Occurrence of Rutin and Chlorogenic Acid in Elderberry Leaf, Flower, and Stem in Response to Genotype, Environment, and Season

The American elderberry (Sambucus nigra subsp. canadensis) is being increasingly consumed as a nutraceutical. In Europe, tea made from flowers of the elderberry subspecies nigra is a popular herbal treatment for respiratory infections such as colds and influenza, but little is known about the medicinal attributes of the subspecies canadensis. Potentially-active compounds in elderberry include phenolics such as rutin, chlorogenic acid, and quercetin. Genetic, environmental and seasonal influences on the concentration of such metabolites in the fruit and non-fruit tissues of American elderberry are unknown. The objective of this study was to quantify the production of medicinal compounds in various non-fruit elderberry tissues in response to genotype, environment and season. In 2003, an experiment was established at Mt. Vernon and Mountain Grove, Missouri, and Corvallis, Oregon (USA) to evaluate the performance of 10 new elderberry selections compared with two older standard cultivars. In 2005, tissue samples were collected from each site in early June (at peak anthesis) and late July (at initiation of fruit ripening). Representative samples of leaf, flower, new green stem, and year-old woody stem were collected from all plots. Dried samples were analyzed by HPLC for the phenolic compounds chlorogenic acid and rutin. Mean levels of chlorogenic acid were 3,367 mg kg in leaf, 2,064 mg kg in flower, 584 mg kg in green stem, and 1,111 mg kg in woody stem, whereas mean levels of rutin were 6,746 mg kg in leaf, 5,546 mg kg in flower, 187 mg kg in green stem, and 44 mg kg in woody stem. Levels of chlorogenic acid and rutin generally varied among the 12 cultivars in leaf tissues only, with the cultivar ‘Johns’ showing very high levels of both compounds. Significant, but somewhat inconclusive, differences in rutin and chlorogenic acid levels were detected among the three locations; the two Missouri sites tended to have higher levels of rutin. In both leaf and green stem tissues, higher phytochemical levels were detected within tissues harvested earlier in the season. Such genotypic, environmental, and seasonal variations in occurrence of these two representative compounds point to the possibility of focused agricultural production of specific phytochemicals. Proc. XXVII IHC Plants as Food and Medicine Eds.-in-Chief: G. Gardner and L.E. Craker Acta Hort. 765, ISHS 2008 198 INTRODUCTION The American elderberry (Sambucus nigra L. subsp. canadensis R. Bolli) is an attractive shrub native to central and eastern North America that produces edible flowers and berries. The fruit and flowers have traditionally been used to make jams, jellies, syrups and wines on a non-commercial scale. Commercial production of high-quality wines in the USA is now rapidly developing, but probably the fastest-growing market for elderberry products is fruit taken as a nutraceutical in the form of a juice concentrate or a processed dietary supplement. A Kansas (USA) winery now sells over $500,000 worth of non-fermented juice concentrate as a health tonic annually (J. Brewer, pers. comm.). American elderberry is one of six subspecies of S. nigra (Bolli, 1994); the European subspecies nigra is a very popular herbal treatment for upper respiratory infections such as colds and influenza. In European herbal tradition, elder flower tea is most commonly used; however, elderberry fruit syrups such as Sambucol have become popular in recent years and have demonstrated efficacy against influenza in clinical trials (Zakay-Rones et al., 1995, 2004). The medicinal properties of elderberry products may be attributed to direct antiviral activity (Zakay-Rones et al., 1995) and to immunostimulant activity resulting in increased production of inflammatory and anti-inflammatory cytokines (Yesilada et al., 1997; Barak et al., 2002). Potential active compounds include several anthocyanins, some novel (Johansen et al., 1991; Nakatani et al., 1995), as well as common flavonoids including quercetin and rutin (Kolodynska and Pasieczna, 1967). However, elderberry is chemically complex and other compound classes, including sterols, lectins and triterpenoids, might be partially responsible for bioactivity (e.g., Gray et al., 2000). In investigating the plant’s traditional use as a treatment for diabetes, Gray et al. (2000) discovered that insulin-like and insulin-releasing bioactivities could not be attributed to a known constituent and were associated with two solvent fractions, therefore resulting from the activities of multiple compounds. While the demand for elderberry products is thus increasing, very few elderberries are presently under managed cultivation in North America. Only a handful of minor studies (Ritter, 1958; Ritter and McKee, 1964; Hill, 1969; Skirvin and Otterbacher, 1977; Craig, 1978; Way, 1981) provide horticultural information to potential producers of American elderberry. Even less is known about genetic, environmental or seasonal influences on the production or concentration of putatively active elderberry metabolites, particularly in the non-fruit tissues. While some elderberry anthocyanins have been studied and quantified in multiple samples (e.g., Kaack and Austed, 1998; Chandra et al., 2001; Wu et al., 2004), none has been sampled from a true replicated genotype by environment planting. The objective of this study, therefore, was to quantify the occurrence of two representative medicinal compounds, rutin and chlorogenic acid, in various elderberry tissues in response to genotype, environment and season of harvest. Both of these compounds, which are present in a wide variety of distantly-related angiosperm lineages, have antioxidant and antimicrobial activities (e.g., Basile et al., 2000; Grace and Logan, 2000; van der Watt and Pretorius, 2001; Zhu et al., 2004; Cushnie and Lamb, 2005). Suites of flavonoids act synergistically; rutin in particular has been found to potentiate the activities of other related and unrelated compounds, even in assays in which it is not itself particularly potent (e.g., Arima et al., 2002; Noldner and Schotz, 2002; Grassmann, 2005). Chlorogenic acid has antiviral activity, notably against adenoviruses, which are among the causes of the common cold (Chiang et al., 2002), and has additionally shown some cancer-preventive activity in rodent studies (Conney et al., 1991; Mori et al., 2000). MATERIALS AND METHODS Field In 2003, an experiment was established at two locations in southern Missouri, USA and a third location in northwest Oregon, USA to evaluate the horticultural and phenological performance of 12 elderberry cultivars growing in different environments.