Discovery of guaiane-type sesquiterpenoids from the roots of Daphne genkwa with neuroprotective effects

Journal Pre-proofs Discovery of guaiane-type sesquiterpenoids from the roots of Daphne genkwa with neuroprotective effects Jie Wang, Qing-Bo Liu, Zi-Lin Hou, Shao-Chun Shi, Hui Ren, Guo-Dong Yao, Bin Lin, Xiao-Xiao Huang, Shao-Jiang Song PII: DOI: Reference:

S0045-2068(19)31643-8 https://doi.org/10.1016/j.bioorg.2019.103545 YBIOO 103545

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

2 October 2019 9 December 2019 21 December 2019

Please cite this article as: J. Wang, Q-B. Liu, Z-L. Hou, S-C. Shi, H. Ren, G-D. Yao, B. Lin, X-X. Huang, S-J. Song, Discovery of guaiane-type sesquiterpenoids from the roots of Daphne genkwa with neuroprotective effects, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103545

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© 2019 Published by Elsevier Inc.

Discovery of guaiane-type sesquiterpenoids from the roots of Daphne genkwa with neuroprotective effects Jie Wanga,1, Qing-Bo Liua,1, Zi-Lin Houa, Shao-Chun Shia, Hui Rena, Guo-Dong Yaoa, Bin Linb, Xiao-Xiao Huanga,*, Shao-Jiang Songa,* aKey

Laboratory of Computational Chemistry-Based Natural Antitumor Drug

Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People′s Republic of China bSchool

of

Pharmaceutical

Engineering,

Liaoning

Province,

Shenyang

Pharmaceutical University, Shenyang 110016, People’s Republic of China *Corresponding author. E-mail addresses: [email protected] (X.-X. Huang), [email protected] (S.-J. Song). 1Both

authors contributed equally to this work.

Abstract A phytochemical study on the roots of Daphne genkwa yielded seven new guaianetype sesquiterpenoids (1-7), daphne A-G. Their structures were elucidated through comprehensive spectroscopic analyses. The absolute configurations were determined by comparison between experimental electronic circular dichroism (ECD) and calculated ECD spectra via time-dependent density functional theory (TDDFT) and the modified Mosher’s method. Furthermore, all isolates were evaluated for their neuroprotective activities against H2O2-induced injury in human neuroblastoma SHSY5Y cells. Among them, compounds 1 (78.42%) and 4 (79.34%) exhibited potent neuroprotective effects against H2O2-induced neurotoxicity at 25 μM. Further Annexin V-FITC/ propidium iodide (PI) doubling staining exhibited that the neuroprotective effects of compounds 1 and 4 appeared to be mediated via suppressing cell apoptosis. Flow cytometry assays also proved that compounds 1 and 4 could attenuate mitochondrial dysfunction in SH-SY5Y cells.

Keywords: Daphne genkwa; Sesquiterpenoids; Neuroprotective effects; Apoptosis

1. Introduction Daphne genkwa Siebold et Zucc., belonging to the family Thymelaeaceae, is a deciduous shrub widely distributed and cultivated in the Yangtze River and the Yellow River of China and Korea [1-3]. The flower buds and roots of this plant are mainly used for anticancer, anti-inflammatory, antitussive, diuretic, analgesia, and abortifacient effects [4-6]. Extensive phytochemical studies of D. genkwa have led to the isolation of a series of constituents, including flavonoids [7], diterpenoids [8], coumarins [9], caffeotannic acids [10] and lignans [11]. Neurodegenerative diseases are a major problem in modern aging society, such as Parkinson's, Alzheimer's, and multiple sclerosis [12,13]. In the past decades, exploring effective treatment to alleviate neurodegenerative diseases attracted lots of attention but it ended up with disappointing effects. Oxidative stress is the major underlying contributor associated with the etiology of these neurodegenerative diseases [14-16]. Oxidative stress from reactive oxygen species (ROS) plays a significant role for the initiation and progression of metabolic disorders and neurodegenerative disorders [17,18]. Modern pharmacological studies have shown that sesquiterpenoids have neuroprotective [19-21]. As part of our continuing efforts to discover compounds with potent neuroprotective activities, seven new guaiane-type sesquiterpenoids daphne A-G (1-7) were isolated and identified from the roots of D. genkwa. Their structures including the absolute configurations were established by comprehensive spectroscopic analyses and ECD calculations. In addition, the protective effects of all compounds were screened against human neuroblastoma SH-SY5Y cells injury induced by H2O2. 2. Experimental 2.1. General experimental procedures

Optical rotations were recorded on a JASCO DIP-370 digital polarimeter (Jasco, Tokyo, Japan). UV spectra were measured on a Shimadzu UV-1700 spectrometer (Shimadzu, Kyoto, Japan). Electronic circular dichroism (ECD) experiments were recorded on a MOS 450 spectrometer (Bio-Logic Science Instruments, SeyssinetPariset, France). The high-resolution electrospray ionization mass spectrometry (HRESIMS) data were performed on a Micro Q-TOF spectrometer (Bruker Daltonics, Billerica, USA). The NMR spectra were obtained on Bruker AVIII-600 (Bruker Corporation, Bremen, Germany) with TMS as an internal standard. Semi-preparative RP-HPLC separations were performed on instruments equipped with a LC-6AD highpressure pump with a SPD-20A ultraviolet-visible (UV-vis) light absorbance detector using a YMC C18 semi-preparative column (250 mm × 10 mm, 5 μm, Shimadzu, Tokyo, Japan). Column chromatography were performed on silica gel (100–200 or 200–300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of China), HP-20 macroporous resin (75–150 μm, Tokyo, Japan), and octadecyl silica gel (ODS) (60–80 μm YMC Co. Ltd., Kyoto, Japan). MTT assay was carried out on a Varioskan Flash Multimode Reader (Thermo Scientific Co. Ltd, Massachusetts, USA). 2.2. Plant material The dried roots of D. genkwa were collected from Xingyi City, Guizhou Province, People’s Republic of China, in July 2017, and were authenticated by Prof. Jin-Cai Lu (Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, People’s Republic of China). A voucher specimen (No. 20170721) has been deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University for further reference. 2.3. Extraction and isolation

The dried roots of D. genkwa (50 kg) were chopped and soaked in 70% EtOH extracted three times at room temperature. The EtOH extract was concentrated under vacuum to yield crude extract. Then, the crude extract was partitioned successively with petroleum ether, ethyl acetate (EtOAc) and n-butanol. The EtOAc extract (1000 g) was subjected to a silica gel column and eluted with a step gradient of CH2Cl2/MeOH (100:1 → 1:1, v/v) of Four fractions, A-D. Fraction B (140.0 g) was separated by HP-20 macroporous resin eluted with EtOH/H2O (30%, 60%, 90%) and afforded four fractions, B1 ~ B4. Subfractions Fr. B2 (30 g) and Fr. B3 (70 g) were further purified by ODS using EtOH/H2O as a mobile phase gradient (from 20:80 to 90:10) to yield fractions, Fr. B2.1 ~ Fr. B2.2 and Fr. B3.1 ~ Fr. B3.3, respectively. Fr. B2.2 (15.0 g) was fractioned over silica gel eluted with CH2Cl2/ MeOH (50:1 → 1:1, v/v) of increasing polarity to obtain 12 fractions, Fr. B2.1.1 ~ Fr. B2.1.12. Fr. B3.2 (20.0 g) was subjected to further silica gel column chromatography and eluted with CH2Cl2/ MeOH (50:1 → 1:1) separately yield fractions Fr. B3.2.1 ~ Fr. B3.2.13. Fr. B3.2.2 (0.4 g) was subsequently purified by semi-preparative RP-HPLC (CH3CNH2O, 25:75, v/v, 2.5 mL/min) over a YMC C18 column to afford compound 1 (13.0 mg, tR = 41.1 min). Compound 2 (4.0 mg, tR = 49.1 min) and compound 3 (10.0 mg, tR = 63.3 min) were obtained from fraction Fr. B3.2.3 (0.5 g) by semi-preparative RPHPLC (CH3CN-H2O, 23:77, v/v, 2.5 mL/min). Subfraction Fr. B3.2.4 (0.6 g) was also purified by semi-preparative RP-HPLC with the mobile phase CH3CN-H2O (27:73, v/v, 2.5 mL/min) to obtain compound 4 (30.0 mg, tR = 48.5 min). Then, compound 5 (15.1 mg, tR = 48.9 min) was isolated from fraction Fr. B3.2.6 (0.5 g) eluted with CH3CN-H2O (24:76, v/v, 2.5 mL/min). Purification of Fr. B3.2.7 (0.4 g) by semipreparative RP-HPLC with CH3CN-H2O (33:67, v/v, 2.5 mL/min) gave compound 6 (5.2 mg, tR = 49.2 min). Compound 7 (15.0 mg, tR = 33.4 min) was subjected to

fraction Fr. B2.1.3 (0.3 g) (CH3CN-H2O, 20:80, v/v, 2.5 mL/min). All isolated compounds were > 95% pure. Daphne A (1): yellow oil (methanol); [α] 20D +16.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 205 (1.42) nm; ECD (MeOH) λmax (Δε) 216 (-11.2), 227 (+2.5) nm; 1H and

13C

NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion

HRESIMS m/z 273.1459 [M + Na]+ (calcd for C15H22O3Na, 273.1461). Daphne B (2): yellow oil (methanol); [α] 20D +18.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (0.89) nm; ECD (MeOH) λmax (Δε) 213 (-23.4) nm; 1H and 13C NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion HRESIMS m/z 275.1612 [M + Na]+ (calcd for C15H24O3 Na 275.1618). Daphne C (3): yellow oil (methanol); [α] 20D +12.2 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 204 (0.96) nm; ECD (MeOH) λmax (Δε) 215 (-30.3) nm; 1H and 13C NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion HRESIMS m/z 291.1561 [M + Na]+ (calcd for C15H24O4 Na 291.1567). Daphne D (4): yellow oil (methanol); [α] 20D +28.8 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (0.65) nm; ECD (MeOH) λmax (Δε) 201 (+13.6), 222 (-34.3) nm; 1H and

13C

NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion

HRESIMS m/z 273.1438 [M + Na]+ (calcd for C15H22O3 Na 273.1461). Daphne E (5): yellow oil (methanol); [α] 20D +46.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 205 (1.19) nm; ECD (MeOH) λmax (Δε) 191 (-5.9), 216 (-25.4) nm; 1H and 13C

NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion

HRESIMS m/z 251.1654 [M + H]+ (calcd for C15H23O3 251.1642). Daphne F (6): yellow oil (methanol); [α] 20D -12.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 253 (0.31) nm; ECD (MeOH) λmax (Δε) 224 (+31.5) 263 (-4.7), 335 (-7.4) nm;

1H

and 13C NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion

HRESIMS m/z 287.1257 [M + Na]+ (calcd for C15H20O4Na 287.1254). Daphne G (7): yellow oil (methanol); [α] 20D -3.5 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 202 (0.25) nm; ECD (MeOH) λmax (Δε) 228 (+11.1), 306 (-4.7) nm; 1H and 13C NMR (600 and 150 MHz, CDCl3), shown in Tables 1 and 2; positive-ion HRESIMS m/z 289.1408 [M + Na]+ (calcd for C15H22O4Na 289.1410). 2.3.1. General procedure for esterification with (S/R)-MTPA. Compounds 1a and 1b (MTPA esters of 1) were prepared by the method described by Hoye et al [22]. Compound 1 (2.3 mg) was dissolved in 400 μL of dried CH2Cl2 and separated into two equal portions at room temperature. compound 1 (1a and 1b), 10

μL

of

(S)-

and

(R)-Mosher

acid

(S-

and

R-

(−)-α-methoxy-α-

(trifluoromethyl)phenylacetic acid) were respective added to initiate the reaction, while 15 mg of EDCI was added as the dehydrating agent, and 10 mg of 4dimethylaminopyridine was used as the catalyst. After stirred at room temperature for 24 h, the mixture was evaporated to dryness and then subjected to semi-preparative HPLC for purification, eluted with 80% CH3CN, to obtain 1.0 mg of 1a (tR = 15.29 min) and 1b (tR = 15.50 min). (S)-MTPA ester of 1 (1a). 1HNMR (600MHz, CDCl3): δH 4.67 (1H, t, H-2), 1.90 (1H, m, H-3α), 1.60 (1H, overlap, H-3β), 2.25 (1H, m, H-4), 2.35 (1H, m, H-5), 0.69 (3H, d, H-15), 1.60 (1H, overlap, H-6a), 1.09 (1H, m, H-6b), 2.62 (1H, m, H-7), 4.10 (1H, d, H-9α), 4.40 (1H, d, H-9β), 4.34 (1H, d, H-13α), 4.49 (1H, d, H-13β), 4.95 (1H, d, H-12a), 5.03 (1H, d, H-12b), 1.79 (3H, d, H-14). (R)-MTPA ester of 1 (1b). 1HNMR (600MHz, CDCl3): δH 4.70 (1H, t, H-2), 1.93 (1H, m, H-3α), 1.60 (1H, m, H-3β), 2.39 (1H, m, H-4), 2.47 (1H, m, H-5), 0.81 (3H, d, H-15), 1.60 (1H, overlap, H-6a), 1.05 (1H, m, H-6b), 2.55 (1H, m, H-7), 4.16 (1H, d,

H-9α), 4.32 (1H, d, H-9β), 4.33 (1H, d, H-13α), 4.48 (1H, d, H-13β), 4.94 (1H, d, H12a), 5.02 (1H, d, H-12b), 1.68 (3H, d, H-14). 2.4. ECD calculations The absolute configurations were determined by using time-dependent density functional theory (TDDFT) calculations [23]. First, the conformational searches were carried out by SPARTAN (11 conformations for compound 1, 17 conformations for compound 2, 13 conformations for compound 3, 8 conformations for compound 4, 10 conformations for compound 5, 8 conformations for compound 6, and 12 conformations for compound 7) [24]. Next, these conformations were further optimized by the density functional theory method at the B3LYP/6-31G(d) level in Gaussian 09 program package [25]. The theoretical ECD calculations were performed using TDDFT method at the B3LYP/6-31G(d,p) and B3LYP/6-311++G(2d,p) levels in MeOH solution. The ECD curves were generated by SpecDis 1.51 software on the basis of the Boltzmann weighting of each conformer [26]. 2.5. Cell culture Human neuroblastoma SH-SY5Y cell line (American Type Culture Collection, Manassas, USA) was cultured in DMEM medium (Hyclone, Logan, USA), which was supplemented with 10% FBS (Gibco, Gaithersburg, USA), 100 μg/mL streptomycin, 100 U/ml penicillin maintained a humidified atmosphere containing 5% CO2 at 37 °C. Logarithmically growing cells were used in all the experiments. 2.6. Cell viability assay Compounds were screened for neuroprotective activities by MTT assay [27]. Briefly, the SH-SY5Y cells were incubated with tested compounds (25, 50 and 100 μM) for 1 h and then exposure to 200 μM H2O2 for 4 h. Then, 20 μL MTT (5 mg/mL) was added to each well for 4 h, and added DMSO to dissolve the formazan crystals.

The absorbance was read at 490 nm with a Thermo microplate reader (Thermo, Waltham, USA). The cell viability was expressed as a percentage with the control group as 100%. 2.7. Annexin V-FITC/PI analysis Annexin V-FITC and PI apoptosis assay kit was applied to evaluate apoptotic ratio of cells [28]. The treated cells were stained with PI followed by Annexin V−FITC at room temperature for 15 min. The samples were then analyzed using flow cytometer (Becton Dickinson, Franklin Lakes, USA) and quantifed with Flow Jo 7.6.1 (Oregon, USA). 2.8. Mitochondrial membrane potential assay Cells treated by compounds 1 and 4 were evaluated for the mitochondrial membrane potential (∆Ψm) by JC-1 assay kit. The loss of ΔΨm was examined with a florescence microscope. The experimental method were conducted on the reported literature [29]. 2.9. Statistical analysis All results and data were confirmed as the mean ± standard deviation (SD) in at least three independent experiments. Statistical comparisons were analyzed by GraphPad Prism 6 software. P values of <0.05 were defined as indicative of significance. 3. Results and discussion 3.1 Phytochemical investigation Compound 1 was obtained as a yellow oil. Its molecular formula was established as C15H22O3 with 5 degrees of unsaturation deduced from its positive-ion HRESIMS at m/z 273.1459 [M + Na]+, (calcd for C15H22O3Na, 273.1461). The 1H NMR data (Table 1) showed characteristic signals for two methyl singlets at δH 0.71 (3H, d, J = 7.3 Hz,

H3-15) and 1.84 (3H, d, J = 2.4 Hz, H3-14), two oxymethylene groups at δH 3.31 (1H, d, J = 10.8 Hz, H-9α), 3.60 (1H, d, J = 10.8 Hz, H-9β), 4.30 (1H, d, J = 13.2 Hz, H13α), and 4.45 (1H, d, J = 13.2 Hz, H-13β), one oxymethine proton at δH 4.77 (1H, t, J = 7.0 Hz, H-2), a terminal double bond at δH 4.93 (1H, d, J = 1.6 Hz, H-12a) and 5.03 (1H, d, J = 1.6 Hz, H-12b). Analysis of the

13C

NMR (Table 2) and HSQC

spectra contained signals for four olefinic (δC 105.5, 129.9, 146.4 and 152.6), two oxymethylene (δC 64.0 and 69.7), one oxymethine (δC 71.1), one oxygenated (δC 85.9), two methylene (δC 30.2 and 43.3), three methine (δC 33.4, 42.8 and 45.5), and two methyl (δC 13.1 and 15.8) carbons. In the HMBC spectrum (Fig. 2), the correlations of H-2 to C-1, C-5, C-10 and H-3 to C-2, C-5, C-15 confirmed that a secondary OH group was fused at C-2. Additionally, a hydroxymethyl group could be determined by the HMBC correlations from H-9 to C-7, C-8, C-10. On the basis of these results, the gross structure of 1 with a 5/6/5 tricyclic skeleton was established (Fig. 1). The relative configuration of 1 was assigned by the NOESY experiment (Fig. 3). The NOESY correlations of H3-15/H-3β, H3-15/H-2, H-3α/H-5, H-5/H-7, H-7/H-9α established the relative configuration of 1 as 2S*, 4S*, 5S*, 7R*, 8S*. To determine the absolute configuration of 1, electronic circular dichroism (ECD) calculations for respective 1a (2S, 4S, 5S, 7R, 8S) and 1b (2R, 4R, 5R, 7S, 8R) were performed using the time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d, p) level with PCM model in MeOH solution. As shown in Fig. 4, the calculated ECD curve of (2S, 4S, 5S, 7R, 8S)-1 matched well with the experimental ECD curve of 1a. The absolute configuration at C-2 of 1 was also determined as S by modified Mosher’s method. Esterification of 1 with (R)- and (S)-α-methoxy-α-(trifluoromethyl) phenylacetic acid (MTPA-OH) yielded (R)- and (S)-MTPA esters of the OH group at C-2. The ΔδH(S−R) values (Fig. 5) calculated from the 1H NMR data defined the

absolute configuration at C-2 as S. Finally, this was consistent with the conclusion drawn from the calculated ECD data and led to the assignment of the absolute configuration of 1 as 2S, 4S, 5S, 7R, 8S. Compound 2 was assigned the molecular formula C15H24O3 based on the positiveion HRESIMS data m/z 275.1612 [M + Na]+, (calcd for C15H24O3Na, 275.1618). The NMR data (Tables 1 and 2) indicated that 2 was similar to 1 except for replacement of a terminal double bond with a hydroxymethyl group at 11 and the absence of a secondary OH group at C-2 in 2. This was confirmed by the HMBC correlations of H11 to C-6, C-7, C-12, C-13 and H-12 to C-7, C-11, C-13 (Fig. 2). The relative configuration of 2 was proposed as 4S*, 5S*, 7R*, 8S*, 11R* in accordance with the NOESY correlations of H3-15/H-3β, H-3α/H-5, H-5/H-7, H-7/H-9, H-11/H-9 (Fig. 3). The absolute configuration of 2 was further determined by the experiment and the calculated ECD spectrum (Fig. 4). The calculated ECD spectrum of 4S, 5S, 7R, 8S, 11R was consistent with the experiment ECD spectrum. Taken together, the absolute configuration of 2 was established as 4S, 5S, 7R, 8S, 11R. The molecular formula of 3 was established as C15H24O4 on the basis of the [M + Na]+ ion peak at m/z 291.1561 (calcd for C15H24O4Na, 291.1567). The analysis of the 1H

and 13C NMR data (Tables 1 and 2) suggested that 3 had an analogous structural

skeleton to 2. The only difference was the additional hydroxyl group at C-11 in 3. The relative configuration of 3 was similar to that of 2 based on the NOESY correlations. The significant NOESY correlations of H3-15/H-3β, H-3α/H-5, H-5/H-7, H-7/H-9α, H-12/H-6 supported the relative configuration of 3 as 4S*, 5S*, 7R*, 8S*, 11S* (Fig. 3). Similarly, based on the calculation of ECD, the theoretical ECD curve of 4S, 5S, 7R, 8R, 11S-3 matched well the experimental ECD curve (Fig. 4). Thus, the absolute configuration of 3 was assigned as 4S, 5S, 7R, 8R, 11S.

Compound 4 gave a positive HRESIMS m/z 273.1438 [M + Na]+ (calcd for C15H22O3Na, 273.1461), consistent with the molecular formula C15H22O3. The 1D NMR spectra (Tables 1 and 2) of 4 displayed a similarity to those of 1 with the main disparity being a 5/7/6 tricyclic skeleton with an oxygen bridge. It was corroborated by the HMBC correlations (Fig. 2) of H-8 to C-6, C-9, C-10 and H-9 to C-1, C-7, C-8, C-10, C-13, C-14. The relative configuration of 4 was proposed as 2S*, 4S*, 5S*, 7R*, 8R*, 9R* in accordance with the NOESY cross-peaks of H3-15/H-3β, H3-15/H-2, H3α/H-5, H-5/H-7, H-8/H-13β (Fig. 3). In the same way, the absolute configuration of 4 was determined by comparing experimental and calculated ECD spectra using TDDFT (Fig. 4). Therefore, the absolute configuration of 4 was determined as 2S, 4S, 5S, 7R, 8R, 9R. Compound 5 displayed a (+)-HRESIMS ion peak at m/z 251.1654 [M + Na]+ (calcd for C15H22O3Na, 251.1642), implying a molecular formula of C15H22O3. Compounds 5 and 4 were found to share an identical carbon skeleton except the different position of the secondary OH group in 5. A secondary OH group at C-3 was supported by the HMBC correlations from H-3 to C-1, C-5, C-15 (Fig. 2). The NOESY correlations between H3-15/H-3, H-5/H-7, H-8/H-13β indicated 3R*, 4R*, 5S*, 7R*, 8R*, 9R* configuration in 5 as shown (Fig. 3). To further unambiguously confirm the absolute configuration of 5, a comparison between its experimental and calculated ECD spectra using TDDFT was performed. The experimental ECD spectrum of 5 was in good agreement with the calculated ECD spectrum for the 3R, 4R, 5S, 7R, 8R, 9R-5 (Fig. 4). Thus, the absolute configuration of 5 was defined as 3R, 4R, 5S, 7R, 8R, 9R. Compound 6 had a molecular formula of C15H20O4 according to its HRESIMS spectrum (m/z 287.1257 [M + Na]+, calcd for C15H20O4Na, 287.1254). The

comparison of the 1D NMR spectra (Tables 1 and 2) of 6 and 4 revealed that the OH group of 4 was substituted with a keto carbonyl group in 6 and the additional a OH group at C-7 in 6. This was verified by HMBC correlations from H-3 and H-4 to C-2, and H-6, H-8, H-9, H-12 and H-13 to C-7 (Fig. 2). Furthermore, the relative configuration of 6 was established by analysis of the NOESY spectrum (Fig. 3). The correlations of H3-15/H-3β, H-3α/H-5, H-8/H-13β indicated that the relative configuration of 6 were 4S*, 5S*, 7S*, 8S*, 9R*. Subsequently, the absolute configuration of 6 was determined by the TDDFT method at the B3LYP/6311++G(2d,p) level in MeOH solution (Fig. 4). Therefore, the absolute configuration of 6 was defined as 4S, 5S, 7S, 8S, 9R. Compound 7 possessed the molecular formula of C15H22O4 established by HRESIMS at m/z 289.1408 [M + Na]+, calcd for C15H22O4Na, 289.1410). The NMR data (Tables 1 and 2) indicated that 7 was structurally similar to 4, except for the replacement of a hydroxyl group with a keto carbonyl group at C-8 and a terminal double bond with a hydroxymethyl group at C-11 in 7. This deduction was supported by the HMBC correlations (Fig. 2) from H-6, H-7, H-9 and H-12 to C-8, and from H13 to C-7, C-11 and C-12. The relative configuration of 7 was established by analysis of the NOESY spectrum of H3-15/H-3β, H-3α/H-5, H-5/H-7, H-11/H-6 (Fig. 3). Further, the calculated ECD curve of 2S, 4S, 5S, 7R, 9R, 12S-7 agreed with the experimental ECD curve of 7 using TDDFT method at the B3LYP/6-311++G(2d,p) level in MeOH solution (Fig. 4). Thus, the absolute configuration of 7 was defined as 2S, 4S, 5S, 7R, 9R, 12S. 3.2. Neuroprotective activity study in vitro Compounds 1-7 were tested for their potential neuroprotective effects against H2O2induced SH-SY5Y cells injury, using the MTT assay (Fig. 6). The results exhibited

that the cell viability of SH-SY5Y cells was suppressed at 64.95% by H2O2 (200 μM) treatment. Additionally, compounds 1 (78.42%) and 4 (79.34%) showed moderate protective activities on SH-SY5Y cells with H2O2-induced injury at 25 μM in comparison with the positive control trolox (72.67% at 25 μM). Further Annexin V-FITC/PI doubling staining was used to further explore the protection of compounds 1 and 4 from H2O2-induced apoptosis in SH-SY5Y cells. As illustrated in Fig. 7, the apoptosis ratio in model group reached to 19.52% compared to the control group (7.14%). Then, when pretreated with 1 and 4 at the concentration of 25 μM, the percentage of apoptotic cells was decreased to 12.76% and 13.47%, respectively. Therefore, the observed neuroprotective effects of 1 and 4 using SHSY5Y cells were mediated by inhibiting cell apoptosis. At present, new strategies targeting mitochondrial dysfunction are emerging as potential alternatives to current treatment options for neurodegenerative diseases [30]. Mitochondria is a significant source of reactive oxygen species, and the increased ROS intensifies mitochondria dysfunction and cell damage [31]. The decreased mitochondrial membrane potential (∆Ψm) was considered as an important indicator of mitochondrial dysfunction. Reduction of ΔΨm was reflected by the change in JC-1 metachromatic dye from a dimeric fluorescent red form (representing high ΔΨm) to a monomeric fluorescent green form (representing low ΔΨm). As the result in Fig. 8, treatment with H2O2 of SHSY5Y cells resulted in a decrease in red fluorescence, indicating a loss of ΔΨm. The presence of compounds 1 and 4 increased more red fluorescence when compared to H2O2 treatment. The result suggested that compounds 1 and 4 could protect SH-SY5Y cells from mitochondrial dysfunction induced by H2O2. 4. Conclusion

In the present study, seven new guaiane-type sesquiterpenoids (1-7), daphne A-G, were isolated from the roots of D. genkwa, and their gross structures and absolute configurations were established by HRESIMS, NMR, together with the comparison of their experimental and calculated ECD spectra. In addition, all compounds were evaluated

for

their

neuroprotective

activities

using

H2O2-induced

human

neuroblastoma SH-SY5Y cells by MTT assay. Further flow cytometry analysis exhibited that 1 and 4 exerted protective effects through inhibiting cell apoptosis which probably implicated in mitochondria damage. ORCID Xiao-Xiao Huang: 0000-0003-0099-1118 Shao-Jiang Song: 0000-0002-9074-2467 Acknowledgements This project was funded by the National Nature Science Foundation of China (81872766 and 81573319), the Young and Middle-aged Science and Technology Innovative Talent Support Program of Shenyang City (RC180250) and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University (ZQN2018006) and the Project of Innovation Team (LT2015027) of Liaoning of P. R. China. Conflict of interest The authors declare no competing financial interest. References [1] R. Wang, J.Y. Li, H.Y. Qi, Y.P. Shi, Two new tigliane diterpene esters from the flower buds of Daphne genkwa, J. Asian Nat. Prod. Res. 15 (2013) 502-506.

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Figure caption Fig. 1. The structures of compounds 1-7. Fig. 2. Key HMBC correlations (H → C), and the key 1H−1H COSY correlations of compounds 1-7. Fig. 3. Key NOESY correlations of compounds 1-7. Fig. 4. Calculated and experimental ECD spectra of 1-7. Fig. 5. δH(S−R) values for the MTPA esters of 1. Fig. 6. Neuroprotective effects of 1-7 on H2O2-induced (200 μM) human neuroblastoma SH-SY5Y cell injury. The percentage of surviving cells is presented as the percentage of viable cells compared to the control group (cell viability 100%). All data are presented as the means ± SD of three independent experiments. Trolox was used as a positive control. *P < 0.05, **P < 0.01, and

***P

< 0.001 compared with the

H2O2. Fig. 7. Flow cytometry analysis of the effects of 1 and 4 (25 μM) on H2O2-induced (200 μM) SH-SY5Y cell apoptosis. The percentage of apoptotic cells was calculated in the right. **p < 0.01 compared with the control group. *p < 0.05 compared with the H2O2-treated group. Fig. 8. Compounds 1 and 4 attenuated H2O2-induced mitochondrial dysfunction in SH-SY5Y cells. Change of ΔΨm was determined by JC-1 staining. The scale bar represents 50 µm. 1 (25 µM) + H2O2 (200 µM) group and 4 (25 µM) + H2O2 (200 µM) group.

Fig. 1. The structures of compounds 1-7.

Fig. 2. Key HMBC correlations (H → C), and the key 1H−1H COSY correlations of compounds 17.

Fig. 3. Key NOESY correlations of compounds 1-7.

Fig. 4. Calculated and experimental ECD spectra of 1-7.

Fig. 5. δH(S−R) values for the MTPA esters of 1.

Fig. 6. Neuroprotective effects of 1-7 on H2O2-induced (200 μM) human neuroblastoma SHSY5Y cell injury. The percentage of surviving cells is presented as the percentage of viable cells compared to the control group (cell viability 100%). All data are presented as the means ± SD of three independent experiments. Trolox was used as a positive control. *P < 0.05, **P < 0.01, and ***P

< 0.001 compared with the H2O2.

Fig. 7. Flow cytometry analysis of the effects of 1 and 4 (25 μM) on H2O2-induced (200 μM) SHSY5Y cell apoptosis. The percentage of apoptotic cells was calculated in the right. **p < 0.01 compared with the control group. *p < 0.05 compared with the H2O2-treated group.

Fig. 8. Compounds 1 and 4 attenuated H2O2-induced mitochondrial dysfunction in SH-SY5Y cells. Change of ΔΨm was determined by JC-1 staining. The scale bar represents 50 µm. 1 (25 µM) + H2O2 (200 µM) group and 4 (25 µM) + H2O2 (200 µM) group.

Table caption Table 1. 1H NMR data (600 MHz, J in Hz) of compounds 1-7 in CDCl3 (δ in ppm). Table 2. 13C NMR data (150 MHz) of compounds 1-7 in CDCl3 (δ in ppm).

Table 1. 1H NMR data (600 MHz, J in Hz) of compounds 1-7 in CDCl3 (δ in ppm). posit ion 2

6 1 4.77, t, 7.0

2 a 2.23, o b 2.34, m

3

4 5 6

7 8

α 1.72, m β 1.90, m 2.30, m 2.51, m a 1.05, m b 1.68, m 2.85, m

3 a 2.16, dd, 8.70, 17.00 b 2.32, dd, 8.70, 17.00

α 1.74, m

1.72, m

β 1.50, m

1.46, m

2.23, o 2.14, m

2.26, o 2.02, m

a 0.92, m

a 0.70, m

b 1.59, m

b 1.92, m

2.41, m

2.26, o

4 4.77, d, 6.5 α 1.66, m β 1.79, m 2.21, m 3.51, m a 1.33, m b 2.03, m 2.87, m 4.02, t,

5 a 2.31, d, 17.5 b 2.70, d, 17.5 3.91, d, 4.7

7 4.78, t, 4.9

2.06, o 3.67, m

α 2.53, d, 16.6 β 2.11, dd, 7.7, 16.6 2.29, m 3.97, m

a 1.42, m

a 1.96, m

a 1.31, m

b 2.06, o

b 2.20,m

b 2.11, m

2.90, m 4.04, t, 5.0

3.95, d, 5.6

α 1.72, m β 1.64, m 2.41, m 2.47, m

2.76, m

α 3.31, d, 10.8 β 3.60, d, 10.8

9

11 a 4.93, d, 1.6 b 5.03, d, 1.6 α 4.30, d, 13.2 β 4.45, d, 13.2 1.84, d, 2.4 0.71, d, 7.3

12

13

14 15

3.45, s

α 3.17, d, 11.5 β 3.87, d, 11.5

2.81, m a 3.66, dd, 7.5, 10.5 b 3.82, dd, 7.5, 10.5 α 3.52, dd, 8.0, 11.0 β 4.01, t, 8.0

a, 3.61, d, 11.3 b, 3.70, d, 11.3 α 3.55, d, 9.3 β 3.74, d, 9.3

1.68, s

1.66, s

0.73, d, 7.1

0.75, d, 7.2

5.0 4.20, d, 5.0

4.20, d, 5.0

4.43, d, 5.6

4.30, s

2.05, m a, 4.72, brs b, 4.75, brs α 4.08, d, 12.7 β 3.74, d, 12.7 1.88, d, 2.0 0.75, d, 7.2

a 4.73, d, 1.3

5.20, s

b 4.76, brs

4.92, s

α 4.09, d, 12.6 β 3.72, d, 12.6

α 4.05, d, 13 β 4.18, d, 13

1.71, s

2.27, d, 2.6

0.71, d, 7.3

0.79, d, 7.2

3.64, m

α 3.68, m β 3.98, m 1.92, d, 1.9 0.88, d, 7.0

Overlapping signals are expressed as o.

Table 2. 13C NMR data (150 MHz) of compounds 1-7 in CDCl3 (δ in ppm). positio n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1

2

3

4

5

6

7

146.4 71.1 43.3 33.4 42.8 30.2 45.5 85.9 64.0 129.9 152.6 105.5 69.7 13.1 15.8

146.0 27.6 32.5 36.2 43.8 23.4 41.9 86.6 66.5 124.2 45.2 61.1 68.8 13.8 14.8

145.6 28.1 32.7 36.2 42.6 26.6 50.6 87.1 67.5 123.9 83.0 62.9 74.3 14.2 14.8

149.6 73.7 42.9 35.1 40.7 33.1 42.6 74.0 79.7 127.2 148.4 109.4 61.9 19.0 15.6

144.5 40.3 77.1 46.5 38.5 32.8 42.5 74.2 79.9 122.8 148.7 109.3 61.7 20.0 13.6

137.4 207.9 49.1 31.2 39.2 43.2 74.4 78.5 80.8 142.8 150.4 107.2 65.9 19.1 15.9

151.1 73.2 42.1 34.8 41.4 29.1 46.8 210.2 86.6 128.8 58.8 47.6 63.5 17.7 15.7

GA

Highlights ►Seven new guaiane-type sesquiterpenoids were isolated from the roots of Daphne genkwa. ► The absolute configurations of isolated compounds were determined by theoretical ECD calculation and the modified Mosher’s method. ► Their neuroprotective activities against H2O2-treated SH-SY5Y cells were tested.

Conflict of Interest Dear Editor, The authors declare that no conflict of interest exists in the submission of this manuscript entitled “Discovery of guaiane-type sesquiterpenoids from the roots of Daphne genkwa with neuroprotective effects”, and manuscript is read and approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. Best regards, Thank you!

Sincerely yours, Shao-Jiang Song, Dr., Prof. School of Traditional Chinese Materia Medica Shenyang Pharmaceutical University Tel./Fax: +024 43520707 E-mail: [email protected]

Xiao-Xiao Huang School of Traditional Chinese Materia Medica Shenyang Pharmaceutical University Tel./Fax: +024 43520793 E-mail: [email protected]