Prenylated Xanthones from the Bark of Garcinia xanthochymus and Their 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activities

Garcinia xanthochymus has been widely used in traditional Chinese medicine for expelling worms and removing food toxins. Bioassay-guided fractionation of an EtOAc-soluble extract of G. xanthochymus stem bark led to the isolation of six new xanthones. Their structures were elucidated by spectroscopic methods, especially 2D-NMR techniques. Free-radical-scavenging activities of the isolated compounds were elucidated through DPPH method. Most of the isolated compounds showed considerable free radical scavenging activity on DPPH assay. Compound 1 exhibited effective antioxidant scavenging activity against DPPH radical with an IC50 value of 19.64 μM, and compound 6 showed the lowest activity among all the tested molecules, with an IC50 value of 66.88 μM. These findings support the notion that the plant genus Garcinia is a good source of bioactive compounds.

The genus Garcinia belongs to the Guttiferae family, which comprises 200 species confined to the tropics as trees or shrubs, and rarely subshrubs. It is well known to be a rich source of oxygenated and prenylated xanthones [5]. Xanthones are a class of polyphenolics that exhibit well-documented pharmacological properties, such as antioxidative, antileukaemic, antitumour, antiulcer, antimicrobial, antihepatotoxic, and CNS depressant activities [6], mainly due to their oxygenated heterocyclic nature and diversity of functional groups [7]. Garcinia xanthochymus is a traditional Dai medicine native to the south and southwest of Yunnan Province, P. R. China which can grow up to 10-20 m. It has been widely used as a traditional medicine for expelling worms and removing food toxins [8]. Previous phytochemical studies of the leaves, seeds, fruits, twig bark, and wood have demonstrated the presence of benzophenones [9][10][11][12][13][14][15], flavonoids [16,17], triterpenes [18] and xanthones [19][20][21]. In order to clarify the bioactive components, bioassay-guided fractionation has led to the isolation of six novel xanthones 1-6 ( Figure 1). Herein we report the isolation and structural elucidation of these new xanthones and DPPH-radical scavenging activities of the isolated compounds.

Structural elucidations of xanthones
Compound 1 was obtained as a yellow powder. Its molecular formula C 25  . The presence of the fused furan ring was substantiated by the methine carbons (δ c 104.9 and 144.8) in the 13 C-NMR spectrum. The HMBC correlations of the hydrogen-bonded proton (1-OH) with an oxygenated aromatic carbon at δ c 160.9, a quaternary aromatic carbon at δ c 105.8 and a methine aromatic carbon at δ c 94.1 corresponding to an aromatic proton [δ H 6.85 (1H, s)] in HSQC spectrum. It suggested that this proton may be attributed to H-2. The position of the furan ring was determined as follow. In the HMBC spectrum, one proton signal at δ H 7.39 (1H , br s) of the furan ring showed correlations with a quaternary aromatic carbon at δ c 108.5 (C-4) and an oxygenated aromatic carbon at δ c 160.6 (C-3). The signals at δ c 108.5 and 160.6 also correlated with the aromatic proton signal at δ H 6.85 (1H, s, H-2). Therefore, the furan ring was fused at C-4 through an oxygen at C-3. The locations of other substituents were determined as follows. In the 13 C-NMR spectrum, the aromatic carbons with an oxygen function were observed at δ c 149.4, 130.3 and 150.5, which suggested the presence of a 1, 2, 3-trioxygenated benzene ring in partial structure B. In HMBC spectrum (Figure 2), the correlations of H 2 -1′′/C-6 (δ C 150.5), C-7 (δ C 126.1) and C-8 (δ C 136.4) indicated that one 3-methyl-2-butenyl group was located at C-7. Thus, the remaining 3hydroxy-3-methylbutyl group should be located at C-8. Compound 1 was thus identified to be 1,5,6trihydroxy-7-(3-methyl-2-butenyl)-8-(3-hydroxy-3-methylbutyl)furano(2′,3′:3,4) xanthone.

DPPH radical-scavenging activities of the purified compounds
The six xanthones were evaluated for their antioxidant activities by DPPH free radical scavenging method ( Table 4). Most of the isolated compounds showed considerable free radical scavenging activity on DPPH assay. The potency of DPPH radical-scavenging activity was in a decreasing order: 1 > 3 > 2 > 5> 4 > 6. Compound 1 exhibited effective antioxidant scavenging activity against DPPH radical, with an IC 50 value of 19.64 μM, and compound 6 showed the lowest activity with an IC 50 value of 66.88μM among all the tested molecules. The DPPH radical scavenging activities of these compounds seemed to be related to the number of phenol-like OH groups at the xanthone skeleton. It was reported previously that the radical scavenging activity was increased in the presence of an increasing number of phenol like OH groups in a molecule [23]. However, compound 5, having four phenol-like OH groups, showed a lower radical scavenging activity compared to that of compound 1 having three phenol-like OH groups. This was because the presence of furan ring in compound 1 extended the conjugation system to participate in stabilizing the phenoxy radical by resonances, therefore increasing the radicalscavenging activity of compound 1 [24]. From above the data, it can be deduced that the main components responsible for the antioxidant activities of Garcinia xanthochymus were the phenolic compounds, such as xanthone derivatives.

General
UV spectra were measured on an SP-2102UVPC spectrometer using MeOH as the solvent. NMR spectra were run in DMSO-d 6 or Me 2 CO-d 6 on a Bruker AM-400 (1D) or Varian Inova-600 (2D) spectrometer with TMS as an internal standard. EIMS and HREIMS measurements were conducted with a Finnigan MAT 95 instrument. Thin-layer chromatography (TLC) was performed on silica gel 60 GF 254 , while column chromatography was carried out using silica gel (200-300 mesh) from Qingdao Haiyang Chemical Group Co., P. R. China and C 18 reversed-phase silica gel from YMC CO., LTD., Japan.

Plant material
The bark of Garcinia xanthochymus was collected from Xishuangbanna Prefecture, Yunnan Province, P.R. China and identified by Xishuangbanna Prefecture National Medicine Research Institute. The voucher specimen (06061201) was deposited with the Herbarium of College of Pharmacy, South Central University for Nationalities.

DPPH radical scavenging activity
Scavenging activities of the purified compounds from G. xanthochymus towards DPPH radical were assessed by using the method described by Scherer and Godoy with a slight modification [25,26]. Briefly, a 0.08 mM solution of DPPH radical solution in methanol was prepared and then, the solvent extracts and purified compounds at different concentrations (0.1 mL) were added to the prepared DPPH radical solution (3.9 mL); the mixture was shaken vigorously, after a 30 min incubation period at 37 ºC in the dark, the absorbance was measured at 517 nm by using a UV-visible spectrophotometer. Obviously, decreasing of the DPPH solution absorbance indicates an increase of the DPPH radicalscavenging activity. The radical scavenging activity is given as DPPH radical scavenging effect that is calculated using equation (1): DPPH radical scavenging effect (%) = [(A 0 -A 1 )/A 0 ] × 100 (1) where A 0 was the absorbance of control and A 1 was the absorbance in the presence of the standard, solvent extracts or purified compounds at different concentrations. Ascorbic acid (V C ) and gallic acid were used as positive controls, respectively. All the tests were performed in triplicate. The scavenging activities of the purified compounds towards DPPH radical were expressed as IC 50 , which was determined to be the effective concentration at which DPPH radical was scavenged by 50%. The IC 50 value was obtained by interpolation from linear regression analysis.

Statistical analyses of results of activity studies
The results were performed as mean ± standard deviation (SD) of three determinations. Analysis of significance differences among means were tested by one-way analysis of variance. The IC 50 values were calculated by linear regression analysis.

Conclusions
In the course of our ongoing research project on bioactive natural products from G. xanthochymus, an EtOAc-soluble partition of the EtOH extract of the bark of G. xanthochymus displayed significant antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging bioassay. This prompted us to perform a detailed bioassay-guided isolation from this plant, which led to the isolation of six new xanthones. Notably, most of the isolated compounds showed considerable free radical scavenging activity in the DPPH assay. Compound 1 exhibited effective antioxidant scavenging activity against DPPH radicals with an IC 50 value of 19.64 μM, and compound 6 showed the lowest activity among all the tested molecules, with an IC 50 value of 66.88 μM. These findings support that plant genus Garcinia is a good source of bioactive compounds.