DSpace at VNU: A new lupane triterpene from Tetracera scandens L., xanthine oxidase inhibitor

This article was downloaded by: [University of Edinburgh] On: 28 June 2013, At: 02:28 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20 A new lupane triterpene from Tetracera scandens L., xanthine oxidase inhibitor a Mai Thanh Thi Nguyen & Nhan Trung Nguyen a a Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City, Vietnam Published online: 19 Jan 2012 To cite this article: Mai Thanh Thi Nguyen & Nhan Trung Nguyen (2013): A new lupane triterpene from Tetracera scandens L., xanthine oxidase inhibitor, Natural Product Research: Formerly Natural Product Letters, 27:1, 61-67 To link to this article: http://dx.doi.org/10.1080/14786419.2011.652960 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions 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 The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material Natural Product Natural ProductResearch, Research2013 Vol 27, No.iFirst 1, 61–67, http://dx.doi.org/10.1080/14786419.2011.652960 2012, 1–7, SHORT COMMUNICATION A new lupane triterpene from Tetracera scandens L., xanthine oxidase inhibitor Mai Thanh Thi Nguyen* and Nhan Trung Nguyen Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City, Vietnam Downloaded by [University of Edinburgh] at 02:28 28 June 2013 (Received 14 June 2011; final version received October 2011) From the MeOH extract of the stem of Tetracera scandens L., a new nor-lupane triterpene, 28-O-�-D-glucopyranosyl ester of platanic acid (1), has been isolated together with six known compounds Their structures were elucidated on the basis of spectroscopic data Compounds 1–6 displayed significant xanthine oxidase inhibitory activity in a concentration-dependent manner, and compound showed more potent inhibitory activity with an IC50 value of 1.9 mM than that of a positive control allopurinol (IC50 2.5 mM) Keywords: Tetracera scandens; 28-O-�-D-glucopyranosyl ester of platanic acid; xanthine oxidase inhibition Introduction Xanthine oxidase (XO) is a key enzyme that catalyses the last step in the conversion of purines to uric acid, and plays a vital role in producing hyperuricemia and gout (Borges, Fernandes, & Roleira, 2002) Allopurinol, the medication prescribed for gout prevention, is a XO inhibitor (Oettl & Reibnegger, 1999) However, due to unwanted side effects of allopurinol, such as hepatitis, nephropathy and allergic reactions, new alternatives with increased therapeutic activity and fewer side effects are desired Moreover, superoxide anion radicals generated by XO are involved in various pathological states such as hepatitis, inflammation, ischemia-reperfusion, carcinogenesis and ageing (Cos et al., 1998) Thus, the search for novel XO inhibitors would be beneficial not only to treat gout but also to combat various other diseases Tetracera scandens L (Dilleniaceae) is an evergreen woody climber, used in Vietnamese traditional medicine for the treatment of rheumatism, hepatitis and inflammatory diseases (Do, 2001) Our preliminary screening study revealed that the methanolic extract of the stem of T scandens exhibited significant XO inhibitory activity (Nguyen et al., 2004) Therefore, we carried out activity-guided fractionation of the MeOH extract and isolated a new nor-lupane triterpene, together with six known compounds In this article, we report the isolation and structure elucidation of the new compound by spectroscopic techniques, together with the XO inhibitory activity of the isolated compounds *Corresponding author Email: nttmai@hcmus.edu.vn ISSN 1478–6419 print/ISSN 1478–6427 online *Corresponding author Email: nttmai@hcmus.edu.vn � 2012 Taylor & Francis http://dx.doi.org/10.1080/14786419.2011.652960 http://www.tandfonline.com © 2013 Taylor & Francis M.T Thi Thi Nguyen 262   M.T Nguyen and andN.T N.T.Nguyen Nguyen R1 R1 OH H HO H H HO H R1 = O, R2 = Glc R1 = O, R2 = H R1 = CH2, R2 = H OH O OH O O O R2 OR2 OH H3C O R1 = OH, R2 = H R1 = R2 = H R1 = H, R2 = Glc-(6''-p-coumaroyl) OH O Downloaded by [University of Edinburgh] at 02:28 28 June 2013 Figure Structures of the compounds isolated from T scandens Results and discussion The dried stem of T scandens was extracted by refluxing MeOH; the MeOH solution was evaporated under pressure to give a MeOH extract The MeOH extract was suspended in H2O and partitioned successively with petroleum ether, CH2Cl2 and EtOAc to yield petroleum ether, CH2Cl2, EtOAc and H2O fractions Among them, the CH2Cl2 and EtOAc fractions showed strong XO inhibitory activity with IC50 values of 10.7 and 6.8 mg mL�1, respectively Further separation and purification of these fractions led to the isolation of a new nor-lupane triterpene, 28-O-�-D-glucopyranosyl ester of platanic acid (1), together with six known compounds The known compounds were identified to be platanic acid (2) (Lunardi et al., 2001), betulinic acid (3) (Shashi, Mahato, Asish, & Kundu, 1994), quercetin (4) (Nessa, Ismail, Mohamed, & Haris, 2004), kaempferol (5) (Harborne & Mabdry, 1982), tiliroside (6) (Jung & Park, 2007) and emodin (7) (Ayo, Amupitan, & Zhao, 2007; Figure 1) by the analysis of their spectroscopic data and comparing with the literature data Compound was obtained as an amorphous solid and showed the quasi-molecular ion at m/z 619.1, corresponding to the molecular formula C35H55O9 in ESI–MS The IR spectrum of showed absorption of hydroxyl (3500 cm�1), ester carbonyl (1730 cm�1) and carbonyl (1660 cm�1) groups The 1H-NMR spectrum of displayed signals due to five quaternary methyls (�H 0.95, s, H-23; �H 0.75, s, H-24; �H 0.81, s, H-25; �H 0.91, s, H-26; �H 0.99, s, H-27), an acetyl (�H 2.19, s, H-29) and signals of a sugar unit at �H 3.3–3.6 ppm On the other hand, the 13C-NMR spectrum showed 35 carbons signals including a ketone (�C 213.1, C-20), an ester carbonyl carbon (�C 175.0, C-28), six methyl carbons, 11 aliphatic methylene carbons and aliphatic quaternary carbons These data were similar to those of platanic acid (3) (Lunardi et al., 2001), the nor-lupane triterpene isolated from the same extract, but they were characterised by the presence of additional signals due to a sugar unit in The location of sugar moiety was determined to be at C-28 on the basis of the high-field shift of C-28 (�C 175.0) compared to that of (�C 179.1), which was confirmed by the HMBC correlation of H-10 with C-28 (Figure 2) Moreover, the GC analysis of chiral derivatives of an acid hydrolysate of showed its sugar moiety to be �-D-glucopyranose (Hara, Okabe, & Mihashi, 1987) The relative stereochemistry of was assigned on the basis of the ROESY correlations and the coupling constant data The ROESY correlations H-3/H-5, H-3/H-23 and H-5/H-9 indicated rings A and B to be trans-fused with a �-axial orientation of H-24 and �-axial orientation of H-3, H-5 and H-9, while the correlations H-27/H-9 and H-27/H-18 Natural Natural Product ProductResearch   Research 633 O (a) H O O OH O HO OH OH HO H H (b) 25 Downloaded by [University of Edinburgh] at 02:28 28 June 2013 CH3 A HO H H3C 23 26 CH3 24 B 11 CH3 13 19 29 14 H COO-Glc H3C D C 28 O 18 H 21 E 22 16 CH3 27 H Figure (a) Connectivities (bold line) deduced by the COSY spectrum and significant HMBC correlations (arrow) and (b) ROESY correlations observed for Table XO inhibitory activity of the isolated compounds XO inhibitory activity of the isolated compounds IC50 (mM) Compounds IC50 (mL) 65.9 42.3 20.8 1.9 Allopurinol 4.0 10.7 4100 2.5 together with the large coupling constant (J ¼ 11 Hz) between H-19 and H-18 indicated rings C and D to be trans-fused with an �-axial orientation of H-18 and H-27 These ROESY correlations also indicated rings A-D all to have the chair conformation (Figure 2) As for ring E, the ROESY correlation between H-19/H13 and H-19/H-29 indicated the boat conformation of ring E Thus, compound was deduced to be 28-O-�-D-glucopyranosyl ester of platanic acid The isolated compounds were tested for their XO inhibitory activity (Table 1) The assay was carried out at five different concentrations ranging from to 100 mM Compounds 1–6 possessed significant XO inhibitory activity in a concentration-dependent manner, and compound showed more potent inhibitory activity, with an IC50 value of 1.9 mM, than that of a positive control allopurinol (IC50, 2.5 mM), a well-known XO inhibitor used clinically for the treatment of gout (Figure 3) Experimental 3.1 General experimental procedures IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solutions NMR spectra were taken on a Bruker Advance III 500 MHz spectrometer with M.T Thi Thi Nguyen 464   M.T Nguyen and andN.T N.T.Nguyen Nguyen 100 Inhibition (%) 80 60 40 20 Downloaded by [University of Edinburgh] at 02:28 28 June 2013 0.2 20 Concentration (mM) All 50 100 Figure Dose-dependent inhibition of XO by and allopurinol The data represent the mean � SD of four different experiments tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in � values ESI–MS was preformed on an Agilent 6310 Ion Trap mass spectrometer Analytical and preparative TLC were carried out on precoated Merck Kieselgel 60F254 or RP-18F254 plates (0.25 or 0.5 mm thickness) XO (EC 1.2.3.2) from bovine milk (10 units mL�1) and xanthine were obtained from Sigma Chemical Co (St Louis, MO, USA) Allopurinol was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan) Other reagents were of the highest grade available 3.2 Plant material The stem of T scandens was collected at Nha Trang province, Vietnam, in October 2007 and was identified by Dr Hoang Viet, Faculty of Biology, University of Science, National University Ho Chi Minh City A voucher sample of the aerial part has been deposited with the number AN-2983 at the Department of Analytical Chemistry of the University of Science, National University-Ho Chi Minh City, Vietnam 3.3 Extraction and isolation Dried stem (3.4 kg) of T scandens was extracted with MeOH (12 L, reflux, h � 3) to yield a MeOH extract (150 g; IC50, 18.5 mg mL�1) The MeOH extract was suspended in H2O and partitioned successively with petroleum ether, CH2Cl2 and EtOAc to yield petroleum ether (57 g; IC50 100 mg mL�1), CH2Cl2 (32 g; IC50, 10.7 mg mL�1), EtOAc (16 g; IC50, 6.8 mg mL�1) and H2O (20 g; IC50 100 mg mL�1) fractions, respectively The CH2Cl2 fraction (30 g) was subjected to silica gel column (9 � 40 cm2) chromatography eluted with MeOH–CHCl3 (0–30%) to give four fractions: fr 1–5% MeOH–CHCl3 eluate, 5.7 g; fr 2–10% MeOH–CHCl3 eluate, 3.4 g; fr 3–20% MeOH–CHCl3 eluate, 4.2 g; fr 4–30% MeOH–CHCl3 eluate, 15.2 g These fractions were examined for XO inhibitory activity, and active fractions, fr (IC50, 9.4 mg mL�1) and fr (IC50, 11.7 mg mL�1), were subjected for further separation Fraction was crystallised in MeOH : acetone (9 : 1) to give betulinic acid (3, 100 mg; Shashi et al., 1994), while fraction was further separated by silica gel column Downloaded by [University of Edinburgh] at 02:28 28 June 2013 Natural Natural Product ProductResearch   Research 655 chromatography, followed by preparative TLC with MeOH : CHCl3 (5 : 95), to give platanic acid (2, 6.5 mg; Lunardi et al., 2001) and 28-O-�-D-glucopyranosyl ester of platanic acid (1, 4.2 mg) The EtOAc fraction (15 g) was subjected to silica gel column (5 � 40 cm2) chromatography eluted with MeOH–CHCl3 (0–30%) to give four fractions: fr 1–7% MeOH–CHCl3 eluate, 4.2 g; fr 2–10% MeOH–CHCl3 eluate, 2.4 g; fr 3–18% MeOH–CHCl3 eluate, 3.6 g; fr 4–30% MeOH–CHCl3 eluate, 3.5 g These fractions were examined for XO inhibitory activity, and active fractions, fr (IC50, 3.5 mg mL�1); fr (IC50, 6.0 mg mL�1) and fr (IC50, 45.5 mg mL�1), were subjected for further separation Fraction was subjected to silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN : MeOH : H2O ¼ : : 3, to yield quercetin (4, 15.2 mg; Nessa et al., 2004) and kaempferol (5, 12.8 mg; Harborne & Mabdry, 1982) Fraction was separated by silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with MeOH : H2O ¼ : 5, to yield tiliroside (6, 5.2 mg; Jung & Park, 2007) Fraction was separated by silica gel column chromatography with MeOH–CHCl3, followed by TLC with MeOH : CHCl3 (10 : 90), to give emodin (7, 5.3 mg; Ayo et al., 2007) 28-O-b-D-glucopyranosyl ester of platanic acid (1): White powder, IR �max (CHCl3) 3500, 1730, 1660, 1455, 1170 and 976 cm�1, ESI–MS m/z 619.1, 1H-NMR (CDCl3–CD3OD, 500 MHz) �: 0.68 (1H, d, J ¼ 10.0, H-5), 0.75 (3H, s, H-24), 0.81 (3H, s, H-25), 0.89 (1H, m, H-1ax), 0.91 (3H, s, H-26), 0.95 (3H, s, H-23), 0.99 (3H, s, H-27), 1.07 (2H, m, H-12), 1.18 (1H, m, H-15ax), 1.28 (2H, m, H-9 and H-11ax), 1.36 (2H, m, H-7), 1.37 (1H, m, H-6ax), 1.44 (1H, m, H-11eq), 1.48 (2H, m, H-15eq & H-16ax), 1.50 (1H, m, H-6eq), 1.51 (1H, m, H-21ax), 1.56 (1H, m, H-22ax), 1.58 (2H, m, H-2), 1.66 (1H, m, H-1eq), 1.99 (1H, m, H-22eq), 2.04 (1H, m, H-21eq), 2.05 (1H, m, H-13), 2.12 (1H, m, H-18), 2.19 (3H, s, H-29), 2.32 (1H, m, H-16eq), 3.17 (1H, dd, J ¼ 12.0, 6.0 Hz, H-3), 3.25 (1H, ddd, J ¼ 5.0, 11.0, 11.0 Hz, H-19), 3.43 (1H, m, H-20 ), 3.44 (1H, m, H-50 ), 3.49 (1H, m, H-40 ), 3.51 (1H, m, H-30 ), 3.77 (1H, dd, J ¼ 12.0, 4.0 Hz, H-60 ), 3.85 (1H, dd, J ¼ 12.0, 3.0 Hz, H-60 ) and 5.53 (1H, d, J ¼ 8.0 Hz, H-10 ), 13C-NMR (CDCl3–CD3OD, 500 MHz) �: 14.7 (C-27), 15.4 (C-24), 15.8 (C-26), 16.1 (C-25), 18.3 (C-6), 21.0 (C-11), 27.2 (C-2), 27.3 (C-12), 28.0 (C-23), 28.2 (C-21), 29.6 (C-15), 29.9 (C-29), 31.3 (C-16), 34.3 (C-7), 36.4 (C-22), 37.2 (C-10), 37.3 (C-13), 38.8 (C-1), 38.9 (C-4), 40.7 (C-8), 42.3 (C-14), 49.6 (C-18), 50.5 (C-9), 51.4 (C-19), 55.4 (C-5), 56.7 (C-17), 61.8 (C-60 ), 69.9 (C-40 ), 72.6 (C-20 ), 76.6 (C-50 ), 77.2 (C-30 ), 78.9 (C-3), 93.8 (C-10 ), 175.0 (C-28) and 213.1 (C-20) 3.4 XO inhibitory assay The XO inhibitory activity was assayed spectrophotometrically under aerobic conditions The assay mixture consisting of 50 mL of test solution, 35 mL of 70 mM phosphate buffer (pH 7.5) and 30 mL of enzyme solution (0.01 units mL�1 in 70 mM phosphate buffer, pH 7.5) was prepared immediately before use After preincubation at 25� C for 15 min, the reaction was initiated by the addition of 60 mL of substrate solution (150 mM xanthine in the same buffer) The assay mixture was incubated at 25� C for 30 The reaction was stopped by adding 25 mL of 1N HCl, and the absorbance at 290 nm was measured with a Perkin Elmer HTS-7000 Bio Assay Reader (Norwalk, CT, USA) A blank was prepared in the same way, but the enzyme solution was added to the assay mixture after adding 1N HCl One unit of XO is defined as the amount of enzyme required to produce mmol of uric acid min�1 at 25� C XO inhibitory activity was expressed as the percentage inhibition M.T Thi Thi Nguyen 666   M.T Nguyen and andN.T N.T.Nguyen Nguyen of XO in the above assay system, calculated as (1 � B/A) � 100, where A and B are the activities of the enzyme without and with test material IC50 values were calculated from the mean values of data from four determinations Allopurinol, a known inhibitor of XO, was used as a positive control Downloaded by [University of Edinburgh] at 02:28 28 June 2013 3.5 Sugar analysis A solution of (1 mg) in 1N HCl (dioxan–H2O, : 1; 0.5 mL) was heated at 80� C for h (Hara et al., 1987) The reaction mixture was neutralised with Amberlite IRA67 (OH� form), and the filtrate was concentrated to dryness in vacuo The residue was dissolved in pyridine (0.1 mL), and 0.1 M of L-cysteine methyl ester hydrochloride in pyridine (0.1 mL) was added to it After the mixture was heated at 60� C for h, trimethylsilimidazole (0.1 mL) was added, and the mixture was heated at 60� C for h The reaction mixture was partitioned between hexane and water (each 0.15 mL), and the organic layer was analysed on a Shimadzu GC-17A; DB-1 column (0.25 mm � 30 m); JMS-AMII20 detector; column temperature, 210� C; injection temperature, 240� C Standard sugars gave peaks at tR (min) 24.97 and 25.98 for D- and L-glucose, respectively Conclusions In this article, we have reported the new nor-lupane triterpene together with the six known compounds Compounds 1–6 showed XO inhibitory activity in a concentration-dependent manner Interestingly, betulinic acid (3) – a naturally occurring pentacyclic triterpenoid which has been shown to exhibit a variety of biological activities including inhibition of human immunodeficiency virus, antibacterial, antimalarial, antiinflammatory, anthelmintic and antioxidant properties (Yogeeswari & Sriram, 2005) – was found in high yield from this plant These results suggested that the traditional use of T scandens for the treatment of rheumatism and inflammatory diseases in Vietnam may be attributable to the XO inhibitory activity of lupane triterpene and flavonoid constituents Supplementary material Supplementary material for this article is available online, including Figures S1–S7 Acknowledgements This study was supported by grant no 104.01.68.09 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) References Ayo, R.G., Amupitan, J.O., & Zhao, Y (2007) Cytotoxicity and antimicrobial studies of 1,6,8-trihydroxy3-methylanthraquinone (emodin) isolated from the leaves of Cassia nigricans Vahl African Journal of Biotechnology, 6, 1276�1279 Borges, F., Fernandes, E., & Roleira, F (2002) Progress towards the discovery of xanthine oxidase inhibitors Current Medicinal Chemistry, 9, 195�217 Cos, P., Ying, L., Calomme, M., Hu, J.P, Cimanga, K., Van Poel, B., & Vanden Berghe, D (1998) Structure–activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers Journal of Natural Products, 61, 71�76 Do, T.L (2001) Vietnamese medicinal plant Hanoi: Medicine Publisher Hara, S., Okabe, H., & Mihashi, K (1987) Gas–liquid chromatography separation of aldose enantiomers as trimethylsilyl ethers of methyl 2-(polyhydrocyalkyl)-thiazolidine-4(R)-carboxylates Chemical and Pharmaceutical Bulletin, 35, 501–506 Harborne, J.B., & Mabdry, T.J (1982) The flavonoids: Advances in research London, New York: Chapman and Hall Natural Natural Product ProductResearch   Research 677 Downloaded by [University of Edinburgh] at 02:28 28 June 2013 Jung, M., & Park, M (2007) Acetylcholinesterase inhibition by flavonoids from Agrimonia pilosa Molecules, 12, 2130�2139 Lunardi, I., Peixoto, J.L.B., Silva, C.C., Shuquel, I.T.A., Basso, E.A., & Vidotti, G.J (2001) Triterpenic acids from Eugenia moraviana Journal of Brazil Chemical Society, 12, 180�183 Nessa, F., Ismail, Z., Mohamed, N., & Haris, M (2004) Free radical-scavenging activity of organic extracts and of pure flavonoids of Blumea Balsamifera DC leaves Food Chemistry, 88, 243�252 Nguyen, M.T.T., Awale, S., Tezuka, Y., Tran, L.Q., Watanabe, H., & Kadota, S (2004) Xanthine oxidase inhibitory activity of Vietnamese medicinal plants Biological & Pharmaceutical Bullletin, 27, 1414�1421 Oettl, K., & Reibnegger, G (1999) Protein structure and molecular enzymology Biochimica et Biophysica Acta, 1430, 387�395 Mahato, S.B., & Kundu, A.P (1994) 13C NMR spectra of pentacyclic triterpenoids – a complication and some salient features Phytochemistry, 37, 32�51 Yogeeswari, P., & Sriram, D (2005) Betulinic acid and its derivatives: a review on their biological properties Current Medicinal Chemistry, 12, 657�666  ... suggested that the traditional use of T scandens for the treatment of rheumatism and inflammatory diseases in Vietnam may be attributable to the XO inhibitory activity of lupane triterpene and flavonoid... carbonyl carbon (�C 175.0, C-28), six methyl carbons, 11 aliphatic methylene carbons and aliphatic quaternary carbons These data were similar to those of platanic acid (3) (Lunardi et al., 2001),... control allopurinol (IC50 2.5 mM) Keywords: Tetracera scandens; 28-O-�-D-glucopyranosyl ester of platanic acid; xanthine oxidase inhibition Introduction Xanthine oxidase (XO) is a key enzyme that catalyses