Inhibition of DNA Topoisomerases I and II and Cytotoxicity of Compounds from Polygonum aviculare L .

Six flavonoid glycosides ( 1 – 3 , 6 – 8 ), one lignan ( 4 ), and one galloyl glycoside ( 5 ) were isolated based on the phytochemical study for the MeOH extract of Polygonum aviculare L. herb. The eight isolated compounds' structures were determined by comparing their physicochemical and spectral data with published ones. Compounds 5 and 6 were first isolated from this plant, and all the compounds were evaluated for DNA topoisomerase inhibitory activities and cytotoxicities. Among the purified compounds, 5 showed stronger inhibitory activity against topoisomerase I with an IC 50 value of 12.0 μM, while the value of positive control camptothecin (CPT) was 17.0 μM. For compounds 1 , 3 , and 5 – 8 , the value ranged from 0.054 to 17.0 µM, suggesting more potent inhibitory activity against topoisomerase II, and that of etoposide (VP-16) was 28.0 µM. Particularly, compounds 6 and 8 (IC 50 = 0.054 and 0.077 µM, respectively) inhibited Topo II about 500 times stronger than VP-16, making them safer agents of DNA topoisomerase. Furthermore, all compounds demonstrated no cytotoxic activity against four cancer cell lines. This study is the first to report the inhibition of DNA topoisomerases of compounds from P. aviculare , suggesting that these compounds may be potential candidates for treating cancer.


Introduction
Various treatments are available against life-threatening cancer, including surgery, chemotherapy or radiation, or combinations of these [1], among which chemotherapy is considered the most effective. As targets of chemotherapy drugs, DNA topoisomerases are enzymes that regulate DNA topology in the cell [2]. Topoisomerase Ι (Topo Ι) and topoisomerase II (Topo II) catalyze the passage of the DNA strand and double strands through a transient single-strand break without any high-energy cofactor and a transitory double-strand break with ATP, respectively [3]. CPT and VP-16 are wellknown Topo Ι and II inhibitors, respectively [4], but their side effects have been reported, known as DNA damage in cancer cells and fast-growing normal cells [5], which highlights the necessity of finding a safer agent of DNA topoisomerase (without or with fewer side effects).
Studies have shown that many chemotherapeutic agents are derived from natural sources, especially classical medicinal plants. For thousands of years, natural products purified from natural sources have shown different therapeutic effects with few side effects based on pharmacological studies and clinical trials and can be crucial to searching topoisomerase inhibition [2,6]. According to previous research, several natural medicinal plants possess Topo I, II inhibitory activity and cytotoxicity, and some activity compounds have been screened out, such as Illicium verum Hook. Fil. [4], Inula japonica Thumb. [7], Ulmus davidiana var. Japonica (Rehd.) Nakai [2], Polygonum multiflorum Thumb. [8], Tilia amurensis Rupr. [9], and Saururus chinensis (Lour.) Baill. [10]. Furthermore, we found that the MeOH extract of Polygonum aviculare L.
(Polygonaceae) showed Topo I and II inhibitory activities, which was never reported previously. P. aviculare is a perennial herbaceous plant widely distributed in countries like China, Korea, and Japan [11] and has been used for diuretic, antidotal, antiphlogistic, antipyretic, antiparasitic, and antidiarrheal effects in Chinese folk medicine to treat nephritis, urinary system infection, and gingivitis [12,13]. Notably, in previous studies, several secondary metabolites of P. aviculare have been identified, such as flavonoids, phenols, and lignins [12,14]. P. aviculare is characterized by a diversity of biological activities. Based on this, further research regarding the bioactivities of primary metabolites is essential. This study described the isolation and identification of eight compounds (1-8) from P. aviculare in detail. In addition, the solvent fractions and all purified compounds of P. aviculare were evaluated for their DNA Topo I and II inhibitory activities and anti-proliferation activities on four cancer cells.
The conversions of supercoiled pBR322 plasmid DNA to the relaxed DNA were investigated using calf thymus Topo I and II in the presence of solvent fractions and 1-8. Table 1 shows that all solvent fractions had potent inhibitory activity against Topo II but only weak inhibitory activity against Topo I. Among the purified compounds, 4, 5, had been reported previously [25][26][27].
Secondary metabolites are critical in controlling anticancer response pathways, such as structurally diverse flavonoids with significant bioactivity. Abundant in both traditional medicinal herbs and marine life, the secondary metabolites possess various biological and chemical properties like antioxidants [28], antimicrobial, antiinflammatory [29], antimutagenic [30]. In particular, studies have been conducted to elucidate the role of flavonoids as topoisomerase inhibitors, contributing to studying highly selective chemotherapeutic agents.
Topoisomerase research on flavonoids has been widely concerned in recent years, and some scholars suggested that flavonoids inhibit both Topo I and II [31]. For instance, quercetin and kaempferol were reported as traditional Topo II poisons, and the C4'-OH was believed to be essential for the compound to function as a traditional poison. The addition of -OH groups at C3' and C5' could increase the redox activity of the B ring, enabling the compound to function as a redox-dependent poison [32]. In this study, compounds (1, 3, 6-7) reported moderate inhibitory activity against Topo II, and the experimental results were consistent with the previous literature. Compound 8 showed strong inhibitory activity toward Topo II without -OH group at C3' and C5', which could result from the purity of the isolated compounds and/or the nature of topoisomerase and different concentrations test methods.
Tests against four cancer cell lines were conducted (A549, SK-OV-3, HepG2, and HT-29) to investigate whether the solvent fractions and all compounds from P. aviculare medicated the DNA topoisomerase inhibitory activity to induce cell death, whose results showed that n-hexane extract and H2O extract exhibited cytotoxicity, while none of the compounds showed cytotoxicity against the selected cell lines (Tables 1 and 2).
No noticeable correlation could be noticed between the cytotoxicity and DNA relaxation and decatenation inhibitory activity by DNA Topo I and II. It might be hypothesized that these compounds may inhibit Topo I and II reactions in enzyme assays, but transportation of these compounds via cell and nucleus membranes to the target Topo I and II may be possible barriers in the MTT assay. Based on this newly reported biological activity, the studied plant could be a source of new anticancer agents, and further phytochemical inquiry should clarify the mechanism of action for the sake of drug discovery.

Conclusion
This is the first study to report the compounds 5 (1, 6-di-O-galloyl-β-D-glucopyranose) and 6 (agetiin) isolated from P. aviculare, and it also informs the inhibition of DNA Topo I and II of the plant. The inhibitory activities of all purified compounds against Topo I and II were reported for the first time, except that 1, 3, and 8 of Topo II were reported previously. Interestingly, compound 5 showed more potent inhibitory activity against both DNA Topo I and II than the positive control (CPT and VP-16). In addition, compounds 6 and 8 (astragalin) (IC50 = 0.054 and 0.077 µM, respectively) inhibited Topo II about 500 times stronger than VP-16, which enabled them to be safer agents of DNA topoisomerase and serve as potential candidates for treating cancer illness. This experiment enriched and clarified the chemical compositions and bioactivities of P.
aviculare, providing a scientific theoretical basis for further in-depth research and rational development and utilization of this plant in the future.

General experimental procedures
The melting point was determined using the Fisher-Johns melting point apparatus and is unadjusted. The JASCO DIP-1000 (Tokyo, Japan) automatic digital polarimeter was used to detect optical rotations. The NMR spectra was acquired on a Bruker 250 MHz

Assay for DNA Topo I inhibition in vitro
The relaxation of supercoiled pBR322 plasmid DNA was measured using the isolated compounds in the DNA Topo I inhibition experiment, which was slightly modified from the formerly described procedure. The reaction mixture contains 72 mM KCl, 5 mM were scanned directly with an image analyzer for visualization and quantification of DNA Topo I activity, with the area representing supercoiled DNA being computed [33].
Each sample was tested in triplicate.

Assay for DNA Topo II inhibition in vitro
The relaxation of supercoiled pBR322 plasmid DNA was used to assess DNA Topo II were stained with 0.5 μg/mL ethidium bromide for 30 min before being destained in water for 30 min. The gels were scanned directly with an image analyzer to visualize and quantify DNA Topo II activity, and the area representing supercoiled DNA was then measured [33]. Each sample was tested three times.