Zhang et al. / J Zhejiang Univ Sci B 2008 9(5):407-415 407 Journal of Zhejiang University SCIENCE B ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.zju.edu.cn/jzus; www.springerlink.com E-mail: [email protected] Tannins from Canarium album with potent antioxidant activity* Liang-liang ZHANG1, Yi-ming LIN†‡1,2 (1Department of Biology, School of Life Sciences, Xiamen University, Xiamen 361005, China) (2Research Centre for Wetlands and Ecological Engineering, Xiamen University, Xiamen 361005, China) †E-mail: [email protected] Received Jan. 1, 2008; revision accepted Mar. 18, 2008 Abstract: The contents of total phenolics and extractable condensed tannins in the leaves, twigs and stem bark of Canarium album were determined. The structural heterogeneity of condensed tannins from stem bark was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) analyses. The results show the predominance of signals representative of procyanidins and prodelphinidins. In addition, epicatechin and epigallocatechin polymers with galloylated procyanidin or prodelphinidin were also observed. The tannins were screened for their potential antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) model systems. Tannins extracted from leaves, twigs and stem bark all showed a very good DPPH radical scavenging activity and ferric reducing power. Key words: Canarium album, Tannins, Antioxidant capacity, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) doi:10.1631/jzus.B0820002 Document code: A CLC number: Q946.84 INTRODUCTION lymerization (DPs). The molecules are water- soluble and can form complexes with proteins and Tannins known as the group of phenolic com- polysaccharides (Haslam, 1998). PAs are of great pounds are the significant plant secondary metabo- interest in nutrition and medicine because of their lites. Tannins in vascular plants occur as two types, potent antioxidant capacity and possible protective the condensed and the hydrolysable (Hernes et al., effects on human health (Santos-Buelga and Scalbert, 2001). Condensed tannins are also known as proan- 2000). They have antioxidant properties related to thocyanidins (PAs), the oligomeric and polymeric their radical scavenging capacity (Ricarda Da Silva et flavan-3-ols, which are linked through C4-C8 or al., 1991), and these properties have been used C4-C6 linkages. The diversity of condensed tannins is against heart disease through reducing lipid oxidation. given by the structural variability of the monomer It was hypothesized that the free radical scavenging units (different hydroxylation patterns of the aromatic properties of PAs may reduce the risk of cardiovas- rings A and B, and different configurations at the cular diseases, cancer (Bagchi et al., 2000) and blood chiral centers C2 and C3) (Fig.1). The size of PA clotting, and certain types of trimeric PAs may protect molecules can be described by their degrees of po- against urinary tract infections (Santos-Buelga and Scalbert, 2000). However, tannins are diverse com- pounds with great variation in structure and concen- ‡ Corresponding author tration within and among plant species. Therefore, * Project supported by the National Natural Science Foundation of biomedical researches on the health benefits and risks China (No. 30671646), the Program for New Century Excellent of increased tannins consumption are severely limited Talents in University, China (No. NCET-07-0725), and the Program for Innovative Research Team in Science and Technology in Fujian by lack of methods for rapid characterization and Province University, China standardization. 408 Zhang et al. / J Zhejiang Univ Sci B 2008 9(5):407-415 Fig.1 Chemical structures of flavan-3-ol (a) monomers and (b) polymers Canarium album or Chinese olive (Burseraceae), MATERIALS AND METHODS is the native species in the southeast of China. It is a good fruit species because of its tolerance to poor Samples soils (e.g., rocky hillsides, saline or alkaline soils) Leaves, twigs and stem bark samples of C. al- (Wei et al., 1999). The dried fruits are a traditional bum were collected in Fuzhou in the southeast of medicine material that has some pharmacological China. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,4,6- functions such as anti-bacterium, anti-virus, anti- tripyridyl-S-triazine (TPTZ), ascorbic acid (AA), inflammation and detoxification (Ding, 1999). The butylated hydroxyanisole (BHA), gallic acid and fresh fruits are widely used in food industry, while the ellagic acid were purchased from Sigma (USA). leaves and stem bark of the plants are discarded as Sephadex LH-20 was purchased from Amersham waste products. (USA). All chemicals were of analytical reagent (AR) Polyphenols make a major contribution to free purity grade. radical scavenging capacities of Mediterranean olives species (Visioli et al., 1998). Previous studies indi- Methods cated that there was a direct relationship between 1. Solvent extraction antioxidant activity and total phenolics content in Ten grams of fresh materials were weighed and selected herbs, vegetables and fruits (Oszmianski et extracted with 100 ml acetone-water solution (70:30, al., 2007; Wu, 2007). In this study, contents of total v/v) twice at room temperature. The extract was fil- phenolics and extractable condensed tannins of leaves, tered and pooled, and the solvent was removed under twigs and stem bark of C. album were determined, reduced pressure by using a rotary vacuum- and the structural heterogeneity of condensed tannins evaporator at 40 °C. The remaining aqueous fraction from stem bark was characterized by matrix-assisted was extracted thrice with hexane in order to remove laser desorption/ionization time-of-flight mass spec- chlorophyll and lipophilic compounds. The remaining trometry (MALDI-TOF MS) and nuclear magnetic crude tannins fraction was chromatographed on an resonance (NMR) analyses. Meanwhile, the free Sephadex LH-20 column (3 cm×22 cm) which was radical scavenging capacities and ferric reducing first eluted with methanol:water (50:50, v/v) to re- power of condensed tannins from leaves, twigs and move oligomeric tannins and then followed by ace- stem bark were also discussed. tone:water (70:30, v/v). The last fraction, containing Zhang et al. / J Zhejiang Univ Sci B 2008 9(5):407-415 409 the polymeric tannins, was freeze-dried and stored at The samples were previously dissolved in mobile −20 °C until required. phase and then filtrated through membrane filter with 2. Determination of total phenolics and con- an aperture size of 0.45 µm. Ten milliliters of the clear densed tannins (PAs) supernatant after centrifugation at 19 000×g for 5 min Established procedures (Lin et al., 2006) were was injected. Separation was performed on a Hypersil used to determine total phenolics and condensed tan- ODS column (4.6 mm×250 mm, 5 μm) thermostatted nins. Total phenolics were measured with the Prus- at 30 °C. The mobile phase was composed of solvent sian blue method (Graham, 1992), and condensed A (0.1% (v/v) trifluoroacetic acid (TFA) in water) and tannins were assayed by the butanol-HCl method solvent B (0.1% (v/v) TFA in acetonitrile). The gra- (Terrill et al., 1992), using purified tannins from C. dient condition was: 0~2nd minutes 100% A, 2nd~6th album and tannic acid as the standards. minutes 100%~95% A, 6th~10th minutes 95% A, 3. NMR analysis 10th~15th minutes 95%~80% A, 15th~20th minutes 13 C NMR spectra were recorded in CD3COCD3- 80% A, and 20th~25th minutes 70% A. Other chro- D2O mixture with a Varian Metcury-600 spectrome- matographic conditions were as follows: flow rate at 1 ter at 150 MHz (proton decoupling mode for carbon). ml/min, detection at 280 nm, and scanning performed 4. MALDI-TOF MS analysis between 200 and 600 nm. The MALDI-TOF MS spectra were recorded on 6. Free-radical scavenging activity a Bruker Reflex III. The irradiation source was a The free-radical scavenging activity was meas- pulsed nitrogen laser with a wavelength of 337 nm, ured according to the method of Braca et al.(2001). A and the duration of the laser pulse was 3 ns. In the 100 µl of sample at different concentration (15~250 positive reflectron mode, an accelerating voltage of µg/ml) was added to 3 ml of DPPH solution (0.004% 20.0 kV and a reflectron voltage of 23.0 kV were used. (w/w) methanolic solution). Thirty minutes later, the The spectra of condensed tannins were obtained from absorbance was measured at 517 nm. Lower absorb- a sum of 100~150 shots and calibrated using Angio- ance of the reaction mixture indicates higher free tensin II (1046.5 MW), Bombesin (1619.8 MW), radical scavenging activity. The IC50 value, defined as ACTHclip1839 (2465.2 MW), and Somatostatin28 the amount of antioxidant necessary to decrease the (3147.47 MW) as external standards. 2,5-Dihydroxy initial DPPH concentration by 50%, was calculated benzoic acid (DHB, 10 mg/ml aqueous solution) was from the results and used for comparison. The capa- used as the matrix. The sample solutions (7.5 mg/ml bility to scavenge the DPPH radical was calculated by aqueous) were mixed with the matrix solution at a using the following equation: volumetric ratio of 1:3. The mixture (1 μl) was ap- plied to the steel target. Amberlite IRP-64 cation- DPPH scavenging effect (%)=[(A1–A2)/A1]×100, exchange resin (Sigma-Aldrich, USA), equilibrated in deionized water, was used to deionize the ana- where A1 is the absorbance of the control reaction, and lyte-matrix solution thrice. Cesium trifluoroacetate (1 A2 is the absorbance in the presence of the sample. mg/ml) was mixed with the analyte-matrix solution BHA and ascorbic acid were used as the controls. (1:3, v/v) to promote the formation of a single type of 7.