Stilbenes from Veratrum maackii Regel. Protect against Ethanol

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Stilbenes from Veratrum maackii Regel. Protect against EthanolInduced DNA Damage in Mouse Cerebellum and Cerebral Cortex Yantong Wu, shasha Li, Jinjin Liu, xiping Liu, Weimin Ruan, Jeng-Wei Lu, Yong Liu, Tom Lawson, Olga Shimoni, David Lovejoy, Adam K. Walker, yue cong, and Bingyang Shi ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00006 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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ACS Chemical Neuroscience

Stilbenes from Veratrum maackii Regel. Protect against Ethanol-Induced DNA Damage in Mouse Cerebellum and Cerebral Cortex Yantong Wu1#, Shasha Li1#, Jinjin Liu2#, Xiping Liu1, Weimin Ruan3, Jengwei Lu3, Yong Liu4, Tom Lawson5, Olga Shimoni6, David Lovejoy7, Adam K. Walker7, Yue Cong1*，Bingyang Shi3* 1

Institute of Pharmacy, Pharmaceutical College, Henan University, Kaifeng, 475004, China 2 Zhengzhou Translational Medicine Research Center, Zhengzhou Sixth's People's Hospital, Zhengzhou, 450000, China 3 International Joint Center for Biomedical Innovation, College of Life Sciences, Henan University, Jin Ming Avenue, Kaifeng, Henan, 475004, China. 4 Laboratory of Nanoscale Biosensing and Bioimaging, Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China 5 Centre for Nanoscale BioPhotonics, Macquarie University, North Ryde, NSW, 2109, 6 School of Mathematical and Physical Sciences, University of Technology Sydney (UTS) 7 Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW, 2109, Australia. *Correspondence: YC ([email protected]) or BS ([email protected]) Equal contributors

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ABSTRACT: Ethanol is a principle ingredient of alcoholic beverages with potential neurotoxicity and genotoxicity, and the ethanol-associated oxidative DNA damage in the central nervous system is well documented. Natural source compounds may offer new options to protect the brain against ethanol-induced genotoxicity. Veratrum maackii Regel is a toxic rangeland plant linked to teratogenicity which is also used in traditional Chinese medicine as ‘Lilu’ and is reported to contain a family of compounds called stilbenes that can have positive biological activity. In this study, nine stilbenes were isolated from the aerial parts of V. maackii Regel and their structures were identified as cis-mulberroside A (1), resveratrol-4, 3′-O-β-D-digluco pyranoside (2), mulberroside A (3), gentifolin K (4), resveratrol-3, 5-O-β- D-digluco pyranoside (5), oxyresveratrol- 4'-O-β-D-glucopyrano side (6), oxyresveratrol-3-Oβ-D -glucopyranoside (7), oxyresveratrol (8) and resveratrol (9) using ESI-MS and NMR techniques. The total concentration of extracted compounds 2-9 was 2.04 mg/g, suggesting that V. maackii Regel is a novel viable source of these compounds. In an in vivo comet assay, compounds 1～9 were observed to decrease the DNA damage in mouse cerebellum and cerebral cortex caused by acute ethanol administration. Histological observation also revealed decreased brain injury in mice administered with compounds 1~9 after acute ethanol administration. The protective effects of compound 6 were associated with increasing T-SOD and GSH-PX activities and a decrease in NO and MDA concentrations. These findings suggest that these compounds are potent inhibitors of ethanol-induced brain injury possibly via the inhibition of oxidative stress and may be valuable leads for future therapeutic development.

KEYWORDS:

Veratrum maackii Regel, stilbenes, DNA damage, oxidative stress,

oxyresveratrol-4'-O-β-D-glucoside, ethanol, cerebellum, cerebral cortex 2


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INTRODUCTION Disease associated with excessive ethanol intake is a costly health problem in many countries.1 One of the main toxic targets of ethanol is the central nervous system. The genotoxic and neurotoxic effects of ethanol are well-known.2 In consideration of diseases related to ethanol induced genotoxicity in the brain cells, new natural products may be effective for the prevention of ethanol-induced oxidative DNA damage. Veratrum plants grow in high mountain ranges and their grazing is associated with cyclopic and other craniofacial malformations in the offspring of sheep.3-6 The plant Veratrum maackii Regel (Liliaceae) is an example from this family of toxic rangeland plants and it is also widely distributed in the Jilin, Henan and Shandong provinces of China. V. maackii Regel was used as the Chinese traditional medicine ‘Lilu’, which was first recorded in the ancient document ‘Shen Nong Ben Cao Jing’ (early third centuries B.C.). Lilu was traditionally used to treat aphasia symptoms arising from apoplexy, wind type dysentery, jaundice, headache, scabies, chronic malaria and other disorders,7 and it’s active ingredients include alkaloids, stilbenes, flavonoids, phenols, glyceride, etc. The preparation and biological activity of veratrum alkaloids extracted from the roots and rhizomes of Veratrum plants was extensively reported in the literature.8-10 Stilbene compounds in the aerial parts of Veratrum species were, however, rarely reported, and no quantitative analytical techniques have comprehensively characterized these constituents. At present, only resveratrol,

oxyresveratrol,

polydatin

and

oxyresveratrol-3-O-glucoside

were

separated from these parts and identified.11,12 Notable

health-promoting

properties

associated

3


with

stilbenes

include


cardiovascular disease, cancer prevention,13 antioxidation, neuroprotective benefits, anti-inflammatory activities etc.14,15 and these benefits are linked to its powerful antioxidant and anti-radical action. Contrary to the detailed accounts in reports elsewhere listing various activities after the intake of resveratrol, the understanding of physiological effects of other stilbenes remains limited. Given that two reports11,12 demonstrated the presence of stilbenes in the aerial part of Veratrum, this work aimed to extend these findings to Veratrum maackii Regel and preparatively separate stilbenes to determine the concentrations of these constituents in this species. Other reports have established the protective effects of resveratrol against ethanol-induced oxidative DNA damage in mouse brain,16 and so this work expands these initial findings by evaluating stilbenes for similar biological properties. On this basis, this work further measured both oxidant and antioxidant effects of the stilbene in vivo to establish whether the proposed protective effects of stilbenes against ethanol-induced brain injury are related with the inhibition of oxidative stress. RESULTS AND DISCUSSION Elucidation of Compound Structures. The 60% ethanol extract of the aerial parts of Veratrum maackii Regel was successively chromatographed with a MCI Gel, Silica Gel, ODS and then purified with a semipreparative HPLC to gain the 9 stilbenes. The chemical structures of these stilbenes are listed in Figure 1. Compounds 1～9 were subsequently identified as cis-mulberroside A (1), resveratrol-4, 3′-O-β-Ddiglucopyranoside (2), mulberroside A (3), gentifolin K (4), resveratrol-3, 5-O-β-Ddiglucopyranoside (5), oxyresveratrol-4'- O-β-D-glucopyranoside (6), oxyresveratrol -3-O-β-D-glucopyranoside (7), oxyresvera trol (8) and resveratrol (9) by means of electrospray ionization mass spectrometry (ESI-MS) and NMR techniques (see

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Supplementary Information Figures S2～S21 for the data collected), and the results compared with data published in the literature (Wang et al, 2008; Dai et al, 2009; Nie et al, 2008; Piao et al, 2006; Hano et al, 1997; Xu et al, 1997; Larronde et al, 2005).12,17-22 To best of the authors’ knowledge, it is the first time that compounds 1, 4 and 5 have been described from Veratrum plant and compounds 1～7 from V. maackii Regel. Determination of Compound Concentration by HPLC-UV Quantitative Analysis. The proposed HPLC method for the determination of the eight analytes (Figure 2) showed good linearity (r > 0.9996) (Supplementary Information Tables S1). The precision, stability, and repeatability were all less than 3.0%. The overall recoveries were between 98.48 and 102.24% with RSD less than 1.82% (Supplementary Information Tables S2 and S3). The developed method was applied to determine the sample (n=3). The average amounts of compounds 2～9 were 0.108, 0.065, 0.053, 0.125, 0.092, 0.183, 0.942 and 0.478 mg·g-1, respectively. The total concentration of the eight compounds was close to 2.04 mg/g extracted from the aerial parts of V. maackii Regel. Compounds 1～～ 9 Decrease Brain Cell DNA Damage Induced by Acute Ethanol Administration. The tail moment length of control ethanol group verged on that of blank group with no significant difference (Figure 3). To analyze the potential protective effects of compounds 1-9 in vivo in a model of acute ethanol exposure, DNA damage was investigated in isolated cells from brains of mice following acute ethanol administration with or without treatment with each of the compounds. Vitamin E treatment was included as a positive control, since this has been shown previously to decrease ethanol-induced DNA damage.16 Treatment with each of the isolated compounds 1～9 decreased ethanol-induced DNA damage in the mouse 5



cerebellum and cerebral cortex, as shown by significantly lowered tail moment length when ethanol was administered (Figure 3). These results show that these compounds at doses of both 6 and 24 µmol/kg are protective against ethanol-induced DNA damage. Compounds 1-9 decrease neuron cell damage. The histological slices confirmed from the control ethanol group had the normal number and arrangement neurons in the cerebral cortex and in the cerebellum with the nuclei also appearing morphologically normal in their size, shape and arrangement (Figures 4, 5). In mice administered ethanol alone, neurons were pyknotic with darkly stained and shrunken nuclei in H&E stained sections. Mice in the positive control group, treated with vitamin E prior to ethanol administration, were observed to decreased neuronal damage by morphological observation, with fewer abnormal pyknotic neurons. Similarly, the cerebelli and cerebral cortices of mice administered each of compounds 1~9 prior to ethanol administration also showed decreased presence of abnormal pyknotic neurons, with more neurons appearing normal in size, shape and arrangement (Figure 4, 5). Effects of Compound 6 on Levels of NO and MDA in Brain Cell and Plasma After Acute Ethanol Administration. The production levels of NO and MDA in mouse cerebellum, cerebral cortex and plasma after acute ethanol treatment, are shown in Figure 6. Significant increases of NO and MDA levels in the ethanol group, compared with the control ethanol group, were observed after ethanol administration. When the mice were pretreated with compound 6 at the doses of 6 and 24 µmol/kg before the ethanol treatment, in compound 6 plus ethanol groups the levels of NO and MDA in the mouse cerebellum, cerebral cortex and plasma was less than that seen in the ethanol group.

6


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Effects of compound 6 on activities of T-SOD and GSH-PX in Brain Cell and plasma after Acute Ethanol Administration. T-SOD and GSH-PX capacities in mouse cerebellum, cerebral cortex and plasma after acute ethanol treatment are shown in Figure 7. Compared with the control ethanol group, T-SOD and GSH-PX activities of the ethanol group were significantly decreased in mouse cerebellum, cerebral cortex and plasma. T-SOD and GSH-PX activities in compound 6 plus ethanol groups had a marked increase compared to the ethanol group. Veratrum maackii Regel is widely distributed across Japan, Korea, Northern China, and Eastern Siberia and also used as a traditional medicine in many Asian countries. However, the Veratrum plant can also cause birth defects such as cyclopic-type craniofacial defect through the action of its alkaloids. In this work, stilbenes were separated from the aerial parts of Veratrum maackii (Regel) without the alkaloids, and this process revealed that they contain high levels of stilbenes, and so the contents of these compounds were further determined in this plant. The results demonstrated that the aerial parts contained high concentrations of resveratrol and oxyresveratrol. The concentration of oxyresveratrol was almost twice that of resveratrol, and nearly seventeen times that of compound 4 with the lowest level detected in this species. The total concentration of the eight stilbenes identified (compound 2-9) was 2.04 mg/g, close to the total stilbenes concentration in grapevine canes of twenty-two grape cultivars (2.5mg/g～5.8 mg/g dw).23 The stilbene family of compounds is important to the plant’s defense mechanism.24 Among these, resveratrol is the best known stilbene. However, bi/mono-glycoside derivatives are also bioactive and present in large amounts in the plant. Compounds 2, 3, 5, 6 and 7, as bi/mono-glycosylated stilbenes were derived from their precursors resveratrol (9) and oxyresveratrol (8). But there was no direct

7



correlation between the levels of compounds 2, 3, 5, 6 and 7 and the levels of compounds 8 and 9. This finding suggests strict control over the metabolite flow through the plant biosynthetic pathways leading to the formation of these bi/mono-glycoside derivatives.25 It was also elsewhere reported that resveratrol and its glycoside derivatives, such as polydatin, can mutually transform in rats both with the ability of anti-oxidative stress in vivo,26 and this hints that bi/mono-glycosylated stilbenes may be as an antioxidant substitute for resveratrol in the clinic. Since phytoalexins exist in dozens of foods and in medicinal plants, stilbenes have many uses related to health and medicine and as such have become the focus of much research. The biological properties of stilbenes include anti-oxidative activity, neuroprotection, anti-tumor, anti-bacterial, and other biological effects. Amongst these, what is notable is resveratrol’s well known protective activity against ethanol-induce oxidative DNA damage in mice brain cells. Because of the similarity between their structures and resveratrol, it was hypothesised that compounds 1～8 would also have this protective property. This is the first study on the protective effect of compounds 1～8 on mouse brain cell DNA damage induced by acute ethanol administration. The results presented in Figure 3 show that this group of compounds significantly reduced tail moment lengths seen after ethanol administration, and demonstrated that compounds 1～9 markedly decrease the levels of DNA single-strand breaks in mouse cerebellum and cerebral cortex after ethanol was administered at 6.0g/kg i.p., and this is especially so for compounds 6 and 9 (Figure 8). Resveratrol (compound 9) displayed the strongest inhibition of ethanol-induced DNA damage, indicating that the structural characteristics of small polarity may contribute to its protective ability. Compounds 1～7 are stilbenes glycosides and so have greater polarity; while unlike resveratrol, compound 8 contains an additional hydroxyl group in its structure. It is 8


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consistent with reports elsewhere that the more lipophilicity structure of resveratrol may help its ability to cross cell membranes.27 Because the sum concentrations of resveratrol and oxyresveratrol account for about 70% of the total stilbene concentration extracted from aerial parts of Veratrum maackii Regel., it is possible that these play a core role in the protective effect described in this work. In support of these notions, histological observation demonstrates that brain injury after administration of compounds 1~9 was significantly less than in untreated mice after ethanol administration. The outcomes seen in the comet experiment also supports this conclusion: that compounds 1~9 decrease damage caused by ethanol in the cerebral cortex and cerebellum neurons. The suppression of ethanol-induced DNA damage in mice after the addition of resveratrol might be due to its ability to inhibit ethanol-induced oxidative-stress.16 This suggests it might be worthwhile investigating other stilbene compounds for a possible role in protecting the brain from ethanol-induced genotoxicity. It is well-known that acute intake of ethanol induces oxidative DNA damage in certain brain regions through oxidative stress. To relieve this stress, brain tissue activates an endogenous anti-oxidant defense system that includes enzymatic and non-enzymatic anti-oxidant mechanisms. The enzymatic anti-oxidant mechanism is mediated through SOD, GSH-PX and thioredoxin reductase, and the non-enzymatic anti-oxidant defense is including anti-oxidants such as vitamins, phenols etc.28-30 Since there are no reports elsewhere of stilbene glycoside’s ability to reduce DNA damage in the brain, in this work the most active compound 6 was selected for further investigation into its effect on the levels of NO and MDA and the activities of SOD and GSH-PX in the cerebellum, cerebral cortex and blood of mice under stress. The results demonstrated compound 6’s antioxidant activities via its promotion of

9



SOD and GSH-PX activity and its inhibition of NO and MDA production. According to hydroxyl groups in the benzene ring making compounds easy to inactivate oxidative radicals,31 it was assumed that the other seven stilbenes would have the same antioxidant properties, since they all belong to the 1, 2-diphenylethylene congener with the hydroxyl groups. Drugs usually enter the bloodstream first and then cross into the brain, so compound 6 might synchronously exert antioxidant actions in the peripheral system and in brain tissue improving the antioxidant capacity of the whole organism. The findings imply that compounds 1～9 suppress oxidative stress induced by ethanol via its potent antioxidant action. This is the first systemic study to identify and evaluate bioactive components extracted from the aerial parts of Veratrum maackii Regel. for their anti-oxidative neuroprotective properties. It is also the first time for compounds 1～7 to be obtained from Veratrum maackii Regel, and for compounds 1, 4 and 5 to be extracted from the genus Veratrum. The compounds were observed to decrease ethanol-induced oxidative DNA damage in mouse brain cells, possibly via inhibition of oxidative stress. Veratrum maackii Regel should therefore be regarded not just as a toxic grassland plant species with teratogenicity properties, but as a major source of stilbenes with potentially useful properties. METHODS General Experimental Procedures. Deionized water was prepared using a Milli-Q reagent water system (Millipore, Billerica, MA, USA). HPLC grade methanol was purchased from Tianjin Siyou Chemical Agencies (Tianjin, China). Stilbenes were isolated using a silica gel (SiO2, 200 to 300 mesh, Qingdao Haiyang Chemical Group Co., Qingdao China) and MCIGel CHP20 (Mitsubishi, Japan) column. A nitric oxide (NO) assay kit, methane dicarboxylic aldehyde (MDA) assay kit, methane 10


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dicarboxylic aldehyde (SOD) assay kit, and Glutathione Peroxidase (GSH-PX) assay kit were purchased from the Nanjing Jiancheng Bioengineering Institute (China). All other organic and inorganic reagents (of analytical grade) were provided by local or international suppliers. NMR spectra were acquired using CD3OD at 25°C on an Avance III HD 400 MHz instrument (H, 400 MHz, C, 100 MHz, Bruker BioSpin, Rheinstetten, Germany) equipped with a broadband inverse (BBI) probe. Standard pulse sequences and parameters were used to obtain the following: 1D H spectra, 1D C spectra, heteronuclear

multiple-bond

correlation

(HMBC)

spectra,

band-selective

heteronuclear single quantum coherence (HSQC) spectra. Chemical shift referencing was carried out using internal solvent resonances at δH 3.31 and δC 49.0 (calibrated to tetramethylsilane, TMS, at 0.00 ppm). Data processing was performed with Topspin software (version 3.5pl2, Bruker BioSpin, Rheinstetten, Germany). Material. The aerial parts of Veratrum maackii Regel were obtained from Longyuwan National Forest Park (Luoyang, China) and their identification confirmed by associate Prof. Wang-Jun Yuan at Henan University and a voucher specimen (2012007) was deposited at the Institute of Pharmacy, Pharmaceutical College, Henan University, China. Male Kunming mice (22 g ± 2 ) were sourced from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China). Animal experiments were conducted in accordance with the guidelines established by the National Research Council’s (USA) document “NIH Guide for the Care and Use of Laboratory Animals” and the protocol approved first by the animal care and use committee of Henan University before its implementation. Isolation of Stilbenes. Veratrum maackii Regel aerial parts were finely crushed to powder and extracted with ethanol [95% (v/v) ethanol in water] under reflux. The

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ethanol extract was purified in a stepwise manner using various chromatographic columns (CC); The extract was applied to a CC (SiO2, CHCl3/MeOH 100:1→1:1) to yield 10 combined fractions. Fr. 5 (14 g) was repeatedly separated by CC (SiO2, CHCl3/MeOH 10:1) to yield compound 8 (42 mg) and compound 9 (31 mg). Fr. 7 (17 g) was further subjected to MCIGEL (CHP20, MeOH/H2O 40:60) to get subfr.7-3. The semi-preparative HPLC was carried out using a Shim-pack CLC-ODS/H column (250mm×20mm, 10µm, Shimadzu Co., Japan) attached to a SHIMADZU SPD-10A UV detector. Subfr. 7-3 was then applied to a semi-preparative HPLC (RP-18， MeOH/H2O 33:67) to give compound 4 (20 mg), compound 5 (36 mg), compound 6 (37 mg) and compound 7 (19 mg). Fr. 8 (15 g) was further subjected to MCIGEL (CHP20, MeOH/H2O 30:70) to get subfr.8-2, which was then applied to a semi-preparative HPLC (RP-18, MeOH/H2O 28:78) to give compound 1 (9 mg), compound 2 (13 mg) and compound 3 (27 mg). Determination of Compounds 2~9 Concentrations in the Aerial Parts of Veratrum Maackii Regel. Experiments were performed using a SHIMADZU LC-20AT series LC system (SHIMADZU Corporation, Japan) that consisted of a vacuum degasser, quaternary pump, auto-sampler, a UV(SPD-20A) and a DAD(SPD-M10AVP) detector. The chromatography was carried out using a Cosmosil 5C18-MS-Ⅱ column (5 µm, 250 mm×4.6 mm i.d., Nacalai Tesque inc., Japan) at a temperature of 25℃. The HPLC linear gradient profile was as follows: acetonitrile:water (containing 0.1% Acetic acid) 10:90 v/v -38:62 (0-20 min), 38:62-100:0 (20-30 min) and 100:0 (30-38 min) run at a flow rate of 0.8 mL/min. The UV detection wavelength was set to 308 nm on the basis of the result of ultraviolet full-wavelength scan. The injection volume used was 5µL. All 8 peaks were identified by comparing the retention time and UV spectra (Supplementary Information Figure 12


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S1) with isolated compounds. The aerial parts of Veratrum maackii Regel were ground into powder and sized by passing it through an 80 mesh. 0.5 g of the processed dried powder was then added to 10 mL of a 60% methanol-water mix held in a triangle bottle with a stopper and then weighed. The powder was extracted ultrasonically using a KQ 3200 apparatus (Kunshan Ultrasonic Co., China) for 30 min at 25℃, weighed to add weight loss and mixed. The triangle bottle was placed on the bench for 10 min to settle. 2 mL of the sample was then filtered through a 0.22µm microporous membrane for its analysis with LC. A standard stock of compounds 2～9 in 0.5 mg/mL solutions were respectively prepared with methanol. Calibration curves were prepared by diluting the stock solutions of eight standards to form a series at the appropriate concentrations. The precision of the dilutes was evaluated with intra- and inter-day variations of the standard mixed solution. The stability and repeatability were tested with the same sample, and its accuracy evaluated by running recovery tests. Dosage and Treatment. Comet Assay. Eighty-eight mice were randomly divided into twenty-two groups each with four mice and included a blank group, a control ethanol group, an ethanol group, a positive group and compounds 1～9 plus ethanol groups. Seventy-two animals from compounds 1～9 plus ethanol groups were orally treated with compounds 1～9 at doses of 6 and 24 µmol/kg for 3 consecutive days. Animals in the positive group were treated using a gavage with vitamin E (VE) at 50 mg/kg for 3 consecutive days. Thirty minutes after the last treatment, the compounds 1～9 plus ethanol groups, positive group and the ethanol only group were treated with an i.p. injection of ethanol at 6.0 g/kg (0.2 mL/10 g b.wt.), and control ethanol group mice were given an i.p. injection of saline. Blank group mice were administered with saline orally. Ethanol was diluted to 30% (v/v) with saline for i.p. injection. Four 13



hours after the administration of ethanol, the animals were culled via decapitation. Histological Observation. Dosage and treatment were the same as that used for the comet assay tests except the blank group and the compounds 1～9 at the low dose plus the ethanol groups were not included. MDA, NO, T-SOD and GSH-PX Assays. Eighty mice were randomly divided into five groups each with sixteen mice. Each group included a control ethanol group, an ethanol group, a positive group and compound 6 plus the ethanol groups. Thirty-two animals in compound 6 plus ethanol groups were orally treated with compound 6 at the doses of 6 and 24 µmol/kg for 3 consecutive days. Animals in positive group were treated using a gavage with vitamin E at 50 mg/kg for 3 consecutive days. Thirty minutes after the last treatment, compound 6 plus ethanol groups, positive group and the ethanol only group were treated with an i.p. injection of 30% (v/v) ethanol at 6.0 g/kg (0.2 mL/10 g b.wt.), and control ethanol group mice were given an i.p. injection of saline. Four hours after ethanol administration, blood was collected to a heparinized tube from the orbital vessels of each animal, before culling the animals via decapitation. Comet Assay. The brains from the mice were removed and dissected immediately on ice. Two regions, the cerebellum and the cerebral cortex, were taken for analysis. The brain regions were minced, then suspended at 1 mL/g in chilled homogenizing buffer (containing PBS/NaCl, 8.01 g, KCl, 0.2 g, Na2HPO4, 2.9 g, KH2PO4, 0.2 g/L), before homogenizing gently manually. To obtain nuclei, the homogenate was centrifuged at 1000 r·min-1 for 5 min, and the precipitate resuspended in chilled homogenizing buffer for its analysis in the comet assay. The comet assay was performed under alkaline conditions according to a procedure described elsewhere.16 Tail moment was used as a parameter to evaluate the DNA

14


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damage. Objects were observed at 200×magnification using an IX53 fluorescence Microscope (Olympus, Japan) connected to image analysis software on a Windows PC (CASP Comet Assay software). For each treatment group, 150 cells were examined. The parameters were calculated using the automatic CASP image analysis system. HE Dye method. Brain tissues were first fixed by soaking in 4% paraformaldehyde for more than a day. They were then fixed with xylene before roasting for its dewaxing, and dehydrated using a gradient ethanol procedure. Paraffin embedding was performed and paraffin sections were hematoxylin stained, with 70% alcohol used to separate the color when the specimens became blue. Specimens were rinsed with distilled water, before staining with 1% eosin for 3 to 5 min, dehydrated with a 70%, 80%, 90% ethanol gradient, immersed in transparent xylene and then sealed with a neutral resin. MDA, NO, T-SOD and GSH-PX assays. Brain regions were minced, suspended at a 9 mL/g concentration in chilled physiological saline using an ice water-bath, and then homogenized manually. The homogenate was centrifuged at 3000 r·min-1 for 10 min to obtain the supernatant. The blood was also centrifuged at 3000 r·min-1 for 15 min to harvest the plasma. The supernatant and plasma were then tested with NO, MDA, T-SOD and GSH-PX assays using commercial kits. All the procedures complied with the manufacturer’s instructions. NO reacts with oxygen and water to produce nitrate and nitrite, which then reacts with a chromogenic agent to produce a pink azo compound. The NO content can be calculated indirectly by measuring the OD value of the solution under 550 nm excitation. The MDA content was determined using a thiobarbituric acid method, which forms a red product that has a maximum absorbance under 532 nm excitation.

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The T-SOD assay provides an indication of a compound’s ability to inhibit oxidation of oxymine using a xanthine-xanthine oxidase system that was excited at 550 nm. GSH-PX can catalyze H2O2 and reduced glutathione (GSH) to produce H2O and oxidized glutathione (GSSG), which is used to calculate enzyme activity by measuring the GSH consumption in an enzymatic reaction excited at 412 nm. Statistical Analysis. Data was collected and used to calculate with a mean ± SD. Statistical comparisons were made with a one-way analysis of variance (ANOVA) test and a Fisher’s least significant difference (LSD) test. The level of significance was set at P< 0.05. Statistical procedures were performed with Graphpad Prism version 6.0 software on Windows. ASSOCIATED CONTENT Supporting Information UV spectra of compounds 2-9(A) and corresponding 8 peaks are shown in Figure S1; NMR Spectrum of compound 1～9 are shown in Figures S2～S21; Calibration curves, linear range, precision, repeatability, stability and recovery tests of eight analytes are shown in Tables S1～S3. AUTHO R INFORMATION Author Contributions Yue Cong and Bingyang Shi conceived and designed the experiments; Yantong Wu, Shasha Li, Jinjin Liu and Xiping Liu performed the experiments; Weimin Ruan, Jeng-Wei Lu and Yong Liu analyzed the data; Tom Lawson, Olga Shimoni, David Lovejoy and Adam K. Walker proofread the writing; Yantong Wu, Shasha Li, Yue Cong and Bingyang Shi wrote the paper. Corresponding Authors *E-mail: [email protected]. Tel: (371) 2388-0680 16


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Figure captions Figure 1. Structures of compounds 1-9. Compound 1: cis-mulberroside A; Compound 2: resveratrol-4, 3′-O-β-D-diglucopyranoside; Compound 3: mulberroside A; Compound 4: gentifolin K; Compound 5: resveratrol-3, 5-O-β-D-diglucopyranoside; Compound 6: oxyresveratrol-4'-O-β-D-glucopyranoside; Compound 7: oxyresveratrol -3-O-β-D-glucopyranoside; Compound 8: oxyresveratrol; Compound 9: resveratrol

Figure 2. HPLC chromatograms of reference substances (compounds 2-9) (A) and a sample of the aerial part of Veratrum maackii Regel (B) at 308nm.

Figure 3. Effects of compounds 1～9 against ethanol-induced oxidative DNA damage in the cerebellum (A) and the cerebral cortex (B) of mice. Tail moments are shown as mean ± SD. ***, P

Stilbenes from Veratrum maackii Regel. Protect against Ethanol

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