Chemical Constituents from Daphne Tangutica and Their Cytotoxicity Against Nasopharyngeal Carcinoma Cells T

Fitoterapia 130 (2018) 105–111

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Fitoterapia

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Chemical constituents from Daphne tangutica and their cytotoxicity against nasopharyngeal carcinoma cells T

Zhi-Yong Yin, Yan-Fang Cheng, Jun-Kang Wei, Xiang-Kun Luo, Pan Luo, Shao-Nan Liu, Jun Xu, ⁎ ⁎ Hao Chen , Qiong Gu

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People's Republic of China

ARTICLE INFO ABSTRACT

Keywords: Two new sesquiterpenoids (1−2), together with 30 known compounds including one sesquiterpenoid (3), six Daphne tangutica diterpenoids (4−9), fourteen lignans (10−23), and nine other kinds of compounds (24−32), were isolated Sesquiterpenoids from the stems of Daphne tangutica Maxim. Their structures were determined through extensive spectroscopic Nasopharyngeal carcinoma analyses, and the absolute conﬁguration of daphnoid A (1) and B (2) were determined by the experimental and Cytotoxicity calculated electron circular dichroism (ECD) spectra. All the isolates were evaluated against two human nasopharyngeal carcinoma cells (HONE-1 and SUNE-1). Compound 25 (daphnenone) showed potent cytotoxicity

toward HONE-1 and SUNE-1with IC50 values of 2.23 and 1.43 μM, respectively. Further studies indicated that compound 25 exhibited cytotoxic eﬀects by inducing tumor cell apoptosis and arresting the cell cycle at G2/M phases in HONE-1 cells.

1. Introduction and a highly metastatic human nasopharyngeal carcinoma (SUNE-1) cell line. Daphne genus (Thymelaeaceae) comprises of almost 90 species, of which 44 species are distributed in West of China. Besides being grown for 2. Results and discussion ornamental purposes, D. tangutica is traditional used for the treatment of rheumatoid arthritis and apoplexia [1,2]. Previous phytochemical work Chromatographic separation of the EtOAc extract from the stems of resulted in the isolation of sesquiterpenoids, diterpenoids, flavonoids, D. tangutica yielded two new guaiane-type sesquiterpenoids (1–2)and30 coumarins, and lignans [2–5]. These secondary metabolites have been known compounds (3−32), anhydrogeigerin (3)[11], daphnetoxin (4) reported having a broad range of biological activity [6], including anti- [12], 9,13,14-ortho-(2,4,6-decatrienoate) of 5β-hydroxyresiniferonol- tumor [7], anti-HIV [4], and anti-inflammatory activity [5]. 6α,7α-oxide (5)[13], excoecariatoxin (6)[13], gniditrin (7)[14], Nasopharyngeal carcinoma (NPC) is the most common malignant yuanhuacin (8)[15], yuanhuajine (9)[16], (8S,9S,8′S)-4,4′-dihydroxy- tumor of thenasopharynx. Geographically, Southeast Asia, Southern 3,3′,9-trimetoxy-9,9′-epoxylignan (10)[17], (8S,9R,8′S)-4,4′-dihydroxy- China, and North African countries have the highest prevalence of NPC. 3,3′,9-trimetoxy-9,9′-epoxylignan (11)[17], (7′S,8′R,8R)-isolariciresinol There are 80,000 new cases per year, making NPC the 23rd most (12)[18], secoisolariciresinol-9,9′-acetonide (13)[19], matairesinol (14) common of all new cancers worldwide [8]. Although cisplatin-based [20], (−)-nortrachelogenin (15)[21], 3,3′-bis(3,4-dihydro-4-hydroxy-6- concurrent chemoradiotherapy is currently considered to be the stan- methoxy-2H-1-benzopyran) (16)[22], acuminatin (17)[23], 4,4′-dihy- dard treatment regimen for patients with advanced NPC. Recently, it droxy-3,3′-dimethoxy-9-ethoxy-9,9′-epoxylignan (18)[24], daphnetone was reported that cisplatin does not improve survival after concurrent (19 )[25], pluviatolide (20)[26], piperitol (21)[27], isocubebin (22) chemoradiotherapy in patients [9]. It is necessary to develop novel [24], epipinoresinol (23)[28], daphneolone (24)[25], daphnenone (25) agents for the treatment of NPC. Natural products play a key role in [29], daphneone (26)[29], S-(+)-1-(4-hydroxy-3-methoxyphenyl)-3- looking for lead compounds [10]. Herein, we report the isolation of 32 hydroxy-5-phenyl-1-pentanone (27)[30], threo-1,5-diphenylpentane- compounds from the stems of D. tangutica and elucidation of their 1,3-diol (28)[31], (−)-(2S)-sakuranetin (29)[32], daphnetin (30)[33], structures, as well as evaluation of their cytotoxicity toward a poorly aurantiamide acetate (31)[34], and syringaldehyde (32)[35] Fig. 1. differentiated human nasopharyngeal carcinoma (HONE-1) cell line Compound 1 (daphnoid A) was isolated as yellow oil. Its molecular

⁎ Corresponding authors. E-mail addresses: [email protected] (H. Chen), [email protected] (Q. Gu). https://doi.org/10.1016/j.ﬁtote.2018.08.012 Received 13 July 2018; Received in revised form 22 August 2018; Accepted 22 August 2018 Available online 24 August 2018 0367-326X/ © 2018 Published by Elsevier B.V. Z.-Y. Yin et al. Fitoterapia 130 (2018) 105–111

Fig. 1. Chemical structures of compounds 1−32 isolated from the stems of D. tangutica.

15 carbon signals, including one aldehyde carbonyl group (δC 207.3), two ketone carbonyl groups (δC 204.8 and 215.0), two double-bond carbons (δC 142.6 and 175.0), one hydroxylated tertiary carbon (δC 81.3), two quaternary carbons (δC 42.6 and 60.6), one methine (δC 34.5), three methylenes (δC 36.3, 45.2, and 50.7), and three methyl groups (δC 9.0, 15.0, and 18.8). The above mentioned revealed a modiﬁed guaiane-type sesquiterpenoid [3]. The NMR data of compound 1 were similar to those of the known compound wiksphyllamin

A[36], except for the presence of an olefinic bond (δC 142.6, C-1; 175.0, C-5) instead of two methines at C-1 (δC 60.1) and C-5 (δC 36.7) in wiksphyllamin A. The planar structure of compound 1 was further confirmed through HMBC correlations from H-3, H-4, H-6, H-9, and Fig. 2. Key 1H−1H COSY (), HMBC () correlations of compound 1. H3–14 to C-1 (δC 142.6), and from H-3, H-4, H-6, and H3–15 to C-5 (δC 175.0). Thus, the planar structure of compound 1 was determined as formula was established as C15H18O4 based on HREIMS ion at m/z shown in Fig. 2. 262.1201 [M]+ (calcd 262.1200). The IR spectrum showed absorption Biogenetically, compound 1 had the same stereo centers at C-4, C-7, − − bands for hydroxy (3401 cm 1), conjugated carbonyl (1701 cm 1, and C-10, and C-11 as those of wiksphyllamin A. Hence, the absolute con- − formyl (1757, 2766 cm 1) groups. In the 1H NMR spectrum, a singlet figuration of compound 1 was assigned as 4S,7S,10R, and 11S, which δ proton appeared at H 9.76, and this signal exhibited a correlation with was further confirmed by comparing the experimental and calculated δ a carbonyl group signal at C 207.3 in the HSQC spectrum, which im- electron circular dichroism (ECD) spectra (Fig. 3). plied the presence of a formyl group. The 13C NMR spectrum displayed Compound 2(daphnoid B) was obtained as a yellow oil. Its molecular

106 Z.-Y. Yin et al. Fitoterapia 130 (2018) 105–111

Fig. 3. ECD spectra for compounds 1 (left) and 2 (right).

+ formula was assigned as C18H26O5 based on the [M + Na] ion peak at had a rare distorted guaiane skeleton. Among of them, six compounds m/z 345.16723 (calcd 345.16725) in the HRESIMS. The IR spectrum of (4, 6, 7, 8, 9, and 25, Table 2) showed cytotoxic activity against two − compound 2 exhibited characteristic absorptions of hydroxy (3394 cm 1), human nasopharyngeal carcinoma cells HONE-1 and SUNE-1 −1 −1 1 carbonyl (1739 cm ,andolefinic bond (1644 cm groups. The HNMR (IC50 <20μM). Specially, compound 25 (IC50 = 2.23 μM) exhibited spectrum displayed four methyl groups at δH 0.95, 1.68, 2.02, and 3.24, better activity than the positive control cisplatin (IC50 = 5.16 μM) in 13 and two olefinic protons at δH 4.86 and 5.16. The CNMRdataof HONE-1 cells. Compounds 24–28 possess C6-C5-C6 scaffold. In com- compound 2 showed 18 carbon signals including four olefinic bond car- parison to 24 and 26–28, compound 25, having an unsaturated ketone bons (δC 103.1, 122.3, 141.9, and 154.4), one carbonyl carbon (δC 171.0), moiety, showed the most potent cytotoxic activity. These results in- 3 two oxygenated sp carbons (δC 81.5 and 103.6), one oxygenated tertiary dicated the unsaturated ketone moiety plays a key role for cytotoxicity. carbon (δC 78.7), two methines (δC 39.4 and 41.2), four methylenes (δC Annexin V-FITC/PI analysis showed compound 25 significantly induced 34.8, 37.1, 37.1, and 67.7), one methoxy carbon (δC 48.1), and three apoptosis in HONE-1 cells (Fig. 5). Furthermore, compound 25 inter- methyl carbons (δC 10.4, 21.5, and 22.4). The NMR data of compound 2 fered with cell cycle progression and led to an increasing number of were comparable to those of auranticanol F, isolated from the stems of cells in the G2/M region in HONE-1 cells (Fig. 6). These findings in- Daphne aurantiaca [4,37]. Comparison of the 1D and 2D NMR data of dicated that compound 25 (daphnenone) may be a promising lead compound 2 with those of auranticanol F revealed major changes at C-3 compound for development of anti-NPC drug. and C-8. And the chemical shift of C-3 at δC 33.6 being downfield to δC 78.7 in compound 2.The13C NMR data of compound 2 also revealed an 3. Experimental section acetoxy group (δC 21.5 and 171.0), which was connected to C-3 based on the HMBC correlation from H-3 (δH 5.15, 1H, m) to 171.0 (carbon of 3.1. General experimental procedures acetoxyl). The methoxy group (δC 48.1) substituted at C-8 was deduced from the HMBC correlation of the methoxy group hydrogen (δH 3.24, 3H, Optical rotations data were obtained with the Anton Paar MCP 200 s) with C-8 (δC 103.6) (Fig. 4). automatic polarimeter. UV spectra were recorded using a Shimadzu UV- The NOESY cross peaks of H-3/H3–15 and H-5/H3–15 suggested 2450 spectrophotometer. ECD spectra were acquired on an Applied that CH3–15, H-3, and H-5 are co-facial and assigned as β-oriented. The Photophysics Chirascan spectrometer. IR spectra were determined from NOESY cross peaks of H3–15/H-6β (δH 1.73, 1H, m), H-6α (δH 1.43, 1H, KBr pellets on a Fourier transformation infrared spectrometer coupled 1 13 m)/H -13α (δH 4.42, 1H, m), and H-13β (δH 4.29, 1H, m)/H3-OMe (δH with infrared microscope. The H (400 MHz), C (100 MHz), and 2D NMR 3.24, 3H) indicated that H3-OMe was β-oriented. The β-orientation of 7- spectra were obtained on a Bruker AM-400 NMR spectrometer with TMS OH was determined by ECD spectra (Fig. 3). Thus, the absolute con- as an internal reference. HREIMS were measured on a Thermo MAT95XP figuration of compound 2 was defined as 3R,4R,5R,7R, and 8S. high-resolution mass spectrometer and EIMS on a Thermo DSQEIMS Biosynthetic pathways for compounds 1 and 2 were proposed in spectrometer. HRESIMS were acquired on a Shimadzu LCMS-IT-TOF, and Scheme 1. Since the guaiane-type sesquiterpenoids are the main ses- the ESIMS data were measured on an Agilent 1200 series LC-MS/MS quiterpenoids of Daphne genus [4], the precursor of compounds 1 and 2 system. RP-C18 silica gel (Fuji, 40–75 μm), MCI gel (CHP20P, 75–150 μm, maybe the fundamental sesquiterpene precursor farnesyl diphosphate Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (200–300 Mesh (FPP). FPP can produce the guaiyl cation by biological catalysis. Marine Chemical Ltd., Qingdao, People's Republic of China), and Compound 1 is formed from the guaiyl cation by electrophilic addition, Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for dehydrogenation, and oxidation (Scheme 1, rote i). Compound 2 is also column chromatography (CC). Preparative and semi-preparative HPLC formed from guaiyl cation by series of dehydrogenation, oxidation, separations were carried out on a LC-20AT Shimadzu liquid chromato- aethrization, oxidation, methylation, and acetylation (Scheme 1, rote ii) graphy system with a YMC ODS-A column (250 × 4.6 mm, 5 μm) con- [37,38]. nected with an SPD-M20A diode array detector. TLC analysis was carried Thirty-two compounds, including two new guaiane-type sesqui- out on silica gel plates (Marine Chemical Ltd., Qingdao, China). Fractions terpenoids, were isolated from the stems of D. tangutica. Compound 1 weremonitoredbyTLCandvisualizedbyheatingplatessprayedwith5%

Fig. 4. Key 1H−1H COSY (), HMBC (), and NOESY () correlations of compound 2.

107 Z.-Y. Yin et al. Fitoterapia 130 (2018) 105–111

bio-cat -H+ OPP ii farnesyl PP (FPP) guaiyl cation [O]

electrophilic addition i

O OH CHO + [O] -H OH O

OH -H2O aethrization 1

OMe OH O [O] O acetylation methylation O AcO HO H OH HO 2

Scheme 1. Plausible biosynthetic pathway of 1 and 2.

H2SO4 in EtOH. The human nasopharyngeal carcinoma cell lines (HONE- Chinese Academy of Sciences. A voucher specimen (2016002G) was 1) and (SUNE-1) were cultured on RPMI-1640 medium (Gibco, China) deposited at the School of Pharmaceutical Sciences, Sun Yat-sen containing 5% FBS (Gibco, America), 100 IU/mL penicillin, and 100 mg/ University. mL streptomycin (Hyclone, Shanghai). MTT (Sigama, America) assay was measured on Microplate reader (Thermo, Electroncorporation, China). Cell apoptosis assay, using Annexin V-FITC Apoptosis Detection Kit 3.3. Extraction and isolation (Beyotime, China). And cell circle assay, detecting with Cell Cycle and Apoptosis Analysis Kit (Beyotime, China) were analyzed on a Flow cyto- The air-dried and powdered stems of D. tangutica (8.0 kg) were ex- metry (Beckman Coulter, EPICS XL, USA). tracted with 90% EtOH to obtain an extract that was partitioned with EtOAc(3×3L)andH2O. The EtOAc fraction (380 g) was subjected to silica gel column chromatography (CC) using dichloromethane/acetone 3.2. Plant material (1:0, 50:1, 9:1, 3:1, 0:1 v/v) to aﬀord fractions A−E. Fraction B (21 g) was

puriﬁed using an MCI gel column with MeOH−H2O(60%–100%) as The stems of D. tangutica (8.0 kg) were collected on August 11, 2016 eluting solvent to give three subfractions B1−B3. Fraction B1 (5.4 g) was in Puan District, Guizhou Province, People's Republic of China, and loaded onto a Sephadex LH-20 column and eluted with CH2Cl2/MeOH identiﬁed by Dr. Chunyan Han from Kunming Institute of Botany, the (1:1)toyieldtwosubfractionsB1A1−B1A2. B1A1 (1.8 g)

Fig. 5. Eﬀects of compound 25 on cell apoptosis in HONE-1 cells. (A) Cells were treated with compound 25 (0, 1, 3, and 10 μM) for 48 h, and stained with Annexin-V FITC/PI, then analyzed by ﬂow cytometry to evaluate apoptosis. (B) The histogram analysis of (A).

108 Z.-Y. Yin et al. Fitoterapia 130 (2018) 105–111

Fig. 6. Compound 25 induced G2/M arrest in HONE-1 cells. (A) Cells were treated with 25 (0, 1, 3, and 10 μM) for 48 h, and stained with PI, then analyzed by ﬂow cytometry to determine cell-cycle phases and distribution of cells (%). (B) The histogram analysis of (A). chromatographed over silica gel CC with cyclohexane/EtOAc (from 4:1 to obtain 31 (8 mg). C1A2B2 (80 mg) was puriﬁed with semi-preparative

1:2) as the eluent to give six subfractions, B1A1C1−B1A1C6. B1A1C1 HPLC (90% MeCN in H2O, 2 mL/min) to yield 7 (14 mg), C1A2B3 (120 mg) was puriﬁed with semi-preparative HPLC (70% MeCN in H2O, (112 mg) was puriﬁed with semi-preparative HPLC (90% MeCN in H2O, 2mL/min)toyield 25 (32 mg), 26 (2 mg), 29 (5 mg). Fraction B1A1C2 2mL/min)toyield 4 (14 mg), 5 (12 mg), 6 (8 mg). C1A2B4 (32 mg) was

(54 mg) was purified with semi-preparative HPLC (50% MeCN in H2O, purified with semi-preparative HPLC (90% MeCN in H2O, 2 mL/min) to 2mL/min)toyield 1 (4 mg), 3 (4 mg), 19 (5 mg). B1A2 (3.4 g) chroma- yield 8 (7 mg), 9 (4 mg). C1A2B7 (50 mg) was purified with semi-pre- tographed over silica gel CC with cyclohexane/EtOAc (from 5:1 to 1:2) as parative HPLC (65% MeCN in H2O, 2 mL/min) to yield 28 (8 mg). E (32 g) the eluent to give sixteen subfractions, B1A1C1−B1A1C16. B1A1C12 was purified using an MCI gel column with MeOH−H2O(60%−100%) as (93 mg) was purified with semi-preparative HPLC (70% MeOH in H2O, eluting solvent to give three subfractions E1−E3. Fraction E2 (7.3 g) was 2 mL/min) to yield 17 (15 mg), 18 (5 mg), B1A1C13 (120 mg) was pur- loaded onto a Sephadex LH-20 column and eluted with CH2Cl2/MeOH ified with semi-preparative HPLC (70% MeOH in H2O, 2 mL/min) to yield (1:1)toyieldtwosubfractionsE1A1−E1A2. E1A1 (3.2 g) chromato- 20 (2 mg), 32 (20 mg). B1A1C14 (50 mg) was purified with semi-pre- graphed over silica gel CC with hexane/EtOAc (from 5:1 to 1:2) as the parative HPLC (70% MeOH in H2O, 2 mL/min) to yield 27 (7 mg), eluent to give ten subfractions, E1A1B1−E1A1B10. E1A1B1 (87 mg) was B1A1C15 (144 mg) was purified with semi-preparative HPLC (70% MeCN purified with semi-preparative HPLC (60% MeCN in H2O, 2 mL/min) to in H2O, 2 mL/min) to yield 21 (4 mg), 22 (55 mg). B1A1C16 (132 mg) was yield 10 (12 mg), 11 (8 mg), 12 (2 mg). E1A1B3 (77 mg) was purified with purified with semi-preparative HPLC (70% MeCN in H2O, 2 mL/min) to semi-preparative HPLC (60% MeCN in H2O, 2 mL/min) to yield 5 (6 mg), yield 16 (24 mg), 23 (15 mg). Fraction C (13 g) was purified using an MCI 24 (10 mg). E1A1B6 (180 mg) was purified with semi-preparative HPLC gel column with MeOH−H2O(80%–100%) as eluting solvent to give two (65% MeCN in H2O, 2 mL/min) to yield 13 (8 mg), 14 (10 mg), 15 subfractions, C1−C2. Fraction C1 (10 g)was further fractionated over si- (12 mg). lica gel CC (CH2Cl2/MeOH, 300:1 to 95:5) to give two subfractions C1A1−C1A3. C1A1 (900 mg) was chromatographed over Sephadex LH- 3.3.1. Daphnoid A (1) 20 20 with MeOH as eluent and further purified by semi-preparative HPLC Yellow oil; [α]D −17.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 237 with 85% MeCN−H2Otogive2 (4 mg). C1A2 (3.3 g) was chromato- (1.033) nm; CD (MeOH) λmax (log ε) 209 (+2.68), 236 (−2.87) nm; IR graphed over silica gel CC with cyclohexane/acetone (from 20:1 to 0:1) as (KBr) νmax 3401, 3001, 2965, 2921, 2876, 2852, 2766, 1757, 1701, the eluent to give twelve subfractions C1A2B1−C1A2B12, C1A2B1 1662, 1622, 1514, 1452, 1364, 1312, 1293, 1262, 1197, 1150, 1091, −1 1 13 (44 mg) finally purified by HPLC (55% MeOH in H2O, 2 mL/min) to 1038, 991, 934 and 899 cm ; H and C NMR data, see Table 1;

109 Z.-Y. Yin et al. Fitoterapia 130 (2018) 105–111

Table 1 reading at 492 nm. All IC50 values were calculated by nonlinear re- 1H NMR (400 MHz) and 13C NMR (100 MHz) data of Compounds 1 and 2 in gression analysis (GraphPad Prism). CDCl3.

Position 1 2 3.4.2. Cell cycle and cell apoptosis assay HONE-1 cells were seeded into 6-well cell culture plates at a density δ δ δ δ 5 C H (J in Hz) C H (J in Hz) of 2 × 10 cells/well overnight. After 48 h treatment with compound 25 (0, 1, 3 and 10 μM), HONE-1 cells were harvested. To measure the 1 142.6 C 141.9 C ratio of apoptotic cells, the cells stained with Annexin V-FITC/PI at 2 204.8 C 37.1 CH2 2.49 brs fl 3 45.2 CH2 2.68 m 78.7 CH 5.15 m room temperature in the dark for 25 min and analyzed by ow cyto- 2.08 d (16.6) metry. To examine the cellular DNA content, the cells fixed with 1 mL 4 34.5 CH 2.69 m 41.2 CH 2.29 m 70% EtOH overnight then stained with propidium iodide (PI) solution, 5 175.0 C 39.4 CH 2.30 m at 4 °C in the dark for 30 min and then analyzed by flow cytometry. 6 36.3 CH2 2.74 m 37.1 CH2 1.73 m 2.34 d (15.8) 1.43 dd (14.4, 11.9) 7 81.3 C 81.5 C Conflict of interest 8 215.0 C 103.6 C 9 50.7 CH2 2.50 d (18.9) 34.8 CH2 2.63 d (15.2) The authors declare no competing financial interests. 2.39 d (18.9) 2.36 dd (15.2, 1.7) 10 42.6 C 122.3 C 11 60.6 C 154.4 C Acknowledgments

12 207.3 CH 9.76 s 103.1 CH2 5.16 m 4.86 d (2.1) This study was supported in part by the Science and Technology 13 9.0 CH3 1.35 s 67.7 CH2 4.42 dd (13.2, 2.3) 4.29 dd (13.2, 2.1) Program of Guangzhou (201604020109), the Administration of

14 15.0 CH3 1.63 s 22.4 CH3 1.68 s Traditional Chinese Medicine of Guangdong Province (20171048), and 15 18.8 CH3 1.20 d (6.9) 10.4 CH3 0.95 d (7.2) the National Science and Technology Major Project of the Ministry of OAc 171.0 C Science and Technology of China (2018ZX09735010). OAc 21.5 CH3 2.02 s

OMe 48.1 CH3 3.24 s Appendix A. Supplementary data

Supplementary data to this article can be found online at https:// Table 2 doi.org/10.1016/j.ﬁtote.2018.08.012. Cytotoxicity of Compounds 4, 6, 7, 8, 9, and 25 in HONE-1 and SUNE-1.

a a References Compounds IC50 (μM) Compounds IC50 (μM)

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