Ribemansides A and B, TRPC6 Inhibitors from Ribes manshuricum

Feb 22, 2018 - Two new acylated β-hydroxynitrile glycosides, ribemansides A (1) and B (2), were isolated from the aerial parts of Ribes manshuricum. ...
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Article Cite This: J. Nat. Prod. 2018, 81, 913−917

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Ribemansides A and B, TRPC6 Inhibitors from Ribes manshuricum That Suppress TGF-β1-Induced Fibrogenesis in HK‑2 Cells Baoping Zhou,§ Yange Wang,§ Chunlei Zhang,§ Guolin Yang, Fan Zhang, Boyang Yu, Chengzhi Chai,* and Zhengyu Cao* Department of TCM Pharmacology, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 211198, People’s Republic of China S Supporting Information *

ABSTRACT: Two new acylated β-hydroxynitrile glycosides, ribemansides A (1) and B (2), were isolated from the aerial parts of Ribes manshuricum. Their structures were elucidated by comprehensive spectroscopic analysis. Ribemansides A and B inhibited transforming growth factor β1 (TGF-β1)-induced expression of α-smooth muscle actin, fibronectin release, and changes in cell morphology in the human proximal tubular epithelial cell line (human kidney-2, HK-2). Further biological evaluation demonstrated that both 1 and 2 inhibit the activity of canonical transient receptor potential cation channel 6 (TRPC6), with IC50 values of 24.5 and 25.6 μM, respectively. The antifibrogenic effect of these compounds appears to be mediated through TRPC6 inhibition, since the TRPC6 inhibitor, SAR7334, also suppressed TGF-β1-induced fibrogenesis in HK-2 cells.

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the antifibrogenic activities of these two purified compounds were evaluated using HK-2 cells. The two new isolated compounds were identified as ribemanside A [2β-D-(6′-O-phydroxybenzoyl)glucopyranosyloxymethy-4-hydroxy-2(E)-butenenitrile] and ribemanside B [2β- D -(6′-O-vanilloyl)glucopyranosyloxymethy-4-hydroxy-2(E)-butenenitrile].

he genus of Saxifragaceae Ribes is distributed worldwide including Asia and North and South America.1 Due to their perceived beneficial health effects, the seeds and fruits of Ribes species have attracted considerable attention2 and have been reported to be a rich source of unsaturated fatty acids, phenolic acids, anthocyanins and other flavonoids, vitamins, and amino acids.3−5 An aqueous extract of Ribes diacanthum is used in Mongolian folk medicine to treat urinary complaints such as bladder infections, cystitis, and edema, exerts diuretic effects,6 and has also been found to protect against cisplatininduced kidney injury, possibly through anti-inflammatory effects.4 Ribes manshuricum (Maxim.) Kom., a deciduous shrub, is distributed mainly in the northeast area of the Inner Mongolia Autonomous Region in the People’s Republic of China. When compared to the relatively extensive research on other species of the genus, little is known regarding the chemical constituents and the biological activity of this species. Unpublished pilot studies found that an ethanolic extract of R. manshuricum could protect against unilateral ureteral occlusion (UUO)-induced kidney injury in mice and inhibit transforming growth factor β1 (TGF-β1)-induced fibrogenesis in a human proximal tubular epithelial cell line (human kidney-2, HK-2). In the present study, two new acylated β-hydroxynitrile glycosides (1 and 2) were isolated from the ethanolic extract of R. manshuricum, and © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The HRESIMS of ribemanside A (1) yielded a [M + H] + ion with m/z 396.1287, consistent with a molecular formula of C18H21NO9, with nine degrees of unsaturation. The characteristic UV absorptions at 206 and 258 nm suggested the presence of a phenyl ring. The IR absorptions at 3394, 1693, and 2226 cm−1 indicated the presence of hydroxy, carbonyl, and cyano Received: December 10, 2017 Published: February 22, 2018 913

DOI: 10.1021/acs.jnatprod.7b01037 J. Nat. Prod. 2018, 81, 913−917

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groups, respectively. The 1H NMR spectrum showed a typical AA′BB′ system at δH 7.98 (2H, d, J = 8.5 Hz) and 6.90 (2H, d, J = 8.5 Hz), indicating the presence of a p-substituted phenyl ring. An olefinic proton signal at δH 6.71 (1H, t, J = 6.5 Hz) suggested a trisubstituted double bond to be present in the molecule. An anomeric proton signal occurring at δH 4.45 (1H, d, J = 8.5 Hz) implied a β-linkage of the sugar unit to the aglycon. The 13C NMR spectrum showed a total of 18 carbon signals comprising an ester carbonyl, six aromatic carbons, two olefinic carbons, a cyano carbon, and eight oxygenated aliphatic carbons. Among them, seven signals at δC 168.1, 163.6, 132.9 (2C), 122.2, and 116.3 (2C) supported the presence of a phydroxybenzoyl moiety. Six signals at δC 104.2, 77.9, 75.7, 75.0, 71.7, and 64.7 gave evidence of a sugar unit being present. The downfield chemical shift of the tertiary olefinic carbon signal at δC 144.6 suggested the presence of an electrophilic group connected to a double bond. The carbon signal at δC 118.2 was assigned to the cyano group, which was supported by the chemical formula of 1, which contained a nitrogen atom.7,8 The structure of the aglycon was determined as 2-hydroxymethyl-4hydroxy-2-butenenitrile by 2D NMR spectroscopic analysis (Figure 1). The NOE correlation between H-5 (δH 4.59) and

Table 1. 1H NMR and 13C NMR Spectroscopic Data for Compounds 1 and 2a 1 δH

position 1 2 3 4 5 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ −OCH3

6.71, 4.17, 4.59, 4.45, 3.32, 3.48, 3.48, 3.66, 4.72, 4.45,

t (6.5) d (6.5) s d (8.0) m m m m m m

7.98, d (8.5) 6.90, d (8.5) 6.90, d (8.5) 7.98, d (8.5)

2 δC 118.2 116.7 144.6 63.2 68.2 104.2 75.0 77.9 71.7 75.7 64.7 122.2 132.9 116.3 163.6 116.3 132.9 168.1

δH

6.73, 4.20, 4.61, 4.47, 3.33, 3.50, 3.50, 3.70, 4.74, 4.50,

t (6.5) d (6.5) m d (7.5) m m m m m m

7.66, d (2.0)

6.94, d (8.0) 7.69, dd (8.0, 2.0) 3.99 s

δC 118.2 116.0 144.5 63.1 68.2 104.2 74.9 77.8 71.7 75.7 64.8 122.5 113.7 148.8 152.9 116.0 125.3 168.0 56.5

a1

H NMR data (δ) were measured in CD3OD at 500 MHz; 13C NMR data (δ) were measured in CD3OD at 125 MHz.

myofibroblasts. Injured epithelial cells have been reported to serve as the primary source for myofibroblasts through a process known as epithelial mesenchymal transition (EMT).13−15 During this so-called trans-differentiation process, the cells acquire a spindle-like shape and start producing αSMA (a marker of myofibroblasts in the kidney) and matrix proteins and show enhanced cell migration and invasion.15 Our unpublished preliminary data demonstrated that the ethanolic extract of the aerial parts of R. manshuricum protected mice from UUO-induced kidney injury. We therefore tested whether ribemansides A (1) and B (2) are capable of mitigating EMTmediated fibrogenesis in HK-2 cells. TGF-β1 exposure (5 ng/ mL, 36 h) induced a spindle-like morphology of HK-2 cells (Figure 2A). The ratio of length/width was increased significantly from 1.63 ± 0.04 to 2.56 ± 0.05 (p < 0.01, n = 40) (Figure 2B). Pretreatment with 1 or 2 (30 μM) significantly reversed the HK-2 cell morphological changes (Figure 2B). Consistent with the previous study, the expression level of α-SMA was significantly increased by 1.82-fold (p < 0.01, n = 4) (Figure 2C and D). Compounds 1 and 2 (30 μM) both inhibited TGF-β1-induced increase of α-SMA expression (p < 0.01, n = 4) (Figure 2C and D). Similarly, 1 and 2 both inhibited TGF-β1-induced fibronectin release (p < 0.01, n = 4) (Figure 2E). TRPC6 channels are Ca2+-selective members of the TRPC subfamily.16,17 Mutations in TRPC6 have been demonstrated to be associated with kidney dysfunction. A single point gain-offunction mutation (P112Q) in TRPC6 is sufficient to cause focal segmental glomerular sclerosis,18 and increased TRPC6 expression has been reported in several proteinuric kidney diseases in humans.19 A more recent proof-of-concept study further demonstrated that UUO-induced kidney injury resulted in increased TRPC6 expression, while TRPC6 knockout ameliorated UUO-induced kidney fibrosis in tubular epithelia cells in mice.20 We therefore tested the effects of 1 and 2 on

Figure 1. Selected H−H COSY (−), HMBC (→), and NOESY (↔) correlations of ribemanside A (1).

H-3 (δH 6.71) suggested an E-configuration of the Δ2(3)-double bond. To determine the structure of the sugar moiety, ribemanside A (1) was hydrolyzed with acid, and the sugar was found to be D-glucose by comparing the optical rotation with an authentic sample. HMBC correlations between H-1′ (δH 4.45) and C-5 (δC 68.2) and between H-5 (δH 4.59) and C1′ (δC 104.2) demonstrated that the D-glucose unit is linked to C-5. In turn, HMBC correlations of H-6′ (δH 4.72 and 4.45) with C-7″ (δC 168.1), coupled with the downfield chemical shift of C-6′, demonstrated that the p-hydroxybenzoyl moiety is connected to C-6′. Consequently, the structure of ribemanside A (1) was determined to be 2β-D-(6′-O-p-hydroxybenzoyl)glucopyranosyloxymethy-4-hydroxy-2(E)-butenenitrile. The HRESIMS of ribemanside B (2) gave a [M + H] + mass at m/z 426.1375, consistent with a molecular formula of C19H23NO10. The 1H NMR data of 2 (Table 1) resembled those of compound 1, but contained an extra methoxy group signal at δH 3.99 (3H, s) and a typical ABX-coupling system comprising three aromatic protons resonating at δH 7.69 (1H, dd, J = 8.0, 1.5 Hz), 7.66 (1H, d, J = 2.0 Hz), and 6.94 (1H, d, J = 8.0 Hz). The 1H NMR data together with the 13C NMR (Table 1) spectrum indicated the presence of a vanilloyl group, attached to the C-6′ position, based on HMBC correlations of H-6′ (δH 4.76, 4.52) with the ester carbonyl at δC 168.0. Consequently, ribemanside B (2) was identified as 2β-D-(6′-Ovanilloyl)glucopyranosyloxymethy-4-hydroxy-2(E)-butenenitrile. Fibrosis is a key step of renal repair following injury or in response to cytokines and other factors9−12 and is characterized by the proliferation of fibroblasts and their transformation into 914

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Figure 2. Ribemansides A (1) and B (2) suppress TGF-β1-induced fibrogenesis in HK-2 cells. (A) Representative images for TGF-β1-induced HK-2 cellular morphological changes in the presence and absence of 1 or 2. (B) Quantification of the ratio of HK-2 cell length/width in the presence and absence of 1 or 2 after TGF-β1 exposure. (C) Representative Western blot for TGF-β1-induced α-SMA expression in HK-2 cells in the presence and absence of 1 or 2. (D) Quantification of α-SMA expression in the presence and absence of 1 or 2 after TGF-β1 exposure. (E) Ribemansides A (1) and B (2) inhibit the TGF-β1-induced secretion of fibronectin. The experiments were performed twice each in duplicates (**, p < 0.01, TGF-β1 vs vehicle (Veh); ##, p < 0.01, TGF-β1 + 1 or 2 vs TGF-β1). measured on a UV−vis spectrophotometer (UV-2550, Shimadzu). IR spectra were recorded on an FT-IR microscope spectrometer (Nicolet 5700). NMR spectra were recorded on a Bruker-500 spectrometer. HRESIMS data were recorded on an Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies 6520). RP-HPLC separations were performed using a Welch Ultimate XB-C18 column (250 × 10 mm, 5 μm) on an Agilent 1260 instrument coupled to a VWD detector. Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden), ODS (45−70 μm, Welch, Shanghai, People’s Republic of China), and silica gel (200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People’s Republic of China) were used for column chromatography. Thin-layer chromatography (TLC) was carried out on precoated GF254 silica gel plates (Qingdao Marine Chemical Ltd.). Plant Material. The aerial parts of Ribes manshuricum were acquired in September 2015 from the Changbai Mountain area of Jilin Province, People’s Republic of China, and were identified by one of the authors (B.Y.). A voucher specimen (RM-201509) was deposited at the School of Traditional Chinese Pharmacy, China Pharmaceutical University. Extraction and Isolation. The dried aerial parts of R. manshuricum (50 kg) were extracted three times with 95% EtOH (5 × 50 L) heated at reflux for 4 h each. The solvent was evaporated under reduced pressure to give a crude extract (1570 g), which was suspended in 5 L of water and first extracted with petroleum ether (15 L), then ethyl acetate (12 L) and then n-butanol (8 L). The n-butanol extract (320 g) was applied to a D101 macroporous resin column (160 × 12 cm) eluted with 0%, 20%, 30%, 40%, and 60% EtOH in H2O, successively, to give five fractions (I−V). Fraction II (50 g) was subjected to reversed-phase C18 silica gel column chromatography (60 × 4 cm), conducted with a stepwise gradient of MeOH in H2O (5%, 20%, and 40%), to afford three subfractions (IIa−IIc). Fraction IIc was

TRPC6 channel activity. Both 1 and 2 suppressed the TRPC6 agonist M08521 and induced Ca2+ influx in HEK-293 cells expressing TRPC6 at micromolar concentrations (Figure 3A and B). The IC50 values for 1 and 2 in terms of suppressing TRPC6 activity were 24.5 and 25.6 μM, respectively (Figure 3C). In order to determine whether inhibition of TRPC6 contributed to the antifibrogenic effect of 1 and 2, the effect of SAR7334, a TRPC6 inhibitor, was evaluated on TGF-β1stimulated fibrogenesis in HK-2 cells. SAR7334 (1 μM) alone had no effect on the cellular morphological changes (Figure S19A and B, Supporting Information). However, SAR7334 did reverse TGF-β1-induced spindle-like morphology in a concentration-dependent manner (Figure S19A and B, Supporting Information). SAR7334 also concentration-dependently decreased the TGF-β1-induced increase of α-SMA expression without any effects on basal α-SMA expression (Figure S19C and D, Supporting Information). Similar to 1 and 2, SAR7334 also concentration-dependently reduced TGF-β1-induced secretion of fibronectin (Figure S19E, Supporting Information). Taken together, these data suggest that TRPC6 channels play a significant role in fibrogenesis in HK-2 cells and that 1 and 2 may be valuable as lead compounds to design novel TRPC6 inhibitors for the treatment of renal fibrosis.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were taken on an Autopol-IV automatic digital polarimeter. UV spectra were 915

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measuring their optical rotations and by co-TLC with an authentic sample.22 HK-2 Cell Culture. The HK-2 cell line was generously provided by Professor Bicheng Liu (Zhongda Hospital, Southeast University, Nanjing, People’s Republic of China) and grown in RPMI 1640 medium containing 2 mg/mL NaHCO3, 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA), 10 mM HEPES, 100 U/ mL penicillin, and 0.1 mg/mL streptomycin (ThermoFisher Scientific, Waltham, MA, USA). The cells at approximately 80% confluency were digested with 0.05% trypsin−EDTA (ThermoFisher Scientific) and seeded in 96-well or 12-well plates (Corning; Corning, NY, USA) at densities of 6000 cells/well or 40 000 cells/well. The cells were then cultured in serum-free medium and stimulated with TGF-β1 (5 ng/ mL, R&D Systems, Minneapolis, MN, USA) in the absence and presence of ribemanside A (1) or B (2) or SAR7334 for 36 h. TRPC6-HEK-293 Cell Culture. HEK-293 cells stably expressing mouse TRPC6 were provided by Professor Michael X. Zhu at the University of Texas Health Science Center at Houston and cultured as described previously.21 Cells at approximately 80% confluency were digested with 0.05% trypsin−EDTA and seeded in poly-D-lysinecoated 96-well plates at a density of ∼20 000 cells/well. The cells were cultured for 6 h before use. Intracellular Ca2+ Concentration Determination. The intracellular Ca2+ concentration was determined as described previously.21 Briefly, after incubation with 4 μM Fluo-8/AM (TEFlabs, Austin, TX, USA) for 45 min, the TRPC6-HEK-293 cells were gently washed four times and loaded into the chamber of a fluorescent imaging plate reader (FLIPRTetra; Molecular Devices, Sunnyvale, CA, USA). Basal fluorescence units (F0) were recorded for 30 s followed by the addition of vehicle or compounds, and the fluorescent signals (F) were recorded for an additional 5 min before addition of M085 (1 μM) (ethyl 4-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a]pyrimidin-5-yl)piperidine-1 carboxylate).21 Data are presented as F/ F0. To analyze the concentration−response relationship, the area under the curve (AUC) was calculated from a time period of 300 s right after addition of M085. Western Blotting. The Western blotting experiments were performed as described previously.23 Equal amounts (30 μg) of protein were mixed with loading buffer, and the samples were loaded onto a 12% SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane by electroblotting. Membranes were blocked with 5% skimmed milk in phosphatebuffered saline for 1 h at room temperature and then were incubated overnight at 4 °C with anti-α-SMA (1:1500) (Abcam, Cambridge, MA, USA) and anti-tubulin (1:5000) (Bioworld, Shanghai, People’s Republic of China) antibodies. After washing, the blots were incubated with the IRDye (680RD or 800CW)-labeled secondary antibodies (1:10 000) for 1 h at room temperature and then were scanned with the LI-COR Odyssey infrared imaging system (LI-COR Biotechnology, Lincoln, NE, USA). Densitometry was performed using the LICOR Odyssey infrared imaging system application software (version 2.1). Measurement of Fibronectin. Fibronectin secretion was determined by a commercial ELISA kit (Jin-Yi-Bai Biological Technology Co. Ltd., Nanjing, People’s Republic of China) according to the instructions of the manufacturer. The optical density value was detected using a Tecan Infinite 200 Pro microplate reader (Tecan Trading AG, Männedorf, Switzerland) at a wavelength of 450 nm. Data Analysis. Data plotting and statistical analysis were performed with GraphPad Prism software (version 5.0, GraphPad Software Inc., San Diego, CA, USA). Concentration response curves were fit by nonlinear regression using a three-parameter logistic equation. Statistical significance between groups was calculated using ANOVA and, where appropriate, a Dunnett’s multiple comparison test; p values of less than 0.05 were considered statistically significant.

Figure 3. Ribemansides A (1) and B (2) suppress the TRPC6 agonist (M085)-induced Ca2+ response in HEK-293 cells expressing TRPC6. (A) Representative traces for 1 suppressing the M085-induced Ca2+ response in HEK-293 cells, with TRPC6 expressed as a function of time. (B) Representative traces for 2 suppressing the M085-induced Ca2+ response in HEK-293 cells, with TRPC6 expressed as a function of time. (C) Concentration−response relationships for 1 and 2 in suppressing TRPC6 activity. The experiments were performed twice, with each in triplicate.

further separated by HPLC [CH3CN: 0.1% TFA−H2O (14:86, v/v, 2 mL/min)] to yield 1 (tR = 14.1 min, 16.0 mg) and 2 (tR = 29.4 min, 14.7 mg). Ribemanside A (1): brown paste; [α]20 D −11.3 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 258 (3.89) nm; IR νmax 3394, 2911, 2226, 1693, 1608, 1515, 1280, 1168, 1066 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 396.1287 [M + H]+ (calcd for 396.1289, C18H22NO9). Ribemanside B (2): brown paste; [α]20 D −7.1 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 263 (3.83), 288 (3.65) nm; IR νmax 3390, 2924, 2225, 1696, 1598, 1515, 1287, 1222, 1080 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 426.1375 [M + H]+ (calcd for 426.1370, C19H24NO10). Acid Hydrolysis. An aliquot of 6 mg of ribemanside A (1) or ribemanside B (2) was refluxed in 6% HCl (4.0 mL) at 80 °C for 2 h. The reaction mixture was extracted with CHCl3 (3 × 5 mL), and the aqueous layer was dried by a N2 stream. The residue was loaded onto a silica gel column and eluted with EtOAc−EtOH−H2O (7:4:1) to yield 20 D-glucose (1.7 mg) from 1, [α]D +47 (c 0.09, H2O), and D-glucose 20 (1.5 mg) from 2, [α]D +42 (c 0.08, H2O), respectively, identified by 916

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(19) Moeller, C. C.; Wei, C.; Altintas, M. M.; Li, J.; Greka, A.; Ohse, T.; Pippin, J. W.; Rastaldi, M. P.; Wawersik, S.; Schiavi, S.; Henger, A.; Kretzler, M.; Shankland, S. J.; Reiser, J. J. Am. Soc. Nephrol. 2007, 18, 29−36. (20) Wu, Y. L.; Xie, J.; An, S. W.; Oliver, N.; Barrezueta, N. X.; Lin, M. H.; Birnbaumer, L.; Huang, C. L. Kidney Int. 2017, 91, 830−841. (21) Qu, C. R.; Ding, M. M.; Zhu, Y. M.; Lu, Y. G.; Du, J.; Miller, M.; Tian, J. B.; Zhu, J. M.; Xui, J.; Wen, M.; Er-Bu, A.; Wan, J. L.; Xiao, Y. L.; Wu, M.; McManus, O. B.; Li, M.; Wu, J. L.; Luo, H. R.; Cao, Z. Y.; Shen, B.; Wang, H. B.; Zhu, M. X.; Hong, X. C. J. Med. Chem. 2017, 60, 4680−4692. (22) Liu, Y. F.; Shi, G. R.; Wang, X.; Zhang, C. L.; Wang, Y.; Chen, R. Y.; Yu, D. Q. J. Nat. Prod. 2016, 79, 428−433. (23) Cao, Z. Y.; George, J.; Baden, D. G.; Murray, T. F. Brain Res. 2007, 1184, 17−27.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01037. 1D and 2D NMR, UV, IR, and mass spectra for compounds 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86 25 86185158. E-mail: [email protected] (C. Z. Chai). *Tel/Fax: +86 25 86185158. E-mail: [email protected] (Z. Y. Cao). ORCID

Zhengyu Cao: 0000-0002-2692-2949 Author Contributions §

B. P. Zhou, Y. G. Wang, and C. L. Zhang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of the People’s Republic of China (81603389, 21777192, and 81473539), the Innovative Drug Development Program from Ministry of Science and Technology (2017ZX09101003-004-002), and the Natural Science Foundation of Jiangsu Province (CN) (BK20160764, BK20160754). We thank Prof. H. Wulff at UCDavis for English editing.

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DOI: 10.1021/acs.jnatprod.7b01037 J. Nat. Prod. 2018, 81, 913−917