Flavonoids from the Roots of Sophora flavescens and Their Potential Anti-Inflammatory and Antiproliferative Activities

The phytochemical investigation of the roots of the traditional Chinese medicinal plant Sophora flavescens led to the isolation of two novel prenylflavonoids with an unusual cyclohexyl substituent instead of the common aromatic ring B, named 4′,4′-dimethoxy-sophvein (17) and sophvein-4′-one (18), and 34 known compounds (1–16, 19–36). The structures of these chemical compounds were determined by spectroscopic techniques, including 1D-, 2D-NMR, and HRESIMS data. Furthermore, evaluations of nitric oxide (NO) production inhibitory activity against lipopolysaccharide (LPS)-treated RAW264.7 cells indicated that some compounds exhibited obvious inhibition effects, with IC50 ranged from 4.6 ± 1.1 to 14.4 ± 0.4 μM. Moreover, additional research demonstrated that some compounds inhibited the growth of HepG2 cells, with an IC50 ranging from 0.46 ± 0.1 to 48.6 ± 0.8 μM. These results suggest that flavonoid derivatives from the roots of S. flavescens can be used as a latent source of antiproliferative or anti-inflammatory agents.


Introduction
The roots of Sophora flavescens are ordinarily served in the traditional Chinese medicine (TCM), "Ku Shen", for the curing of skin diseases, cancer, dysentery, hematochezia, jaundice, pruritus vulvae, eczema, and hepatitis [1]. Modern pharmacological research shows that it exhibits outstanding activities toward tumors, inflammation, diabetes, and microbial infections [2][3][4][5][6][7]. Phytochemical studies demonstrated that alkaloids and flavonoids are the major chemical classes of S. flavescens compounds [8][9][10]. Matrine, as a reflective component of the alkaloids of S. flavescens, has been used as an antitumor drug (Compound matrine injection) in China. With more in-depth research on the alkaloids of S. flavescens, the anticancer effects of some alkaloids have been shown to be more potent than those of flavonoids of S. flavescens in vitro and in vivo [11].
In order to discover new compounds with antiproliferative and anti-inflammatory activities from S. flavescens, we isolated 36 flavonoids from this medicinal plant (Figure 1), including flavanones, isoflavones, flavonols, flavanonols, and chalcones. Among them, compounds 17 and 18 were two new dihydroflavones with an unusual cyclohexyl substituent instead of the common aromatic ring B. Many studies have shown an interesting link between chronic inflammation and cancers [12]. Thus, all isolated components were evaluated for their anti-inflammatory activity by acting on LPS-stimulated macrophage (RAW 264.7) cell lines in vitro. In addition, all components were assessed for their antiproliferative activities against HepG2 cell lines.
Here, we characterize the isolation, chemical structure elucidation, antiproliferative activity against HepG2 cells, and NO production inhibitory activity of all isolates.

Results and Discussion
The comprehensive use of separation materials and chromatographic methods such as normal silica gel, hydroxypropyl dextran gel (Sephadex LH-20), MPLC, TLC, MCI reversed-phase column, and HPLC, with the help of MS and NMR and other spectroscopy methods, was carried out to isolate and identify 36 compounds, including 2 new ones: 4′,4′-dimethoxy-sophvein (17) and sophvein-4′-one (18).

Results and Discussion
The comprehensive use of separation materials and chromatographic methods such as normal silica gel, hydroxypropyl dextran gel (Sephadex LH-20), MPLC, TLC, MCI reversed-phase column, and HPLC, with the help of MS and NMR and other spectroscopy methods, was carried out to isolate and identify 36 compounds, including 2 new ones: 4 ,4 -dimethoxy-sophvein (17) and sophvein-4 -one (18).

Biological Studies
The potential cytotoxicity of compounds on RAW 264.7 macrophages cells was determined before executing further studies. Macrophage cells were treated with compounds, and a mitochondria colorimetric (MTT) assay was used to test cell survival. Cell viability , and SPSS version 21.0 (probit analysis) was used for calculating CC 50 extract. Table 2 shows that some compounds exhibited obvious cytotoxicity on macrophages. Among them, compound 22 was the most toxic composition with a CC 50 value of 13.8 ± 0.6 µM. NO is one of the immune effectors used by macrophages to defend our bodies against intracellular pathogens. To determine if compounds 1-36 can modulate the NO production by macrophages, the Griess reagent was used to analyze the NO levels. The cells were incubated with compounds 1-36 ( (Table 3), while others were inactive (IC 50 > 50 µM). Compound 22 exhibited the best antiproliferative effects on HepG2 cell lines, with an IC 50 value of 0.46 ± 0.1 µM. The present work revealed that many secondary metabolites of S. flavescens have antiproliferative and NO production inhibitory activities, indicating its application in traditional Chinese medicine.

General Experimental Procedure
The optical rotation value was recorded with a Jasco DIP-370 polarimeter (JASCO Corporation, Tokyo, Japan). The ultraviolet (UV) spectrum was recorded by a UV2700 spectrophotometer (Shimadzu, Kyoto, Japan). The infrared (IR) spectrum was obtained by an FT-IR spectrophotometer (PerkinElmer, Waltham, MA, USA) using KBr pellets. High-resolution electrospray ionization mass spectroscopy (HRESIMS) was obtained with an Agilent 6500 LC/Q-TOF mass spectrometer (Agilent, Waldbronn, Germany). The 1 H NMR, 13  . Macrophage cells were placed in a constant temperature incubator and cultured at a temperature of 37 • C and a concentration of 5% CO 2 . Human hepatoma HepG2 cell lines were also obtained from the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). HepG2 cells were cultured at 37 • C under 5% CO 2 . Additionally, an inverted phase contrast microscope was used to observe cell morphology.

Plant Material
The roots of S. flavescens were collected in Honghe, Yunnan Province, People's Republic of China, in June 2019. The sample was identified by one of the authors (Xuan-Qin Chen). A voucher specimen (number: KUMST20190628) was conserved at the Key Laboratory of Phytochemistry, Kunming University of Science and Technology, China.

Extraction and Isolation
The air-dried roots of S. flavescens (20 Kg) were crushed and extracted with 95% aq. ethanol (24 h × 3 times). The ethanol extracts were percolated and evaporated in vacuo to obtain a residue. This residue was suspended in water and then extracted with ethyl acetate (3 times) and concentrated to produce an ethyl acetate phase (493 g). The ethyl acetate phase was segmented and enriched by macroporous resin to obtain 308 g of total flavonoids. The total flavonoid extract (300 g) was segmented using a normal phase silica gel column chromatography (CC) and stepwise gradient elution with a gradient of petroleum ether-ethyl acetate (4:1-1:1) to obtain 7 subfractions (Fr.1-7).

Cell Viability Examination
To measure cell viability, an MTT assay was performed [41]. Macrophages (4 × 10 4 per well) were cultured in 96-well plates for 24 h; then, they were treated with several concentrations of compounds 1-36 (3.12, 6.25, 12.5, 25, and 50 µM) for 1 h, and then they were stimulated with LPS (1 µg/mL) for another 24 h. After washing twice with a PBS buffer, 20 µL of the MTT solution was added to each well, and incubation continued for 4 h. Optical density (O.D) was measured at 490 nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The relative cell viability was calculated in contrast to the normal control group.

NO Production Measurement
Activated macrophage cells could express inducible nitric oxide synthase (iNOS), catalyzing the production of NO from L-arginine. To determine the effect of compounds 1-36 on treatments on NO production, the accumulation of nitrites (NO 2− ) in the culture medium was recorded as an indicator of NO production [42,43]. Macrophages (8 × 10 4 per well) were seeded onto 96-well plates and pretreated by compounds 1-36 (3.12, 6.25, 12.5, 25, and 50 µM) 1 h prior to treatment by LPS (1 µg/mL). Afterward, costimulation for 24 h at 37 • C was carried out in an incubator under 5% CO 2 . Then, Griess reagents I and II (100 µL) were mixed with cell culture medium (70 µL). Prior to measuring the optical density, plates were incubated at room temperature for 10 min, and the absorbance at 540 nm was measured using a Thermo Fisher Scientific microplate reader. N(G)-monomethyl-Larginine, monoacetate salt (L-NMMA), and the medium were used as positive and negative controls, respectively.

Antiproliferative Assay
Briefly, the cells were incubated in 96-well microplates (1 × 10 5 per well) and allowed to adhere for 24 h before drug administration. Then, the cell lines were treated with test compounds 1-36 at five concentrations (3.12, 6.25, 12.5, 25, and 50 µM). At 24 and 48 h of incubation, cells were treated with MTT (200 µL, 5.0 mg/mL) and dissolved in the culture medium, for 1 h under at 37 • C under a 5% CO 2 humidified atmosphere. The MTT was then removed carefully and resolved with DMSO (150 µL/well). Optical density was recorded using a Thermo Fisher Scientific microplate reader at 490 nm. Cisplatin (CP) and medium were used as positive and negative controls, respectively.

Statistical Analysis
The data were analyzed using Statistical Package for Social Sciences (SPSS Version 21.0) software and are presented as the mean ± S.D. values of three different experiments. The results were analyzed via one-way analysis of variance (ANOVA), and statistical significance was defined as p < 0.05.