Bilberry and lingonberry, two shrubs of the Ericaceae family as sources of phenolic secondary metabolites

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General description

Lingonberry, like bilberry, is part of the Ericaceae family, genus Vaccinium. The scientific name of lingonberry is Vaccinium vitis idaea L. and it is also known as cowberry (Andersen, 1985; Pyka et al., 2007; Lee and Finn, 2012). In France and Romania lingonberry is called ―airelle rouge‖ and ―merisor‖, respectively (Blamey and Grey-Wilson, 2003; Fischer, 2000). Lingonberry is native (Figure III-4) to Scandinavia, Europe, Alaska, the U.S., Canadian Pacific Northwest and northeastern Canada (Penhallegon, 2006). In Europe, lingonberry are very popular in Nordic countries, the Baltic states, Germany, Austria, Switzerland, Czech Republic, Poland, Slovenia, Slovakia, Romania, Russia, and Ukraine (http://en.wikipedia.org/wiki/Vaccinium_vitis-idaea). Regarding Romania lingonberry is found throughout the Carpathian mountain chain, mostly in the counties of Suceava, Bistrita-Nasaud, Harghita, Neamt, Brasov, Prahova, Dimbovita, Arges, Sibiu, Vâlcea, Gorj and Hunedoara.
Figure III-4. Distribution of lingonberry in Europe and the Mediterranean region (A) and in North America (B). Source: http://ww2.bgbm.org/EuroPlusMed.
Lingonberry is a small wild shrub with a height of up to 10-30 cm that grows in acidic soils. Lingonberry is a perennial evergreen plant as it keeps its leaves during winter, covering the ground like a carpet. The leaves (Vitis idaea Folium) of the lingonberry are alternate, dark green upper surfaces and light green lower leaf surfaces with black spots on the back. The stem is cylindrical and branched. Campanulate white corollas flowers (Figure III-5A), of 5 to 7 mm long, bloom from May until July. Lingonberry fruits grow in clusters (Figure III-5B) and are dark red in colour, 4 to 10 mm in diameter, with a sour taste due to the arbutin content (Bojor, 2003). Similar to lingonberry is cranberry that refers to the species Vaccinium oxycoccus or the European cranberry, Vaccinium microcarpum or the small cranberry and Vaccinium macrocarpon or the large cranberry, industrially cultivated in North America (Baroffio et al., 2012; Lee and Finn, 2012).

Photo by Oana-Crina Bujor.

Because of its high nutritional value, lingonberries are used as food source being considered as a health promoting natural fruit. The fruits have a variety of applications such as jams, jellies, syrups, purees, sauces, fruit juices, beverage concentrates as well ingredient for chocolates, tarts, cookies, ice cream, cereals and yogurt. Dried fruits are commercially available in whole, powder or ingested in supplemental form (NutriPhy® Bilberry extract of Chr. Hansen SAS, Denmark). Leaves and stems are consumed as infusions and herbal teas.

Chemical composition: phenolic compounds and other constituents

Lingonberry is known as a natural source of food, beverage and dietary supplements due to its richness in nutritional and bioactive compounds. Although lingonberry constituents have multiple biological activities, most of the research has focused on the polyphenolic compounds (Mane et al., 2011; Kylli et al., 2011).
– Phenolic compounds:
The most abundant polyphenols in the fruit of lingonberry (Vaccinium vitis-idaea L.) are glycosides of quercetin, monomers and oligomers of catechin and epicatechin, caffeic acid derivatives and anthocyanins, all known to be powerful antioxidants that act by direct trapping of reactive oxygen species, binding of transition metal ions and inhibition of enzymes involved in the oxidative stress (Lorrain et al., 2012; Goupy et al., 2009; Volf et al., 2014). Many phenolic compounds identified in Vaccinium vitis-idaea L. are presented in Table III-4.
– Other constituents:
Lingonberries contain also carotenoids, terpenoids, fatty acids, vitamins C, A and E, minerals, citric (18.2 g/l), malic (4.2 g/l) and benzoic (0.7 g/l) acids (Viljakainen et al., 2002; Bojor, 2003; Radulović et al., 2010; Seeram, 2008). Benzoic acid is known to contribute to the acidity of the lingonberries as well to prevent the fermentation of lingonberry juice due to its microbiocidal properties (Viljakainen et al., 2002). Among vitamins, niacin (0.6 mg/100 g), vitamin C (7.5 mg/100 g), vitamin A (0.8 µg/100 g), vitamin E (1.5 mg/100 g) and vitamin K (9.0 µg/100 g) are mainly found in fruits (Fineli Database, www.fineli.fi). Lingonberry fruits also contain minerals in small concentrations: calcium – 22 mg/100 g, potassium – 80 mg/100 g, phosphorus – 17 mg/100g and magnesium – 9 mg/100 g.
As carotenoids, Lashmanova et al., (2012) found in the fruits neoxanthin, violaxanthin, antheraxanthin, lutein, zeaxanthin and β-carotene in the concentration range of 6 to 76 mg/100 g dry weight.
Both the leaves and the seeds are rich in oil. Seed oil content is approximately 15% of the dry matter. The oils from seeds and leaves have quite different fatty acid compositions. The seed oil is rich in linoleic (46.4%), α-linolenic (27.3%), oleic (15.1%), palmitic (6.7%), stearic (1.9%) and myristic (2.6% ) acids (Yang et al., 2003), whereas the oil from leaves is rich in α-terpineol (17.0%), pentacosane (6.4%), (E,E)-α-farnesene (4.9%), linalool (4.7%) and (Z)-hex-3-en-1-ol (4.4%) (Radulović et al., 2010). Comparative studies of the triterpenoid content of fruit and leaves of Vaccinium vitis-idaea from Finland and Poland showed the presence of the following compoundsμ α-β-amirin, amirin, betulin, campesterol, cicloartanol, eritrodiol, fern-7-en-3-β-ol, friedelin, lupeol, sitosterol, stigmasterol, stigmasta-3,5-dien-7-one, swert-9 (11)-en-3-ol, β-taraxasterol, bear-12-en-29-al, uvaol, ursolic and oleanolic acids (Szakiel et al., 2012). Ursolic acid was identified as the main triterpenoid in fruits, while sitosterol is the major compound in leaves.

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Extraction and analysis of bilberry and lingonberry phenolic compounds

For the extraction of bilberry and lingonberry phenolics are used methods that are generally applied for the analysis of phenolic compounds and that are presented in chapter II.1.2. Generally the most common extraction methods use methanol (Hokkanen et al., 2009; Ek et al., 2006; Burdulis et al., 2009; Jovančević et al. 2011) or acetone (Zheng and Wang, 2003; Kylli et al., 2011; Jungfer et al., 2012; Lee and Finn, 2012; Liu et al., 2014; ) as extraction agents of bilberry/lingonberry phenolic compounds but in terms of the utilisation for food and cosmetic industry ethanol and water are prefered (Ignat et al., 2011; Denev et al., 2010; Oancea et al., 2012; Ieri et al., 2013; Aaby et al., 2013).

Table of contents :

I. Phenolic compounds as secondary metabolites found in plants and foods 
I.1. Structure and classification
I.1.1. Non-flavonoid polyphenols
I.1.2. Flavonoids
I.2. Methods for extraction and characterisation of phenolic compounds
I.3. Applications of phenolic compounds in different biological systems
I.3.1. Applications of phenolic compounds in the plants development
I.3.1.1. As plants growth bioregulators
I.3.1.2. As amendments in bioremediation
I.3.2. Applications of phenolic compounds in microorganisms development
II. The antioxidant activity of phenolic compounds
II.1. The antioxidant action of phenolic compounds and their mechanisms
II.1.1. Through reducing effects: H-atom and/or electron transfer
II.1.2. Through non-reducing effects
II.1.2.1. Metal-ion chelating activity
II.1.2.2. Inhibition of enzymes implied in the production of ROS
II.1.3. Inhibition of lipid oxidation
II.2. Methods to evaluate the antioxidant activity of phenolic compounds
II.2.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method
II.2.2. Folin–Ciocalteu redox method
II.2.3. Other methods
III. Bilberry and lingonberry, two shrubs of the Ericaceae family as sources of phenolic secondary metabolites
III.1. Bilberry (Vaccinium myrtillus L.)
III.1.1. General description
III.1.2. Chemical composition: phenolic compounds and other constituents
III.2. Lingonberry (Vaccinium vitis-idaea L.)
III.2.1. General description
III.2.2. Chemical composition: phenolic compounds and other constituents
III.3. Extraction and analysis of bilberry and lingonberry phenolic compounds
III.4. Health benefits of bilberry and lingonberry
III.4.1. Cardioprotective activity
III.4.2. Anti-cancer activity
III.4.3. Antidiabetic activity
III.4.4. Vision improvement activity
III.4.5. Bacterial anti-adhesion activity
III.5. Other applications of bilberry and lingonberry extracts
PERSONAL CONTRIBUTION
Chapter I. PHENOLIC CONSTITUENTS IN BILBERRY (VACCINIUM MYRTILLUS L.): ACCUMULATION IN LEAVES, STEMS AND FRUITS AT DIFFERENT HARVEST PERIODS AND ANTIOXIDANT ACTIVITY 
1. Background
2. Methodology
2.1 Materials
2.1.1. Bilberry samples
2.1.2. Chemicals and solvents
2.2. Extraction of phenolic compounds
2.3. Qualitative and quantitative analyses of phenolic compounds
2.3.1. Identification of phenolic compounds by UPLC/MS
2.3.2. Quantification of phenolic compounds
2.4. Analysis of procyanidins using thioacidolysis
2.4.1. Freeze-dried extracts
2.4.1.1. HPLC analysis without thiolysis
2.4.1.2. HPLC analysis after thiolysis
2.4.2. Freeze-dried fruits
2.4.2.1. HPLC analysis without thiolysis
2.4.2.2. HPLC analysis with thiolysis
2.5. Antioxidant activity by applying spectrophotometric methods
2.5.1. Total Phenolic Contents by the Folin Ciocalteu method
2.5.2. DPPH (2,2-diphenyl- 1-picrylhydrazyl) radical scavenging test
2.6. Statistical analyses
3. Results and discussion
3.1. Optimal extraction conditions: the preliminary test
3.2. Phenolic profile and content of bilberry extracts
3.2.1. Caffeic acid derivatives
3.2.2. Coumaric acid derivatives
3.2.3. Flavonol glycosides
3.2.4. Flavanols
3.2.5. Anthocyanins

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