Seladoeflavones A–F, six novel flavonoids from Selaginella doederleinii

    Seladoeflavones A–F, six novel flavonoids from Selaginella doederleinii ZhenXing Zou, KangPing Xu, PingSheng Xu, XiaoMin Li, Fei Cheng, Jing Li, Xia Yu, DongSheng Cao, Dan Li, Wei Zeng, GuoGang Zhang, GuiShan Tan PII: DOI: Reference:

S0367-326X(16)30545-7 doi:10.1016/j.fitote.2016.11.014 FITOTE 3533

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

9 October 2016 11 November 2016 19 November 2016

Please cite this article as: ZhenXing Zou, KangPing Xu, PingSheng Xu, XiaoMin Li, Fei Cheng, Jing Li, Xia Yu, DongSheng Cao, Dan Li, Wei Zeng, GuoGang Zhang, GuiShan Tan, Seladoeflavones A–F, six novel flavonoids from Selaginella doederleinii, Fitoterapia (2016), doi:10.1016/j.fitote.2016.11.014

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ACCEPTED MANUSCRIPT Seladoeflavones A-F, six novel flavonoids from Selaginella doederleinii

ZhenXing Zou a,b, KangPing Xu b, PingSheng Xu a, XiaoMin Li a, Fei Cheng b, Jing Li b, Xia Yu b,

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DongSheng Cao b, Dan Li a, Wei Zeng a, GuoGang Zhang a*, GuiShan Tan a,b,**

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Xiangya Hospital of Central South University, Changsha 410008, PR China

School of Pharmaceutical Sciences, Central South University, Changsha 410013, PR China

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Corresponding author. E-mail address: [email protected] (G.G. Zhang)

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Corresponding author.

E-mail address: [email protected] (G.S. Tan) 1

ACCEPTED MANUSCRIPT Abstract Six new flavonoids, seladoeflavones A˗F (1˗6), were isolated from the whole herbs of Selaginella doederleinii, together with one known flavonoid (7). Their structures including absolute

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configuration were characterized on the basis of extensive spectroscopic methods including NMR, HRMS, and electronic circular dichroism (ECD). All compounds consist of an aryl substituent at

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the C-3' position of naringenin or apigenin skeletons, and compounds 1 and 6 were identified as R configurations, which are uncommonly encountered in nature. A possible biosynthetic pathway was postulated. In addition, bioassay of the isolates revealed that 5˗7 exhibited moderate

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cytotoxicity against three human cancer cell lines NCI-H460, A549, and K562 in vitro with IC50

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values ranging from 8.17 to 18.66 μM.

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Keywords: Selaginella, Selaginella doederleinii, unique flavonoids, cytotoxicity

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ACCEPTED MANUSCRIPT 1. Introduction Selaginella is the only genus in the plant family Selaginellaceae and comprises approximately 700 species widely distributed all over the world, of which approximately 70 species mainly in

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Southern China [1-2]. Selaginella doederleinii is a perennial herb that is distributed mainly in Guangxi Zhuang Autonomous Region and Yunnan province of mainland China, the whole plant

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has been used as folk medicine by local people for treatment of cancer and cardiovascular disease [3]. Previous phytochemical investigations of S. doederleinii have led to reports of biflavonoids, lignans, and alkaloids, of which some exhibit antitumor, antioxidative, cytotoxic, and hypertensive

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activities [4-11].

Selaginella is well-known rich in flavonoids. In previous work, several classes of unique

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flavonoids were isolated from some species of this genus including S. tamariscina, S. uncinata, S. involvens, and S. moellendorffii [12-21]. In our continued research on the discovery of structurally

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unique flavonoids, six novel flavonoids, seladoeflavones A-F with 3'-aryl substituent together with

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one known flavonoid, were isolated from the 75% aqueous ethanol extract of the whole herbs of S. doederleinii. In this paper, we report the isolation and structural elucidation of the novel

vitro.

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flavonoids, along with the evaluation of their cytotoxicity against three human cancer cell lines in

2. Experimental

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2.1. General experimental procedures Optical rotations were measured with a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). UV spectra were measured on a UV-2450 spectrometer (SHIMADZU, Kyoto, Japan). ECD spectra were recorded with an Applied Photophysics spectrometer (Chirascan, New Haven, USA). HRESIMS data were collected with a Finnigan LTQ-FT (Thermo Fisher, MA, USA). NMR spectra were recorded on a Bruker AV-500 MHz spectrometer and a Bruker AVHD˗800 MHz spectrometer (Bruker, Karlsruhe, Germany) with tetramethylsilane (TMS) as an internal standard. Silica gel GF-254 (Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC) and Thin Layer Chromatography (TLC). Sephadex LH-20 (TOYOPEARL TOSOH, Tokyo, Japan). The spots were visualized using UV light at 254 and 365 nm and by spraying with AlCl3-EtOH (1:99, v/v) followed by heating. Semipreparative HPLC was performed on an Agilent 1200 unit equipped with a DAD detector, utilizing a preparative YMC Pack ODS-A 3

ACCEPTED MANUSCRIPT RP-18 column (10 μm, 250×10 mm, YMC Co. Ltd., Kyoto, Japan). All solvents were analytical grade. 2.2. Plant material

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The whole herbs of S. doederleinii were collected in the town of Wutong, Lingui district, Guangxi Zhuang Autonomous Region, China, in July 2013. A botanical specimen of this species

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(20130710) was deposited at the School of Pharmaceutical Sciences, Central South University. The plant was identified by Prof. ZhenJi Li (Xiamen University, Xiamen, China). 2.3. Extraction and isolation

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Whole herbs of S. doederleinii (10.0 kg) were exhaustively extracted with 75% EtOH twice under reflux (2/60 L, 3 h/each). After removing the solvents under vacuum, the extract (150 g) was

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suspended in H2O, followed by successive partitions with petroleum ether, EtOAc and n-BuOH. The EtOAc fraction (90 g) was applied subsequently to silica gel CC and eluted with

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CH2Cl2˗MeOH˗H2O (from 1:0:0 to 0:1:1) to obtain eight fractions (Fr. 1˗8). Applying Fr. 6 (16.8 g)

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to Sephadex LH˗20 CC and eluted with H2O˗MeOH (from 40:60 to 0:100) to yield nine subfractions (Fr. A˗I). Fr. C was directly purified by semipreparative RP˗HPLC using a mobile

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phase of ACN˗H2O (40:60, v/v) to yield compounds 1 (3.5 mg, tR = 16.6 min) and 2 (7.9 mg, tR = 20.2 min). Fr. D was performed on Sephadex LH-20 (MeOH/H2O in gradient) and semi-preparative HPLC (YMC-Pack ODS-A; 250*10 mm 10 m; 3 mL/min; 50% ACN/H2O)

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sequentially, compounds 3 (7.5 mg, tR = 14.4 min), 4 (7.2 mg, tR = 16.7 min), and 7 (14.0 mg, tR = 20.7 min) were obtained. Compounds 6 (4.8 mg, tR = 23.5 min) and 5 (6.1 mg, tR = 29.3 min) were obtained from Fr. F by repeated semipreparative RP˗HPLC [ACN˗H2O (65:35, v/v)]. 2.3.1. Seladoeflavone A (1) Yellow amorphous powder; [α]25D –11.4 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 290 (6.29), 330 (6.15) nm; CD (MeOH, c 0.57 mM) λmax (Δ ε): 289 (+0.40), 330 (–0.33) nm; 1H NMR (800 MHz in DMSO-d6) and

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C NMR (200 MHz in DMSO-d6) data are shown in Tables 1 and 2;

HRESIMS m/z 433.1272 [M + H]+, calculated for C25H21O7, 433.1282. 2.3.2. Seladoeflavone B (2) Yellow amorphous powder; HPLC-UV (ACN-H2O) λmax: 265 nm, 330 nm; 1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6) data are shown in Tables 1 and 2; HRESIMS m/z 431.1123 [M + H]+, calculated for C25H19O7, 431.1125. 4

ACCEPTED MANUSCRIPT 2.3.3. Seladoeflavone C (3) Light yellow amorphous powder; [α]25D –15.7 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 287 (5.13), 330 (4.59) nm; CD (MeOH, c 0.13 mM) λmax (Δ ε): 286 (–6.33), 330 (+1.37) nm; 1H NMR

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HRESIMS m/z 393.0966 [M + H]+, calculated for C22H17O7, 393.0969.

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(500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6) data are shown in Tables 1 and 2;

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2.3.4. Seladoeflavone D (4)

Yellow amorphous powder; HPLC-UV (ACN-H2O) λmax: 266 nm, 295 nm, 340 nm; 1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6) data are shown in Tables 1 and 2;

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HRESIMS m/z 391.0823 [M + H]+, calculated for C22H15O7, 391.0821. 2.3.5. Seladoeflavone E (5)

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Yellow amorphous powder; [α]25D –17.2 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 288 (6.12), 330 (5.51) nm; CD (MeOH, c 0.12 mM) λmax (Δ ε): 287 (–2.16), 329 (+0.62) nm; 1H NMR (500 13

C NMR (125 MHz in DMSO-d6) data are shown in Tables 1 and 2;

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HRESIMS m/z 409.0919 [M + H]+, calculated for C22H17O8, 409.0918. 2.3.6. Seladoeflavone F (6)

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Light yellow amorphous powder; [α]25D –8.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 286 (5.83), 330 (5.06) nm; CD (MeOH, c 0.14 mM) λmax (Δ ε): 293 (+0.59), 330 (–0.44) nm; 1H NMR (500 MHz in DMSO-d6) and 13C NMR (125 MHz in DMSO-d6) data are shown in Tables 1 and 2;

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HRESIMS m/z 365.1018 [M + H]+, calculated for C22H17O6, 365.1020. 2.3.7. 3'-phenol-apigenin (7) Yellow amorphous powder; HPLC-UV (ACN-H2O) λmax: 266 nm, 295 nm, 340 nm; 1H NMR (500 MHz, DMSO-d6) δH: 13.00 (1H, s, 5-OH), 10.84 (1H, s, 7-OH), 10.41 (1H, s, 4'-OH), 9.51 (1H, s, 4"-OH), 7.88 (1H, d, J = 2.0, H-2'), 7.85 (1H, dd, J = 8.5, 2.0, H-6'), 7.46 (2H, d, J = 8.5, H-2"/6"), 7.07 (1H, d, J = 8.5, H-5'), 6.88 (1H, s, H-3), 6.82 (2H, d, J = 8.5, H-3"/5"), 6.50 (1H, d, J = 2.0, H-8), 6.20 (1H, d, J = 2.0, H-6); 13C NMR (125 MHz, DMSO-d6) δC: 181.9 (C-4), 164.2 (C-7), 164.0 (C-2), 161.6 (C-5), 158.1 (C-4'), 157.5 (C-4"), 156.8 (C-9), 130.5 (C-2"/6"), 128.8 (C-3'), 128.5 (C-2'), 128.1 (C-1"), 126.6 (C-6'), 121.8 (C-1'), 116.6 (C-5'), 115.0 (C-3"/5"), 103.9 (C-10), 103.3 (C-3), 99.0 (C-6), 94.1 (C-8). 2.4. Cytotoxicity test Three human cancer cell lines NCI-H460, A549, and K562 were used in the cytotoxicity assay. 5

ACCEPTED MANUSCRIPT All cells were cultured in DMEM medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) in a 5% CO2 atmosphere at 37 °C. The cytotoxicity assay was performed using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method in 96-well

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microplates. Then, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just

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before drug addition with an initial density of 5 × 104 cells/mL. Each cancer cell was exposed to a test compound at concentrations of 0.3, 1, 3, 10, 30, 100 μM in DMSO in triplicate for 72 h, with cisplatin (DDP) (Qilu pharmaceutical, China) as a positive control. The OD value of each well

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was measured at a microplate spectrophotometer. The IC50 value of each compound was calculated by the Reed and Muench method [22].

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3. Results and Discussion

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3.1. Chemistry

Fig. 1 Structures of compounds 1-7.

Compound 1 was obtained as a yellow amorphous powder. The molecular formula was suggested as C25H21O7 by the positive HR-ESI-MS at m/z 433.1272 [M + H]+ (calcd. 433.1282). The UV spectrum showed the Ⅰ band of 330 nm and Ⅱ band of 290 nm, and the positive result 6

ACCEPTED MANUSCRIPT of the spay reagent of AlCl3, implying a flavone-like compound [23]. The 1H NMR spectrum of 1 (Table 1) showed signals for two sets of 1, 3, 4-trisubstituted aromatic protons [δH 7.52 (1H, dd, J = 10.4, 2.4 Hz, H-6'), δH 7.49 (1H, d, J = 2.4 Hz, H-2') and δH 6.88 (1H, d, J = 10.4 Hz, H-5') (ring

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B)], [δH 7.31 (1H, m, H-2''), δH 7.30 (1H, d, J = 1.6 Hz, H-6'') and δH 6.89 (1H, d, J = 8.8 Hz, H-5'')

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(ring D)], a set of meta-coupled aromatic signals at δH 5.90 (1H, d, J = 2.4 Hz, H-8) and δH 5.89

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(1H, d, J = 2.4 Hz, H-6), two olefinic protons [δH 7.57 (1H, d, J = 16.0 Hz, H-7") and δH 6.60 (1H, d, J = 16.0 Hz, H-8")], as well as three aliphatic protons including an oxygenated methyne signal at δH 5.48 (1H, dd, J = 12.8, 3.2 Hz, H-2), a methylene signal [δH 3.31 (1H, m, H-3ax), 2.71 (1H,

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dd, J = 17.6, 3.2 Hz, H-3eq)], and an acetylated methyl signal at δH 2.28 (3H, s, H-10"). These signals are characteristic of naringenin derivatives [24]. The

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C NMR spectrum (Table 2)

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confirmed the presence of 25 carbon resonances, in addition to a naringenin skeleton (15 carbons), one keto-carbonyl, six aromatic, two olefinic, and one methyl carbons were observed. HMBC

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correlations were used to establish the structure of the side chains. Correlations between H-2' (δH

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7.49) to C-1" (δC 126.1) and H-2"/6" (δH 7.30, 7.31) to C-3' (δC 127.2) confirmed the presence of the C-3'-C-1"-linked in rings B and D. The trans double-bond was located at C-7"/C-8", which

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was further supported by the key HMBC correlations between H-8" (δH 6.60) to C-5" (δC 124.5) and H-7" (δH 7.57) to C-9" (δC 198.2) (Fig. 2). The ECD spectrum of 1 showed a negative Cotton effect due to the n → π* transition around 330 nm and a positive Cotton effect in the π → π*

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region around 290 nm (Fig. 3), indicating an R configuration [25], which is rare in nature. Hence, the structure of 1 was determined, as depicted, and named seladoeflavone A. Compound 2 was isolated as a yellow amorphous powder. Its HR-ESI-MS exhibited a hydrogen adduct ion peak at m/z 431.1123 [M + H]+ (calcd. for C25H19O7, 431.1125), corresponding to the molecular formula C25H18O7. The 1H NMR and 13C NMR data (Tables 1 and 2) of 2 were similar to those of 1, the main differences between the two compounds included the oxidation of two sp3 carbons in ring C of 2 to a double bond (δC 164.3, C˗2; δC 103.4, C˗3) and the characteristic resonance of a conjugated carbonyl at δC 182.1 (C˗4). Thus, the structure of 2 was established as (E)-2-(4', 6- dihydroxy-3'-(3-oxobut-1-en-1-yl)-[1, 1'-biphenyl]-3-yl)-5, 7-dihydroxy-4H-chromen -4-one, and named seladoeflavone B.

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Fig. 2 1H-1H COSY (H─H), key HMBC (H→C) correlations of compounds 1-6.

Fig. 3 ECD spectra of compounds 1, 3, 5 and 6.

Compound 3 was isolated as a light yellow amorphous powder. Its molecular formula was suggested as C22H17O7 by the positive HR-ESI-MS at m/z 393.0966 [M + H]+ (calcd. 393.0969). Comparing the NMR data (Tables 1 and 2) of 3 with those of 1, it was found that one more aldehyde group was observed in 3. HMBC correlations between H-7" (δH 9.82) to C-5" (δC 128.3) and H-6" (δH 7.71) to C-7" (δC 191.3) confirmed the aldehyde group was attached to C-5" of ring D (Fig. 2). The absolute configuration of 3 was determined as S, which was confirmed by the positive Cotton effect at approximately 330 nm and negative Cotton effect at approximately 290 nm in ECD spectrum (Fig. 3). Consequently, the structure of 3 was elucidated as (S)-5'-(5, 7-dihydroxy-4-oxochroman-2-yl)-2', 4-dihydroxy-[1, 1'-biphenyl]-3-carbaldehyde, and named seladoeflavone C. 8

ACCEPTED MANUSCRIPT Compound 4 was obtained as a yellow amorphous powder. The HR-ESI-MS data of 4 supported a molecular formula of C22H15O7 for molecular ion cluster at m/z 391.0823 [M + H]+ (calcd. 391.0821). The 1H NMR and 13C NMR data (Tables 1 and 2) of 4 were quite similar to those of 1

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except for two sp3 carbons in ring C were oxidized to a double bond (δC 164.3, C˗2; δC 103.4, C˗3),

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and the characteristic resonance of a conjugated carbonyl at δC 182.2 (C˗4). Therefore, the

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structure of 4 was identified as shown, and named seladoeflavone D.

Compound 5 was obtained as a yellow amorphous powder. Its HR-ESI-MS indicated a pseudo molecular ion peak at m/z 409.0919 [M + H]+ (calcd. 409.0918) giving the molecular formula

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C22H17O8. Further analysis of the 1H NMR and 13C NMR data (Tables 1 and 2) of 5 indicated that were similar to those of 3. The NMR data of 5 showed the absence of an aldehyde group, and the

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present of an additional carboxyl group was attached to C-5" (δC 138.8) of ring D, as deduced by key HMBC correlations from H-6" (δH 7.33) to C-7" (δC 167.4) (Fig. 2). The ECD spectrum of 5

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showed a negative Cotton effect and a positive Cotton effect around 290 nm and 330 nm,

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respectively, (Fig. 3), indicating an S configuration [25]. Thus, compound 5 was identified as (S)-5'-(5, 7-dihydroxy-4-oxochroman-2-yl)-2', 4-dihydroxy-[1, 1'-biphenyl]-3-carboxylic acid, and

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named seladoeflavone E.

Compound 6 was obtained in the form of a light yellow amorphous powder. Its molecular formula of C22H17O6 was determined by the HR-ESI-MS ion peak at m/z 365.1018 [M + H]+

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(calcd. 365.1020). The 1H NMR and 13C NMR data (Tables 1 and 2) of 6 are very similar to those of 5 except for absence of a carboxyl group attached to C-5". Comparing the ECD spectra confirmed that 6 and 1 had the same stereochemistry (Fig. 3) [25]. Consequently, compound 6 was identified as (R)-2-(4', 6-dihydroxy-[1, 1'-biphenyl]-3-yl)-5, 7-dihydroxychroman-4-one, and named seladoeflavone F. A known flavonoid named 3'-phenol apigenin (7) was also identified on the basis of a spectroscopic experiment and a comparison of its spectroscopic data with those in the literature [18]. A tentative biosynthetic pathway for compounds 1-7 was proposed as shown in Scheme 1. These compounds cloud be plausibly traced back to (±)-naringenin or apigenin [26, 27], which are coupled [28] with a polyketide product compound 8 [29] to give compounds 1 or 2, respectively. Enzymatic double-bond cleavage on 1 or 2 results in the corresponding aldehydes 3 or 4 [30], 9

ACCEPTED MANUSCRIPT followed by the formation of 5 through an aldehyde oxidation [31]. Finally, the decarboxylation

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was performed to yield compounds 6 and 7 [32].

Scheme 1. Putative biosynthetic pathway of compounds 1-7.

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3.2. Cytotoxicity assay

All compounds were obtained in sufficient amounts to be evaluated for their cytotoxicity activity against human lung cancer (NCI-H460 and A549) and human myeloid leukemia (K562)

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cell lines. Of the substances tested, only compounds 5˗7 exhibited moderate cytotoxicity against

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3.3. Conclusions

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three human cancer cell lines, with IC50 values in the range 8.17˗18.66 μM (Table 3).

The phytochemical investigation of the S. doederleinii extract yielded six new flavonoids (1˗6); together with one previously known flavonoid (7). All the isolated flavonoids were biologically

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evaluated their cytotoxicity activity against three human cancer cell lines. The findings could enrich the flavonoids diversity and be regarded as some further insight into the chemotaxonomic diversity of natural products in Selaginella.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This research was financially supported by the National Natural Science Foundation of China (No. 31370370), Key Project of Application Technology Research and Development of Haikou (No. 2015-039), Project of Strategic Emerging Industry Technology Research and Major Scientific and Technological Achievements of Hunan (No. 2015GK1053), Key Project of Science and Technology of Hunan (No. 2014SK2002, No. 2013SK5077), and Innovation Project for Graduate Students of Hunan (No. CX2013B100). We are also very grateful to Dr. BangShao Yin (College of 10

ACCEPTED MANUSCRIPT Chemistry and Chemical Engineering in Hunan Normal University), Prof. GuoGen Liu (Modern Analysis and Testing Centre in Central South University), and Prof. Zheng Li (High Resolution Mass Spectrometry Laboratory of Advanced Research Center in Central South University) for the

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NMR and HRESIMS measurement.

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macrocyclic biflavone from Selaginella uncinata (Desv.) Spring, Tetrahedron Lett. 57 (2016) 892˗894.

[20] H. Zou, M.L. Yi, K.P. Xu, X.F. Sheng, G.S. Tan. Two new flavonoids from Selaginella uncinata, J. Asian Nat. Prod. Res. 18 (2016) 248˗252. [21] Z.X. Zou, P.S. Xu, C.R. Wu, W.X. Zhu, G.Z. Zhu, X.A. He, et al., Carboxymethyl flavonoids and a chromone with antimicrobial activity from Selaginella moellendorffii Hieron, Fitoterapia 111 (2016) 124˗129. [22] L.J. Reed, H. Muench. A simple method of estimating fifty percent endpoints, Am. J. Hyg. 27 (1938) 493˗497. [23] T.J. Mabry, K.R. Markham, M.B. Thomas, The ultraviolet spectra of flavones and flavonols, Springer, Berlin Heidelberg, 1970 44˗71. [24] E. Giorgio, N. Parrinello, S. Caccamese, C. Rosini, Non-empirical assignment of the absolute 12

ACCEPTED MANUSCRIPT configuration of (-)-naringenin, by coupling the exciton analysis of the circular dichroism spectrum and the ab initio calculation of the optical rotatory power, Org. Biomol. Chem. 2 (2004) 3602˗3607.

IP

T

[25] D. Slade, D. Ferreira, J.P.J. Marais, Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids, Phytochemistry 66 (2005) 2177˗2215.

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[26] M.L.F. Ferreyra, S.P. Rius, P. Casati, Flavonoids: biosynthesis, biological functions, and biotechnological applications, Front. Plant Sci. 3 (2012) 279˗286.

[27] J. Mcnulty, J.J. Nair, E. Bollareddy, K. Keskar, A. Thorat, D.J. Crankshaw, et al., Isolation of

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flavonoids from the heartwood and resin of Prunus avium, and some preliminary biological investigations, Phytochemistry, 70 (2009) 2040˗2046.

MA

[28] P. Belin, M.H. Le Du, A. Fielding, O. Lequin, M. Jacquet, J.B. Charbonnier, et al., Identification and structural basis of the reaction catalyzed by CYP121, an essential

D

cytochrome P450 in Mycobacterium tuberculosis, P. Natl. Acad. Sci. USA. 106 (2009)

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7426˗7431.

[29] I.J. Flores Sanchez, R. Verpoorte, Plant polyketide synthases: a fascinating group of enzymes,

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Plant Physiol. Bioch. 47 (2009) 167˗174. [30] R. Aashrita, L. Miguel, K. Wolfgang, Oxidative alkene cleavage by chemical and enzymatic methods, Adv. Synth. Catal. 45 (2014) 3321˗3335.

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[31] P. Ferreira, A. Hernándezortega, B. Herguedas, J. Rencoret, A. Gutiérrez, M.J. Martínez, et al., Kinetic and chemical characterization of aldehyde oxidation by fungal aryl-alcohol oxidase, Biochem. J. 425 (2009) 585˗593. [32] Y.X. Ren, S. Yang, Q.P. Yuan, X.X. Sun, Microbial production of phenol via salicylate decarboxylation, Rsc Adv. 5 (2015) 92685˗92689.

13

ACCEPTED MANUSCRIPT Table 1 1

H NMR data for compounds 1-6. 1a

2b

3b

4b

5b

6b

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

5.47 (dd, 13.0,

5.48 (dd, 13.0,

3.0) 2.70 (dd, 17.0,

2.71 (dd, 17.6, 3

3.2)

3.0)

6.82 (s)

3.33 (dd, 17.0,

3.31 (m)

3.0)

5.89 (d, 2.4)

6.19 (d, 2.0)

5.89 (d, 2.0)

8

5.90 (d, 2.4)

6.48 (d, 2.0)

5.90 (d, 2.0)

2'

7.49 (d, 2.4)

7.85 (d, 2.5)

7.31 (d, 2.0)

5'

6.88 (d, 10.4)

7.00 (d, 8.5)

7.52

7.90

(dd, 10.4, 2.4)

(dd, 8.5, 2.5)

3.0)

2.70 (dd, 17.0,

6.03 (s)

3.0)

3.31 (d, 13.0)

3.0) 2.73 (dd, 17.0, 3.0) 3.33 (dd, 17.0, 3.0)

6.19 (d, 2.0)

5.88 (d, 2.0)

5.89 (d, 2.0)

6.49 (d, 2.0)

5.90 (d, 2.0)

5.90 (d, 2.0)

7.90 (d, 2.5)

7.28 (d, 2.0)

7.34 (d, 2.0)

6.95 (d, 8.5)

7.04 (d, 8.5)

6.94 (d, 8.5)

6.94 (d, 8.5)

7.35

7.93

7.32

7.24

(dd, 8.5, 2.0)

(dd, 8.5, 2.5)

(dd, 8.5, 2.0)

(dd, 8.5, 2.0)

7.56

7.74

7.76

7.75

(dd, 8.5, 2.0)

(dd, 8.5, 2.0)

(dd, 8.5, 2.0)

(dd, 8.5, 2.0)

MA

NU

6

6'

-

T

3.2)

5.48 (dd, 13.0,

-

IP

5.48 (dd, 12.8,

2

SC R

Positon

7.31 (m)

3"

6.89 (d, 8.8)

6.92 (d, 8.5)

7.07 (d, 8.0)

7.05 (d, 8.5)

6.96 (d, 8.5)

6.80 (d, 8.5)

5"

-

-

-

-

-

6.80 (d, 8.5)

6"

7.30 (d, 1.6)

7.57 (d, 2.0)

7.71 (d, 2.0)

7.78 (d, 2.0)

7.33 (d, 2.0)

7.39 (d, 8.5)

7"

7.57 (d, 16.0)

7.60 (d, 16.5)

9.82 (s)

9.84 (s)

12.43 (br.s)

-

8"

6.60 (d, 16.0)

6.65 (d, 16.5)

-

-

-

-

10"

2.28 (s)

2.29 (s)

-

-

5-OH

12.17 (s)

13.03 (s)

12.17 (s)

13.01 (s)

12.16 (s)

12.16 (s)

7-OH

10.81 (br.s)

10.80 (br.s)

10.82 (br.s)

10.82 (br.s)

10.83 (br.s)

10.81 (br.s)

4'-OH

10.11 (br.s)

-

10.54 (br.s)

10.82 (br.s)

10.13 (s)

9.62 (br.s)

-

9.60 (br.s)

-

9.53 (s)

-

TE

CE P

-

AC

4"-OH

D

2"

a

Measured in DMSO-d6 at 800 MHz.

b

Measured in DMSO-d6 at 500 MHz.

14

7.39 (d, 8.5)

-

ACCEPTED MANUSCRIPT Table 2 13

2b

3b

4b

5b

6b

δC

δC

δC

δC

δC

δC

2

79.1

164.3

78.7

164.3

78.7

78.7

3

42.5

103.4

42.2

103.4

42.2

42.3

4

197.0

182.1

196.6

182.2

196.6

5

163.9

161.8

163.7

161.9

163.6

163.7

6

96.2

99.3

96.0

7

167.1

164.7

166.8

8

95.4

94.4

95.2

9

163.5

157.9

163.2

10

102.2

104.3

102.0

1'

128.7

121.6

128.9

2'

132.8

130.1

130.6

3'

127.2

127.9

124.7

4'

162.1

160.0

5'

116.4

117.1

6'

129.3

127.6

1"

126.1

126.5

2"

127.5

129.6

3"

116.4

4"

IP

99.3

95.9

96.0

164.6

166.8

166.8

94.5

95.1

95.2

157.8

163.1

163.1

104.2

102.0

102.0

121.1

128.7

129.5

130.1

130.3

129.0

128.2

125.0

128.0

155.4

160.9

155.3

154.7

115.7

117.0

115.6

116.1

127.7

127.9

127.4

126.4

126.4

126.3

121.2

129.0

130.4

130.9

133.4

130.3

117.1

116.2

117.0

115.7

115.0

155.8

156.0

161.1

162.5

159.2

156.5

5"

124.5

124.6

128.3

126.2

125.5

-

6"

130.5

132.8

134.2

134.7

130.2

-

7"

144.5

144.3

191.3

191.5

167.4

-

8"

123.9

124.2

-

-

-

-

9"

198.2

198.1

-

-

-

-

27.6

-

-

-

-

D

TE

CE P

AC

10"

MA

196.6

SC R

Positon

T

1a

NU

C NMR data for compounds 1-6.

27.6

a

Measured in DMSO-d6 at 200 MHz.

b

Measured in DMSO-d6 at 125 MHz.

Table 3 Cytotoxicity activity (IC50, μM) of selected compounds for cancer cell lines. Compound

NCI-H460

A549

K562

5

12.60

17.21

8.17

6

13.22

14.31

10.64

7

18.66

15.45

14.76

DDP

0.09

0.38

>50

15

ACCEPTED MANUSCRIPT Graphical Abstract

T

Seladoeflavones A-F, six novel flavonoids from Selaginella doederleinii

IP

ZhenXing Zou a,b, KangPing Xu b, PingSheng Xu a, XiaoMin Li a, Fei Cheng b, Jing Li b, Xia Yu b,

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DongSheng Cao b, Dan Li a, Wei Zeng a, GuoGang Zhang a*, GuiShan Tan a,b,**

a

Xiangya Hospital of Central South University, Changsha 410008, PR China School of Pharmaceutical Sciences, Central South University, Changsha 410013, PR China

AC

CE P

TE

D

MA

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b

*

Corresponding author. E-mail address: [email protected] (G.G. Zhang)

**

Corresponding author.

E-mail address: [email protected] (G.S. Tan) 16