Chemopreventive flavonoids from Millettia pulchra Kurz var-laxior (Dunn) Z.Wei (Yulangsan) function as Michael reaction acceptors

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1078–1081

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Chemopreventive flavonoids from Millettia pulchra Kurz var-laxior (Dunn) Z.Wei (Yulangsan) function as Michael reaction acceptors Wenli Wang a,b, Jian Wang b,c, Ning Li a,b,⇑, Xiangrong Zhang a, Weihong Zhao a,b, Jiayuan Li a,b, Yingying Si a,b a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, Wenhua Road 103, Shenyang 110016, PR China c School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China b

a r t i c l e

i n f o

Article history: Received 15 October 2014 Revised 21 December 2014 Accepted 6 January 2015 Available online 13 January 2015 Keywords: Millettia pulchra Kurz var-laxior (Dunn) Z.Wei Yulangsan Flavonoids NQO1 Cancer chemoprevention Molecular docking

a b s t r a c t Natural NQO1 [NAD(P)H quinine oxidoreductase 1] inducing agents play a critical role in cancer chemoprevention. The expression of NQO1 is regulated by Michael reaction acceptors (MRAs) via the Keap1/ Nrf2/ARE signaling pathway. The aims of this study were to identify and characterize novel effective chemopreventive agents from naturally occurring products. Using bioassay-guided isolation approaches 16 bioactive MRAs from Millettia pulchra Kurz var-laxior (Dunn) Z.Wei, also called Yulangsan as a famous Zhuang medicine. The structures were elucidated as chalcone (1–7), flavonone (8–14), flavanone (15) and isoflavan (16). Their electrophilic abilities and NQO1 inducing activity were assessed using GSH (glutathione) rapid screening, and in vitro cell-based (Hepa 1c1c7 cells) assay, respectively. Compounds 3, 4, 6, 13, and 14 showed to have NQO1 inducing activity. Among them, compounds 4 and 14 interact with NQO1 at Gly 149, Gly 150, Phe 106, Typ 105 and His 161, revealed by molecular docking studies. Ó 2015 Elsevier Ltd. All rights reserved.

Cancer chemoprevention plays an integral role in the overall strategy geared toward controlling the incidence of cancer.1 It refers to the use of chemical agents that occur naturally in food or administrated as pharmaceuticals to inhibit or reverse the process of carcinogenesis. Epidemiological studies have suggested that consumption of food of plant-origin can decrease the incidence of many cancers.2 This effect appears to be associated with up-regulation of factors, known as phase II metabolism enzymes. Phase II metabolism enzymes, including carcinogen and oxidant detoxifying enzymes, quench carcinogens, which are activated by phase I metabolism enzymes, and inhaled endogenous oxidants. The current strategies for effective cancer chemoprevention are to develop agents to modulate the metabolism and disposition of endogenous and environmental carcinogens and oxidants through up-regulation of phase II enzymes.3,4 NQO1 [NAD(P)H quinine oxidoreductase 1], a phase II metabolism enzyme, can catalyze diverse reactions that collectively result in broad protection against electrophiles and oxidants.5–7 Many studies have revealed that elevation of NQO1 is correlated with protection against chemicalinduced carcinogenesis in animal models.8,9 Knockout of the NQO1 gene in mice shows significant increases in both carcinogen-induced and spontaneous tumorigenesis.10–13 The expression ⇑ Corresponding author. Tel.: +86 24 23986475; fax: +86 24 31509368. E-mail address: [email protected] (N. Li). http://dx.doi.org/10.1016/j.bmcl.2015.01.009 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

of NQO1 is regulated through the Keap1/Nrf2/ARE signaling pathway, which is regulated by Michael reaction acceptors (MRAs). MRAs are a class of active molecules, directly or indirectly involved in various cellular processes in cancer chemoprevention. MRAs contain olefins or acetylenes conjugated to electron-withdrawing functional groups, which confer the ability to conduct the Michael reaction with critical nucleophilic amino acids in electrophilesensitive proteins, including Keap1. It has been shown that naturally occurring phytochemicals possess anticancer properties. Among them, flavonoids found in fruits, vegetables, tea, and wine have received considerable attention in recent years.14,15 They also have high bio-availability in various tissues after being ingested, and, thus, potentially exert their biological effects efficiently. Recently, we have focused on screening potential natural cancer chemopreventive agents from natural food and herbal medicines, using GSH-conjugation experiment and cell-based (Hepa 1c1c7 cells) bioassays. We have selected a rich source of effective flavonoids, Millettia pulchra Kurz var-laxior (Dunn) Z.Wei (Yulangsan), out of 40 candidate plants. Then the bioactivity-guided identification, bioassay and molecular docking of effective constituents were carried out successively. Yulangsan, Millettia pulchra Kurz var-laxior (Dunn) Z.Wei, belongs to the Leguminosae family. The roots of this plant, also called Yulangsan or Longyanshen, are used as a folk medicine for

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postpartum women and people with certain health conditions, presumably to replenish blood to treat postpartum frail and malnutrition.16 Modern pharmacological research has revealed that Yulangsan also has properties in cardiovascular protection, antitumor, and hepatoprotective action. The aerial parts of Millettia pulchra Kurz var-laxior (Dunn) Z.Wei, called Daluosan, is another traditional herbal medicine to eliminate inflammation, alleviate pain, increase blood circulation, and relax and activate tendons in rheumatic arthralgia.17–19 NMR spectra were recorded on a Bruker ARX-600 and ARX-400 spectrometer, using TMS as an internal standard. Silica gel for chromatography was produced by Qingdao Ocean Chemical Group Co. of China. HPLC separations were performed on a Hypersil PREPODS column (5 lm, 250  20 mm) equipped with a Shimadzu SPD10A UV-detector and a Shimadzu LC-10AT series pumping system (Co., Ltd Japan). The UPLC–DAD analysis was performed on Waters system coupled with a DAD detection, conducting on a acquity Hclass BEH-C18 column (1.7 lm, 100  2.1 mm) (Waters, America). GSH was produced by Bailingwei Group Co. of China. Specific rotation were recorded on a Polarimeter (Model 341, Perkin Elmer, America). The roots of Millettia pulchra Kurz var-laxior (Dunn) Z.Wei. was collected from Guangxi province of China, in June 2012. The plant

O

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material was identified by the professor Jincai Lu. Voucher specimen (no. 20120619) was deposited in the School of Traditional Chinese Material Medica, Shenyang Pharmaceutical University. The dried roots (4.5 kg) were crushed to small pieces fitting for 10 L flask and reflux extracted with 8 L of 95% ethanol at 100 °C (2 h  3times) to give a total crude 1040 g. The extract was partitioned into ethyl acetate part (49.5 g) and n-butyl alcohol part (55.5 g) successively.20 Through the preparative reverse-phase HPLC, fr.1 yielded (E)-1(4-hydroxybenzofuran-5-yl)-3-phenylprop-2-en-1-one21 (1) (tR = 58 min, 1.4 mg), lonchocarpin22 (5) (tR = 114 min, 8.4 mg) and pongachalconel23 (6) (tR = 131 min, 1.7 mg) and eluted with 80% methanol. Purpurenone24 (7) (8.5 mg) was isolated from fr.2 through Sephedax LH-20. Pongamol25 (2) (1143.7 mg) and () isolonchocarpin26 (15) (9.7 mg) were recrystallizated from methanol and dichloromethane from fr.3. Through column chromatography on silica gel and recrystallizated obtained ovalitenin A27 (3) (86.8 mg) and (R)-ovalitenin B28,29 (4) (18.2 mg) from fr.4. Through recrystallizated (PE) obtained karanjin30(8) (3993.2 mg) from fr.5. From fr.7 200 ,200 -dimethylchromene-[500 ,600 :7,8]-flavone24 (11) (165.5 mg) and lanceolatin B28 (9) (9.6 mg) were obtained by recrystallizated (MeOH) and Sephedax LH-20. From fr.9 [2]benzopyrano[4,3-b]-furo[2,3h][1]benzopyran-6(8H)-one31 (10) (40 mg),

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Figure 1. Structures of the isolated compounds of Yulangsan.

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Figure 2. Overlay graphs of UPLC analysis before and after incubation with GSH (recorded at 210 nm).

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Table 1 NQO1-inducing activity of compounds 1–15a and 40 -bromaflavone on hepa 1c1c7 cells Sample name

C (lM)

Cell viability (%)

IRb (mean±SE)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 40 -Bromaflavonec

20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 10

90 78 81 92 84 83 77 100 100 97 99 106 79 71 114 109

1.75 ± 0.07 1.69 ± 0.17 2.01 ± 0.09 2.07 ± 0.06 1.70 ± 0.12 2.14 ± 0.04 1.90 ± 0.11 1.57 ± 0.14 1.64 ± 0.09 1.67 ± 0.12 1.60 ± 0.07 1.40 ± 0.11 2.02 ± 0.06 2.14 ± 0.04 1.54 ± 0.06 2.17 ± 0.08

a The result of compound 16 was listed in Table 1 because it was used up for structure elucidation. b Each IR value represents the means ± SE of three independent experiments. c 40 -Bromaflavone was used as positive control at 10 lM.

6-methoxy-200 ,200 -dimethylpyrano-[500 ,600 :8,7]flavone32 (12) (20.0 mg) and () (6aR,11aR) Maackiain33 (16) (3.7 mg) through recrystallizated (MeOH) and Sephdax LH-20. 5-methoxy-2,2dimethylpyrano-[5,6:8,7]flavones34 (13) (6.3 mg), 6-hydroxy-2,2dimethylpyrano-[5,6:8,7]flavones30 (14) (8.0 mg) were obtained through column chromatography on silica gel, Sephedax LH-20 and recrystallizated (MeOH) from fr.11. The structures of the isolated compounds were shown in Figure 1. Glutathione (GSH) binding property test was performed by UPLC–DAD method to detect the MRAs (Michael reaction acceptors) in the Yulangsan EtOAc extract. The components with high binding ability with GSH might be potential MRAs, which could be possible to change the space structure of Keap1 protein so as to activate the Keap1/Nrf2/ARE signaling pathway. The extract

was incubated with 5 mM GSH for 2 h at 37 °C in total volume of 200 lL of 50 mM Tris–HCl buffer (pH 8.0). The final concentration of extract was 2 mg/mL.35,36 The NQO1 activity was determined by measuring NADPH dependent menadiol mediated of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) to blue formazan. NQO1 induction activity was determined by comparing NQO1 activity of sample treated cells with that of DMSO treated cells.37–39 AutoDock4.240was used for molecular docking study. The crystal structure of NQO1 in complex with a coumarin-based inhibitor (PDB code 3JSX)41 was used. The co-crystal ligand was extracted to define active site, and polar hydrogen atoms were added to protein geometrically. The docking area was assigned around the active site with AutoDock Tools (ADT). A grid of 30 Å  30 Å  30 Å with 0.175 Å spacing was calculated around the docking area for ligand atom types using AutoGrid. Nine separate docking calculations were performed for both compounds 4 and 14, as each docking calculation consisted of 25 million energy evaluations using Lamarckian genetic algorithm local search method, with a population size of 200, and 3000 rounds of Solis and Wets local search. The docking results from each of the nine calculations were clustered on the basis of root-mean-square deviation (rmsd) and were ranked on the basis of free binding energy. The top-ranked compounds were visually inspected with Accelrys Discovery Studio Visualizer. As shown in Figure 2, some peaks greatly weakened after reacting with GSH in the UPLC chromatogram of the Yulangsan EtOAc extract. The result indicated that the constituents of the EtOAc extract might be potential Michael reaction acceptors, which could combine with Keap1 to activate Keap1/Nrf2/ARE pathway and upgrade the expression of NQO1 accordingly. So, the EtOAc extract of Yulangsan was selected as effective extract. After the phytochemical work, the peaks in the UPLC chromatogram were determined as compounds 2, 3, 4, 8, 11, 12, 13, 14, 15, respectively. All the compounds, isolated from the bioactive EtOAc extract of Yulangsan, were assayed for cytotoxicities and NQO1 inducing activities using Hepa 1c1c7 cells (Table 1). Compounds 3, 4, 6, 13, and 14 exhibited significant NQO1 inducing activities with

Figure 3. Interaction modes of compounds within NQO1 binding pocket. (A) H-bonds between compound 4 and NQO1 pocket; (B) hydrophobicity between compound 4 and pockets atoms; (C) H-bonds between compound 14 and NQO1 pocket; (D) hydrophobicity between compound 14 and pockets atoms.

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the IR >2.0 at 20 lM (IR means induction ratio, calculated as: NQO1sample/NQO1control). Compounds 1, 2, 5, 7–12, and 15 showed moderate effects with the IR 1.4–1.9 at 20 lM. All the tested samples did not display evident toxicity in Hepa 1c1c7 cells at the effective concentration (20 lM). Moreover the brief structure and activity relationship was concluded. Firstly, for chalcones (1–7) the substitution of 2-methoxyl group might improve the NQO1 inducing activity, while the b-hydroxyl group could reduce the effect. Secondly, the methoxyl group substituted at C-5 and hydroxyl group at C-6 should be beneficial to the activity of flavonones (8–14). In order to clarify the mode of action of the bioactive flavonoids on NQO1, we carried out molecular docking study to measure the relative binding energies and localize binding sites in compounds and NQO1. Based on the NQO1 inducing activity and binding energies, compounds 4 and 14 were selected as representative constituents to discuss the mode of action as shown in Figure 3. The hydrogen-bonds were observed between compounds 4, 14 and NQO1 at Gly-149, 150, Phe-106 and His-161. Phytochemicals from Yulangsan are promising candidates for cancer prevention. They possess significant NQO1 inducing activity and lower toxicity. Based on the chemical and bioactivity assays, the chemopreventive properties are suggested to be associated with ovalitenin A (3), (R)-ovalitenin B (4), pongachalconel (6), 5methoxy-2,2-dimethylpyrano-[5,6:8,7]flavones (13), and 6hydroxy-2,2-dimethylpyrano-[5,6:8,7]flavones (14). Molecular docking revealed similar binding sites of the effective compounds. Comparing the bioactivities, binding energy and binding sites of the tested compounds, it is suggested that the compounds with significant activities bind with NQO1 with lower energy. Moreover, Gly149, Gly150, Phe106, Typ105, and His161 are considered as key active residues interacting with the flavonoids. Acknowledgments The work was supported partially by National Natural Science Foundation of China (81173531, U1403102), Training Programme Foundation for the Distinguished Young Scholars of University in Liaoning Province (LJQ2012088), Fund for long-term training of young teachers in Shenyang Pharmaceutical University (ZCJJ2013409), Scientific Research Foundation for the Returned Overseas Scholars in Shenyang Pharmaceutical University (GGJJ2013103). Supplementary data Supplementary data (these data include the NMR data of all the compounds) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.01.009. References and notes 1. Gary, J. K.; Ernest, T. H.; Caroline, C. S. Cancer chemoprevention In Strategies for cancer chemoprevention; Totowa Humana press: New Jersey, 2005; Vol. 2, p 22. 2. Tan, X. L.; Shi, M.; Tang, H.; Han, W. G.; Spivack, S. D. J. Nutr. 2010, 140, 1404. 3. Hong, W. K.; Sporn, M. B. Science 1997, 278, 1073. 4. Talalay, P. Biofactors 2000, 12, 5. 5. Dinkova-Kostova, A. T.; Talalay, P. Free Radical Biol. Med. 2000, 29, 231. 6. Nioi, P.; Hayes, J. D. Mutat. Res. 2004, 555, 149. 7. Cuendet, M.; Otehan, C. P.; Moon, R. C.; Pezzuto, J. M. J. Nat. Prod. 2006, 69, 460. 8. Boone, C. W.; Steele, V. E.; Kelloff, G. J. Mutat. Res. 1992, 267, 251.

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