New iridoid from Valeriana hardwickii Wall

Cite this paper: Vietnam J. Chem., 2021, 59(1), 12-16 Article DOI: 10.1002/vjch.202000073 12 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH New iridoid from Valeriana hardwickii Wall. Le Thi Thu Hong 1 , Tran Thi Huyen 2* , Le Minh Tri 2 , Tran Duy Hien 3 , Huynh Loi 4 1 Department of Pharmacognosy, Faculty of Pharmacy, Lac Hong University, 10 Huynh Van Nghe street, Bien Hoa city, Dong Nai Province 76108, Viet Nam 2 School of Medicine, Vietnam National University - HCMC, Quarter 6, Linh Trung Ward, Thu Duc District, Ho Chi Minh City 70000, Viet Nam 3 Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Ho Chi Minh City, 41 Dinh Tien Hoang street, District 1, Ho Chi Minh City 70000, Viet Nam 4 Department of Pharmacognosy, School of Medicine and Pharmacy, University of Da Nang, Hoa Quy ward, Ngu Hanh Son District, Da Nang City 55000, Viet Nam Submitted May 7, 2020; Accepted August 18, 2020 Abstract A new iridoid, (4S,5R,6S,7S,9S)-7,10,11-trihydroxy dihydronepetalactone (1) together with four known flavonoids including linarin, neodiosmin, neobudofficide and rhoifolin were isolated from aerial parts of Valeriana hardwickii Wall.. Chemical structures were elucidated by extensive spectroscopic analyses including ESI-HRMS and 1D, 2D- NMR spectral data as well as by comparison with those reported in the literatures. Compound 1 showed no cytotoxic activity toward three human cancer cell lines (CCRF-CEM, MDA-MB-231 and HCT-116). Keywords. Valeriana hardwickii, (4S,5R,6S,7S,9S)-7,10,11-trihydroxy dihydronepetalactone, iridoid, flavonoid. 1. INTRODUCTION Valeriana, belonging to Caprifoliaceae family, is traditionally used as sedative, tranquilizing and anxiolytic. Valeria hardwickii grows in many Asian countries. In Vietnam, V. hardwickii, vernacular name as “ an ”, is used for medicinal above purposes. [1] The chemical constitunents from V. hardwickii include sesquiterpenoids, triterpenoids, iridoids, and flavonoids. [2-6] The chemical investigation on the aerial parts of V. hardwickii led to isolate a new iridoid (1), along with four known flavonoids including linarin, rhoifolin, neobudofficide, and neodiosmin. This research reports the isolation, structural elucidation of these compounds, and cytotoxic identification of compound 1. 2. MATERIALS AND METHODS 2.1. Plant materials The aerial parts of V. hardwickii were collected in Bidoup - Nui Ba mountain, Lam Dong province, Vietnam, in September 2011. The plant was identified by Dr. Vo Van Chi and confirmed using gemomic analysis by Dr. Huynh Ky, Can Tho University, Vietnam. The voucher specimen (No. 01-2011-BMDL) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy, Ho Chi Minh city, Vietnam. 2.2. General experimental procedures NMR spectra with a Bruker Avance 500 spectrometer, with tetramethyl silane (TMS) as an internal standard. ESI-HRMS data were measured on a Shimadzu LCMS-IT-TOF spectrometer. Column chromatography was performed by using silica gel (40-60 µm, Merck), Sephadex LH-20 (Dowex ® 50WX2-100, Sigma-Aldrich). Analytical TLC was performed on pre-coated silica gel 60 F254 (0.25 mm thickness, Merck). 2.3. Extraction and isolation The dried aerial parts of V. hardwikii (2.5 kg) were percolated with 96 % ethanol at room temperature to yield a crude extract (0.5 L). This crude extract was partitioned sequentially with petroleum ether (2.5 L×3 times), ethyl acetate (2 L×4 times) and n- Vietnam Journal of Chemistry Tran Thi Huyen et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 13 butanol (2 L×3 times affording petroleum ether (VH-PE, 32.88 g), ethyl acetate (VH-EA, 70.64 g), n-butanol (VH-Bu, 38.73 g) extracts. VH-Bu extract (28 g) was separated by silica gel column chromatography (7×60 cm), eluted with gradient mixtures of EtOAc-MeOH (10:0 → 7:3, v/v) to yield 10 fractions (BB1-BB10). Fraction BB- 6 was recrystallized in EtOAc-MeOH (8:2, v/v) to give compound 1 (730 mg). Fraction BB-7 was precipitated in MeOH to furnish compound 2 (450 mg). Fraction BB-8 was separated by Sephadex LH- 20 column chromatography (2.5×60 cm) using MeOH as mobile phase to obtain compound 3 (80 mg). Fraction BB-10 was also applied to Sephadex LH-20 column chromatography (2.5×60 cm), eluted with MeOH, affording compound 4 (40 mg) and 5 (8 mg). Compound 1: Colorless crystal; mp. 127-128 o C; = +47.3 (MeOH, 0.1 mg/mL); IR (KBr) νmax: 3320 (-OH), 2940 (C-H), 1700 (C=O), 1480, 1400, 1300, 1190, 900 cm -1 ; UV (MeOH) λmax: 205 nm; ESI-HRMS positive mode m/z 239.0919 [M+Na] + calcd for (C10H16O5); 1D- and 2D-NMR, see table 1. Compound 2: White amorphous powder; UV (MeOH) λmax: 267 and 322 nm; ESI-MS (m/z): 593.59 [M+H] + (C28H32O14); 1 H-NMR (DMSO-d6, 400 MHz): 12.90 (1H, s, 5-OH), 8.04 (2H, d, 9.0 Hz, H-2′/6′), 7.14 (2H, d, 9.0 Hz, H-3′/5′), 6.93 (1H, s, H-3), 6.78 (1H, d, 2.0 Hz, H-8), 6.44 (1H, d, 2.0 Hz, H-6), 5.05 (1H, d, 7.0 Hz, H-1′′), 4.55 (1H, d, 1.0 Hz, H-1′′′), 3.85 (3H, s, 4′-OCH3), 1.07 (3H, d, 7.0 Hz, H-6′′′); 13C-NMR (DMSO-d6, 100 MHz): 182.1 (C-4), 164.1 (C-2), 163.0 (C-7), 162.5 (C-4′), 161.3 (C-5), 157.1 (C-8a), 128.6 (C-2′/6′), 122.8 (C-1′), 114.8 (C-3′/5′), 105.6 (C-4a), 103.9 (C-3), 100.6 (C- 1′′′), 100.0 (C-1′′), 99.8 (C-6), 94.9 (C-8), 76.4 (C- 3′′), 75.8 (C-5′′), 73.2 (C-2′′), 72.2 (C-4′′′), 70.9 (C- 3′′′), 70.5 (C-2′′′), 69.7 (C-4′′), 68.4 (C-5′′′), 66.2 (C- 6′′), 55.7 (4’-OCH3), 17.9 (C-6′′′). Compound 3: Yellow amorphous powder; UV (MeOH) λmax: 267 and 335 nm; ESI-MS (m/z): 601.9 [M+Na] + (C27H30O14); 1 H-NMR (DMSO-d6, 400 MHz): 12.97 (1H, s, 5-OH), 7.92 (2H, d, 9.0 Hz, H- 2′/6′), 6.93 (2H, d, 9.0 Hz, H-3′/5′), 6.85 (1H, s, H- 3), 6.78 (1H, br s, H-8), 6.36 (1H, br s, H-6), 5.22 (1H, d, 7.0 Hz, H-1′′), 5.12 (1H, s, H-1′′′), 1.19 (3H, d, 6.0 Hz, H-6′′′); 13C-NMR (DMSO-d6, 100 MHz): 182.1 (C-4), 164.4 (C-2), 162.6 (C-7), 161.6 (C-4′), 161.2 (C-5), 157.1 (C-8a), 128.7 (C-2′/6′), 121.0 (C- 1′), 116.2 (C-3′/5′), 105.5 (C-4a), 103.3 (C-3), 100.6 (C-1′′′), 99.5 (C-6), 97.9 (C-1′′), 94.6 (C-8), 77.3 (C- 3′′), 77.1 (C-5′′), 76.4 (C-2′′), 71.2 (C-4′′′), 70.6 (C- 3′′′), 70.5 (C-2′′′), 69.8 (C-4′′), 68.5 (C-5′′′), 60.6 (C- 6′′), 18.2 (C-6′′′). Compound 4: Yellow amorphous powder; UV (MeOH) λmax: 267 and 335 nm; ESI-MS (m/z): 739.54 [M+H] + (C34H42O18); 1 H-NMR (DMSO-d6, 400 MHz): 12.91 (1H, s, 5-OH), 8.02 (2H, d, 9.0 Hz, H-2′/6′), 7.15 (2H, d, 9.0 Hz, H-3′/5′), 6.94 (1H, s, H-3), 6.72 (1H, d, 2.0 Hz, H-8), 6.38 (1H, d, 2.0 Hz, H-6), 5.22 (1H, d, 7.0 Hz, H-1′′), 5.12 (1H, s, H- 1′′′′), 4.54 (1H, s, H-1′′′), 3.85 (3H, s, 4′-OCH3), 1.20 (3H, d, 6.0 Hz, H-6′′′′), 1.07 (3H, d, 6.0 Hz, H-6′′′); 13 C-NMR (DMSO-d6, 100 MHz): 182.1 (C-4), 164.1 (C-2), 162.6 (C-7), 162.5 (C-4′), 161.3 (C-5), 157.1 (C-8a), 128.5 (C-2′/6′), 122.8 (C-1′), 114.9 (C-3′/5′), 105.7 (C-4a), 104.0 (C-3), 100.6 (C-1′′′/1′′′′), 99.5 (C-6), 97.9 (C-1′′), 94.5 (C-8), 77.1 (C-3′′), 76.3 (C- 2′′), 75.6 (C-5′′), 72.1 (C-4′′′), 71.9 (C-4′′′′), 70.8 (C- 3′′′), 70.5 (C-3′′′′), 70.4 (C-2′′′), 70.1 (C-2′′′′), 69.8 (C-4′′), 68.4 (C-5′′′/5′′′′), 66.1 (C-6′′), 55.7 (4′- OCH3), 18.2 (C-6′′′′), 17.9 (C-6′′′). Compound 5: Yellow amorphous powder; UV (MeOH) λmax: 267 and 322 nm; ESI-MS (m/z): 609.00 [M+H] + (C28H32O15); 1 H-NMR (DMSO-d6, 500 MHz): 12.92 (1H, s, 5-OH), 7.52 (1H, dd, 9.0 and 2.0 Hz, H-6′), 7.42 (1H, d, 9.0 Hz, H-2′), 7.07 (1H, d, 2.0 Hz, H-5′), 6.86 (1H, s, H-3), 6.77 (1H, br s, H-8), 6.38 (1H, br s, H-6), 5.21 (1H, d, 7.0 Hz, H- 1′′), 5.11 (1H, s, H-1′′′), 3.84 (3H, s, 4′-OCH3), 1.18 (3H, d, 6.0 Hz, H-6′′′); 13C-NMR (DMSO-d6, 125 MHz): 182.9 (C-4), 164.2 (C-2), 162.6 (C-7), 161.1 (C-5), 157.0 (C-8a), 151.4 (C-4′), 146.9 (C-3′), 122.9 (C-1′), 118.8 (C-6′), 113.1 (C-2′), 112.2 (C- 5′), 105.5 (C-4a), 103.9 (C-3), 100.5 (C-1′′′), 99.4 (C-6), 97.8 (C-1′′), 94.5 (C-8), 77.1 (C-5′′), 77.0 (C- 3′′), 76.3 (C-2′′), 71.9 (C-4′′′), 70.5 (C-4′′), 70.4 (C- 2′′′), 69.7 (C-3′′′), 68.3 (C-5′′′), 60.5 (C-6′′), 55.8 (4′- OCH3), 18.0 (C-6′′′). 2.4. XTT assay 2.4.1. Sample preparation, cell culture and XTT viability assay The protocol for experiment on sample preparation, cell culture (CCRF-CEM, MDA-MB-231 and HCT- 116 cell line) and XTT assay was described in the literature. [7] 2.4.2. Statistical analysis The data is expressed in terms of mean (Mean) ± standard deviation (SEM). Results are processed by MS Excel and statistical Statview 4.5 software. Data were statistically analyzed using the non-parametric Kruskal-Wallis (H) -test and Chi square (χ2) test. The difference was statistically significant when P < 0.05. Data were compared against the control Vietnam Journal of Chemistry New iridoid from Valeriana hardwickii Wall. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 14 sample. 3. RESULTS AND DISCUSSION The n-butanol extract from aerial parts of V. hardwickii was separated and purified that led to the isolation of five compounds (1-5). Compound 1 was obtained as a colorless crystal. The ESI-HRMS of 1 gave an [M+Na] + ion at m/z 239.0919 which was calculated for C10H16O5. The 1 H-NMR spectrum of compound 1 showed six protons of three oxygenated methylene groups at H- 3 (4.69 dd (10.5, 3.0) 1H and 4.37 t (10.5 Hz) 1H), H-10 (4.53 dd (10.5, 4.5 Hz) 1H and 4,47 dd (10.5, 7.5 Hz) 1H), H-11 (3.95 dd (11.0, 4.5 Hz) 1H and 3.74 dd (11.0, 7,5 Hz) 1H); one proton of oxygenated methine group at H-7 (4.81 t (3.5 Hz) 1H). The 13 C-NMR spectrum of 1 indicated 10 signals, including one carbonyl carbon (δc 176.6), three oxygenated methylene carbons (δc 70.6, 62.4 and 62.2), one oxygenated methine carbon (δc 73.5), four methine carbons (δc 51.8, 44.9, 42.8 and 37.7) and one methylene carbon (δc 42.4). HMBC correlations from H-4 to C-6/C-9, H-5 to C-1/C-3/C- 11, H-9 to C-C-4/C-6/C-10 and COSY correlations between H-4 with H-3/H-5/H-11, H-8 with H-7/H- 9/H-10 identified that compound 1 had dihydronepetalactone skeleton with bicyclic structure, including a lactone 6-sided round and a 5- sides one. On the other hand, the addition of three hydroxyl groups were determined by downfield chemical shifts of two methylene carbons (C-10 and C-11) and one methine carbon (C-7). The NOESY correlation between H-5 with H-3β/H-6β/H-9, H-4 with H-3α/H-6α, H-8 with H-6α/H-7 (figure 2) and coupling constant of JH-5/H-9 = 11.5 Hz, JH-7/H-8 = 3.5 Hz show that configuration of compound 1 is similar to that of compound isolated from V. laxifolia. [8] Detail NMR spectral data analyses led to the identification of (4S,5R,6S,7S,9S)-7,10,11- trihydroxy dihydronepetalactone for compound 1. This metabolite is reported for the first time from nature (according to searching on www.scifinder.cas.gov, April 27 th 2020). Table 1: NMR spectral data of compound 1 Position δC a (ppm) δH b (ppm) mult (J, Hz) nH HMBC (HCn) COSY (HHn) NOESY (HHn) 1 176.7 - - - - 3α 70.6 4.69 dd (10.5, 3.0) 1H 1, 4, 5, 11 4 4 3β 4.37 t (10.5) 1H 4, 5, 9, 11 4 44.9 1.96 m 1H 3, 5, 6, 9, 11 3, 5, 11 3α, 6α, 11 5 37.7 3,08 tdd (11.5, 9.5, 7.0) 1H 1, 3, 4, 6, 9, 11 4, 6, 9 3β, 6β, 9, 11 6α 42.4 1.54 ddd (13.0, 11.0; 3.5) 1H 4, 5, 7, 8, 9 5, 7 4, 7, 8 6β 2.37 ddd (13.0, 7.0, 1.5) 1H 5, 7 7 73.5 4.81 t (3.5) 1H 5, 9 6, 8 6α, 6β, 8 8 51.8 2.89 ddd (9.0, 7.5, 3.5) 1H 1, 6, 10 7, 9, 10 6α, 7, 10 9 42.8 3.33 dd (11.5, 9.5) 1H 1, 4, 5, 6, 8, 10 5, 8 3β, 5, 10 10 62.4 4.53 dd (10.5, 4.5) 1H 7, 8, 9 8 8, 9 4,47 dd (10.5, 7.5) 1H 11 62.2 3.95 dd (11.0, 4.5) 1H 3, 4, 5 4 3β, 4, 5 3.74 dd (11.0, 7,5) 1H a : 13 C-NMR (pyridine-d5, 125 MHz); b : 1 H-NMR (pyridine-d5, 500 MHz). Cytotoxic activity indicates that compound 1 is not toxic on all three cell lines (CCRF-CEM, MDA- MB-231 and HCT-116) at trial concentrations of 5 and 50 mg/mL. The chemical structures of isolated compounds were elucidated by the analyses of their spectral data together comparison with those in the published literatures (figure 1). The remaining compounds were characterized as linarin (2), rhoifolin (3), neobudofficide (4), and neodiosmin (5) by comparison of their NMR and MS spectral data with those literature data. [9][10][11][12] Linarin, rhoifolin, neobudofficide, valechlorine and isovaleroxyvaltrate hydrine were reported from MeOH extract of V. hardwickii by our research group. [5] Neodiosmin (5) was firstly isolated from this herb. Neodiosmin is used to debitter. It has also a high antioxidant activity. [13] Ning Sun et al. (2020) used molecular docking to screen neodiosmin on 3CL Pro protein of SARS-CoV-2 showed that six hydrogen bonds at HIS41, LEU141, GLY143, HIS163, THR190 and carbon hydrogen bonds at Vietnam Journal of Chemistry Tran Thi Huyen et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 15 HIS41, LEU141, ASN142, ASP187. [14] Figure 1: Chemical structure of compounds 1-5 Figure 2: Main HMBC, COSY and NOESY correlations of compound 1 4. CONCLUSION Using various chromatography methods, five compounds were isolated from the n-butanol extract from the aerial parts of Valeria hardwickii Wall. These isolated compounds were elucidated by spectral data analyses and comparison with those in the literatures as (4S,5R,6S,7S,9S)-7,10,11- trihydroxy dihydronepetalactone (1), linarin (2), rhoifolin (3), neobudofficide (4), and neodiosmin (5). 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