Antioxidative Carbohydrate Polymer from Enhydra fluctuans and Its Interaction with Bovine Serum Albumin Debjani Ghosh, † Sayani Ray, † Kanika Ghosh, † Vale ́ rie Micard, ‡ Udipta R. Chatterjee, † Pradyot K. Ghosal, † and Bimalendu Ray* ,† † Natural Products Laboratory, Department of Chemistry, The University of Burdwan, West Bengal 713 104, India ‡ MontpellierSupAgro-INRA-UMII-CIRAD, UMR IATE, 2, Place Pierre Viala, 34060 Montpellier Cedex 01, France ABSTRACT: Enhydra fluctuans leaves are traditionally sold on Indian markets for various health benefits. However, no phytochemical study on its high molecular weight compound has so far been performed. Chemical, chromatographic, ESI- TOF-MS, and NMR analyses of the water extracted carbohydrate polymer (CP) of E. fluctuans leaves showed the presence of a 24 kDa arabinogalactan having a (1,3)-linked β- D-Galp main chain, substituted at O-6 by (1,6)-linked β-D-Galp side chains. The latter residues were substituted at O-3 by (1,3)-, (1,5)-, and (1,3,5)-linked α-L-Araf chains, and nonreducing end-units of α-L-Araf and β-D-Galp. This polymer contained esterified phenolic acids. Biochemical analysis revealed similarity in antioxidative potential between the identified carbohydrate polymer and known standard antioxidants. The highly branched side chains and the phenolic acid residues of the arabinogalactan might be the functional sites. Fluorimetric and ultraviolet spectrometric analyses showed that the studied carbohydrate polymer can form complex with bovine serum albumin having binding constant K = 2.42 × 10 6 /M and changes its microenvironment. Thus, traditional aqueous extraction method provides a carbohydrate polymer, which stimulates a fair biological response: this could represent an interesting approach in phytotherapeutic treatments. ■ INTRODUCTION The free radical theory of aging, conceived in 1956, has turned 57 and is rapidly attracting the interest of the mainstream of biochemical research. 1,2 Reactive oxygen species (ROS) are recently evidenced to be closely linked to degenerative diseases such as Alzheimer’s disease, neuronal death including ischemic and hemorrhagic stroke, and acute and chronic degenerative cardiac myocyte death. 2-5 Many drugs are proven to be successful in the management of these diseases, but their use is often limited because of toxic side-effects, and the development of drug resistance. Thus, there is a need to develop new compounds with strong antioxidative effects accompanied by favorable pharmacological and clinical properties. Notably, numerous drugs and drug precursors in the current pharmacopoeia originate from plant sources. 6 Enhydra fluctuans (Asteraceae), a marshy herb, is used for the treatment of various diseases throughout Indian subcontinent since ancient times. 7,8 Therefore, researchers have tested biological activities of extracts and pure compounds from different parts of this herb. Various extracts of E. fluctuans possess a range of pharmacological effects, such as anticancer, antidiarrheal, antifungal, antimicrobial, antioxidative, and analgesic, supporting its use in traditional medicine. 9-14 Up to this time, only secondary metabolites, namely, isoflavone glycoside, 14 sesquiterpene lactones, 15-17 diterpenoids, 18 ster- ols, 19 and gibberellins, 20 have been chemically characterized from this herb. Many polysaccharides stimulate a range of biological effects, 21-29 but no report exists on the carbohydrate polymer from E. fluctuans. Complex formation between macromolecules such as anionic polysaccharides and proteins finds their application in a diversity of fields including pharmacy. 30 The formed complexes can be either soluble or insoluble. The development of complexes retaining water solubility and stability over a wide pH range should be of interest as it would enlarge their potential applications. We report herein the isolation, purification and structural analysis of an arabinogalactan from E. fluctuans leaves. Also, we have evaluated the antioxidative activity of this polymer using the free radical scavenging and the ferric reducing capacity measurement. In addition, the interaction between bovine serum albumin (BSA) and carbohydrate polymer (CP) has been studied for elucidating the influence of pH, concentration of CP, and charge density of BSA, on complex formation. UV spectrometric and fluorescence quenching studies show that the formation of water-soluble protein-polysaccharide complexes is favorable particularly when the protein molecules are positively charged (pH < isoelectric point, pI). Received: January 27, 2013 Revised: May 1, 2013 Article pubs.acs.org/Biomac © XXXX American Chemical Society A dx.doi.org/10.1021/bm4001316 | Biomacromolecules XXXX, XXX, XXX-XXX