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Natural Product Research: Formerly Natural Product Letters

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A new lupane triterpene from Tetracera scandens L., xanthine oxidase inhibitor

Mai Thanh Thi Nguyen a & 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

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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

(Received 14 June 2011; final version received 1 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 4 showed more potent inhibitory activity with an IC50value of 1.9mM than that of

a positive control allopurinol (IC502.5mM)

Keywords: Tetracera scandens; 28-O-�-D-glucopyranosyl ester of platanic acid;

xanthine oxidase inhibition

1 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

� 2012 Taylor & Francis

http://dx.doi.org/10.1080/14786419.2011.652960

http://www.tandfonline.com

© 2013 Taylor & Francis

*Corresponding author Email: nttmai@hcmus.edu.vn

2 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 6.8mg 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 1 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 1 showed absorption of hydroxyl (3500 cm�1), ester carbonyl (1730 cm�1) and carbonyl (1660 cm�1) groups The1H-NMR spectrum of 1 displayed signals due to five quaternary methyls (�H0.95, s, H-23; �H0.75, s, H-24; �H0.81, s, H-25; �H0.91, s, H-26; �H 0.99, s, H-27), an acetyl (�H2.19, s, H-29) and signals of a sugar unit at �H3.3–3.6 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 1 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 3 (�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 1 showed its sugar moiety to be

�-D-glucopyranose (Hara, Okabe, & Mihashi, 1987)

The relative stereochemistry of 1 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

R 1

HO

O O H

H

H H

R 2

O

O

H3C

OH

OH OH

1 R1 = O, R 2 = Glc

2 R1 = O, R 2 = H

3 R1 = CH2, R 2 = H

O

OH

HO

OR 2

R 1

OH

O

4 R1 = OH, R 2 = H

5 R1 = R 2 = H

6 R1 = H, R 2 = Glc-(6''-p-coumaroyl)

7

Figure 1 Structures of the compounds isolated from T scandens

2 M.T Thi Nguyen and N.T Nguyen

62 M.T Thi Nguyen and N.T Nguyen

together with the large coupling constant (J¼ 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 1 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 1 to 100mM Compounds 1–6 possessed significant XO inhibitory activity in a concentration-dependent manner, and compound 4 showed more potent inhibitory activity, with an IC50 value of 1.9mM, than that of a positive control allopurinol (IC50, 2.5mM), a well-known XO inhibitor used clinically for the treatment of gout (Figure 3)

3 Experimental

3.1 General experimental procedures

IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3solutions NMR spectra were taken on a Bruker Advance III 500 MHz spectrometer with

O

HO

O O H

OH HO

OH

HO

H 3 C

CH 3

CH 3 CH 3

CH3

COO-Glc

H O

H 3 C

H

H

1

9

18

22

23

24

27

28 29

H

E

(a)

(b)

Figure 2 (a) Connectivities (bold line) deduced by the COSY spectrum and significant HMBC correlations (arrow) and (b) ROESY correlations observed for 1

Table 1 XO inhibitory activity of the isolated compounds

XO inhibitory activity of the isolated compounds IC50(mM) Compounds IC50(mL)

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 60F254or 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, 3 h� 3) to yield

a MeOH extract (150 g; IC50, 18.5mg mL�1) The MeOH extract was suspended in H2O and partitioned successively with petroleum ether, CH2Cl2and EtOAc to yield petroleum ether (57 g; IC504 100 mg mL�1), CH2Cl2(32 g; IC50, 10.7mg mL�1), EtOAc (16 g; IC50, 6.8mg mL�1) and H2O (20 g; IC504 100 mg mL�1) fractions, respectively

The CH2Cl2 fraction (30 g) was subjected to silica gel column (9� 40 cm2) chroma-tography 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 2 (IC50, 9.4mg mL�1) and fr 3 (IC50, 11.7mg mL�1), were subjected for further separation

Fraction 2 was crystallised in MeOH : acetone (9 : 1) to give betulinic acid (3, 100 mg; Shashi et al., 1994), while fraction 3 was further separated by silica gel column

0 20 40 60 80 100

Concentration (mM)

0.2

Figure 3 Dose-dependent inhibition of XO by 4 and allopurinol The data represent the mean� SD

of four different experiments

4 M.T Thi Nguyen and N.T Nguyen

64 M.T Thi Nguyen and N.T Nguyen

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) chromatog-raphy 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 2 (IC50, 3.5mg mL�1); fr 3 (IC50, 6.0mg mL�1) and fr 4 (IC50, 45.5mg mL�1), were subjected for further separation

Fraction 2 was subjected to silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN : MeOH : H2O¼ 2 : 1 : 3, to yield quercetin (4, 15.2 mg; Nessa et al., 2004) and kaempferol (5, 12.8 mg; Harborne & Mabdry, 1982)

Fraction 3 was separated by silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with MeOH : H2O¼ 5 : 5, to yield tiliroside (6, 5.2 mg; Jung & Park, 2007)

Fraction 4 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, J¼ 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, J¼ 12.0, 6.0 Hz, H-3), 3.25 (1H, ddd, J¼ 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, J¼ 12.0, 4.0 Hz, H-60), 3.85 (1H, dd, J¼ 12.0, 3.0 Hz, H-60) and 5.53 (1H, d, J¼ 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 50mL of test solution, 35 mL of 70 mM phosphate buffer (pH 7.5) and 30mL of enzyme solution (0.01 units mL�1in 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 60mL of substrate solution (150 mM xanthine in the same buffer) The assay mixture was incubated at 25�C for 30 min The reaction was stopped by adding 25mL 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 1mmol of uric acid min�1at 25�C XO inhibitory activity was expressed as the percentage inhibition

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 IC50values were calculated from the mean values of data from four determinations Allopurinol, a known inhibitor of XO, was used as a positive control

3.5 Sugar analysis

A solution of 1 (1 mg) in 1N HCl (dioxan–H2O, 1 : 1; 0.5 mL) was heated at 80�C for 4 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 2 h, trimethylsilimidazole (0.1 mL) was added, and the mixture was heated at 60�C for 1 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 (0.25mm � 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 forD- andL-glucose, respectively

4 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, anthelmin-tic 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)

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