the bottom of the wells. Then the supernatant was discard, the compounds (10 µM, 100 µL/well) were added to the culture and incubated for 24 h before cell viability measurement. 10 µL MTS reagent (0.5 mg/mL) were added and further incubated for 4 h.
Finally, absorbance was read using an automatic micro-plate reader (Molecular Devices, USA) at 490 nm to test the cell viabilities, and the results were presented as the percent of non-treated control for each concentration. Irinotecan was used as the positive control. All measurements were repeated in triplicate. Data were expressed as the mean ± SD.

Conclusions
To sum up, a total of 28 compounds were isolated from the EtOAc extract of S. flavescens and two of them were new structures. They were identified mainly by NMR, UV and MS analyses. NMR spectroscopy was a powerful tool for the analysis of structure, the known frameworks mostly used 1D 1 H-NMR spectrum, which simply showed signals for each of the hydrogen atoms in a compound. For the new ones, we also used 2D NMR spectrum, which revealed more signals between protons, adjacent 13 C-1 H and remote 13 C-1 H and was more useful and sensitive. The main contribution of UV spectrum in the structural identification of compounds was to determine the main skeleton. Kurarinol A-B were deduced to have flavonoid skeletons with maximum UV absorption at 303 and 295 nm, respectively. Additionally, the molecular weight of the compounds can be accurately determined through HRESIMS analyses. Furthermore, a number of isoprenoid flavonoids were found to be significant antioxidant inhibitors, and showed protective activities on ABTS, PTIO and DPPH free radical scavenging, including kurarinol A (1), kurarinol B (2), kushenol H (3), kushenol L (4), kuraridine (6), kushenol Q (9), kurarinol (12), sophoflavescenol (14), noranhyoicaritin (15), quercetin (16), 7,3 -di-O-methyl (17) and luteolin (23). Two new isoprenoid derivatives 1-2 (kurarinol A-B), exhibited the most potent antioxidant capacities against the ABTS enzyme with IC 50 of 5.51 µg/mL and 2.70 µg/mL, respectively. Furthermore, the new compound 1 (kurarinol A) demonstrated significant cytotoxicity against the HepG2, A549 and MCF7 cell lines with IC 50 values ranging from 7.50 to 10.55 µM. There were two lavandulyl groups which might remarkably improve the cytotoxicities. These compounds could be promising antioxidant and anti-tumor natural agents.