T: Toxicology & Chemical Food Safety Sea Buckthorn (Hippophae rhamnoides) Proanthocyanidins Inhibit In Vitro Enzymatic Hydrolysis of Protein Ranjith Arimboor and C. Arumughan Abstract: Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 μg, respectively. Relative enzymatic hydrolysis of the protein-proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty. Keywords: average degree of polymerization, Hippophae rhamnoides, pepsin, proanthocyanidins, trypsin Introduction Sea buckthorn (SB) (Hippophae rhamnoides) is a temperate, thorny, nitrogen fixing, and deciduous shrub grows in Central Asia and Europe. This plant has a long history of applications in Tibetan, Mongolian, and Ayurveda systems of medicines. A wide spectrum of physiological activities including antioxidant (Fan and others 2007), cytoprotective, immunomodulatory (Geetha and others 2002), and cardioprotective (Johansson and others 2000) efficacies are attributed to SB berries. SB berries consists of a fairly tough skin and juicy pulp enveloping a small and hard, oval seed. SB berries are excellent source of bioactive phytochemicals such as carotenoids, tocopherols, sterols, vitamin C, organic acids, and polyphenols (Arimboor and others 2006; Ranjith and others 2006). A wide array of phenolic compounds coming under the classes of flavanols, flavones, phenolic acids, and tannins are re- ported in SB berries and leaves (Rosch and others 2004a, 2004b; Arimboor and others 2006, 2008; Fan and others 2007). Some low molecular weight proanthocyanidins (PA) with significant an- tioxidant efficacies are reported in SB berries (Rosch and others 2004a, 2004b; Fan and others 2007). Tannins, the secondary plant metabolites formed by the con- densation of simple phenolics with diverse biological activities are the least studied class of phenolics in SB. Tannins in common foods including fruits, wines, vegetables, and cereals affect their texture, color, and taste and render beneficial and adverse nutri- MS 20101071 Submitted 9/22/2010, Accepted 4/13/2011. Authors are with Agroprocessing and Natural Products Division, Natl. Inst. for Interdisciplinary Sci- ence and Technology (CSIR), Thiruvananthapuram, Kerala, India, 695019. Direct inquiries to author Arumughan (E-mail: carumughan@yahoo.com). tional effects. Oligomeric and polymeric flavon-3-ols constituted a major subclass of tannins called condensed tannins or PA. Plant PA possesses a variety of physiological activities and greater parts of these activities are governed by their chemical structure and degree of polymerization (DP). DP of PA has been reported to modulate its antioxidant (Plumb and others 1998) antidiabetic (Lee and others 2007), and antitumor (Schmidt and others 2004) prop- erties. Vidal and others (2003) demonstrated that Astringency of PA increased with DP and galloylation. Intestinal absorption stud- ies show that bioavailability of PA decreased with the increase in their size. Holt and others (2002) reported the absorption of procyanindin dimmers in human substances. Apple procyanidin oligomers up to pentamers are absorbed in the rat (Shoji and others 2006). In an in vitro study, it has been shown that pro- cyanidin oligomers (dimers and trimers) passed through the hu- man epithelial Caco-2 cell monolayer, whereas polymers did not (Deprez and others 2001). Phenolics form soluble and insoluble complexes with proteins that exert both beneficial and detrimen- tal biological effects. Its beneficial and adverse effects on diges- tion and protein bioavailability are reported by many researchers (Butler and others 1982; Robbins and others 1987; Hanley and others 1992). Protein polyphenol complexes are demonstrated as effective antioxidants in digestive tract (Riedi and Hangerman 2001). Higher molecular weight PA has higher affinity towards proteins (Asquith and Butler 1986; Artz and others 1987). SB berries and leaves are extensively used in food and medicinal applications and leaves also serve as a major source of cattle feed in SB growing areas. Phenolics, which are present in high proportions in SB berries and leaves may interact with other food components and enzymes. Studies on SB phenolics on this perspective have not been conducted. Therefore, the main aim of this study was to evaluate the ability of SB PAs to precipitate proteins and to C  2011 Institute of Food Technologists R  T130 Journal of Food Science  Vol. 76, Nr. 6, 2011 doi: 10.1111/j.1750-3841.2011.02238.x Further reproduction without permission is prohibited