Biophenols and antioxidant activity in wild and cultivated heather

Abstract Over the last decade, there has been an increasing interest to study the antioxidants from natural sources which can be applied to replace the synthetic compounds commonly used in food or as cosmetic ingredients. Calluna vulgaris (L.) Hull (heather), a plant grown in most parts of Europe and Northern America, has been used in ethnopharmacology. The content of selected biophenols and the antioxidant capacity of the extracts of wild heather collected from natural environmental localities of central Poland were assessed in this study and compared with cultivated plant. Chlorogenic acid was the major biophenolic compound present in the extracts, followed by a high amount of catechins. The reducing power of the extracts evaluated by Folin–Ciocalteau assay was in the range of 75.7–89.1 mg GA/g dry flowers. The cultivated plant extract showed the largest radical scavenging on 1,1-diphenyl-2-pirylhydrazyl (DPPH) radicals.


Introduction
Calluna vulgaris L. Hull (heather), a member of the Ericaceae family, can be found in most parts of Europe and Northern America. The flowers emerge in late summer and wild species are usually in purple or mauve shades. Garden cultivars come in colours ranging from white, through pink and a wide range of purples. The existence of several groups of natural chemicals has been identified in heather (Szakiel et al. 2013). The plant material is a part of traditional folk medicine for treating urinary tract disturbances and inflammatory related disorders (Monschein et al. 2010). Pharmacological studies showed antiseptic, antioxidant and MAO-A inhibitory effects of that plant extracts (Hooper & Cassidy 2006;Deliorman-Orhan et al. 2009;Saaby et al. 2009).
The aim of this work was to determine the content of selected biophenols as well as the antioxidant capacity of the extracts of wild heather collected from natural environmental localities of central Poland and to compare them with cultivated plant. We selected a limited area, the central part of Poland, in order to have uniform climatic conditions as well as altitude. The review of literature revealed very little data on antioxidant properties of different C. vulgaris flowers extracts (Deliorman-Orhan et al. 2009).

Results and discussion
The ethanol-water mixture (60:40 v/v) was used for biophenols isolation from heather flowers. The presence of water is helpful to enhance swelling of plant material, which is favourable to increase the contact surface area between the plant matrix and the solvent, resulting in increase of the extraction yield (Hemwimon et al. 2007). The data from the HPLC analysis are presented in Table 1 and Figure 1 presents the extracted ion chromatogram of main flavonoids in the extract of greenhouse heather (sample S4).
Chlorogenic acid was the major biophenolic compound present in the extracts, which is in accordance with the previous studies (Deliorman-Orhan et al. 2009;Dias et al. 2011). The influence of the wild plant collection locality on this hydroxycinnamic acid content was significant (Table 1). Ferulic acid was found at the similar level in all samples. The presence of tricetin (5,7,3′,4′,5′-pentahydroxy flavone) with potent anti-inflammatory and anticancer activities (Hsu et al. 2010) was not reported yet. The extracts of C. vulgaris plant, either cultivated or wild, particularly sample S1, contained also a high amount of catechins (flavan-3ols) with strong antioxidant activity and numerous potentially beneficial effects on living organisms (Lambert & Elias 2010).
The results of total flavonoid contents in tested extracts of C. vulgaris flowers, based on aluminium complex formation, are presented in Table 2. The highest value was obtained for sample S1 (wild plant) and the garden cultivar (sample S4). Both these samples exhibited the highest content of biophenols (Table 1). The total reducing capacity of examined extracts was evaluated by Folin-Ciocalteu (FC) method, which is very often called 'total phenolic content' . However, the FC reagent could simultaneously oxidise several non-phenolic organic compounds as well as some inorganic substances to give elevated apparent phenolic content (Everette et al. 2010). The reducing power of C. vulgaris extracts was in the range of 75.7-89.1 mg GA/g dry flowers ( Table 2). The highest value was obtained for cultivated garden plant which probably, except determined biophenols, contains also other compounds with reducing properties. Deliorman-Orhan et al. (2009) reported that the ethyl acetate extract of aerial part of heather collected in northeaster part of Turkey gave the highest results in FC assay (29.7 ± 0.1 mg/g of fraction), followed by the n-butanol (15.9 ± 0.1 mg/g), chloroform (6.7 ± 0.1 mg/g) and water (5.8 ± 0.1 mg/g).
The antioxidant capacity of the studied extracts was also assessed on the basis of their scavenging effect on the stable 1,1-diphenyl-2-pirylhydrazyl (DPPH) radical. The scavenging effect on DPPH radicals measured after 30 min of reaction, expressed as trolox equivalent, increased in the order: sample S3 < S1 < S2 < S4 (Table 2). Thus, the cultivated plant showed the largest radical scavenging capacity among the C. vulgaris extracts. The extracts of this plant exhibited also the highest reducing power evaluated by FC assay, although the total  flavonoid content as well as the concentration of the individual biophenols was lower than in other studied samples. The leaves of Laurus nobilis L. (Conforti et al. 2006) and Myrtus communis L. fruits (Uzun & Bayir 2011) from wild plants also showed greater radical scavenging activity and reducing power (measured in FC assay) than those from cultivated plants.
In summary, our results showed that C. vulgaris extracts contained high content of biophenols and in vitro antioxidant activities with potential pharmaceutical and cosmetics applications.