Unusual Immuno-Modulatory Triterpene-Caffeates in the Skins of Russeted Varieties of Apples and Pears Christelle M. Andre, †,⊥ Lesley Larsen, ‡,⊥ Elaine J. Burgess, ‡,⊥ Dwayne J. Jensen, §,⊥ Janine M. Cooney, §,⊥ Danie ̀ le Evers, ∥ Jingli Zhang, † Nigel B. Perry, ‡,⊥ and William A. Laing* ,†,⊥ † The New Zealand Institute for Plant & Food Research Limited, Mt Albert Research Centre, Private Bag 92 169, Auckland 1142, New Zealand ‡ The New Zealand Institute for Plant & Food Research Limited, and Chemistry Department, Otago University, Dunedin, New Zealand § The New Zealand Institute for Plant & Food Research Limited, Ruakura, Hamilton 3240, New Zealand ∥ Department “Environment and Agro-biotechnologies”, Centre de Recherche Public-Gabriel Lippmann, 41, rue du Brill, L-4422 Belvaux, Luxembourg ⊥ Nutrigenomics, Ruakura, Hamilton 3240, New Zealand ABSTRACT: Three triterpene-caffeates have been isolated from skins of a russeted apple cultivar “Merton Russet” and identified by LC−MS and NMR as betulinic acid-3-cis-caffeate, betulinic acid-3-trans-caffeate, and oleanolic acid-3-trans-caffeate. Betulinic acid-3-trans-caffeate and oleanolic acid-3-trans-caffeate were also found in russeted pear skins. These compounds have not been previously reported in apples or pears, or in any other foods. Their presence was related to suberized tissue as they were only found in russet portions of the partially russeted apple cultivar “Cox’s Orange Pippin” and were not detected in the waxy apple cultivar “Royal Gala”. High concentrations of betulinic acid-3-trans-caffeate were found in the bark of both “Merton Russet” and “Royal Gala” trees. The three triterpene-caffeates showed anti-inflammatory activity in vitro, inhibiting NF-κB activation with IC 50 ’s of 6−9 μM. Betulinic acid-3-trans-caffeate, the predominant compound in the apples, was immuno-modulatory at around 10 μM in the in vitro and ex vivo bioassays, boosting production of the pro-inflammatory cytokine TNFα in cells stimulated with bacterial lipopolysaccharides. KEYWORDS: apple, pear, russet, triterpene, betulinic acid, betulinic acid-3-O-caffeate, inflammation, NF-κB, TNFα ■ INTRODUCTION Apple (Malus X domestica Borkh.) is one of the most popular fruits around the world, 1 and epidemiological studies have linked the consumption of apples with reduced risk of various diseases. 2 Apple skin is known to contain large amounts of triterpenes (up to 60 mg per apple). 3 Although these triterpenes occur in apples at concentrations similar to those of phenolic compounds, 3 their likely contribution to the health benefits associated with apple consumption is often ignored or underestimated. Pentacyclic triterpenes have been associated with numerous pharmacological properties, including modu- lation of inflammation and anticancer effects in vitro and in vivo. 4−7 The potential of some triterpenes as therapeutic anticancer agents is currently being evaluated in clinical trials. 5,6 Triterpenes are terpenoids produced in plants by cyclization of 2,3-oxidosqualene, 8 with approximately 20 000 triterpenes identified to date. The ursane (ursolic acid, uvaol, α-amyrin), oleanane (oleanolic acid, erythrodiol, β-amyrin) and lupane (betulinic acid, betulin, lupeol) series of pentacyclic triterpenes derived from α-amyrin, β-amyrin, and lupeol, respectively, are the most widely distributed pentacyclic triterpenes in plants. 9 They occur particularly in the waxy coating of leaves and fruits, along with long chain fatty acids and additional secondary metabolites, such as sterols and phenolic compounds. 10 Triterpenes are included in the epicuticular and intracuticular wax (embedded in cutin polymer) of the plant cuticle. 10 Apple skin with its cuticle plays an important functional role in the protection of the fruit, acting as the interface with the external environment. The waxy cuticle protects the plant from water loss and bacterial and fungal attacks 10−12 and provides a physical and chemical barrier against pests. 13 The skin of apples from most cultivars is composed of several layers including an outer cuticular wax layer and one or more underlying layers of epidermal cells. 14 However, some old heritage cultivars such as “Merton Russet” or “Egremont Russet” present a russeted skin (pear-type skin), characterized by a thinner wax layer and the presence of cork (suberin) in epidermal cells as a reaction to the death of epidermal tissue. 14,15 Although it appears naturally on those varieties, russeting is seen as a defect in most commercial cultivars. It results in significant economic losses for the growers, as russeted fruit is downgraded during packing and marketing because consumers prefer smooth- or waxy- skinned apples. In our previous study on the triterpene concentrations (ursolic, oleanolic, and betulinic acid) in 109 apple cultivars, 3 we noted that all of the russeted cultivars had a different triterpene pattern from their waxy counterparts, characterized Received: December 4, 2012 Revised: February 13, 2013 Accepted: February 19, 2013 Published: February 19, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 2773 dx.doi.org/10.1021/jf305190e | J. Agric. Food Chem. 2013, 61, 2773−2779