Appl Biochem Biotechnol (2014) 173:179–192 DOI 10.1007/s12010-014-0828-z

Isolation and Purification of Condensed Tannins from Flamboyant Tree and Their Antioxidant and Antityrosinase Activities Hui-Ling Feng & Ling Tian & Wei-Ming Chai & Xiao-Xin Chen & Yan Shi & Yu-Sen Gao & Chong-Ling Yan & Qing-Xi Chen

Received: 28 November 2013 / Accepted: 19 February 2014 / Published online: 27 March 2014 # Springer Science+Business Media New York 2014

Abstract Flamboyant tree, a kind of medicinal plant, was studied as a source of condensed tannins. The antioxidant activities of the condensed tannins from the leaf, fruit, and stem bark of flamboyant tree were screened by ABTS radical and hydroxyl radical scavenging activity methods. The results indicated that these compounds possessed potent antioxidant activity. Their structures were then characterized by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) after thiolytic degradation. The results showed that the leaf condensed tannins were composed of afzelechin/epiafzelechin, catechin/ epicatechin, and gallocatechin/epigallocatechin, while the fruit and stem bark condensed tannins had afzelechin/epiafzelechin and catechin/epicatechin. In addition, the condensed tannins were evaluated for their antityrosinase ability. They were found to have significant antityrosinase activity. The IC 50 values were 35 ± 2.0 and 40 ± 0.5 μg/ml for the condensed tannins of fruit and stem bark, respectively. Further, fluorescence quenching and copper interacting techniques were utilized to unravel the molecular mechanisms of the inhibition. The results showed that the hydroxyl group of the condensed tannins could chelate the dicopper center of the enzyme and interact with tryptophan residues. Our studies revealed that condensed tannins might be suitable for use in food, agriculture, cosmetic, nutraceutical, and pharmaceutical applications. Keywords Flamboyant tree . Condensed tannins . ABTS . Hydroxyl radical scavenging activity . HPLC-ESI-MS . Antityrosinase activity

Hui-Ling Feng and Ling Tian contributed equally to this work.

H. leaf. To sum up, the condensed tannins from different parts of the flamboyant tree all had inhibitory effects on the oxidation of L-Tyr by the mushroom tyrosinase. Two copper ions exist in the active center of tyrosinase, and a slight change in the dicopper center may lead to activity loss of tyrosinase [35]. The previous study clarified that condensed tannins inhibit tyrosinase activity by binding to its copper ions with hydroxyl groups, and the inhibition ability was affected by the substituted position of the hydroxyl groups (Fig. 5) [36]. Compared with the fruit and stem bark condensed tannins, the condensed tannins from the leaf showed lower inhibition activity on the tyrosinase. This may be due to the steric hindrance of gallocatechin/ epigallocatechin’s excessive hydroxyl moiety [37], since gallocatechin/epigallocatechin was not found in the fruit and stem bark condensed tannins. Fluorescence Quenching Study The conformational changes of tyrosinase are often investigated based on the fluorescence of tryptophan in tyrosinase molecules [37]. The interaction of tyrosinase with the condensed tannins from the leaf, fruit, and stem bark of the flamboyant tree was evaluated by measuring the intrinsic fluorescence intensity of the protein before and after the addition of the condensed tannins. The fluorescence emission spectra of tyrosinase were recorded from a range of 300–450 nm while excitation wavelength was set at 290 nm. The tyrosinase has a strong fluorescence emission with a peak at 337 nm and that of the condensed tannins at 319 nm on excitation at 290 nm. Conspicuous reduction of the fluorescence intensity and blue shift of the spectra occurred, with increasing concentration of the condensed tannins (Fig. 6). The results indicated the fact that the condensed tannins interacted with the tryptophan of tyrosinase which implied that the tyrosinase became disagglomerated and its structure get loosened. Hence, we concluded that the tryptophan masking properties of the condensed tannins from the leaf, fruit, and stem bark of the flamboyant tree were contributing to the antityrosinase activities.

Fig. 5 Structure of the monomer unit of the condensed tannins

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Fig. 6 Fluorescence emission spectra of tyrosinase in solutions with different concentration of the condensed tannins from the leaf, fruit, and stem bark of the flamboyant tree. The concentrations of the condensed tannins for curves 1–5 are 0, 0.0375, 0.075, 0.1125, and 0.15 mg/ml, respectively

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Fig. 7 UV-Visible spectrum of the complex formed between the condensed tannins and several concentrations of Cu(II)

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Copper Ion Chelating Ability Tyrosinase has a catalytic center formed by dinuclear copper, which can catalyze the orthohydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinine [38]. Condensed tannins have been reported to chelate metal ions [39]. Thus, the copper-chelating properties of the condensed tannins from different part of the flamboyant tree were investigated. Figure 7 showed the UV–vis spectra of the condensed tannins in the presence of different concentration of CuSO4. The absorbance at 280 nm which represents the existence of the condensed tannins increased distinctly with the increase of CuSO4 concentration, showing that binding to Cu(II) occurred. Therefore, we concluded that the inhibitory activities of the condensed tannins on the tyrosinase might be caused by their binding of dinuclear copper center of tyrosinase.

Conclusions The leaf, fruit, and stem bark of the flamboyant tree were measured to have high contents of TP and ECT. The structures of the condensed tannins were firstly characterized by thiolysis with cysteamine followed by reversed-phase HPLC-ESI-MS, which showed that the leaf, fruit, and stem bark of the flamboyant tree all had afzelechin/epiafzelechin and catechin/epicatechin and that the leaf contained gallocatechin/epigallocatechin as well. The condensed tannins from different parts of the flamboyant tree showed obvious antioxidant and tyrosinase inhibition activities. In conclusion, the condensed tannins from the leaf, fruit, and stem bark of the flamboyant tree might be potential sources of natural tyrosinase inhibitors and antioxidants which could be developed into effective agents for usage in food preservation, agriculture, cosmetic, nutraceutical, and pharmaceutical applications. Acknowledgments The present investigation was supported by the Natural Science Foundation of China (No. 31071611; No. 31070522), by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant No.J1310027/J0106), and by the Science and Technology Foundation of Fujian Province (No. 2010 N5013).

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Isolation and purification of condensed tannins from flamboyant tree and their antioxidant and antityrosinase activities.

Flamboyant tree, a kind of medicinal plant, was studied as a source of condensed tannins. The antioxidant activities of the condensed tannins from the...
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