Original Papers

1721

Authors

Lun-Lun Zhu 1, 2*, Wen-Wei Fu 1, 2*, Shimpei Watanabe 3, Yi-Nuo Shao 1, 2, Hong-Sheng Tan 1, 2, Hong Zhang 1, 2, Chang-Heng Tan 4, Yan-Feng Xiu 1, 2, Hisayoshi Norimoto 3, Hong-Xi Xu 1, 2

Affiliations

The affiliations are listed at the end of the article

Key words " Garcinia esculenta l " Clusiaceae l " xanthine oxidase inhibitor l " 1,3,6,7‑tetrahydroxyl xanthone " griffipavixanthone l

Abstract

received revised accepted

June 18, 2014 August 3, 2014 Sept. 17, 2014

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383193 Published online October 23, 2014 Planta Med 2014; 80: 1721–1726 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Hong-Xi Xu School of Pharmacy Shanghai University of Traditional Chinese Medicine Cai Lun Lu 1200 Shanghai 201203 Peopleʼs Republic of China Phone: + 86 21 51 32 30 89 Fax: + 86 21 51 32 30 89 [email protected] Correspondence Dr. Hisayoshi Norimoto Kampo Research Laboratories Kracie Pharma Ltd. Kanebo-machi 3–1 Takaoka 933–0856 Japan Phone: + 81 7 66 28 79 53 ext. 540 Fax: + 81 7 66 28 79 60 [email protected]

the DP4 probability and analysis of its MTPA ester derivatives.

!

The EtOAc-soluble portion of the 80 % (v/v) EtOH extract from the twigs of Garcinia esculenta exhibited strong xanthine oxidase inhibition in vitro. Bioassay-guided purification led to the isolation of 1,3,6,7-tetrahydroxyxanthone (3) and griffipavixanthone (8) as the main xanthine oxidase inhibitors, along with six additional compounds (1, 2, 4–7), including two new compounds (1 and 2). This enzyme inhibition was dose dependent with an IC50 value of approximately 1.2 µM for 3 and 6.3 µM for 8. The inhibitory activity of 3 was stronger than the control allopurinol (IC50 value: 5.3 µM). To our knowledge, compound 8 is the first bixanthone that demonstrated potent XO inhibitory activity in vitro. The structures of the new compounds were established by spectroscopic analysis, and the optical properties and absolute stereochemistry of racemic (±) esculentin A (2) were further determined by the calculation of

Introduction !

Gout is characterized by hyperuricemia, which can result in the deposition of uric acid crystals in joints and kidneys, causing inflammatory arthritis and uric acid nephrolithiasis. It is one of the most painful inflammatory conditions that human beings can experience, and it affects 1– 2 % of the Western population [1]. XO inhibitors are generally used for the treatment of gout and hyperuricemia. These inhibitors hinder the enzyme reactions that are involved in uric acid synthesis, reduce uric acid formation, and relieve symptoms of the aforementioned diseases. Allopurinol is most commonly used to treat gout [2]; however, its use is limited by unwanted side ef-

* These authors contributed equally to this work.

Abbreviations !

CMAD: DFT: GIAO: IR: MTPA: PCM: PPAPs: ROS: XO:

corrected mean absolute deviation density functional theory gauge-independent atomic orbital infrared α-methoxy-α-(trifluoromethyl)-phenylacetic acid polarizable continuum model polycyclic polyprenylated acylphloroglucinols reactive oxygen species xanthine oxidase

Supporting information available online at http://www.thieme-connect.de/products

fects [3]. The identification of novel, efficient, and less toxic XO inhibitors remains an important and challenging task. In addition, XO is one of the major enzymatic sources of ROS, so it is considered a major contributor of free radicals in various pathological conditions. More specifically, XO has been implicated in several diseases, including ischemia-reperfusion injury, myocardial infarction, hypertension, atherosclerosis, diabetes, and cancer [4]. Thus, the search for novel XO inhibitors would be beneficial not only to treat gout but also to combat various other diseases. Garcinia L. (Clusiaceae) is a large genus of polygamous trees or shrubs that is distributed in tropical Asia, Africa, and Polynesia. It consists of 450 species, of which 21 are spread across China [5]. Our research group has reported many novel bioactive xanthone derivatives and PPAPs from Garcinia plants in China [6–14]. In the course of

Zhu L-L et al. Xanthine Oxidase Inhibitors …

Planta Med 2014; 80: 1721–1726

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Xanthine Oxidase Inhibitors from Garcinia esculenta Twigs

Original Papers

Fig. 1 Chemical structures of garcinaxanthone J (1), (±) esculentin A (2), 1,3,6,7-tetrahydroxyxanthone (3), and griffipavixanthone (8).

our screening program for natural XO inhibitors, it was found that the S2 fraction from the Garcinia esculenta twigs exhibited strong inhibitory activity at an IC50 value of 7.8 µg/mL (Fig. 1S and Table 1S, Supporting Information). Bioassay-guided separation of the S2 fraction led to the M2 subfraction, which demonstrated more potent inhibition (with an IC50 value of 5.2 µg/mL) than the S2 fraction (Fig. 2S and Table 2S, Supporting Information). A further phytochemical study of the M2 fraction furnished the isolation of a new prenylated xanthone named garcinaxanthone J and a new xanthonolignoid named (±) esculentin A (1 " Fig. 1) along with six known compounds and 2, repsectively; l (3–8). We herein report the structures of these compounds and their XO inhibitory activity. 1,3,6,7-Tetrahydroxyxanthone (3) and griffipavixanthone (8) were identified as potential XO inhibitors with IC50 values of 1.2 and 6.3 µM, respectively, and compound 3 demonstrated a more potent inhibition than the positive control allopurinol, which had an IC50 value of 5.3 µM. To the best of our knowledge, compound 8 is the first bixanthone that demonstrated potent XO inhibitory activity, and compound 2 is the first xanthonolignoid from the genus Garcina. Compound 2 is also the first xanthonolignoid whose complete structural assignment was established through a combination of computational methods using DP4 probability and analysis of their MTPA ester derivatives. G. esculenta Y. H. Li is an endemic plant of China, which is mainly distributed in the western and northwestern part of Yunnan province. To date, there have been only some compounds with cytotoxic and anti-inflammatory properties from this plant reported recently by our groups [15].

Results and Discussion !

Garcinaxanthone J (1) was obtained as a yellow powder. The IR spectrum of 1 revealed bands at 3421, 1647, and 1612 cm−1, indicating the presence of a hydroxyl, conjugated carbonyl, and an aromatic ring, respectively. The 1H NMR spectrum of 1 indicated

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the presence of a singlet aromatic proton [δH 6.70 (1H, s)], two meta-coupled aromatic protons [δH 6.21 (1H, br s) and 6.10 (1H, d, J = 1.7 Hz)], one methylene group attached to the aromatic ring [δH 3.43 (2H, m)], two methylene protons [δH 1.77 (2H, m)], and two methyl protons [δH 1.32 (each 3H × 2, s)]. Its 13C NMR spec" Table 1) revealed the presence of 18 carbons including trum (l one conjugated carbonyl carbon (δC 183.5), 12 aromatic carbons, one oxygenated quaternary carbon, two methylene carbons, and two methyl carbons. The 1H and 13C NMR data for 1 were similar to those of hyperxanthone E [16]. HRESI‑MS of 1 gave a molecular ion at m/z 345.0976 ([M]−, calcd. 345.0974), suggesting the molecular formula of C18H18O7, which is 18 mass units greater than hyperxanthone E. These data suggest that 1 is a xanthone derivative with a 3-hydroxy-3-methylbutyl side chain. The structure of 1 was further confirmed by DEPT, HSQC, and HMBC experiments. Thus, the structure of 1 was designated as shown in " Fig. 1. l (±) Esculentin A (2) was also obtained as a yellow powder, and it was assigned the formula C24H20O10 on the basis of HRESI‑MS (m/ z 469.1142, calcd. 469.1135) with 15 degrees of unsaturation. The IR spectrum of compound 2 demonstrated absorption bands at 3429, 1656, and 1612 cm−1, indicating the presence of OH, a conjugated carbonyl, and an aromatic ring, respectively. The 1H NMR spectrum displayed signals of a hydrogen-bonded hydroxyl group [δ 12.34 (1H, s)], six aromatic protons [δ 6.36 (s, 1H), 6.72 (2H, br s), 7.30 (1H, dd, J = 9.0, 3.0 Hz), 7.41 (1H, d, J = 3.0 Hz), and 7.53 (1H, d, J = 9.0 Hz)], two methoxyl groups [δ 3.73 (3H × 2, s)], two oxygenated methines [δ 5.10 (1H, d, J = 7.9 Hz) and 4.25 (1H, ddd, J = 7.9, 4.0, 2.6 Hz)], and a hydroxymethyl group [δ 3.65 (br d, J = 15.6 Hz, 1H) and 3.42 (1H, overlap)]. The observation of a deshielded doublet [δ 5.10] measured in DMSO-d6 suggested a benzylic methylene substituted by oxygen, and its trans-diaxial relationship (J = 7.9 Hz) indicated the occurrence of a trans-substituted 1,4-dioxane ring between the xanthone framework and the phenyl ring, and framed a xanthonolignoid skeleton [17–19]. Furthermore, the HMBC and 1H-1H COSY correlations as demon-

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Original Papers

Compound 1a δH

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 1′ 2′ 3′ 4′ 5′ 6′

a

6.10 (d, 1.7) 6.21 (br s) 6.70 s

3.43 m 1.77 m 1.32 s 1.32 s

Position δC 164.7 98.6 165.7 93.9 158.5 101.1 153.6 142.1 130.9 112.1 183.5 104.0 154.3 23.0 44.4 72.1 29.1 29.1

Compound 2b δH

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ OMe×2

δC

6.36 s

7.53 (d, 9.0) 7.30 (dd, 9.0, 3.0) 7.41 (d, 3.0)

6.72 (br s)

6.72 (br s) 5.10 (d, 7.9) 4.25 (ddd, 7.9, 4.0, 2.6) 3.65 (br d, 15.6), 3.42 (o)c 3.73 s

154.5 97.7 150.8 124.2 144.7 119.3 124.9 154.2 108.0 120.4 180.3 103.0 149.1 125.7 105.5 148.0 136.2 148.0 105.5 77.6 77.6 60.0 56.2

Table 1 1H and 13C NMR spectroscopic data for 1 and 2 (δ in ppm and J in Hz).

In CD3OD (1H: 400 or 600 MHz; 13C: 151 MHz); b in DMSO-d6 (1H: 600 Mz; 13C: 151 MHz); c overlap with the signal of DOH

Fig. 2 1H–1H COSY (bold lines) and key HMBCs (arrows) of 1 and 2.

" Fig. 2 also indicated the same skeleton. However, strated in l these data could not warrant a conclusion of the stereochemical structure, especially the orientation of the substituents on the 1,4-dioxane rings. By comparison with those stereochemical known similar structures, the orientation of the substituents on the 1,4-dioxane rings could not be established unequivocally because of the different Δδ value of the key chemical shift from the known structures (Fig. 12S and Table 3S, Supporting Information). The theoretical method of the ab initio DFT-GIAO calculation of the NMR chemical shifts has been proven as a powerful additional tool for the assignments of gross structure and stereochemistry of natural products in the case of only having one set of experimental data [20, 21]. Compound 2 contains two stereocenters and the two orientations of the substituents on the 1,4-dioxane rings. The J value (7.9 Hz) of H-7′ indicated the trans-substituents on the 1,4-dioxane ring. So, it has only four possible diastereomers of different relative configuration (2–1–2–4; Fig. 25S, Supporting Information). All of the diastereomers were submitted to a conformational search using molecular mechanics calculations in Discovery Studio 2.5 Client [22, 23]. The corresponding minimum geome-

tries were fully optimized at the B3LYP/6–31 G(d) level in the gas phase to get more accurate conformers. The 1H and 13C NMR shielding constants of the corresponding optimized configurations were computed using the GIAO technique at the mPW1PW91-SCRF/6–311+G(2 d,p) level of theory in the PCM solvent with DMSO as a solvent [22]. The Boltzmann-calculated population-weighted chemical shifts of the four diastereomers were afforded after corrections using the slope and intercept obtained from linear regression analysis [21]. Because of xanthonolignoids being reported as a racemic mixture [17], the four possible diastereomers were divided into two groups based on the orientation of the substituents on the 1,4-dioxane rings. Comparisons of experimentally measured 1H and 13C NMR resonances for 2 and those of the Boltzmann-weighted DFT-GIAO NMR calculations for the two possible group diastereomers were carried out. The DP4 analysis, recently developed by Smith and Goodman [21], identified group 1 (structures 2–1 and 2–2) as the most likely, with probabilities of 90.3%, 98.2 %, and 99.8 % for the 1H NMR, 13C NMR, and combined 1H and 13C NMR chemical shifts, respectively (Table 6S, Supporting Information). In addition, the lowest values both of the largest deviations and the average er-

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Planta Med 2014; 80: 1721–1726

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Position

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Original Papers

Table 2

1

H NMR spectroscopic data for (7′R,8′R)/(7′S,8′S)-venkatasin 4′,9′-di-(S)-MTPA ester and (±) esculentin A tetra-(S)-MTPA ester (2a and 2b) in CDCl3.

Position

7′R,8′R-venkatasin-4′,9′-

2a

Position

7′S,8′S-venkatasin-4′,9′-

di- (S)-MTPA ester [17, 24] 7′ 8′ 9′

4.97 (d, 7.6) 4.41 (ddd, 7.6, 4.5, 2.4) 4.75 (dd, 12.5, 2.4) 4.24 (dd, 12.5, 4.5)

2b

di- (S)-MTPA ester [17, 24] 4.88 (d, 8.2) 4.32 (m) 4.91 (dd, 12.5, 2.2) 4.22 (dd, 12.5, 4.5)

7′ 8′ 9′

5.00 (d, 8.0) 4.38 (dt, 8.0, 2.5) 4.99 (dd, 12.6, 2.5) 3.98 (dd, 12.6, 2.5)

5.02 (d, 8.2) 4.30 (m) 5.12 (dd, 12.7, 3.5) 4.04 (dd, 12.7, 3.5)

Coupling constants given (J, Hz) in parentheses

Final concentration (µM)

Inhibition (%)

IC50 (µM)

3

8.0 4.0 2.0 1.0 8.0 4.0 2.0 1.0 8.0 4.0 2.0 1.0

78.5 ± 2.0 67.2 ± 2.0 64.4 ± 2.1 45.4 ± 2.7 68.6 ± 4.6 24.5 ± 6.6 0.6 ± 6.1 0.0 ± 0.0 67.2 ± 0.8 41.1 ± 4.0 35.5 ± 0.8 15.8 ± 3.8

1.2

8

Allopurinol (positive control)

a

Table 3 In vitro inhibitory activity of compounds 3 and 8 against xanthine oxidase.

6.3

5.3

All of the samples were dissolved in DMSO; b data are represented as means ± SD (n = 3)

rors (CMAD) of group 1 (Tables 4S, 6S, and 7S, Supporting Information) further supported that compound 2 was likely a mixture of 7′S,8′S and 7′R,8′R. Compound 2 was optically active [[α]D − 4.0 (c 0.025 MeOH)] in spite of many xanthonolignoids being reported as a racemic mixture [18, 24]. In order to elucidate the optical nature for 2, a MTPA ester derivative of 2 was prepared. The 1,7,4′,9′-tetra-MTPA ester derivative obtained from 2 showed two sets of 1H NMR signals in a ratio of 93 : 100. 1H NMR chemical shifts and coupling patterns of H-7′, H-8′, and H2-9′ in two esters (2a and 2b) were similar to those in the 4′,9′-di-(S)-MTPA esters of (7′R, 8′R) and (7′S, 8′S)" Table 2; Fig. 23S, Supporting Invenkatasin [25], respectively (l formation). The ratio of the enantiomers in 2 was estimated to be 48 : 52. The major enantiomers were concluded to be 7′S, 8′S. " Fig. 1. Thus, the structure of 2 was elucidated as shown in l The known compounds were identified as 1,3,6,7-tetrahydroxyxanthone (3) [26], (−)-syringaresinol (4) [27], 2,6-dihydroxy-4methoxybenzophenone (5) [28], (−)-GB‑1a (6) [29], (+)-volkensiflavone (7) [30], and griffipavixanthone (8) [31] by analysis of their spectroscopic data and comparison with data from the literature. The in vitro inhibitory activity of compounds 1–8 against XO was measured spectrophoto-metrically by following uric acid levels at 295 nm [32]. Compounds presenting an inhibitory effect higher than 60 % at 100 µM were further assessed at a wide range of concentrations to determine their IC50 values; the results are " Table 3. Among them, 3 and 8 inhibited XO activity shown in l in a concentration-dependent manner with IC50 values of 1.2 " Table 3). On the basis of the IC and 6.3 µM, respectively (l 50 values, compound 3 exhibited better XO inhibition than the positive control allopurinol (IC50 value: 5.3 µM) [33], and compound 8 exerted a similar inhibitory activity against XO as allopurinol, which is a drug that is clinically used for gout treatment [2]. Compound 1 displayed weak inhibition on XO activity with 31.50 % inhibition at 100 µM in the other isolates (Table 5S, Supporting In-

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formation). The presence of hydroxyl groups at the C1 and C3 positions of the xanthone framework turned out to be essential for the high inhibitory activity in our experiments. Compound 3 exhibited better XO inhibitory activity than the positive control allopurinol, and its C-glycoside mangiferin could significantly reduce serum urate levels in hyperuricemic mice [34]. On the other hand, compound 8 is the first bixanthone that demonstrated potent XO inhibitory activity; it would afford a new chemical scaffold for gout therapeutic agent development. So, compounds 3 and 8 would be desirable candidates for use in the treatment of gout.

Materials and Methods !

General experimental procedures Optical rotations were measured using a Jasco P-1020 digital polarimeter. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer. IR spectra were obtained on a Shimadzu FTIR-8400s spectrophotometer. NMR spectra were recorded on a Bruker AV-400 spectrometer with TMS as the internal standard. ESI‑MS and HR‑ESI‑Q‑TOF‑MS experiments were performed on an Agilent 1100 Series MSD Trap mass spectrometer, Agilent 6210 ESI‑TOF spectrometer, and Agilent 6520 ESI‑Q‑TOF spectrometer, respectively. A Waters 2535 series machine equipped with an Xbridge C18 column (4.6 × 250 mm, 5 µm) was used for HPLC analysis, and a preparative Xbridge Prep C18 OBD column (19 × 250 mm, 5 µm) was used in sample preparation. Column chromatography was performed with CHP20P MCI gel (75– 150 µm, Mitsubishi Chemical), silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd.), Sephadex LH-20 (GE Healthcare BioSciences AB), and reversed-phase C18 silica gel (50 µm, YMC). Analytical and preparative TLC were performed on precoated GF254 plates (0.25 or 0.5 mm thickness, Qingdao Haiyang Chem-

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Compounds

Original Papers

Plant material The G. esculenta Y. H. Li. twigs were collected in Nujiang, Yunnan Province, Peopleʼs Republic of China, in August 2010. The plant material was identified by Prof. Yuanchuan Zhou, Yunnan University of Traditional Chinese Medicine. A voucher specimen (No. 20100801) was deposited at the Innovative Research Laboratory of TCM, Shanghai University of Traditional Chinese Medicine.

Extraction and isolation According to the previous separation process [15], air-dried and powdered plant twigs (4 kg) were extracted with petroleum ether to gain the petroleum ether-soluble S1 portion. The remaining materials were refluxed with 80% EtOH and concentrated to afford a residue. The residue was suspended in H2O and fractionated with EtOAc to obtain S2 and S3, respectively. The remaining materials were refluxed with distilled water to obtain S4. S2 possessed significant inhibition activities against XO in vitro. S2 was subjected to CC on MCI and eluted with 30 %, 60 %, 90%, and 100 % EtOH and EtOAc, successively, to obtain M1– M5 subfractions, as described previously [15]. M2 had significant inhibitory activity against XO in vitro. The details on the bioassayguided isolation are described in Supporting Information. M2 (18.0 g) was chromatographed over C18 MPLC (4 × 60 cm) and eluted with MeOH‑H2O (45 : 55 to 100 : 0 v/v; 30 mL/min; 1000 mL each) in a gradient, as described previously [15]. The fractions were combined and monitored by TLC to yield 1,3,6,7tetrahydroxyxanthone (3) (108 mg) and five subfractions (IIB1– IIB5). Fraction IIB1 (3.0 g) was subjected to separation on a Sephadex LH-20 column (300 g, 3 × 160 cm) and eluted with CHCl3/ MeOH (1 : 1, monitor by TLC) to give seven subfractions (IIB1aIIB1g). Subfraction IIB1b (0.6 g) was purified via preparative TLC using CH2Cl2/MeOH (15 : 1) to produce (−)-syringaresinol (4) (2 mg). Subfraction IIB1f (2.0 g) was passed through a silica gel column (90 g, 3 × 40 cm) using CHCl3/MeOH (13 : 1) to produce 1,3,6,7-tetrahydroxyxanthone (3) (1.0 g). Fraction IIB2 (5.9 g) was chromatographed over a silica gel column (200 g, 5 × 30 cm) using a CH2Cl2/MeOH gradient (20 : 1 to 5 : 1, 50 mL each) to produce 2,6-dihydroxy-4-methoxybenzophenone (5) (6 mg) and subfractions IIB2a-IIB2d. Subfraction IIB2c (0.5 g) was passed through a Sephadex LH-20 CC (300 g, 3 × 160 cm), eluting with acetone (2000 mL), to obtain (−)-GB‑1a (6) (50 mg) and (+)-volkensiflavone (7) (50 mg). Fraction IIB3 (1.5 g) was chromatographed over a silica gel column (90 g, 3 × 40 cm) using a CH2Cl2/ MeOH gradient (20 : 1 to 8 : 1, 20 mL each) and rechromatographed via preparative TLC using CH2Cl2/MeOH (12 : 1) to produce compound 1 (4 mg). Fraction IIB4 (6.3 g) was chromatographed over a silica gel column (300 g, 6 × 60 cm) using a CH2Cl2/MeOH gradient (20 : 1 to 10 : 1, 60 mL each) as a solvent to produce five subfractions (IIB4a-IIB4e). Subfraction IIB4b was further purified by Sephadex LH-20 (50 g, 2 × 100 cm) using MeOH (1000 mL) as a solvent to produce compound 2 (5 mg). Fraction IIB5 (0.141 g) was chromatographed over a silica gel column (60 g, 3.5 × 20 cm) using a CH2Cl2/MeOH gradient (20 : 1, 20 mL each) as a solvent to produce the compound griffipavixanthone (8) (25 mg).

Isolates Garcinaxanthone J (1): yellow powder; IR (KBr) νmax 3421, 2922, 1647, 1612, 1462, 1375, 1263, 1167, 1024, 806 cm−1; UV (MeOH): λmax (log ε) 310 (4.31), 255 (4.49), 210 (4.94) nm; 1H‑(CD3OD, " Table 1; 400 MHz) and 13C‑NMR (CD3OD, 151 MHz) data, see l HRESI‑MS m/z 345.0976, [M]− (calcd. for C18H17O7, 345.0974). (±) Esculentin A (2): yellow powder; [α]25 D − 4.0 (c 0.025 MeOH); IR (KBr) νmax 3429, 2962, 1656, 1612, 1479, 1342, 1261, 1091 cm−1; UV (MeOH): λmax (log ε) 390.0 (3.64), 315.0 (3.93), 274.9 (4.55), 237.5 (4.47), 202.5 (4.86) nm; 1H‑(DMSO-d6, 600 MHz) and 13 " Table 1; HRESI‑MS m/ C‑NMR (DMSO-d6, 151 MHz) data, see l z 469.1142, [M]+ (calcd. for C24H20O10, 469.1135).

Conversion of (±) esculentin A (2) to (±) esculentin A tetra-(S)-MTPA ester (2a and 2b) Compound 2 (0.6 mg) in pyridine (100 µL) was treated with (R)MTPACl (5 µL) for 1 h. MeOH (30 µL) was added to the reaction mixture to destroy the acid chloride. Solvents were removed in vacuo to afford (±) esculentin A tetra-(S)-MTPA ester (2a and 2b).

Biological material Xanthine oxidase from buttermilk (Lot No. 27093203) was purchased from Oriental Yeast Co., Ltd. Allopurinol was purchased from Tokyo Chemical Industry Co., Ltd. Xanthine (Lot No. TGG4402) was purchased from Wako Pure Chemical Industries, Ltd. DMSO was purchased from Sigma-Aldrich Co., LLC. All other chemicals, hydrochloric acid, and PBS were purchased from Sinopharm Chemical Reagent Co., Ltd. The purity of allopurinol and other tested compounds was greater than 98%.

Xanthine oxidase inhibitory assay in vitro XO inhibitory activity was assayed spectrophotometrically at 295 nm under aerobic conditions using 96-well plates, as described previously. XO inhibitory activity is expressed as the percentage of inhibition of XO in the above assay system, which was calculated as (1- B/A)/100, where A and B are the enzyme activities without and with test material, respectively. The IC50 values were calculated using the mean data values from three determinations. Details of the XO inhibition in vitro are described in Supporting Information.

Supporting information The in vitro xanthine oxidase inhibitory activity of different fractions, subfractions, and compounds 1–8 obtained from G. esculenta twigs, as well as UV, IR, MS, 1H‑NMR, 13C‑NMR, and 2D‑NMR data of compounds 1 and 2 are available as Supporting Information.

Acknowledgements !

We would like to acknowledge financial support from the National Natural Science Foundation of China (81173485, 81202863, 81303266, and 81303188) for part of this work.

Conflict of Interest !

The authors declare no conflict of interest.

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ical Co., Ltd.). Detection was performed by spraying the plates with 10% sulfuric acid followed by heating.

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Affiliations 1

2

3 4

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, Peopleʼs Republic of China Engineering Research Center of Shanghai Colleges for TCM New Drug Discovery, Shanghai, Peopleʼs Republic of China Kampo Research Laboratories, Kracie Pharma Ltd., Takaoka, Japan State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, CAS, Shanghai, Peopleʼs Republic of China

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Xanthine oxidase inhibitors from Garcinia esculenta twigs.

The EtOAc-soluble portion of the 80 % (v/v) EtOH extract from the twigs of Garcinia esculenta exhibited strong xanthine oxidase inhibition in vitro. B...
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