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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa a
a
b
c
Thi Hoai Nguyen , Viet Duc Ho , Thi Thao Do , Huu Tai Bui , Van d
e
Kiem Phan , Katrin Sak & Ain Raal
f
a
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen Street, Hue City, Vietnam b
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam c
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea d
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam e
NGO Praeventio, Näituse 22-3, Tartu, Estonia
f
Department of Pharmacy, University of Tartu, 1 Nooruse Street, Tartu, Estonia Published online: 22 Oct 2014.
To cite this article: Thi Hoai Nguyen, Viet Duc Ho, Thi Thao Do, Huu Tai Bui, Van Kiem Phan, Katrin Sak & Ain Raal (2014): A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.971790 To link to this article: http://dx.doi.org/10.1080/14786419.2014.971790
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.971790
A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa
Downloaded by [The University of Manchester Library] at 06:56 11 December 2014
Thi Hoai Nguyena, Viet Duc Hoa, Thi Thao Dob, Huu Tai Buic, Van Kiem Phand, Katrin Sake and Ain Raalf* a
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen Street, Hue City, Vietnam; bInstitute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam; cCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Korea; dInstitute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam; eNGO Praeventio, Na¨ituse 22-3, Tartu, Estonia; f Department of Pharmacy, University of Tartu, 1 Nooruse Street, Tartu, Estonia (Received 2 July 2014; final version received 21 September 2014) Chemical investigation of the aerial parts of Uvaria rufa (Dunal) Blume collected from Vietnam yielded one new lignan glycoside, ufaside (1), along with six known compounds, oxoanolobine (2), ergosta-4,6,8(14),22-tetraen-3-one (3), catechin (4), epicatechin (5), daucosterol (6) and glutin-5-en-3-one (7). Their chemical structures were determined by using NMR, HR-MS spectroscopic analyses and in comparison with the reported data. A cytotoxic analysis of U. rufa herb extracts was performed for the first time using nine human cancer cell lines (MCF-7, MDA-MB-231, LNCaP, MKN7, SW480, KB, LU-1, HepG2 and HL-60) derived from different tumour types. Of these seven constituents, compounds 2 and 3 displayed moderate cytotoxicity against the human lung adenocarcinoma cell line (LU-1) with IC50 values of 9.22 ^ 1.02 mg/mL and 10.21 ^ 1.16 mg/mL, respectively. Keywords: Uvaria rufa; Annonaceae; lignan glycoside; ufaside; cytotoxicity
1. Introduction The genus Uvaria L. (Annonaceae) comprises about 150 species (Leboeuf et al. 1982). It continues to be a rich source of new natural products of varied structures (Leboeuf et al. 1982; Parmar et al. 1994), some of which have shown interesting antibacterial (Hufford et al. 1980), antioxidant (Alali et al. 1999) and antitumour (Jolad et al. 1982; Zhou et al. 1999, 2000) activities. Uvaria rufa (Dunal) Blume (synonym U. rufa (Fin. & Gagnep.) Ast) is a long woody vine, 8 –10 –m tall. It is commonly distributed in the Indochina peninsula as well as in the central part of Vietnam (Ban 2000). Although this plant has been used in Vietnamese traditional medicine to treat tumours, its anticancer action has not yet been thoroughly studied. Also, the knowledge about the chemical composition of U. rufa is rather poor and only few previous works have described the content of some flavonoids and non-flavonoid polyphenols as well as alkaloids in the leaves and stems of this shrub (Huang et al. 1998; Hasan et al. 2001; Asha et al. 2003). In our preliminary screening of anticancer activity of natural products, methanol extract of U. rufa showed potent in vitro cytotoxicity against various cancer cell lines. Therefore, this plant was selected for bioassay-guided fractionation to clarify its active constituents.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Herein, we report the isolation and structural elucidation of a new lignan glycoside, ufaside (1), along with six known compounds, oxoanolobine (2), ergosta-4,6,8(14),22-tetraen-3-one (3), catechin (4), epicatechin (5), daucosterol (6) and glutin-5-en-3-one (7). The cytotoxicity of the isolated compounds towards human lung adenocarcinoma cell line was also evaluated.
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2. Results and discussion Compound 1 was obtained as a colourless amorphous powder. Its molecular formula was determined to be C28H40O12 by HR-ESI-MS at m/z 591.2437 [M þ Na]þ (calcd C28H40O12Na for 591.2417). The FT-IR spectrum exhibited bands at 3356 (hydroxyl groups), 1659 and 1435 cm21 (aromatic nucleus). The 1H NMR spectrum of 1 (CD3OD) showed specific signals of four aromatic protons at dH 6.32 (2H, s, H-20 , H-60 ), 6.30 (2H, s, H-2, H-6), four methoxy groups at dH 3.77 (6H, s), 3.76 (6H, s), one anomeric proton at dH 4.69 (1H, d, J ¼ 1.0 Hz, H-100 ) and one methyl group at dH 1.28 (3H, d, J ¼ 6.0 Hz, H-600 ). The 13C NMR and DEPT spectra (Table S1) revealed 28 carbon signals including four methoxy (dC 56.6 ( £ 2), and 56.7 ( £ 2)), two methylene (dC 36.5, and 36.8), two oxymethylene (dC 62.9, and 69.5), four aromatic methine (dC 107.1 ( £ 2), and 107.2 ( £ 2)), two saturated methine (dC 40.9, and 44.2), eight quaternary carbons (dC 132.8, 133.0, 134.4, 134.5 and 149.0 ( £ 4)) and six carbons of sugar moiety. Sugar carbon signals at dC 102.3 (C-100 ), 72.4 (C-200 ), 72.6 (C-300 ), 73.9 (C-400 ), 70.2 (C-500 ) and 18.0 (C600 ) along with the spin coupling pattern of the sugar proton signals (J100 , 200 ¼ 1.0 Hz, J200 , 300 ¼ 3.5 Hz, J300 , 400 ¼ 10.0 Hz, J400 , 500 ¼ 9.0 Hz) showed an a-L -rhamnopyranosyl unit. The identification of the sugar unit was continued by hydrolysis with 10% HCl to afford L rhamnopyranose, which was confirmed by thin-layer chromatography (TLC) comparison with those of an authentic sample. In general, the 1D NMR spectrum of 1 displayed typical signals of the diarylbutane-type lignan glycoside. The 1H – 1H COSY correlations between H-7 (dH 2.60 and 2.66)/H-8 (dH 2.11) and H-9 (dH 3.39 and 3.84), between H-70 (dH 2.54 and 2.75) and H-80 (dH 1.95)/H-90 (dH 3.52 and 3.77), and between H-8 (dH 2.11) and H-80 (dH 1.95) led to the assignment of connectivities C-7/C-8/C-9, C-70 /C-80 /C-90 , C-8/C-80 (Figure S1). This constitution of 1 was further confirmed by the HMBC correlations from H-7 (dH 2.60 and 2.66), H-70 (dH 2.54 and 2.75), H-9 (dH 3.39 and 3.84) and H90 (dH 3.52 and 3.77) to C-8 (dC 40.9) and C-80 (dC 44.2), from H-7 (dH 2.60 and 2.66) to C-9 (dC 69.5), from H-70 (dH 2.54 and 2.75) to C-90 (dC 62.9). The HMBC correlations between 3-OMe (dH 3.76) and C-3 (dC 149.0), between 5-OMe (dH 3.76) and C-5 (dC 149.0), between 30 -OMe (dH 3.77) and C-30 (dC 149.0), and between 50 -OMe (dH 3.77) and C-50 (dC 149.0) are shown in Figure S1, suggesting the methoxy groups at C-3, C5, C-30 and C-50 . The HMBC correlations from H-2, H-6 (dH 6.30) to C-7 (dC 36.8), from H-20 , H-60 (dH 6.32) to C-70 (dC 36.5), from H-8 (dH 2.11) to C-1 (dC 132.8) and from H-80 (dH 1.95) to C-10 (dC 133.0) suggested that two 1,3,4,5-tetrasubstituted aromatic moieties were at C-7 and C70 , respectively. Furthermore, the a-L -rhamnopyranose moiety at C-9 of aglycone was confirmed by HMBC correlations from H-100 (dH 4.69) to C-9 (dC 69.5) and from H-9 (dH 3.39 and 3.84) to C-100 (dC 102.3). The NMR data of 1 were close to those of ssioriside ((8S,80 S)-4,40 -dihydroxy-3,5,30 ,50 tetramethoxy-8,80 -butyrolignan 9-O-b-D -xylopyranoside) (Yoshinari et al. 1989), (8S,80 S)-4,40 dihydroxy-3,5,30 ,50 -tetramethoxy-8,80 -butyrolignan 9-O-b-D -glucopyranoside (Shibuya et al. 1992), except for difference of sugar moiety. Unfortunately, the attempt to identify absolute configuration of C-8, C-80 was not successful because of a shortage of sample. Based on the previously mentioned evidence, 1 was elucidated to be 4,40 -dihydroxy-3,5,30 ,50 -tetramethoxy8,80 -butyrolignan 9-O-a-L -rhamnopyranoside, named ufaside. The remaining compounds were identified as oxoanolobine (2) (Tip-Pyang et al. 2011), ergosta-4,6,8(14),22-tetraen-3-one (3) (Quang & Bach 2008), catechin (4), epicatechin (5)
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(Martin et al. 2000), daucosterol (6) (Voutquenne et al. 1999) and glutin-5-en-3-one (7) (Duwiejua et al. 1999) (Figure 1). Their structures were determined based on their spectral data in agreement with previous studies. Among these, compounds 3 and 7 were isolated for the first time from the genus Uvaria. To the best of our knowledge, this is the first report on the chemical constituents of U. rufa growing in Vietnam. The in vitro cytotoxicity of isolated compounds (1– 5) was tested by sulforhodamine B (SRB) assay against the growth of human lung adenocarcinoma LU-1 cells. The results are shown in Table S1. These data revealed that compounds 2 and 3 exhibited moderate cytotoxic effects with IC50 values of 9.22 ^ 1.02 mg/mL and 10.21 ^ 1.16 mg/mL, respectively, whereas 1, 4 and 5 showed very weak or no cytotoxic activity against this cell line (IC50 . 50 mg/mL). 3. Experimental 3.1 General experimental procedures UV spectra were recorded on an UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were obtained on an FT-IR Prestige-21 spectrophotometer (Shimadzu). The 1D and 2D
Figure 1. The structure of 1 – 7 from the aerial parts of U. rufa.
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NMR experiments were recorded on a Bruker Avance 500 (Billerica, MA, USA; 500 MHz for 1 H NMR and 125 MHz for 13C NMR), using CD3OD, DMSO-d6 and CDCl3 as solvents, tetramethylsilane as an internal standard. HR-ESI-MS was recorded on the micrOTOF-Q 10187. Silica gel (70 –230, 230– 400 mesh, Merck) and YMC RP-18 resins (30 –50 mm, Fuji Silysia Chemical Ltd, Kasugai, Aichi, Japan) were used as absorbents in the column chromatography (CC). TLC plates (Silica gel 60 F254 and RP-18 F254, 0.25 mm, Merck) were purchased from Merck KGaA (Darmstadt, Germany). Spots were detected under UV radiation (254 nm and 365 nm) and by spraying the plates with 10% sulphuric acid followed by heating with a heat gun.
3.2 Plant material The whole aerial parts of U. rufa were collected in Dakrong, Quangtri province, Vietnam in July 2013 and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (BDR-07-2013) has been deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy.
3.3 Extraction and isolation The dried powder of aerial parts of U. rufa (7.0 kg) was extracted with MeOH three times at room temperature to yield 550.0 g of a dark solid extract. This was suspended in water and successively partitioned with chloroform and ethyl acetate (EtOAc) (each, 5.0 L £ 3 times) to obtain the chloroform (C, 150.0 g), the EtOAc (E, 120.0 g) and the water (W, 250.0 g) layers after removal of solvent in vacuo. The C extract was crudely separated on a silica gel CC using step-wise elution of n-hexane – acetone (100:0, 40:1, 20:1, 10:1 and 5:1, v/v, each, 1.0 L) to obtain corresponding five fractions, C1 – C5. The fraction C1 (20.0 g) was chromatographed on silica gel column, using n-hexane – acetone (50:1, v/v, 1.5 L) as elution solvent, to yield 7 (80.0 mg). The fraction C3 (30.0 g) was subjected to YMC RP-18 CC eluted with acetone –MeOH –water (1.5:1:0.1, v/v, 2.0 L) to yield five sub-fractions, C3A –C3E. The sub-fraction C3D (5.0 g) was further chromatographed on silica gel column eluting with n-hexane – EtOAc (20:1, v/v, 1.0 L) to yield 3 (5.0 mg). Similarly, the E extract was also separated into eight fractions, E1 –E8, on silica gel column eluting with CHCl3 –MeOH gradient system (100:0, 40:1, 20:1, 10:1, 5:1, 2.5:1, 1:1 and 0:100, v/v, each 1.0 L). The sub-fraction E4 (5.0 g) was further purified to afford 2 (30 mg) and 6 (50 mg) by a silica gel CC using CHCl3 – MeOH (20:1, v/v, 1.5 L) as eluent. Chromatography of the sub-fraction E5 (15.0 g) on YMC RP-18 column eluted with MeOH – water (1:1, v/v, 1.2 L) afforded five fractions, E5A –E5E. Fraction E5A (3.0 g) was then purified on silica gel column eluted with CHCl3 –MeOH in a gradient (20:1 ! 0:1, v/v), followed by Sephadex LH-20 CC with MeOH –water (4:1, v/v) to afford 1 (8.0 mg). Purification of sub-fraction E7 (5.0 g) on YMC RP-18 column eluted with MeOH – water (1:2, v/v, 0.5 L) followed by MeOH – water –nBuOH (1/1.5/0.1, v/v/v, 0.5 L) to yield 4 (20.0 mg) and 5 (12.0 mg). Purity of all compounds was assessed by HPLC as . 95%. Ufaside (1): A colourless amorphous powder. UV (MeOH) lmax (nm): 207, 236 shoulder, 273. IR (KBr) nmax (cm21): 3356 (OH), 2947 (CH), 1659, 1435 (aromatic rings), 1033. HR-ESIMS: m/z 591.2437 [M þ Na]þ (calcd C28H40O12Na for 591.2417). 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD) spectral data, see Table S2. Acid hydrolysis of (1): A small amount of 1 in 4 mL of MeOH/H2O (3:1, v/v) was added with 10% HCl (1 mL) and then heated to 608C in a water bath for 3 h. The reaction solution, after dilution with H2O (5 mL), was extracted with CHCl3 (each, 10 mL £ 2 times). The aqueous layer was evaporated and further examined for sugar by direct comparison on silica gel TLC
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with authentic L -rhamnopyranose (Sigma-Aldrich, St Louis, MO, USA) (Rf ¼ 0.75 (CHCl3/ MeOH/H2O, 3:1:0.1, v/v/v)). 3.4 Cell culture The monolayer human cancer cell line LU-1 (lung adenocarcinoma, ATCC number: HTB-57) was used in the cytotoxicity assays. Stock cultures were grown in T-75 flasks containing 50 mL of Dulbecco’s modified Eagle’s medium with 2 mM L -glutamine, 1.5 g/L sodium bicarbonate and 10% foetal bovine serum. Media were changed at 48-h intervals. The cells were dissociated with 0.05% Trypsin –EDTA, sub-cultured every 3 –5 days with the ratio of (1:3) and incubated at 378C under humidified 5% carbon dioxide atmosphere. 3.5 Cytotoxicity assays Tumour cells were cultivated in a humidified atmosphere of 5% CO2 at 378C for 48 h. Cell viability was examined by SRB method for the determination of cell density, based on the measurement of cellular protein content (Monks et al. 1991). Viable cells were seeded in the growth medium (180 mL) into 96-well microwell plates (4 £ 104 cells per well) and allowed to attach overnight. Test samples were added carefully into wells of 96-well plates and the cultivation was continued under the same conditions for another 72 h. Thereafter, the medium was removed and the remaining cell monolayers are fixed with the cold 20% (w/v) trichloroacetic acid for 1 h at 48C and stained by 1X SRB staining solution at room temperature for 30 min, after which the unbound dye was removed by washing repeatedly with 1% (v/v) acetic acid. The proteinbound dye is dissolved in 10 mM Tris base solution for OD determination at 515 nm on an ELISA Plate Reader (Bio-Rad). DMSO 10% was used as blank sample while ellipticine was used as positive control. The cytotoxicity was measured at doses of 100 mg/mL, 20 mg/mL, 4 mg/mL and 0.8 mg/mL and estimated as a half maximal inhibitory concentration (IC50), which was calculated by the program TableCurve Version 4.0. All experiments were prepared in triplicates. The inhibition rate (IR) of cells was calculated by the following formula IR% ¼ {100% – [(absorbancet – absorbance0)/(absorbancec – absorbance0)] £ 100}, where IR indicates inhibition rate of cell growth, absorbancet indicates average optical density value at day 3; absorbance0 indicates average optical density value at time-zero and absorbancec indicates average optical density value of the blank DMSO control sample. 4. Conclusions U. rufa is a medicinal plant in Vietnamese traditional medicine. Chemical investigation of the aerial parts of this plant yielded one new lignan glycoside namely ufaside, along with six known compounds. Therein, ergosta-4,6,8(14),22-tetraen-3-one and glutin-5-en-3-one was isolated from the genus Uvaria for the first time. Oxoanolobine and ergosta-4,6,8(14),22-tetraen-3-one displayed significant cytotoxicity against the human lung adenocarcinoma cell line (LU-1). Supplementary material Supplementary material relating to this paper is available online, alongside Tables S1 and S2 and Figure S1. Acknowledgements This research was funded by the Hue University (Vietnam) under the grant number DHH2014-04-35. The authors are also grateful to the Institute of Chemistry, VAST for spectroscopic measurements.
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References Alali FQ, Liu XX, McLaughlin JL. 1999. Annonaceous acetogenins: recent progress. J Nat Prod. 62:504– 540. Asha KN, Chowdhury R, Hasan CM, Rashid MA. 2003. Antibacterial activity and cytotoxicity of extractives from Uvaria hamiltonii stem bark. Fitoterapia. 74:159–163. Ban NT. 2000. Flora of Vietnam – Annonaceae Juss. Vol. 1. Hanoi: Science and Technology Publishing House. Duwiejua M, Zeitlin IJ, Gray AI, Waterman PG. 1999. The anti-inflammatory compounds of Polygonum bistorta: isolation and characterization. Planta Med. 65:371–374. Hasan CM, Asha KN, Rashid MA. 2001. Aristolactams from the stem bark of Uvaria hamiltonii. Biochem Syst Ecol. 29:207–208. Huang L, Wall ME, Wani MC, Navarro H, Santisuk T, Reutrakul V, Seo EK, Farnsworth NR, Kinghorn AD. 1998. New compounds with DNA strand-scission activity from the combined leaf and stem of Uvaria hamiltonii. J Nat Prod. 61:446–450. Hufford CD, Oguntimein BO, Van Engen D, Muthard D, Clardy J. 1980. Vafzelin and uvafzelin, novel constituents of Uvaria afzelii. J Am Chem Soc. 102:7365–7367. Jolad SD, Hoffmann JJ, Schram KH, Cole JR. 1982. Constituents of Eremocarpus setigerus (Euphorbiaceae) – a new diterpene, eremone, and hautriwaic acid. J Org Chem. 47:3156–3158. Leboeuf M, Cave A, Bhaumik PK, Mukherjee B, Mukherjee R. 1982. The phytochemistry of the Annonaceae. Phytochemistry. 21:2783–2813. Martin TS, Kikuzaki H, Hisamoto M, Nakatani N. 2000. Constituents of Amomum tsao-ko and their radical scavenging and antioxidant activities. J Am Oil Chem Soc. 77:667–673. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigrowolff A, et al. 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor-cell lines. J Natl Cancer Inst. 83:757–766. Parmar VS, Tyagi OD, Malhotra A, Singh SK, Bisht KS, Jain R. 1994. Novel constituents of Uvaria species. Nat Prod Rep. 11:219–224. Quang DN, Bach DD. 2008. Ergosta-4,6,8(14),22-tetraen-3-one from Vietnamese Xylaria sp. possessing inhibitory activity of nitric oxide production. Nat Prod Res. 22:901–906. Shibuya H, Takeda Y, Zhang R, Tanitame A, Tsai Y, Kitagawa I. 1992. Indonesian medicinal plants IV. On the constituents of the bark of Fagara rhetza (Rutaceae). (2). Lignan glycosides and two apioglucosides. Chem Pharm Bull. 40:2639–2646. Tip-Pyang S, Payakarintarungkul K, Sichaem J, Phuwapraisirisan P. 2011. Chemical constituents from the roots of Uvaria rufa. Chem Nat Compd. 47:474–476. Voutquenne L, Lavaud C, Massiot G, Sevenet T, Hadi HA. 1999. Cytotoxic polyisoprenes and glycosides of long-chain fatty alcohols from Dimocarpus fumatus. Phytochemistry. 50:63–69. Yoshinari K, Sashida Y, Shimomura H. 1989. 2 New lignin xylosides from the barks of Prunus ssiori and Prunus padus. Chem Pharm Bull. 37:3301–3303. Zhou GX, Chen RY, Zhang YJ, Yu DQ. 2000. New annonaceous acetogenins from the roots of Uvaria calamistrata. J Nat Prod. 63:1201 –1204. Zhou GX, Zhou LE, Chen RY, Yu DQ. 1999. Calamistrins A and B, two new cytotoxic monotetrahydrofuran annonaceous acetogenins from Uvaria calamistrata. J Nat Prod. 62:261– 264.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa a
a
b
c
Thi Hoai Nguyen , Viet Duc Ho , Thi Thao Do , Huu Tai Bui , Van d
e
Kiem Phan , Katrin Sak & Ain Raal
f
a
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen Street, Hue City, Vietnam b
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam c
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea d
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam e
NGO Praeventio, Näituse 22-3, Tartu, Estonia
f
Department of Pharmacy, University of Tartu, 1 Nooruse Street, Tartu, Estonia Published online: 22 Oct 2014.
To cite this article: Thi Hoai Nguyen, Viet Duc Ho, Thi Thao Do, Huu Tai Bui, Van Kiem Phan, Katrin Sak & Ain Raal (2014): A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.971790 To link to this article: http://dx.doi.org/10.1080/14786419.2014.971790
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.971790
A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa
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Thi Hoai Nguyena, Viet Duc Hoa, Thi Thao Dob, Huu Tai Buic, Van Kiem Phand, Katrin Sake and Ain Raalf* a
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen Street, Hue City, Vietnam; bInstitute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam; cCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Korea; dInstitute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam; eNGO Praeventio, Na¨ituse 22-3, Tartu, Estonia; f Department of Pharmacy, University of Tartu, 1 Nooruse Street, Tartu, Estonia (Received 2 July 2014; final version received 21 September 2014) Chemical investigation of the aerial parts of Uvaria rufa (Dunal) Blume collected from Vietnam yielded one new lignan glycoside, ufaside (1), along with six known compounds, oxoanolobine (2), ergosta-4,6,8(14),22-tetraen-3-one (3), catechin (4), epicatechin (5), daucosterol (6) and glutin-5-en-3-one (7). Their chemical structures were determined by using NMR, HR-MS spectroscopic analyses and in comparison with the reported data. A cytotoxic analysis of U. rufa herb extracts was performed for the first time using nine human cancer cell lines (MCF-7, MDA-MB-231, LNCaP, MKN7, SW480, KB, LU-1, HepG2 and HL-60) derived from different tumour types. Of these seven constituents, compounds 2 and 3 displayed moderate cytotoxicity against the human lung adenocarcinoma cell line (LU-1) with IC50 values of 9.22 ^ 1.02 mg/mL and 10.21 ^ 1.16 mg/mL, respectively. Keywords: Uvaria rufa; Annonaceae; lignan glycoside; ufaside; cytotoxicity
1. Introduction The genus Uvaria L. (Annonaceae) comprises about 150 species (Leboeuf et al. 1982). It continues to be a rich source of new natural products of varied structures (Leboeuf et al. 1982; Parmar et al. 1994), some of which have shown interesting antibacterial (Hufford et al. 1980), antioxidant (Alali et al. 1999) and antitumour (Jolad et al. 1982; Zhou et al. 1999, 2000) activities. Uvaria rufa (Dunal) Blume (synonym U. rufa (Fin. & Gagnep.) Ast) is a long woody vine, 8 –10 –m tall. It is commonly distributed in the Indochina peninsula as well as in the central part of Vietnam (Ban 2000). Although this plant has been used in Vietnamese traditional medicine to treat tumours, its anticancer action has not yet been thoroughly studied. Also, the knowledge about the chemical composition of U. rufa is rather poor and only few previous works have described the content of some flavonoids and non-flavonoid polyphenols as well as alkaloids in the leaves and stems of this shrub (Huang et al. 1998; Hasan et al. 2001; Asha et al. 2003). In our preliminary screening of anticancer activity of natural products, methanol extract of U. rufa showed potent in vitro cytotoxicity against various cancer cell lines. Therefore, this plant was selected for bioassay-guided fractionation to clarify its active constituents.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Herein, we report the isolation and structural elucidation of a new lignan glycoside, ufaside (1), along with six known compounds, oxoanolobine (2), ergosta-4,6,8(14),22-tetraen-3-one (3), catechin (4), epicatechin (5), daucosterol (6) and glutin-5-en-3-one (7). The cytotoxicity of the isolated compounds towards human lung adenocarcinoma cell line was also evaluated.
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2. Results and discussion Compound 1 was obtained as a colourless amorphous powder. Its molecular formula was determined to be C28H40O12 by HR-ESI-MS at m/z 591.2437 [M þ Na]þ (calcd C28H40O12Na for 591.2417). The FT-IR spectrum exhibited bands at 3356 (hydroxyl groups), 1659 and 1435 cm21 (aromatic nucleus). The 1H NMR spectrum of 1 (CD3OD) showed specific signals of four aromatic protons at dH 6.32 (2H, s, H-20 , H-60 ), 6.30 (2H, s, H-2, H-6), four methoxy groups at dH 3.77 (6H, s), 3.76 (6H, s), one anomeric proton at dH 4.69 (1H, d, J ¼ 1.0 Hz, H-100 ) and one methyl group at dH 1.28 (3H, d, J ¼ 6.0 Hz, H-600 ). The 13C NMR and DEPT spectra (Table S1) revealed 28 carbon signals including four methoxy (dC 56.6 ( £ 2), and 56.7 ( £ 2)), two methylene (dC 36.5, and 36.8), two oxymethylene (dC 62.9, and 69.5), four aromatic methine (dC 107.1 ( £ 2), and 107.2 ( £ 2)), two saturated methine (dC 40.9, and 44.2), eight quaternary carbons (dC 132.8, 133.0, 134.4, 134.5 and 149.0 ( £ 4)) and six carbons of sugar moiety. Sugar carbon signals at dC 102.3 (C-100 ), 72.4 (C-200 ), 72.6 (C-300 ), 73.9 (C-400 ), 70.2 (C-500 ) and 18.0 (C600 ) along with the spin coupling pattern of the sugar proton signals (J100 , 200 ¼ 1.0 Hz, J200 , 300 ¼ 3.5 Hz, J300 , 400 ¼ 10.0 Hz, J400 , 500 ¼ 9.0 Hz) showed an a-L -rhamnopyranosyl unit. The identification of the sugar unit was continued by hydrolysis with 10% HCl to afford L rhamnopyranose, which was confirmed by thin-layer chromatography (TLC) comparison with those of an authentic sample. In general, the 1D NMR spectrum of 1 displayed typical signals of the diarylbutane-type lignan glycoside. The 1H – 1H COSY correlations between H-7 (dH 2.60 and 2.66)/H-8 (dH 2.11) and H-9 (dH 3.39 and 3.84), between H-70 (dH 2.54 and 2.75) and H-80 (dH 1.95)/H-90 (dH 3.52 and 3.77), and between H-8 (dH 2.11) and H-80 (dH 1.95) led to the assignment of connectivities C-7/C-8/C-9, C-70 /C-80 /C-90 , C-8/C-80 (Figure S1). This constitution of 1 was further confirmed by the HMBC correlations from H-7 (dH 2.60 and 2.66), H-70 (dH 2.54 and 2.75), H-9 (dH 3.39 and 3.84) and H90 (dH 3.52 and 3.77) to C-8 (dC 40.9) and C-80 (dC 44.2), from H-7 (dH 2.60 and 2.66) to C-9 (dC 69.5), from H-70 (dH 2.54 and 2.75) to C-90 (dC 62.9). The HMBC correlations between 3-OMe (dH 3.76) and C-3 (dC 149.0), between 5-OMe (dH 3.76) and C-5 (dC 149.0), between 30 -OMe (dH 3.77) and C-30 (dC 149.0), and between 50 -OMe (dH 3.77) and C-50 (dC 149.0) are shown in Figure S1, suggesting the methoxy groups at C-3, C5, C-30 and C-50 . The HMBC correlations from H-2, H-6 (dH 6.30) to C-7 (dC 36.8), from H-20 , H-60 (dH 6.32) to C-70 (dC 36.5), from H-8 (dH 2.11) to C-1 (dC 132.8) and from H-80 (dH 1.95) to C-10 (dC 133.0) suggested that two 1,3,4,5-tetrasubstituted aromatic moieties were at C-7 and C70 , respectively. Furthermore, the a-L -rhamnopyranose moiety at C-9 of aglycone was confirmed by HMBC correlations from H-100 (dH 4.69) to C-9 (dC 69.5) and from H-9 (dH 3.39 and 3.84) to C-100 (dC 102.3). The NMR data of 1 were close to those of ssioriside ((8S,80 S)-4,40 -dihydroxy-3,5,30 ,50 tetramethoxy-8,80 -butyrolignan 9-O-b-D -xylopyranoside) (Yoshinari et al. 1989), (8S,80 S)-4,40 dihydroxy-3,5,30 ,50 -tetramethoxy-8,80 -butyrolignan 9-O-b-D -glucopyranoside (Shibuya et al. 1992), except for difference of sugar moiety. Unfortunately, the attempt to identify absolute configuration of C-8, C-80 was not successful because of a shortage of sample. Based on the previously mentioned evidence, 1 was elucidated to be 4,40 -dihydroxy-3,5,30 ,50 -tetramethoxy8,80 -butyrolignan 9-O-a-L -rhamnopyranoside, named ufaside. The remaining compounds were identified as oxoanolobine (2) (Tip-Pyang et al. 2011), ergosta-4,6,8(14),22-tetraen-3-one (3) (Quang & Bach 2008), catechin (4), epicatechin (5)
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(Martin et al. 2000), daucosterol (6) (Voutquenne et al. 1999) and glutin-5-en-3-one (7) (Duwiejua et al. 1999) (Figure 1). Their structures were determined based on their spectral data in agreement with previous studies. Among these, compounds 3 and 7 were isolated for the first time from the genus Uvaria. To the best of our knowledge, this is the first report on the chemical constituents of U. rufa growing in Vietnam. The in vitro cytotoxicity of isolated compounds (1– 5) was tested by sulforhodamine B (SRB) assay against the growth of human lung adenocarcinoma LU-1 cells. The results are shown in Table S1. These data revealed that compounds 2 and 3 exhibited moderate cytotoxic effects with IC50 values of 9.22 ^ 1.02 mg/mL and 10.21 ^ 1.16 mg/mL, respectively, whereas 1, 4 and 5 showed very weak or no cytotoxic activity against this cell line (IC50 . 50 mg/mL). 3. Experimental 3.1 General experimental procedures UV spectra were recorded on an UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were obtained on an FT-IR Prestige-21 spectrophotometer (Shimadzu). The 1D and 2D
Figure 1. The structure of 1 – 7 from the aerial parts of U. rufa.
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NMR experiments were recorded on a Bruker Avance 500 (Billerica, MA, USA; 500 MHz for 1 H NMR and 125 MHz for 13C NMR), using CD3OD, DMSO-d6 and CDCl3 as solvents, tetramethylsilane as an internal standard. HR-ESI-MS was recorded on the micrOTOF-Q 10187. Silica gel (70 –230, 230– 400 mesh, Merck) and YMC RP-18 resins (30 –50 mm, Fuji Silysia Chemical Ltd, Kasugai, Aichi, Japan) were used as absorbents in the column chromatography (CC). TLC plates (Silica gel 60 F254 and RP-18 F254, 0.25 mm, Merck) were purchased from Merck KGaA (Darmstadt, Germany). Spots were detected under UV radiation (254 nm and 365 nm) and by spraying the plates with 10% sulphuric acid followed by heating with a heat gun.
3.2 Plant material The whole aerial parts of U. rufa were collected in Dakrong, Quangtri province, Vietnam in July 2013 and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (BDR-07-2013) has been deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy.
3.3 Extraction and isolation The dried powder of aerial parts of U. rufa (7.0 kg) was extracted with MeOH three times at room temperature to yield 550.0 g of a dark solid extract. This was suspended in water and successively partitioned with chloroform and ethyl acetate (EtOAc) (each, 5.0 L £ 3 times) to obtain the chloroform (C, 150.0 g), the EtOAc (E, 120.0 g) and the water (W, 250.0 g) layers after removal of solvent in vacuo. The C extract was crudely separated on a silica gel CC using step-wise elution of n-hexane – acetone (100:0, 40:1, 20:1, 10:1 and 5:1, v/v, each, 1.0 L) to obtain corresponding five fractions, C1 – C5. The fraction C1 (20.0 g) was chromatographed on silica gel column, using n-hexane – acetone (50:1, v/v, 1.5 L) as elution solvent, to yield 7 (80.0 mg). The fraction C3 (30.0 g) was subjected to YMC RP-18 CC eluted with acetone –MeOH –water (1.5:1:0.1, v/v, 2.0 L) to yield five sub-fractions, C3A –C3E. The sub-fraction C3D (5.0 g) was further chromatographed on silica gel column eluting with n-hexane – EtOAc (20:1, v/v, 1.0 L) to yield 3 (5.0 mg). Similarly, the E extract was also separated into eight fractions, E1 –E8, on silica gel column eluting with CHCl3 –MeOH gradient system (100:0, 40:1, 20:1, 10:1, 5:1, 2.5:1, 1:1 and 0:100, v/v, each 1.0 L). The sub-fraction E4 (5.0 g) was further purified to afford 2 (30 mg) and 6 (50 mg) by a silica gel CC using CHCl3 – MeOH (20:1, v/v, 1.5 L) as eluent. Chromatography of the sub-fraction E5 (15.0 g) on YMC RP-18 column eluted with MeOH – water (1:1, v/v, 1.2 L) afforded five fractions, E5A –E5E. Fraction E5A (3.0 g) was then purified on silica gel column eluted with CHCl3 –MeOH in a gradient (20:1 ! 0:1, v/v), followed by Sephadex LH-20 CC with MeOH –water (4:1, v/v) to afford 1 (8.0 mg). Purification of sub-fraction E7 (5.0 g) on YMC RP-18 column eluted with MeOH – water (1:2, v/v, 0.5 L) followed by MeOH – water –nBuOH (1/1.5/0.1, v/v/v, 0.5 L) to yield 4 (20.0 mg) and 5 (12.0 mg). Purity of all compounds was assessed by HPLC as . 95%. Ufaside (1): A colourless amorphous powder. UV (MeOH) lmax (nm): 207, 236 shoulder, 273. IR (KBr) nmax (cm21): 3356 (OH), 2947 (CH), 1659, 1435 (aromatic rings), 1033. HR-ESIMS: m/z 591.2437 [M þ Na]þ (calcd C28H40O12Na for 591.2417). 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD) spectral data, see Table S2. Acid hydrolysis of (1): A small amount of 1 in 4 mL of MeOH/H2O (3:1, v/v) was added with 10% HCl (1 mL) and then heated to 608C in a water bath for 3 h. The reaction solution, after dilution with H2O (5 mL), was extracted with CHCl3 (each, 10 mL £ 2 times). The aqueous layer was evaporated and further examined for sugar by direct comparison on silica gel TLC
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with authentic L -rhamnopyranose (Sigma-Aldrich, St Louis, MO, USA) (Rf ¼ 0.75 (CHCl3/ MeOH/H2O, 3:1:0.1, v/v/v)). 3.4 Cell culture The monolayer human cancer cell line LU-1 (lung adenocarcinoma, ATCC number: HTB-57) was used in the cytotoxicity assays. Stock cultures were grown in T-75 flasks containing 50 mL of Dulbecco’s modified Eagle’s medium with 2 mM L -glutamine, 1.5 g/L sodium bicarbonate and 10% foetal bovine serum. Media were changed at 48-h intervals. The cells were dissociated with 0.05% Trypsin –EDTA, sub-cultured every 3 –5 days with the ratio of (1:3) and incubated at 378C under humidified 5% carbon dioxide atmosphere. 3.5 Cytotoxicity assays Tumour cells were cultivated in a humidified atmosphere of 5% CO2 at 378C for 48 h. Cell viability was examined by SRB method for the determination of cell density, based on the measurement of cellular protein content (Monks et al. 1991). Viable cells were seeded in the growth medium (180 mL) into 96-well microwell plates (4 £ 104 cells per well) and allowed to attach overnight. Test samples were added carefully into wells of 96-well plates and the cultivation was continued under the same conditions for another 72 h. Thereafter, the medium was removed and the remaining cell monolayers are fixed with the cold 20% (w/v) trichloroacetic acid for 1 h at 48C and stained by 1X SRB staining solution at room temperature for 30 min, after which the unbound dye was removed by washing repeatedly with 1% (v/v) acetic acid. The proteinbound dye is dissolved in 10 mM Tris base solution for OD determination at 515 nm on an ELISA Plate Reader (Bio-Rad). DMSO 10% was used as blank sample while ellipticine was used as positive control. The cytotoxicity was measured at doses of 100 mg/mL, 20 mg/mL, 4 mg/mL and 0.8 mg/mL and estimated as a half maximal inhibitory concentration (IC50), which was calculated by the program TableCurve Version 4.0. All experiments were prepared in triplicates. The inhibition rate (IR) of cells was calculated by the following formula IR% ¼ {100% – [(absorbancet – absorbance0)/(absorbancec – absorbance0)] £ 100}, where IR indicates inhibition rate of cell growth, absorbancet indicates average optical density value at day 3; absorbance0 indicates average optical density value at time-zero and absorbancec indicates average optical density value of the blank DMSO control sample. 4. Conclusions U. rufa is a medicinal plant in Vietnamese traditional medicine. Chemical investigation of the aerial parts of this plant yielded one new lignan glycoside namely ufaside, along with six known compounds. Therein, ergosta-4,6,8(14),22-tetraen-3-one and glutin-5-en-3-one was isolated from the genus Uvaria for the first time. Oxoanolobine and ergosta-4,6,8(14),22-tetraen-3-one displayed significant cytotoxicity against the human lung adenocarcinoma cell line (LU-1). Supplementary material Supplementary material relating to this paper is available online, alongside Tables S1 and S2 and Figure S1. Acknowledgements This research was funded by the Hue University (Vietnam) under the grant number DHH2014-04-35. The authors are also grateful to the Institute of Chemistry, VAST for spectroscopic measurements.
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