Antioxidant activity of C-Glycosidic ellagitannins from the seeds and peel of camu-camu (Myrciaria dubia)

https://doi.org/10.1016/j.lwt.2016.01.024Get rights and content

Highlights

  • Six C-glycosidic ellagitannins were isolated from the seeds and peel of camu-camu.

  • These tannins exhibited strong antioxidant activities in the DPPH and ABTS assays.

  • The activities may depend on the orientation of the C-1 hydroxyl group.

  • The activities in the ORAC assay could not be explained by these structural features.

  • Camu-camu seeds and peel could be a useful resource for functional foods.

Abstract

C-Glycosidic ellagitannins grandinin (1), vescalagin (2), castalagin (3), methylvescalagin (4), stachyurin (5), and casuarinin (6) were isolated from the 50% acetone extract of camu-camu (Myrciaria dubia) seeds and peel. These tannins exhibited antioxidant activities measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, and the oxygen radical absorbance capacity (ORAC) assay. In the DPPH and ABTS assays, stachyurin exhibited the strongest antioxidant activity among the tannins, and the activities were stronger for tannins containing two hexahydroxydiphenoyl (HHDP) groups and a galloyl group than for tannins containing a nonahydroxyterphenoyl group and a HHDP group. The activity of vescalagin was stronger than that of castalagin, and a similar relationship was observed for stachyurin and casuarinin. Thus, the antioxidant activities of these tannins may depend on the conformation of the hydroxyl group at C-1 of the open ring d-glucose. However, in the ORAC assay, casuarinin exhibited the strongest activity, and the respective activities could not be explained by the structural rigidity or conformation of the C-1 hydroxyl group.

The seeds and peel of camu-camu, waste products of juice production, were found to contain C-glycosidic ellagitannins with potent antioxidant activities, and thus, they could be a useful resource for functional foods and food additives.

Introduction

The biological activities of polyphenols have attracted attention as they have proven to be effective in the prevention of lifestyle-related diseases and in the maintenance of human health. Among polyphenols, flavonols, isoflavones, flavan-3-ols, and anthocyanidins have been extensively studied, and several of them have been utilized in a variety of functional foods (Del Rio et al., 2013). Tannins are categorized as polyphenols as they contain many phenolic hydroxyl groups in their structures. Therefore, the number of reports on the biological activities of tannins has steadily increased as researchers have focused their attention on relationships between biological activity and the structures of tannins.

Tannins are classified into two categories, condensed tannins and hydrolysable tannins. Condensed tannins are comprised of flavan-3-ols, and hydrolysable tannins are esters of polyols (mostly d-glucose) and phenolic carboxylic acids such as gallic acid, hexahydroxydiphenoic acid, valoneic acid, and nonahydroxyterphenoic acid. A variety of biological activities of hydrolysable tannins have been reported including cytotoxicity (Del Rio et al., 2013), inhibition of enzymes (Ochir et al., 2010), and antimicrobial activities (Kamijo, Kanazawa, Funaki, Nishizawa, & Yamagishi, 2008). Among hydrolysable tannins, C-glycosidic ellagitannins have structures characterized by a C–C bond between the anomeric carbon of an open ring sugar and the unsubstituted carbon of a hexahydroxydiphenoyl (HHDP) or nonahydroxyterphenoyl (NHTP) group. Recently, C-glycosidic ellagitannins were demonstrated to be sensory-active non-volatiles that migrate from oak barrels into alcoholic beverages such as whisky, brandy, and wine (Glabasnia & Hofmann, 2006). Moreover, biological activities of C-glycosidic ellagitannins including anti-herpes virus activity (Quideau et al., 2004), alleviation of insulin resistance, inhibition of adipocyte differentiation (Chang and Shen, 2013, Chang et al., 2013), inhibition of a human breast cancer cell line growth (MCF-7) and colon cancer cell line growth (Caco-2 and HT-29) (Fernandes et al., 2009, Fridrich et al., 2008), and inhibition of human DNA topoisomerase II (Auzanneau et al., 2012) have been reported.

We have been studying camu-camu (Myrciaria dubia) fruit, a tropical fruit from Peru, because its juice is of interest in Japan and other developed countries as an ingredient of functional foods. Because the fruits are not easy to keep fresh, the juice was processed in Peru, and the residual seeds and peel became an industrial waste. Therefore, utilization of the residual seeds and peel would be beneficial for the camu-camu industry. We have previously reported that the crude extract of camu-camu seeds and peel contains large amounts of polyphenols (seeds: 369.4 mg/g, peel: 203.8 mg/g); (Myoda et al., 2010) approximately four and ten times the amounts from the residues of acerola and passion fruit juice production, respectively (de Oliveira et al., 2009). The extract of camu-camu seeds and peel showed potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 32.2 μg/mL), and C-glycosidic ellagitannins, vescalagin (2) and castalagin (3) were shown to be responsible for the DPPH radical scavenging activity (Kaneshima, Myoda, Toeda, Fujimori, & Nishizawa, 2013). In this paper, we report the isolation and characterization of four additional C-glycosidic ellagitannins and the relationship between their antioxidant activities and structures.

Section snippets

Materials and chemicals

The dried powder of the seeds and peel from camu-camu juice production was obtained from Empresa Agroindustrial del Peru S.A. (Peru). These samples were used after drying at room temperature. Fluorescein sodium salt and (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) were purchased from Sigma–Aldrich Co. (St. Louis, USA). 2,2′-Azobis (S-methylpropionamidine) dihydrochloride (AAPH) and ascorbic acid were purchased from Wako Pure Chemical (Osaka, Japan). Gallic acid,

Isolation and characterization of C-glycosidic ellagitannins

The crude extract of camu-camu seeds or peel was fractionated by chromatography on Sephadex LH-20, and following purification by preparative reverse-phase HPLC resulted in the isolation of grandinin (1), methylvescalagin (4), stachyurin (5), and casuarinin (6) along with the previously reported vescalagin (2) and castalagin (3). Vescalagin (2) and castalagin (3) were found to be the main C-glycosidic ellagitannins, approximately 5% and 10% of the total polyphenols, respectively, as shown in

Conclusion

Six C-glycosidic ellagitannins (compounds 1, 2, 3, 4, 5, and 6) were isolated from the seeds and peel of camu-camu (Myrciaria dubia), and this study represents the first time these tannins have been isolated from fruit seeds and peel. These tannins (compounds 2, 3, 5 and 6) exhibited stronger antioxidant activities measured by both single electron transfer assays and a hydrogen atom transfer assay than gallic acid and ascorbic acid. The peel and seeds of camu-camu are industrial waste products

Acknowledgment

The authors wish to express their gratitude to Empresa Agroindustrial del Peru S.A. for the donation of camu-camu samples, and to Professor Koji Wada of the Hokkaido Pharmaceutical University School of Pharmacy.

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