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Lignans and aromatic Glycosides from piper wallichii and their antithrombotic Activities Yan-Ni Shi, Yi-Ming Shi, Lian Yang, Xing-Cong Li, Jin-Hua Zhao, Yan Qu, Hong-Tao Zhu, Dong Wang, Rong-Rong Cheng, Chong-Ren Yang, Min Xu, Ying-Jun Zhang

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Journal of Ethnopharmacology

Received date: 25 July 2014 Revised date: 3 November 2014 Accepted date: 23 December 2014italic>vivo Cite this article as: Yan-Ni Shi, Yi-Ming Shi, Lian Yang, Xing-Cong Li, Jin-Hua Zhao, Yan Qu, Hong-Tao Zhu, Dong Wang, Rong-Rong Cheng, Chong-Ren Yang, Min Xu, Ying-Jun Zhang, Lignans and aromatic Glycosides from piper wallichii and their antithrombotic Activities, Journal of Ethnopharmacology, http://dx.doi. org/10.1016/j.jep.2014.12.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

 

Lignans and Aromatic Glycosides from Piper wallichii and Their Antithrombotic Activities

Yan-Ni Shi,a,b Yi-Ming Shi,a,b Lian Yang,a Xing-Cong Li,c Jin-Hua Zhao,a Yan Qu,a Hong-Tao Zhu,a Dong Wang,a Rong-Rong Cheng,a Chong-Ren Yang,a Min Xu,*,a and Ying-Jun Zhang*,a a

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China

b

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

c

National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Mississippi 38677, United States

* Corresponding author. Tel: +86 871 65223235; E-mail: address: [email protected] (M. Xu), [email protected] (Y.-J. Zhang)

 

 

ABSTRACT:

Ethnopharmacological relevance: Piper wallichii (Miq.) Hand.-Mazz. is a medicinal plant used widely for the treatment of rheumatoid arthritis, inflammatory diseases, cerebral infarction and angina in China. Previous study showed that lignans and neolignans from Piper spp. had potential inhibitory activities on platelet aggregation. In the present study, we investigated the chemical constituents of P. wallichii and their antithrombotic activities, to support its traditional uses. Materials and methods: The methanolic extract of the air-dried stems of P. wallichii was separated and purified using various chromatographic methods, including semi-preparative HPLC. The chemical structures of the isolates were determined by detailed spectroscopic analysis, and acidic acid hydrolysis in case of the new glycoside 2. Determination of the absolute configurations of the new compound 1 was facilitated by calculated electronic circular dichroism using time-dependent densityfunctional theory. All compounds were tested for their inhibitory effects on platelet aggregation induced by platelet activating factor (PAF) in rabbits' blood model, from which the active ones were further evaluated the in vivo antithrombotic activity in zebrafish model. Results: A new neolignan, piperwalliol A (1), and four new aromatic glycosides, piperwalliosides A–D (2–5) were isolated from the stems of Piper wallichii, along with 25 known compounds, including 13 lignans, six aromatic glycosides, two phenylpropyl aldehydes, and four biphenyls. Five known compounds (6–10) showed in vitro antiplatelet aggregation activities. Among them, (–)-syringaresinol (6) was the  

 

most active compound with an IC50 value of 0.52 mM. It is noted that in zebrafish model, the known lignan 6 showed good in vivo antithrombotic effect with an value of 37% at a concentration of 30 μM, compared with the positive control aspirin with the inhibitory value of 74% at a concentration of 125 μM. Conclusion: This study demonstrated that lignans, phenylpropanoid and biphenyl found in P. wallichii may be responsible for antithrombotic effect of the titled plant.

Keywords Piper wallichii (Miq.) Hand.-Mazz. Lignans Aromatic glycosides Antithrombotic effect

Zebrafish model

 

 

1. Introduction Platelet plays an important role at sites of vascular injury, while over-aggregation of platelets may lead to pathologic thrombosis (Huang et al., 2014). The majority of cardiovascular diseases, including acute coronary syndrome, ischemic stroke and peripheral vascular diseases, are associated with thrombotic disorders, resulting in serious outcomes such as sudden death or long-term disability (Edelstein et al., 2013; Tsai et al., 2014). The inhibition of platelet aggregation represents an important approach for the treatment of thrombotic diseases (Goto and Tomita, 2013). The quest for safe and efficacious natural leaders with antiplatelet aggregation activity thus remains considerable attention (Tsai et al., 2014). Lignans, one of the most ubiquitous natural products in terrestrial plants, are commonly derived from oxidative dimerization of two phenylpropanoid units (Saleem et al., 2005). Due to their diversified chemical structures, lignans are functionally diverse, e.g. platelet activating factor (PAF) receptor antagonistic, antitumor, anti-AD (Alzheimer's disease), antiviral, anti-inflammatory, and antifungal activities (Pan et al., 2014; Saleem et al., 2005; Zhu et al., 2013). It is worth noting that lignans are regarded as one of the vital sources for the discovery of PAF receptor antagonism (Tsai et al., 2014). The Piper genus is belonging to the family Piperaceae. Alkaloids and lignans are the mainly characteristic metabolites in Piper plants (Gutierrez et al., 2013; Pan et al., 2009). Piper wallichii (Miq.) Hand.-Mazz. (Piperaceae) is a perennial herb distributed widely in the Hubei, Hunan, Yunnan, Sichuan, and Gansu provinces of People’s

 

 

Republic of China, as well as Nepal, India, Bengal and Indonesia. The stems have been used for the treatment of rheumatoid arthritis, inflammatory diseases, cerebral infarction and angina by the local people living in China (Duan et al., 2010; Tamuly et al., 2014). Previously, several lignans, alkaloids, and phenylpropanoids have been isolated from this species (Tamuly et al., 2014; Wei et al., 2011). During our search for bioactive lignans with potential antiplatelet aggregation activities, we investigated on the stems of P. wallichii, and led to the isolation of one new neolignan, piperwalliol A (1) and four new aromatic glycosides, piperwalliosides A–D (2–5), together with 13 known lignans, six aromatic glycosides, two phenylpropyl aldehydes, as well as four biphenyls. Compound 1 features in neolignan skeleton (C6-C3-C6) crosslinked with a tetraoxygenated benzene ring to furnish an extra benzofuran moiety. All compounds were tested for their inhibition against platelet aggregation in vitro and the active compounds were further tested in vivo for their antithrombotic activity in zebrafish model. Herein, we described the isolation, structural elucidation, and bioactivity of all isolates. 2. Material and methods 2.1. General The details for different instruments, column chromatography (CC), thin-layer chromatography (TLC), preparative HPLC and semi-preparative HPLC used in this study were shown in the Supporting Information. 2.2. Plant material

 

 

The stems of P. wallichii were collected from Henan Province, People’s Republic of China, in June 2012, and were identified by Mr. Xiao-Ming Fang from South China Botanical Garden, Chinese Academy of Sciences. A voucher specimen (HITBC_015077) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The air-dried stems of P. wallichii (19.0 kg) were powdered and extracted with MeOH at 60 oC. After removal of the solvent under reduced pressure, the crude extract (2.1 kg) was partitioned between EtOAc and H2O (1:1, v/v) to furnish an EtOAc–soluble fraction (600 g) and an aqueous phase (1.4 kg). The EtOAc extract was applied to a silica gel CC, eluting with a CHCl3–MeOH (99:1–5:1, v/v), to give eight fractions A–H. Fr. E (75.2 g) was subjected to a RP-18 column using a step gradient of MeOH–H2O (from 40% to 90% MeOH) to afford eight Fr. E1–E8. Fr. E2 (499 mg) was chromatographed on silica gel with petroleum ether–acetone (5:1, v/v) to afford 18 (1.8 mg). Fr. E3 (3.0 g) was chromatographed over Sephadex LH-20 column, eluting with MeOH to give eight subfractions Fr. E3A–E3H. Fr. E3D (111 mg) was purified by semi-preparative HPLC (CH3CN–H2O, 22:78, v/v) to yield 6 (1.1 mg), 7 (3.8 mg), and 11 (2.0 mg). Fr. E3E (205 mg) was separated by RP-18 column using a step gradient of MeOH–H2O (from 40% to 80% MeOH), followed by semi-preparative HPLC (CH3CN–H2O, 35:65, v/v) to yield compound 8 (2.4 mg), 12 (29.2 mg), 27 (5.1 mg), and 28 (12.7 mg).

 

 

Fr. F (45.3 g) was subjected to Diaion HP20SS CC, using a step gradient of MeOH–H2O (from 40% to 100%) to afford 13 Fr. F1–F13. Fr. F8 (2.6 g) was purified by Sephadex LH-20, eluting with MeOH to yield 19 (4.8 mg). Fr. F9 (14.3 g) was separated by Sephadex LH-20 eluting with MeOH to afford five fractions Fr. F9AF9E. Fr. F9B (500 mg) and Fr. F9D (108 mg) was purified with preparative HPLC (CH3CN–H2O, 30:70, v/v) and semi-preparative HPLC (CH3CN–H2O, 20:80, v/v), respectively, to afford 13 (11.0 mg), 14 (5.2 mg), 15 (9.9 mg), and 20 (2.5 mg), 29 (0.8 mg), 30 (57.0 mg). Fraction H (440.1 g) was subjected to a Diaion HP20SS column using a step gradient of MeOH–H2O (from 20% to 80% MeOH) to afford five Fr. H1–H5. Fr. H1 (70.5 g) was chromatographed on Sephadex LH-20 (from 20% to 50% MeOH) to obtain ten subfractions H1A–H1J. Fr. H1C (5.2 g) was separated by RP-18 with a solvent system of MeOH–H2O (from 25% to 40% MeOH) to afford compound 9 (446.7 mg) and other five fractions Fr. H1C1–H1C5. Fr. H1C2 (135 mg) was purified

by preparative TLC (CHCl3–MeOH, 5:1, v/v) to yield compounds 21 (52.7 mg) and 23 (23.0 mg). Fr. H1C3 (75 mg) was subjected to semi-preparative HPLC (CH3CN–H2O, 14:86, v/v) to afford compounds 1 (2.4 mg), 22 (5.1 mg), and 24 (25.7 mg). Fr. H1J (2.3 g) was separated by preparative HPLC (CH3OH–H2O, 15:85, v/v) to yield compounds 4 (27 mg), 25 (565.7 mg), and a mixture which was further purified by semi-preparative HPLC (CH3OH–H2O, 15:85, v/v) to afford compounds 3 (8.3 mg), 5 (2.4 mg), and 26 (10.1 mg). Fr. H3 (13.4 g) was separated by RP-18 using a step gradient of MeOH–H2O (from 20% to 100% MeOH), followed by Sephadex

 

 

LH-20 eluting with MeOH and preparative TLC (CHCl3: MeOH, 1:1, v/v) and finally purified by semi-preparative HPLC (CH3CN–H2O, 13:87, v/v) to yield 16 (18.4 mg) and 17 (13.1 mg). Fr. H5 (598 mg) was purified by preparative HPLC using a step gradient of CH3CN–H2O (from 5% to 40% for 45 min) to yield compound 2 (98.3 mg), and 10 (1.3 mg). 2.4. Acid hydrolysis of 2. Compound 2 (6 mg) was hydrolyzed by acid to give D-glucose and L-rhamnose as sugar residues, which were determined by GC analysis of their corresponding trimethylsilylated L–cysteine adducts (Supporting Information). 2.5. ECD Calculation The theoretical calculations of compound 1 were performed using Gaussian 09 (Frisch et al., 2010). Conformational analysis was initially carried out using Discovery

Studio

3.5

Client.

The

optimized

conformational

geometries,

thermodynamic parameters, and populations of all conformations were provided in the supporting information (Figures S53–S54 and Tables S1–S8). The conformers were then optimized at B3LYP/6-31G(d) level. Room-temperature equilibrium populations were calculated according to Boltzmann distribution law. The theoretical calculation of ECD was performed using TDDFT (Bringmann et al., 2009) at B3LYP-SCRF/6-31G+(d,p) level with PCM in MeOH solvent. The ECD spectra of compound 1 were obtained by weighing the Boltzmann distribution rate of each geometric conformation.

 

 

The ECD spectra are simulated by overlapping Gaussian functions for each transition according to Δε ( E ) =

1 2.297 × 10

− 39

×

1 2πσ

A

∑ ΔE i Ri e

− [( E − Ei )/(2σ )] 2

i

where σ represents the width of the band at 1/e height, and ΔEi and Ri are the excitation energies and rotational strengths for transition i, respectively. σ = 0.20 eV and Rvelocity have been used in this work. 2.6. MO Analysis The orbital information (NBO plot files) was generated by NBO program of Gaussian 09 (Glendening, 2009). The predominantly populated conformers were selected for MO analysis. NBO plot files were used to generate corresponding Gaussian-type grid file by Multiwfn 2.4 (Lu and Chen, 2012). After that, the isosurface of generated grid date was afforded by VMD software (Humphrey et al., 1996). 2.7. Rabbits’ blood and zebrafish The New Zealand white rabbits were purchased from Experimental Animal Center of Kunming Medical University of China. The rabbits were anaesthetized by pentobarbital sodium, and the blood was collected by cardiac puncture.  The experiments were reviewed and approved by the Animal Ethics Committee of Kunming Institute of Botany, Chinese Academy of Sciences. A breeding stock of healthy mature zebrafish (4 to 5 pairs) was used for naturally embryos production. Each pair can yield up to 200 to 300 embryos and the dead embryos were removed 6 and 24 hours post-fertilization (hpf). The rest suitable  

 

embryos, according to the embryonic stage of development (Kimmel et al., 1995), were selected and then incubated in the fish water (1L reverse osmosis water containing soluble salt (200 mg), with conductivity of 480 - 510 μS/cm, pH 6.9 - 7.2, and hardness of 53.7 - 71.6 mg/L CaCO3) under 28 ˚C. Since the embryos can nutrients obtained from their own yolk sac, it is not necessary to feed them for nine days post-fertilization (dpf). The 3 dpf larval zebrafish were used to the antithrombotic test. Hunter Biotechnology, Inc. is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAA LAC) International. 2.8. Antiplatelet aggregation assay in vitro Turbid metric measurements of platelet aggregation of all the isolates and the positive control ginkgolide B (Sigma, No.129K1405V) were performed on a Chronolog Model 700 Aggregometer (Chronolog Corporation, Havertown, PA, USA) according to Born’s method (Born, 1962; Born and Cross, 1963). The blood from the rabbits by cardiac puncture was anticoagulated with 3.8% sodium citrate (9:1, v/v). Platelet-rich plasma (PRP) was prepared shortly after blood collection by spinning the sample at 180 g for 10 min at 22 °C. The PRP was carefully removed and the remaining blood was centrifuged at 2400 g for 10 min at 22 °C to obtain platelet-poor plasma (PPP). Platelet counts were adjusted by the addition of PPP to the PRP to achieve a count of 500 × 109 L-1. Platelet aggregation studies were completed within 3h of the preparation of PRP. Immediately after the preparation of PRP, 250 μL was transferred into each of prepared test tubes, with 250 μL PPP set as a control. Before addition of inducers, compounds were incubated with PRP at 37°C for 5 min. The

 

 

aggregation of PRP was stimulated by PAF (Sigma, No.041M1291V) 40 ng/mL. The change of optical density as a result of platelet aggregation was recorded, and inhibition percentage of compounds was calculated according to the formula: Inhibition of aggregation (%) = (A–B) / A × 100% [A: maximum change of turbidity in DMSO added. B: maximum change of turbidity in sample added.] 2.9. Antithrombotic bioassay in vivo. (Gregory and Jagadeeswaran, 2002) The arachidonic acid (AA) was used to induce larval zebrafish thrombus as model group. The tested compounds were added separately as the test group with a final concentration of 30 μM. The negative control group was the solvent group, which not only represented the background value of the test group, but also made sure that the cardiac red blood cells of larval zebrafish were normal. Blank control group was used to prove that the solvent did not have harmful effects on the larval zebrafish. Aspirin was used as positive control. The experiment was carried on 96-well cell culture plates, on which it included a blank control, a solvent control, a model, a positive control and the test samples' groups. Each group processed 8 wells, and the larval zebrafish were seeded 4 fish per well. After a treatment period of 48h until 5 dpf. The larval

zebrafish

were

anaesthetized

by

MESAB

(ethyl-m-aminobenzoate

methanesulphonate, Sigma-Aldrich) and then dyed with dianisidine (sigma, MKBG4648V) as staining agent. The quantitative analysis of staining power (S) of cardiac red blood cells was carried on a fluorescence microscope (SMZ645, Nikon Company) and analyzed by Nikon NIS-Elements D3.10 software. The inhibition ratio was calculated by the following equation, and before calculations the S (test group) and S (model group) should minus the background values. The results were shown as

X ± SE. Statistical evaluation included one-way analysis of variance followed by

 

 

Dunnett’s t-test for multiple comparisons. P < 0.05 (*) were taken as significant. antithrombotic ratio（%）= S（test group）- S（model group） ×100% S（solvent group）- S（model group）

3. Results 3.1. Compounds isolated from P. wallichii The methanolic extract of air-dried stems of P. wallichii was suspended into H2O and partitioned with EtOAc. The EtOAc layers were subjected to column chromatography over silica gel, Sephadex LH-20, and reversed-phase C18 to afford five new compounds (1–5). In addition, 25 known compounds were identified as (–)-syringaresinol (6)  (He et al., 2012), (+)-medioresinol (7) (Deyama et al., 1985), (+)-epipinoresinol (8) (Rahman et al., 1990), sinapyl aldehyde (9) (Zhou et al., 2013), 3',5'-dimethoxybiphenyl-3,4'-diol (10) (Bao et al., 2010), (–)-lirioresinol A (11) (Zhou et al., 2007), (+)-pinoresinol (12) (Roy et al., 2002), prinsepiol (13) (Piccinelli et al., 2004), (–)-4-epilyoniresinol (14) (Ohashi et al., 1994), isolariciresinol (15) (Abe and Yamauchi, 1989), (+)-lyoniside (16), (–)-nudiposide (17) (Šmite et al., 1995), (–)-prestegane B (18) (Meragelman et al., 2001), (–)-olivil (19) (Yeo et al., 2004), neoolivil (20) (Schöttner et al., 1997), 3,4,5-trimethoxyphenyl-β-D-glucopyranoside (21) (Jin et al., 2014), kelampayoside A (22) (Miyamura et al., 1983), benzyl β-D-glucopyranoside (23) (Goncalves et al., 2004), prunasinamide (24) (Sendker and Nahrstedt, 2009), prunasin  (25) (Nahrstedt and Rockenbach, 1993), garcimangosone D (26) (Huang et al., 2001), coniferyl aldehyde (27) (El-Hassan et al., 2003), 4'-methoxyaucuparin (28) (Kokubun et al., 1995), aucuparin (29) (Borejsza-Wysocki et al., 1999), and garcibiphenyl C (30) (Chen et al., 2006), respectively, by comparing

 

 

their spectroscopic data with those reported previously in literatures.

Fig. 1. Structures of compounds 1–10 from P. wallichii. 3.2. Structure elucidation of new compound Compound 1 was obtained as a yellow oil. The molecular formula of 1 was determined as C25H24O9 by a quasimolecular ion peak at m/z 467.1337 [M – H]– (calcd. for C25H23O9, 467.1342) in the negative HRESIMS, indicating 14 degrees of unsaturation. The IR spectrum of 1 indicated the presence of hydroxy (3452 cm-1) and aromatic (1631 and 1453 cm-1) groups. The

13

C NMR spectrum displayed the

occurrence of 25 carbon resonances ascribable to four methoxys (δC 56.7, 56.7, 61.3, 57.2), one aliphatic methylene (δC 63.4), two aliphatic methines (δC 89.3, 54.1), and 18 aromatic carbons (δC 97.6–154.3) due to three benzene rings. The 1H NMR  

 

spectroscopic data (Table 1) revealed the presence of a benzofuran-type lignan skeleton [δH 5.71 (1H, d, J = 6.0 Hz, H-7), 3.88 (1H, m, H-8), 4.17 (1H, dd, J = 11.2, 4.0Hz, H-9a), 4.00 (1H, dd, J = 11.2, 7.4 Hz, H-9b)], which was supported by the three aliphatic resonances in the

13

C NMR spectrum [δC 89.3 (CH, C-7), 54.1 (CH,

C-8), 63.4 (CH2, C-9)] (Yang et al., 2007). Further analysis of the 1H NMR spectrum assigned the three aromatic rings to be one symmetrical 1,3,4,5-tetrasubstituted benzene group [δH 6.71 (2H, s, H-2,6)], one AB-aromatic system [δH 7.71, 6.86 (each 1H, d, J = 8.3 Hz, H-5',6')] and a pentasubstituted benzene ring (δH 7.20, 1H, s, H-5 ). The aforementioned data indicated that 1 is a neolignan possessing one more aromatic ring. Apart from the furan ring and the three benzene rings accounting for 13 degrees of unsaturation, 1 should have an additional ring in the molecule. Comparison 1D NMR data of 1 with those of 4,6-dimethoxy-5-hydroxy-3-hydroxymethyl-2-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzofuran isolated from Tarenna attenuate (Yang et al., 2007) suggested that it shared the same A and B rings. This was further confirmed by the 1H–1H COSY correlations of H-7/H-8/H2-9, and the HMBC correlations from H-2,6 to C-3,5 (δC 149.4)/C-4 (δC 136.3)/C-7, from H-7 to C-1 (δC 134.1)/C-2,6 (δC 103.9)/C-9 (Fig. 2). However, the

13

C NMR resonances due to the C ring were

different with those of the known neolignan because 1 had an additional aromatic ring. The structure of C ring and the linkage of the additional aromatic ring was further determined by the 1H–1H COSY correlation of H-5'/H-6', the HMBC correlations from H-7 to C-1' (δC 161.2)/C-2' (δC 110.5), from H-5' to C-1'/C-3'(δC 154.3)/C-6" (δC 117.3), from H-6' to C-2'/C-4' (δC 120.4), and from H-5" to C-4'/C-1" (δC 144.5)/C-3"

 

 

Table 1 The 1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data for compounds 1 and 2 (methanol-d4) 1 2a position δC δH (mult., J in Hz) δC δH (mult., J in Hz) 1 134.1, C 108.8, C 2 103.9, CH 6.71 (s) 159.5, C 3 149.4, C 95.6, CH 6.04 (br s) 4 136.3, C 164.9, C 5 149.4, C 98.3, CH 5.97 (br s) 6 103.9, CH 6.71 (s) 163.2, C 7 89.3, CH 5.71 (d, 6.0 ) 200.1, C 8 54.1, CH 3.88 (m) 141.7, C 4.17 (11.2, 4.0) 130.6, CH 7.60 (br d,7.3) 9 63.4, CH2 4.00 (dd, 11.2, 7.4) 10 128.9, CH 7.30 (t, 7.3) 11 132.9, CH 7.40 (m) 12 128.9, CH 7.30 (t, 7.3) 13 130.6, CH 7.60 (br d, 7.3) 1' 161.2, C 98.3, CH 4.85 (d, 7.8) 2' 110.5, C 80.6, CH 2.64 (dd,8.1, 7.8) 3' 154.3, C 78.3, CH 3.31 (m) 4' 120.4, C 77.9, CH 3.19 (m) 5' 120.9, CH 7.71 (d, 8.3 ) 71.1, CH 3.07 (br t, 9.2) 6' 106.0, CH 6.86 (d, 8.3 ) 62.6, CH2 3.73 (br d, 12.1) 3.55 (dd, 12.1, 5.7) 1" 144.5, C 102.3, CH 4.63 (br s) 2" 134.6, C 72.1, CH 3.50 (dd, 9.5, 3.1) 3'' 139.0, C 72.2, CH 3.72 (overlapped) 4" 147.4, C 74.2, CH 3.19 (overlapped) 5" 97.6, CH 7.20 (s) 69.9, CH 3.39 (dq, 9.2, 6.1) 6" 117.3, C 17.9, CH3 0.88 (d, 6.1) 3.81 (s) 3-OMe 56.7, CH3 5-OMe 56.7, CH3 3.81 (s) 4.11 (s) 2''-OMe 61.3, CH3 3.94 (s) 4''-OMe 57.2, CH3 a

Data were measured at 400 MHz for 1H NMR and 100 MHz for 13C NMR spectra, respectively.

(δC 139.0), and the ROESY correlations of H-5" with H-5' (Fig. 2). The HMBC correlations from OMe-3 (δH 3.81) to C-3, OMe-5 (δH 3.81) to C-5, and OMe-4" (δH 3.94) to C-4" (δC 147.4), together with the ROESY correlations of OMe-3,5 with

 

 

H-2/6 and OMe-4'' with H-5'' clearly revealed the locations of these three methoxy groups (Fig. 2). Although there were no available ROESY correlations for the downfield-shifted methoxy group at δH 4.11, the HMBC correlations of H-2/6 to δC 136.3 (C-4), H-5'' to δC 139.0 (C-3'') and OMe-2" (δH 4.11) to δC 134.6 (C-2") favorably supported this methoxy group was linked to C-2'' and not C-4. Therefore, the planar structure of 1 was established as shown in Fig. 1.

Fig. 2. Key 1H–1H COSY (─), HMBC (H→C) and ROESY correlations of 1. The relative configuration of 1 was elucidated on the basis of the coupling constants observed in the 1H NMR spectrum and ROESY experiment. The value as 6.0 Hz of JH-7, H-8 indicated a 7,8-trans configuration (Yang et al., 2007), which was confirmed by the ROESY correlation of H-7 with H2-9. A theoretical calculation of electronic circular dichroism (ECD) spectrum of 1 was performed to determine the absolute configurations, using time-dependent density-functional theory (TDDFT) method. A pair of enantiomers, (7S,8R)-1 and (7R,8S)-1, were calculated at the

 

 

Fig. 3. Experim mental ECD spectrum of o 1 (black) and calculaated ECD spectra s of nd (7R,8S)-11 (blue). (7S,8R)-1 (red) an YP-SCRF/6-31G+(d,p)///B3LYP/6--31G(d) lev vel with poolarizable continuum B3LY modeel (PCM) in n MeOH. As A illustratedd in Fig. 3, the calcullated ECD curve for (7S,8R)-1 was inn good agreement with thhe experimeental one. Inn order to comprehend g of o the experrimental EC CD spectrum m of 1, moolecular orbiital (MO) the generation analyysis using coonformer 1g as an exampple was carriied out at thee B3LYP/6-331+G(d,p) level with PCM in MeOH (Fig. ( 4). Thee positive Cotton C effectt at 205 nm m could be ascrib bed to the positive p rotatory strenggths at 210.76 and 2111.94 nm, whhich were causeed by the ellectronic traansitions froom MO120 to MO125 and from MO122 M to MO130 involvingg two π → π* π transitionss in the arom matic rings. T The negativee rotatory strenggth at 225.866 nm contribbuted to the nnegative Cottton effect att 223nm, whhich was assocciated with th he electronicc transitions from MO1118 to MO1244 involving a π → π* transiition. Accord dingly, the absolute a conffigurations of o 1 were esttablished as 7S,8R 7 and the coompound waas named pip perwalliol A A. To the bestt of our know wledge, pipeerwalliol A (1)), represents the first exaample possesssing a 5/6/55/6 ring systeem in the liggnan familly.  

 

MO118

MO120

MO122

MO124

MO125

MO130

Fig. 4. Important MOs in the ECD spectrum of the optimized conformer 1g. Compound 2, obtained as a yellow oil, had a molecular formula of C25H30O13, as determined by the HREIMS (m/z 538.1683 [M]+; calcd for C25H30O13, 538.1686). The13C NMR (DEPT) spectra of 2 (Table 1) showed the presence of 12 aromatic carbon signals (δC 95.6–164.9), a conjugated carbonyl group at δC 200.1 (C-7), and 12 carbon signals at δC 98.3 (C-1'), 80.6 (C-2'), 78.3 (C-3'), 77.9 (C-4'), 71.1 (C-5'), 62.6 (C-6'), 102.3 (C-1"), 72.1 (C-2"), 72.2 (C-3''), 74.2 (C-4"), 69.9 (C-5"), and 17.9 (C-6"), arising from two hexosyl groups. The 1H NMR spectrum (Table 1) exhibited a typical mono-substituted benzene ring [δH 7.60 (2H, br d, J = 7.3 Hz, H-9, 13), 7.30 (2H, t, J = 7.3 Hz, H-10, 12), and 7.40 (1H, m, H-11)], two one-proton aromatic broad

 

 

singlets at δH 6.04 (1H, br s, H-3) and 5.97 (1H, br s, H-5) due to an asymmetric 1,2,4,6–tetra-substituted phenyl, two anomeric protons at δH 4.85 (1H, d, J = 7.8 Hz, H-1') and 4.63 (1H, br s, H-1"), and the characteristic rhamnosyl signals at δH 3.39 (1H, dq, J = 9.2, 6.1 Hz, H-5") and 0.88 (3H, d, J = 6.1 Hz, H-6") (Pathak et al., 2004). Acid hydrolysis of 2 gave D-glucose and L-rhamnose as sugar residues that were determined by GC analysis of their corresponding trimethylsilylated L–cysteine adducts. The aforementioned data revealed that compound 2 was a benzophenone glycoside similar to garcimangosone D from the fruit hulls of Garcinia mangostana (Huang et al., 2001). The only difference was the presence of an additional rhamnosyl unit in 2. The benzophenone moiety in 2 was further established on the basis of the HMBC correlations from H-3 to C-1 (δC 108.8)/C-4 (δC 164.9)/C-5 (δC 98.3)/C-7 (δC 200.1) and from H-9 and H-13 to C-7/C-11 (δC 132.9) (Fig. 5). The location and sequence of the sugar moieties were readily confirmed by the HMBC correlations from H-1' to C-2 (δC 159.5) and from H-1" to C-2' (δC 80.6) (Fig. 5), which was supported by the ROESY correlation of H-3 with H-1'. Thus, the structure of 2 was established as 4,6-dihydroxy-2-O-[a-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]benzophenone and named piperwallioside A.

Fig. 5. Key 1H–1H COSY (─) and HMBC (H→C) correlations of 2, 3 and 5.  

 

Compound 3, a colorless oil, [α]D21 –87.9, possessed a molecular formula of C15H20O8, as deduced by the HREIMS at m/z 328.1159 [M]+ (calcd for C15H20O8, 328.1158). The 13C NMR and DEPT spectra of 3 showed a set of signals attributed to a glucopyranosyl moiety (δC 100.9, 74.8, 78.2, 71.6, 77.6 and 62.8), and nine carbon signals comprising of six aromatic (δC 129.1–136.4), one carboxyl (δC 173.3), one methoxy (δC 53.0) and one oxymethine (δC 78.3) carbons. The 1H NMR spectrum of 3 (Table 2) exhibited the presence of one mono-substituted aromatic ring [δH 7.48 (2H, m, H-2,6) and 7.37 (3H, m, H-3,4,5)], one anomeric proton [δH 4.09 (d, J = 7.7 Hz, H-1')] and a methoxy group (δH 3.69). In the HMBC spectrum of 3, correlations from the oxymethine proton (δH 5.51, s, H-7) to the carboxyl carbon at δC 173.3 and aromatic carbons at C-1 (δC 136.4)/C-2 (δC 129.1)/C-6 (δC 129.1)/C-8 (δC 173.3), and the methoxy proton (δH 3.69) to the carboxyl carbon (δC 173.3) established the aglycone moiety as methyl 2-hydroxy-2-phenylacetate (Fig. 5). The glucosyl moiety was determined to be attached to the C-7 hydroxy group by the HMBC correlation from H-1' to C-7 (δC 78.3) (Fig. 5). Further comparison of the NMR and optical rotation data with those of d-mandelic acid β-D-glucopyranoside ([α]D –126) (Kitajima and Tanaka, 1993) concluded that compound 3 was a methyl ester of d-mandelic acid β-D-glucopyranoside, which was given a trivial name of piperwallioside B. Compound 4 was isolated as colorless oil. The molecular formula of 4 was determined as C15H20O8, on the basis of its HREIMS (m/z 328.1150 [M]+, calcd for C15H20O8, 328.1158). The

 

13

C NMR and DEPT spectra of 4 resembled those of 3

 

(Table 2), except for the chemical shift of the anomeric carbon, which was downfield shifted to δC 103.0 in 4. The anomeric carbon of the glucosyl residue was deduced to be β by the coupling constant of anomeric proton (J = 7.6 Hz) and it should have D-form as that of 2 from a biogenic standpoint. The aforementioned data suggested

that 4 should be a C-7 configurational isomer of 3. Since the experimental ECD spectrum of 4 showed an obvious positive Cotton effect at 222 nm being opposite to that of 3 (Fig. 6), compound 4 should possess an S absolute configuration at C-7. Therefore, the structure of piperwallioside C (4) was determined as l-mandelic acid methyl ester β-D-glucopyranoside. Compound 5, a colorless oil, had a molecular formula of C14H19NO7 as deduced from HREIMS at m/z 313.1160 [M] + (calcd for C14H19NO7, 313.1162). The 13C and 1

H NMR data of 5 (Table 2) were similar to those of prunasinamide

[(2R)-2-β-D-glucopyranosyloxymandelamide] from Cyanogenic glucosides, except for the downfield-shifted anomeric carbon at δC 102.8 (Backheet et al., 2003). The HMBC correlations from H-7 (δH 5.21) to C-1 (δC 139.0)/C-2 (δC 128.0)/C-6 (δC 128.0)/C-8 (δC 173.7) and from NH2 (δH7.59, s) to C-8 demonstrated that the planar structure of 5 was consistent with that of prunasinamide (Fig. 5). However, the specific rotation value ([α]D21 –0.88) of 5 was obviously different from that of prunasinamide ([α]D20–141.0) (Sendker and Nahrstedt, 2009). In the ECD spectrum of 5, an obvious positive Cotton Effect at 216 nm was observed, similar to that of 4 (Fig. 6), supporting an S configuration for C-7. Thus, compound 5 was determined as (2S)-2-β-D-glucopyranosyl-oxymandelamide and named piperwallioside D.

 

 

Table 2 The 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data for compounds 3-5 (methanol-d4). 3 4 position δC δH (mult., J in Hz) δC δH (mult., J in Hz) 1 136.4, C 137.8, C 2 129.1, CH 7.48 (m) 128.5, CH 7.47 (m) 3 129.8, CH 7.37 (overlapped) 129.4, CH 7.34 (m) 4 130.1, CH 7.37 (overlapped) 129.6, CH 7.34 (overlap) 5 129.8, CH 7.37 (overlapped) 129.4, CH 7.34 (m) 6 129.1, CH 7.48 (m) 128.5, CH 7.47 (m) 7 78.3, CH 5.51 (s) 79.0, CH 5.54 (s) 8 173.3, C 173.4, C 1' 100.9, CH 4.09 (d, 7.7) 103.0, CH 4.48 (d, 7.6) 2' 74.8, CH 3.33 (m) 75.3, CH 3.28 (m) 3' 78.2, CH 3.09 (m) 78.2, CH 3.26 (m) 4' 71.6, CH 3.27 (m) 71.4, CH 3.26 (m) 5' 77.6, CH 3.24 (br t, 8.9) 77.9, CH 3.39 (br t, 7.4) 3.85 (dd, 11.9, 2.1) 62.7, CH2 3.83 (br d, 11.7) 6' 62.8, CH2 3.63 (dd, 11.9, 6.2) 3.61 (dd, 11.7, 4.7) 3.69 (s) 52.9, CH3 3.71 (s) OMe 53.0, CH3 NH2 a

Data were measured at 600 MHz for 1H NMR and 150 MHz for 13C NMR spectra in acetone-d6, respectively.

 

δC 139.0, C 128.0, CH 128.8, CH 128.6, CH 128.8, CH 128.0, CH 80.5, CH 173.7, C 102.8, CH 74.7, CH 77.8, CH 71.3, CH 77.8, CH 62.6, CH

5a δH (mult., J in Hz) 7.49 (br d, 7.2) 7.32 (m) 7.29 (m) 7.32 (m) 7.49 (br d, 7.2) 5.21 (s) 4.57 (d, 7.86) 3.35 (m) 3.48 (m) 3.33 (m) 3.40 (m) 3.82 (dd, 11.8, 2.5) 3.62 (dd, 11.8, 5.8) 7.59 (br s) 6.82 (br s)

 

Fig. 6. Experimental ECD spectra of compounds 3-5. 3.3. Physical and spectroscopic data of new compound Piperwalliol A (1): yellow oil; [α]D20 –18.7 (c 0.2, MeOH); UV (MeOH) λmax (log ε): 316 (3.65), 209 (4.27) nm; ECD (c 0.06, MeOH) λmax nm (Δε): 223 (–5.32), 205 (+9.00); IR (KBr) νmax: 3452, 1641, 1631, 1134 cm-1; 1H and 13C NMR data, see Table 1; negative ESIMS: m/z 467 [M – H]–; HRESIMS: m/z 467.1337 [M – H]– (calcd for C25H23O9, 467.1342). Piperwallioside A (2): yellow oil; [α]D25 –128.9 (c 0.3, MeOH); UV (MeOH)

λmax (log ε): 303.5 (3.28), 248.5 (3.32), 204.5 (3.94) nm; IR (KBr) νmax: 3440, 1624, 1600, 1451, 1277 cm-1; 1H and 13C NMR data, see Table 1; negative ESIMS: m/z 537 [M – H]–; HREIMS: m/z 538.1683 [M]+ ( calcdfor C25H30O13, 538.1686). Piperwallioside B (3): colorless oil; [α]D21 –87.9 (c 0.3, MeOH); UV (MeOH)

λmax (log ε): 257.6 (1.45), 203.6 (3.11) nm; ECD (c 0.3, MeOH) λmax nm (Δε): 218 (–6.76); IR (KBr) νmax: 3421, 1741, 1631, 1384, 1078 cm-1; 1H and 13C NMR data, see Table 2; positive ESIMS: m/z 351 [M + Na]+; HREIMS: m/z 328.1159 [M]+  

 

( calcd for C15H20O8, 328.1158);. Piperwallioside C (4): colorless oil; [α]D22 +4.94 (c 0.2 MeOH); UV (MeOH)

λmax (log ε): 257.4 (1.69), 204.6 (3.2) nm; ECD (c 0.2, MeOH) λmax nm (Δε): 222 (+10.49); IR (KBr) νmax: 3428, 1741, 1630, 1076, 1038 cm-1; 1H and 13C NMR data, see Table 2; positive ESIMS: m/z 351 [M + Na]+; HREIMS: m/z 328.1150 [M]+ (calcd for C15H20O8, 328.1158);. Piperwallioside D (5): colorless oil; [α]D21 –0.88 (c 0.3, MeOH); UV (MeOH)

λmax (log ε): 257.4 (1.83), 204.2 (3.3) nm; ECD (c 0.2, MeOH) λmax nm (Δε): 216 (+2.06); IR (KBr) νmax: 3441, 3426, 1676, 1641, 1384, 1076, 1039cm-1; 1H and 13C NMR data, see Table 2; negative ESIMS: m/z 312 [M – H]–; HREIMS: m/z 313.1160 [M]+ (calcd for C14H19NO7, 313.1162). 3.4. Inhibition activities on platelet aggregation induced by PAF All the isolates were evaluated for their inhibition activities on platelet aggregation induced by PAF, with ginkgolide B (GB) used as a positive control (IC50 = 0.016 mM). It showed that five known compounds, (–)-syringaresinol (6), (+)-medioresinol (7), (+)-epipinoresinol (8), sinapyl aldehyde (9), and 3',5'-dimethoxybiphenyl-3,4'-diol (10) exhibited antiplatelet aggregation activities in a concentration-dependent manner (see SI, Table S9). Among which, compound 6 was the most active one with an IC50 value of 0.52 mM. (–)-Lirioresinol A (SI, Fig. S52) with different configurations from 6 presented no activity. Compound 7 showed the maximum platelet aggregation and inhibition of aggregation with 52.7 ± 4.0% and 16.8 ± 6.1%, respectively, at a concentration of 0.50 mM, while in the case of 8

 

 

at a concentration of 0.40 mM, it was 46.3 ± 4.0% and 24.1 ± 1.0%, respectively. Moreover, (+)-pinoresinol showed no activity, which might be correlated with the absence of a phenolic methoxy group. Compound 9 and 10 displayed moderate antiplatelet aggregation activities with IC50 values of 2.4 and 2.0 mM, respectively. The aforementioned results implied that configurations and methoxy group of lignans should be crucial for the antiplatelet aggregation activities of these compounds. 3.5. In vivo antithrombotic effect of compounds 6, 9 and 10 The active compounds in platelet aggregation induced by PAF model were further tested for their in vivo antithrombotic effects at a concentration of 30 μM on zebrafish model. Due to limited amounts, only compounds 6, 9 and 10 were further tested. The results were shown in Fig. 7 and Table S10 of SI. (–)-Syringaresinol (6) and sinapyl aldehyde (9) showed good in vivo antithrombotic effect with inhibitory values of 37% and 11%, at a concentration of 30 μM, respectively. Although 3',5'-dimethoxybiphenyl-3,4'-diol (10) displayed better activity than compound 9 in PAF model, compound 10 showed no antithrombotic effect on zebrafish model, at a concentration of 30 μM. Among them, compound 6 was also the most potential active compound on zebrafish model, compared with the positive control aspirin with an inhibitory value of 74% at a concentration of 125 μM. Moreover, all of them showed no cytotoxicity on zebrafish at a concentration of 30 μM and 1.1% DMSO did not affect the thrombus in zebrafish.

 

 

Fig. 7. Antithrombotic effects on zebrafish of compounds 6, 9 and 10

4. Discussion and conclusion This study on P. wallichii led to the isolation and identification of a new neolignan, piperwalliol A (1), and four new aromatic glycosides, piperwalliosides A-D (2-5), along with 25 known compounds. Being the first neolignan with 5/6/5/6 ring system, piperwalliol A (1) features an unique architecture, which represents one of an interesting work during decades of the studies on Piper family. Three known lignans, (–)-syringaresinol (6), (+)-medioresinol (7), and (+)-epipinoresinol (8), and two phenolic compounds, sinapyl aldehyde (9) and 3',5'-dimethoxybiphenyl-3,4'-diol (10) showed in vitro antiplatelet aggregation activities. It is noted that (–)-syringaresinol (6) also displayed in vivo antithrombotic effect on zebrafish model, which represents one new scaffold with antiplatelet aggregation activity. The

 

 

activities of compounds from P. wallichii supported the traditional use of this herbal medicine.

Acknowledgments This work was supported by the NSFC 81273408, the 973 Program of Ministry of Science and Technology of China (2011CB915503), the National Science and Technology Support Program of China (2013BAI11B02), the Fourteenth Candidates of the Young Academic Leaders of Yunnan Province (Min Xu, 2011CI044), and the West Light Foundation of the Chinese Academy of Sciences. The calculation sections were supported by the High Performance Computing Center of Kunming Institute of Botany, Chinese Academy of Sciences.

Appendix A. Supporting information General, Extraction and isolation, and Acid hydrolysis of 2 of Material and methods, 1D and 2D NMR, MS, IR, UV, and ECD spectra for compounds 1–5, ECD calculation details for compound 1, structures of the known compounds and biological evaluation of the isolates are available in the online version at http:// .

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Figure captions 1. Fig. 1. Structures of compounds 1–10 from P. wallichii. 2. Fig. 2. Key 1H–1H COSY, HMBC and ROESYcorrelations of 1. 3. Fig. 3. Experimental ECD spectrum of 1 (black) and calculated ECD spectra of (7S,8R)-1 (red) and (7R,8S)-1 (blue). 4. Fig. 4. Important MOs in the ECD spectrum of the optimized conformer 1g. 5. Fig. 5. Key 1H–1H COSY (─) and HMBC (H→C) correlations of 2, 3, and 5. 6. Fig. 6. Experimental ECD spectra of compounds 3–5. 7. Fig. 7. Antithrombotic effects on zebrafish of compounds 6, 9 and 10  

 

 

 

(–)-sy yringaresinool (PubChem m CID: 3332426); (+)--medioresinool (PubCheem CID: 181681); (+)-epip pinoresinol (PubChem C CID: 637584); sinapyl aldehyde (P PubChem i nol (PubChem CID: 160 0521); (+)-pinoresinol (P PubChem CID: 5280802); isolariciresin Chem CID: 586733); pruunasin (PubChem CID: 120639); CID: 73399); (–)-olivil (PubC m CID: 52800536); aucup parin (PubChhem CID: 4442508) conifferyl aldehydde (PubChem

Grapphical abstraact

Lignans and Aroomatic Glyccosides from m Piper walllichii and Th heir Antithrrombotic Activvities Yan-N Ni Shi, Yi-M Ming Shi, Lian Yang, Xiing-Cong Li, Jin-Hua Zhhao, Yan Quu, Hong g-Tao Zhu, Dong D Wang, Rong-Rongg Cheng, Choong-Ren Yaang, Min Xu,, and Ying-Jun Zhang