Glucaric acids from Leonurus japonicus

    Glucaric acids from Leonurus japonicus Jianshuang Jiang, Yixiu Li, Ziming Feng, Yanan Yang, Peicheng Zhang PII: DOI: Reference:

S0367-326X(15)30101-5 doi: 10.1016/j.fitote.2015.10.007 FITOTE 3280

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

3 August 2015 14 October 2015 21 October 2015

Please cite this article as: Jianshuang Jiang, Yixiu Li, Ziming Feng, Yanan Yang, Peicheng Zhang, Glucaric acids from Leonurus japonicus, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.10.007

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ACCEPTED MANUSCRIPT Glucaric acids from the Leonurus japonicus Jianshuang Jiang a, Yixiu Li b, Ziming Feng a, Yanan Yang a, Peicheng Zhang a,* a

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of

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Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s

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Republic of China

Department of pharmacy, the second affiliated hospital of Nanchang, Nanchang 330000, People’s Republic of China

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b

Corresponding auther. Tel: 86-10-63165231. E-mail address: [email protected]

Abstract: Three new glucaric acids, namely 2-feruloyl-4-syringoyl or 5-feruloyl-3-syringoyl glucaric

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acid (1), 2-syringoyl-4-feruloyl or 5-syringoyl-3-feruloyl glucaric acid (2), and 3-feruloyl-4-syringoyl or 4-feruloyl-3-syringoyl glucaric acid (3), were isolated from the Leonurus japonicus Houtt. Their

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structures were elucidated by detailed spectroscopic means including UV, IR, HR-ESI-MS, 1D and 2D NMR data spectra. The bioactive assays of compounds 1-3 against hepatoprotection activity were determined. The result suggested compound 2 exhibited a moderate hepatoprotection activity and the cell survival rate was 74% (10-5 mol/L), using bicyclol (survival rate: 66%, 10-5 mol/L) as a positive

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control. Furthermore, compound 1-3 were evaluated cytotoxic activities in vitro using HCT-8,

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Bel-7402, BGC-823, A-549, and A2780 model and the results exhibited no obvious cytotoxicity activity.

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Keywords: Leonurus japonicus Houtt; Glucaric acid; Hepatoprotection activity; Cytotoxicity activities

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Introduction Leonurus japonicus Houtt. has been used as a remedy for regulating menstrual disturbance,

invigorating blood circulation, diuretics, and dispel edema [1]. Up to date, many constituents such as

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labdane-type diterpenoids, alkaloids [2-3], phenylethanoid glycosides [4-5], iridoids [5], flavonoids and

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cyclic peptides [7] were isolated from this plant. Among them, alkaloids and diterpenoids were consider as active compounds and have exhibited anti-platelet aggregation. In previous paper, we have

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reported the structural elucidation of two phenylethanoid glycosides and one sesquiterpene glycoside isolated from this plant [8]. Continued research has resulted in three new glucaric acids were isolated, and named 2-feruloyl-4-syringoyl or 5-feruloyl-3-syringoyl glucaric acid (1), 2-syringoyl-4-feruloyl or 5-syringoyl-3-feruloyl glucaric acid (2), and 3-feruloyl-4-syringoyl or 4-feruloyl-3-syringoyl glucaric

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acid (3) (Fig 1). These new glucaric acids were isomers including same moeities of glucaric acid, feruloyl, and syringyl. This was the first report that the natural products with feruloyl and syringoyl groups of glucaric acid were obtained. And compounds 1-3 were evaluated for hepatoprotection and

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cytotoxicity activities. 2. Experimental 2.1 General procedures

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The optical rotations were measured on a Jasco P-2000 polarimeter. IR spectra were recorded on an 13

C NMR (125 MHz), HSQC, and HMBC

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IMPACT 400 (KBr) spectrometer. 1H NMR (500 MHz),

spectra were run on an INOVA-500 spectrometer. HR-ESI-MS spectra were performed on a Finnigan LTQ FT massspectrometer. ESI-MS were recorded on an Agilent 1100 series LC/MSD TOF from

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Agilent Technologies. Column chromatography was performed with Macroporous resin (Diaion HP- 20, Mitsubishi Chemical Corp. Tokyo, Japan), Rp-18 (50 μm; YMC, Kyoto, Japan), and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). Preparative HPLC was carried out on a Shimadzu μm).

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LC-6AD instrument with a SPD-20A detector, using a YMC-Pack ODS-A column (250× 20 mm, 5

2.2 Plant material

The dried aerial part of Leonurus japonicus Houtt. was purchased from Tongrentang Pharmacy, and identified by Professor Lin Ma of the Chinese Academy of Medical Sciences. A voucher specimen (NO. ID-S-2387) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences. 2.3 Extraction and isolation The Leonurus japonicus Houtt. (100 kg) were exhaustively extracted with 30% ethanol under refluxed conditions. The extracts were then concentrated under reduced pressure to give a residue (3000 g). The residue was dissolved in 95% ethanol, and the 95% ethanol was concentrated to give a residue. Then the residue was subjected to chromatographing over macroporous adsorbent resin (HP-20) column. After eluting with H2O, then the adsorbed constituents were eluted with 15% ethanol, 30% ethanol, and 50% ethanol, respectively. The 30% ethanol part (99 g) was chromatographed over Sephadex LH-20 eluting with H2O–MeOH (from 100:0 to 0:100) to give 10 fractions. Fr.2 was purified by reversed-phase preparative HPLC using CH3OH–H2O (10:90) as mobile phase to yield compound 1 (12 mg). Using MeOH–H2O (10:90) as the mobile phase, Fr.5 was further purified by reversed-phase

ACCEPTED MANUSCRIPT preparative HPLC, resulting in 2 (15 mg) and 3 (10 mg). 2-feruloyl-4-syringoyl or 5-feruloyl-3-syringoyl glucaric acid (1). white powder. []

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= -8.67 (c =

0.014, H2O); UV (MeOH) λmax: 216, 278 nm; IR max cm : 3397, 2958, 1696, 1603, 1514, 1464, 1348, -1

1285, 1225, 1117, 1030 cm-1; 1H NMR (D2O, 500 MHz) and 13C NMR (D2O, 125 MHz) see Table 1

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and 2. ESI-MS m/z: 565 [M-H]; HR-ESI-MS m/z: 589.1165 [M+Na]+ (calcd for C25H26O15Na,

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589.1272).

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2-syringoyl-4-feruloyl or 5-syringoyl-3-feruloyl glucaric acid (2). white powder. [] D = -6.28 (c 0.077, H2O), UV (MeOH) λmax: 216, 278 nm; IR max cm-1: 3397, 3016, 1691, 1632, 1517, 1428, 1205, 1133, 1022, 954 cm-1; 1H NMR (D2O, 500 MHz) and +

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C NMR (D2O, 125 MHz) see Table 1 and 2.

HR-ESI-MS m/z 589.1160 [M+Na] (calcd for C25H26O15Na, 589.1272).

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3-feruloyl-4-syringoyl or 4-feruloyl-3-syringoyl glucaric acid (3). white powder. UV (MeOH) λmax: 216, 278 nm; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) see Table 1 and 2. [M+H]+ (calcd for C25H27O15, 567.1350).

2.4 Alkaline hydrolysis of compound 1-3

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HR-ESI-MS m/z 567.1299

A 0.1 M K2CO3/D2O solution of compound (1.0 mg) was heated at 80 °C for 1 h. After cooling, the hydrolysis product was analysed by 1H NMR.

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2.5. Hepatoprotective effects on cytotoxicity induced by Dgalactosamine in HL-7702 cells

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Compounds were tested for hepatoprotective effects using an MTT assay in HL-7702 cells [9]. Each cell suspension of 1×105 cells in 1 mL of Dulbecco’s modified Eagle’s medium containing fetal calf

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serum (10%), penicillin (100 units/mL) and streptomycin (100 μg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium containing bicyclol and the test samples was added, and the cells were cultured for 1 h. The cultured cells were exposed to 25 mM Dgalactosamine for 24 h. The medium was then changed to fresh medium

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containing 0.5 mg/mL MTT. After an incubation of 4 h, the medium was removed, and DMSO was added to dissolve formazan crystals. The optical density (OD) of the formazan solution was measured on a microplate reader at 492 nm. Inhibition (%) was obtained by the following formula: Inhibition (%) = [(OD(sample)－OD(control))/(OD(normal)－OD(control))]× 100. 2.6. Cytotoxicity assay in vitro Cytotoxicity against human tumor cell was measured in a 5-day MTT test for HCT-8 (human ileocecal carcinoma), Bel7402 (human hepatocellular cancer), BGC-823 (stomach adenocarcinoma), A549 (human lung carcinoma), and A2780 (ovary adenocarcinoma) by employing the revised MTT method in literature [10]. Briefly, 1×103 cells/100 μl were seeded in 96-well microplates and preincubated for 24 h to allow cell attachment. This medium was then aspirated, and 100 μl of fresh medium containing various concentrations of the test drug were added to the cultures. The cells were incubated with each drug for 5 days. Cell survival was evaluated by adding 50 μl of MTT reagent (5 mg MTT/ml in RPMI 1640 medium) to each well. After 4 h reincubation at 37°C, 100 μl DMSO were added to dissolve the precipitate of reduced MTT. Microplates were agitated on a rotation platform at room temperature for 15 min, and the absorbance of the reaction mixtures was determined at 570 nm

ACCEPTED MANUSCRIPT with a multiwell scanning spectrophotometer.

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3. Results and discussion

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of glucaric acid, respectively. The unambiguous positional isomers of 1 was not determined by NMR spectra experiments because of the pseudo symmetry of glucaric acid [12]: its non-rigid chain results in many possible conformations in solution. Moreover, the vital dihedral angles were not obtained by the coupling constants in NMR spectra [13], so the 1H and 13C NMR spectra data of the glucaric acid could not be absolutely assigned.

tautomers as

ACCEPTED MANUSCRIPT Compound 2 was isolated as a white powder. The IR spectrum of 2 showed the presence of hydroxyl, carbonyl, and aromatic groups. Compound 2 possessed a molecular formula of C25H26O15, which was determined by HR-ESI-MS. The 1H NMR data (Table 1) showed a feruloyl group signals including a trans olefinic hydrogen signals at  7.07 (1H, d , J = 16.0 Hz) and 6.11 (1H, d, J = 16.0 Hz), a ABX

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system proton signals at  6.90 (1H, d , J = 1.5 Hz), 6.85 (1H, d, J = 8.0 Hz), and 6.83 (1H, dd, J = 8.0,

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1.5 Hz)], and a methyl proton signal at  3.88 (3H, s). Additionally, a syringoyl group signals including a X2 system proton signals at  7.35 (2H, s) and a methyl proton signal at  3.82 (6H, s), and a hexaric

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acid group signals including at  5.36 (1H, d, J = 6.5 Hz), 5.30 (1H, d, J = 1.0 Hz), 4.78 (1H, d, J = 1.0 Hz), and 4.31 (1H, d, J = 6.5 Hz) were observed. And the NMR data of 2 were closely similar to those of compound 1 except for those of the hexaric acid unit in compound 2. The difference of chemical shift of the hexaric acid unit suggested the linkage sites change of feruloyl and syringoyl in compound

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2. This was further confirmed by the detailed HMBC analysis (Fig. 3). The C-7″ of the syringoyl unit was confirmed to link to C-2 or 5 of the hexaric acid by the correlations of C-7″/H-2 or 5 in the HMBC

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experiment. Similarity, The connection between C-9′ of the feruloyl unit and C-4 or 3 of the hexaric acid was verified by the cross-peak between H-4 or 3 and C-9′ in the HMBC experiment. Above all the results, the syringoyl moiety and the feruloyl moiety were linked to the position C-2,4 and C-5,3 of hexaric acid, respectively. Furthermore, the absolute configuration of hexaric acid moiety in 2 was

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determined as glucaric acid of 1 by hydrolysis under alkaline conditions. Therefore, compound 2 was

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elucidated as 2-syringoyl-4-feruloyl or 5-syringoyl-3-feruloyl glucaric acid. Compound 3 was obtained as a white powder. The molecular formula was determined to be C25H26O15 as indicated by HR-ESI-MS. The 1H NMR spectrum of 3 (Table 1) also exhibited a feruloyl

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group signals including a trans olefinic hydrogen signals at  7.42 (1H, d , J = 16.0 Hz) and 6.24 (1H, d, J = 16.0 Hz), a ABX system proton signals at  7.17 (1H, d , J = 2.0 Hz), 6.75 (1H, d, J = 8.5 Hz), and 6.98 (1H, dd, J = 8.5, 2.0 Hz), a phenolic hydroxyl proton signal at  9.59 (1H, brs), and a methyl

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proton signal at  3.77 (3H, s), as well as a syringoyl group signals including a X2 system proton signals at  7.11 (2H, s), a phenolic hydroxyl proton signal at  9.27 (1H, brs), and a methyl proton signal at  3.74 (6H, s). Furthermore, a hexaric acid group signals including at  4.33 (1H, d, J = 3.0 Hz), 5.58 (1H, d, J = 9.0, 3.0 Hz), 5.86 (1H, d, J = 9.0, 3.0 Hz), 4.44 (1H, d, J = 3.0 Hz) were showed in the 1H NMR spectrum. The 13C NMR spectrum of 3 showed 25 carbon signals (Table 2), except for 19 carbons of feruloyl and syringoyl carbons, six carbon signals of a hexaric acid group at  172.6, 172.4, 74.7, 72.1, 69.9, and 69.3 were observed. The absolute configuration of hexaric acid moiety in 3 was confirmed as glucaric acid by the same method as compounds 1 and 2. Since the NMR data of 3 were closely similar to those of compound 1 except for chemical shift and the coupling constant of the glucaric acid unit, it suggested the linkage sites of feruloyl and syringoyl were changed. These were further confirmed by HMBC spectrum experiment (Fig. 4). In HMBC spectrum, the correlations of C-9′ (feruloyl)/H-3 or 4 (glucaric acid), C-7″ (syringoyl)/H-4 or 3 (glucaric acid), suggested that the feruloyl moiety and the syringoyl moiety were linked to C-3,4 or C-4,3 of glucaric acid, respectively. Therefore, compound 3 was determined to be 3-feruloyl-4-syringoyl or 4-feruloyl-3-syringoyl glucaric acid. In the vitro bioactive assays, compounds 1-3 were tested for their hepatoprotection activity.

ACCEPTED MANUSCRIPT Compound 2 exhibited a moderate hepatoprotection activity and the cell survival rate was 74% (10-5 mol/L), using bicyclol (survival rate: 66%, 10-5 mol/L) as a positive control. All these compounds were in vitro evaluated for their cytotoxic potential against five tumor cell lines of HCT-8, Bel-7402, BGC-823, A-549, and A2780 mode by using the revised MTT method. The results showed that

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compounds 1-3 exhibited no obvious cytotoxicity activity.

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Acylation of hexaric acid derivatives have been reported in many literatures, These acyl groups included caffeoyl [12], feruloyl, coumaroyl [14,15], galloyl [16], etc. The acyl substitutions of a

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molecular in most of the reported structures were the same, such as monocaffeoyl [12], monoferuloyl [14], dicaffeoyl [13], diferuloyl [15], tricaffeoyl [17], etc. Intriguingly, compounds 1-3 contained two different acyl substituents (feruloyl and syringoyl) in their structures. This was the first report that the natural products with feruloyl and syringoyl groups of glucaric acid were obtained. However, the

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pseudo symmetry of glucaric acid in the above literatures was not ultimately distinguished linkage positions of acyl groups at C-2 or 5 and at C-3 or 4 by NMR spectra, Thus, such types of glucaric acid

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were only confirmed as tautomers. Acknowledgments

This research was supported by Technology Project of China (No. 2012ZX09301002). The authors

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thank Prof. Lin Ma for the plant identification and Prof. YingHong Wang for recording NMR spectra.

References

[1] National Pharmacopoeia Commission.Chinese Pharmacopoeia. Vol. 1. Beijing: China Medical

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Science Press 2005;203–4.

[2] Savona G, Piozzi F, Bruno M, Rodriguez B. Diterpenoids from Leonurus sibiricus. Phytochemistry 1982;21:2699–701.

[3] Po M-h, En S-w, Stellak M-l, Yuen M-c, Chi M-l, Henry N-c. Preleoheterin and leoheterin, two

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labdane diterpenes from Leonurus heterophyllus. Phytochemistry 1993;33:639–41. [4] Çaliş İ, Ersöz T, Tasdemir D, Ruedi P. Two phenylpropanoid glycosides from Leonurus glaucescens. Phytochemistry 1992;31:357–9. [5] Tasdemir D, Scapozza L, Zerbe O, Linden A, Sticher O, Çaliş İ. Iridoid glycosides of Leonurus persicus. J Nat Prod 1999;62:811–6. [6] Cong Y, Wang J-h, Li X. A new flavonoside from Leonurus heterophyllus. J Asian Nat Prod Res 2005;7:273–7. [7] Morita H, Lizuka T, Gonda A, Itokawa H, Takeya K. Cycloleonuripeptides E and F, Cyclic nonapeptides from Leonurusheterophyllus. J Nat Prod 2006;69:839–41. [8] Li Y-x, Chen Z, Feng Z-m,Yang Y-n, Jiang J-s,Zhang P-c. Hepatoprotective glycosides from Leonurus japonicus Houtt. Carbohydrate Research 2012;348:42–6. [9] Liu Y-f, Liang D, Luo H, Hao Z-y, Chen R-y, Yu D-q. Hepatoprotective iridoid glycosides from the roots of Rehmannia glutinosa. J Nat Prod 2012;75:1625–31. [10] Liu R, Ma S-g, Yu S-s, Pei Y-h, Zhang S, Chen X-g, Zhang J-j. Cytotoxic oleanane triterpene saponins from Albizia chinensis. J Nat Prod 2009;72:632–9.

ACCEPTED MANUSCRIPT [11] Takenaka M, Yan X-j, Ono H, Yoshida M, Nagata, T, Nakanishi T. Caffeic acid derivatives in the roots of Yacon (Smallanthus sonchifolius). J Agric Food Chem 2003;51(3):793–6. [12] Ruiz A, Mardones C, Vergara C, Baer D, Gómez-Alonso S, Gómez M-v, Hermosín-Gutiérrez I.

acids from Calafate Berries. J Agric Food Chem 2014;62:6918–25.

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Isolation and structural elucidation of anthocyanidin 3,7-β‑O‑diglucosides and caffeoyl-glucaric

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[13] Maas M, Petereit F, Hensel A. Caffeic acid derivatives from Eupatorium perfoliatum L. Molecules 2009;14:36–45.

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[14] Risch B, Herrmann K, Wray V, Grotjahn L. 2′-(E)-O-p-Coumaroylgalactaric acid and 2′-(E)-O-feruloylgalactaric acid in citrus. Phytochemistry 1987;26(2):509–10. [15] Risch B, Herrmann K, Wray V. (E)-O-p-Coumaroyl, (E)-O-feruloyl-derivatives of glucaric acid in citrus. Phytochemistry 1988;27(10):3327–9.

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[16] ZhangY-j, Tanaka T, Yang C-r, Kouno I. New phenolic constituents from the fruit juice of Phyllanthus emblica. Chem Pharm Bull 2001;49(5):537–40. [17] Schwaiger S, Cervellati R, Seger C, Ellmerer E, About N, Renimel I, Godenir C, André P, Gafner

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F, Stuppner H. Leontopodic acid—a novel highly substituted glucaric acid derivative from Edelweiss (Leontopodium alpinum Cass.) and its antioxidative and DNA protecting properties.

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Tetrahedron 2005;61:4621–30.

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Fig. 1. Structures of compound 1-3

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Fig. 2. The key HMBC correlations of compound 1 (2-feruloyl-4-syringoyl glucaric acid)

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Fig. 3. The key HMBC correlations of compound 2 (2-syringoyl-4-feruloyl glucaric acid)

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Fig. 4. The key HMBC correlations of compound 3 (3-feruloyl-4-syringoyl glucaric acid)

ACCEPTED MANUSCRIPT Table 1 1H-NMR data of compound 1-3 1 (in D2O)

2 (in D2O)

3 (in DMSO-d6)

2 (5)

5.18 d (2.5)

5.30 d (1.0)

4.33 d (3.0)

3 (4)

4.77 d (2.5)

4.78 d (1.0)

5.58 dd (9.0, 3.0)

4 (3)

5.45 d (5.5)

5.36 d (6.5)

5.86 dd (9.0, 3.0)

5 (2)

4.40 d (5.5)

4.31 d (6.5)

6.81 d (1.5)

6.90 d (1.5)

hexaric acid

Feruloyl 1' 2'

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7.17 d (2.0) 9.59 brs (OH)

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6.83 d (8.0)

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6.77dd (8.0,1.5)

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7.32 d (16.0)

7.07 d (16.0)

7.42 d (16.0)

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5.96 d (16.0)

6.11 d (16.0)

6.24 d (16.0)

3.81 s

3.88 s

3.77 s

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3' 4'

4.44 d (3.0)

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6 (1)

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1 (6)

7.35 s

7.11 s

syringic 1'' 2''

7.25 s

6'' 7''

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3'' 5''

6.75 d (8.5)

6.83 dd (8.0,1.5)

6.98 dd (2.0,8.5)

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6.85 d (8.0)

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9.27 brs (OH)

7.25 s

7.35 s

7.11 s

3.73 s

3.82 s

3.74 s

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177.4 80.5 71.9 79.4 75.3 180.6

177.4 80.4 71.6 78.7 74.6 180.7

172.6 69.9 74.7 72.1 69.3 172.4

129.5 113.3 150.4 150.2 118.1 126.2 149.3 116.2 171.5 58.9

129.4 113.4 150.3 150.1 118.0 126.0 148.9 116.3 171.3 58.9

125.4 110.8 149.4 147.9 123.2 115.4 145.3 114.1 164.9 55.9

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122.6 110.6 149.7 142.4 149.7 110.6 170.0 58.4

122.6 110.6 149.4 142.3 149.4 110.6 170.5 58.4

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2 (in D2O)

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hexaric acid 1 (6) 2 (5) 3 (4) 4 (3) 5 (2) 6 (1) Feruloyl 1' 2' 3' 4' 5' 6' 7' 8' 9' 2×- OCH3 syringic 1'' 2'' 3'' 4'' 5'' 6'' 7'' - OCH3

1 (in D2O)

119.4 107.1 147.3 140.5 147.3 107.1 165.4 55.6

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Graphical abstract

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Conflict of interest