Dried Bilberry fruit – Myrtilli fructus siccus (Vaccinium myrtillus L.)

Latin name of the genus: Myrtilli fructus siccus
Latin name of herbal substance: Vaccinium myrtillus l.
Botanical name of plant: Herbalref.com
English common name of herbal substance: Dried bilberry fruit

Latin name of the genus: Myrtilli fructus siccus
Botanical name of plant: Vaccinium myrtillus L.
English common name of herbal substance: Dried Bilberry fruit

Table of Contents

Introduction

1.1. Description of the herbal substance(s), herbal preparation(s) or combinations thereof

Herbal substances

Vaccinium myrtillus (L.), fructus siccus (bilberry fruit, dried): dried ripe fruit of V. myrtillus (L.) (Ph. Eur. 8.0, 2008:1588). It contains minimum 1.0% of tannins expressed as pyrogallol.

Vaccinium myrtillus (L.), fructus recens (bilberry fruit, fresh): fresh or frozen ripe fruit of V. myrtillus (L.) (Ph. Eur. 8.0, 2008:1602). It contains minimum 0.30% of antocyanins, expressed as cyanidin 3- O-glucoside chloride (chrysanthemin) (dried drug).

Bilberry (V. myrtillus L.) is a species of a shrubby perennial plant of the heather family (Ericaceae) reaching from 15 to 60 cm in height. It has many common names, including blueberry. It is widespread in Asia, Europe and North America in the areas with a temperate and arctic climate. The flowers, blooming from April to June, are pollinated by insects. The time from pollination to full ripeness of the fruit is about two months. The plant occurs from lowlands to high mountain positions (even above the tree line) and plant prefers strongly acidic soil in the pine forests, other coniferous forests, oak woods, beech forests and moors. The fruits are the black berries with a bluish, waxy bloom (Frohne, 2006) with sweet and slightly astringent taste (European Pharmacopoeia 8.0, 2008:1588 and 1602).

Dried fruits are traditionally used in therapy of digestive disorders, particularly in diarrhoea. The traditional use and some not controlled studies from 1960 and 1970 suggested the potential benefits of bilberry preparations for improvement of night vision, but more recent well-designed studies did not show any advantage (Canter and Ernst 2004).

The following substances were found in bilberry fruits:

Polyphenols

Flavan-3-ols and Proanthocyanidins

The amount is oppositely correlated with the degree of fruit ripening. Epicatechin-dimer B-2, catechin- dimer B-3, catechin-epicatechin-dimer B-1, catechin-epicatechin-dimer B-4, and other unidentified dimers or oligomers of procyanidins were found (Hansel et al., 1994).

Anthocyanosides

Anthocyanic compounds are present in plant cells in the form of glycosides (anthocyanins). There are about 400 known anthocyanic glycosides. The most important anthocyanins are the cyanidin glycosides, as they represent 50% of the pigment composition of fruits (Kong et al., 2003). Anthocyanins are present in ripe fruits of Vaccinium species but the highest total anthocyanin content occurs in the bilberry (V. myrtillus L.) (Kalt et al., 1999).

Total anthocyanin amount ranges from 300 to 700 mg per 100 g in ripe fruits of V. myrtillus (Prior et al., 1998; Prior and Cao 2000). There is a great diversity of the total content of anthocyanins in bilberry collected in various geographical areas, from 19.3 to 38.7 mg/g dry weight (Lätti et al., 2008). Moreover, in concentrated bilberry extracts the total content of anthocyanins may amount to 24%

(Zhang et al., 2004). According to the European Pharmacopoeia 8.0 the standardized dry extract of V. myrtillus contains 32.4% to 39.6% of anthocyanins, expressed as cyanidin 3-O-glucoside chloride.

Most of the researchers investigating anthocyanin composition of bilberries have reported mainly 14 or 15 anthocyanins (Lätti et al., 2008; Yue and Xu, 2008). Fifteen anthocyanins have been identified in bilberry fruit, juice, and extract (1 – delphinidin 3-galactoside; 2 – delphinidin 3-glucoside; 3 – cyanidin 3-galactoside; 4 – delphinidin-3-arabinoside; 5 – cyanidin 3-glucoside; 6 – petunidin 3- galactoside; 7 – cyanidin 3-arabinoside; 8 – petunidin 3-glucoside; 9 – peonidin 3-galactoside; 10 – petunidin 3-arabinoside; 11 – peonidin 3-glucoside; 12 – malvidin 3-galactoside; 13 –peonidin 3 arabinoside; 14 – malvidin 3-glucoside; 15 – malvidin 3 – arabinoside).

During ripening process, there is an increase in the quantity of anthocyanidins in fruits. Usually the highest content of anthocyanins is found in berries collected late summer, at the end of August and the beginning of September. The levels are approximately 1.5-fold those of July. Late harvest, collection and location have an impact on anthocyanin content (Hansel et al., 1994; Morazzoni and Bombardelli, 1996; Upton et al., 2001). Burdulis et al. (2007) have shown in their experiments that in all samples of bilberry fruits, cyanidin was found in the highest quantities subsequently followed by delphinidin, petunidin, peonidin and malvidin. Only in fruits harvested in Sweden, malvidin was more abundant (1.5-fold) than delphinidin and petunidin. The amount of malvidin was almost the same throughout the picking (0.016-0.017 g/ml) (Burdulis et al., 2007).

There is a great diversity of the total content of anthocyanins in bilberry collected in various geographical areas of Finland, from 19.3 to 38.7 mg/g dry weight (Lätti et al., 2008). Delphinidin and cyanidin derivatives dominated in the northern and southern berries, respectively (Lätti et al., 2008; Prior and Cao 1998; Bilberry Fruit Extract. Summary of data for chemical selection)

Anthocyanins are pigments highly soluble in water and polar solvents. They are unstable and are oxidized under the influence of various factors (pH, temperature, enzymes, UV radiation, SO2, ascorbic acid, metal ions), resulting in colour change and degradation (Rivas-Gonzalo, 2003). Processing and storage at low temperatures can improve the stability of anthocyanins (Delgado-Vargas et al., 2000).

In the study of Yue and Xu (2008) the thermal stability and degradation of the anthocyanins derivatives conjugated with a variety of sugars (delphinidin, cyanidin, petunidin, peonidin and malvidin) at the heating temperature of 80, 100, and 125°C were alike. However, when the heating temperature was increased to 125oC, degradation of each compound increased sharply, with half-life to be less than eight minutes.

Bilberry anthocyanin content was studied by Kähkönen et al., 2003. Individual compounds were identified and quantified using HPLC and HPLC/ESI–MS techniques (Table 1). The total anthocyanin content in the phenolic extracts of bilberry was 6000 mg/kg of fresh weight. There were 15 dominant compounds in bilberry (monoglycosides of cyanidin, delphinidin, malvidin, peonidin and petunidin) (Kähkönen et al., 2003).

Table 1: Anthocyanin composition in bilberry raw extract, extraction solvent CH3CN/TFA/H2O 49.5:0.5:50 V/V/V (after Kähkönen et al., 2003)

gal, galactoside; glu, glucoside; ara, arabinoside; rut, rutinoside. a–d Corresponding standards not available; quantified as aDp-3-glu, bPt-3-glu,cPn-3-gal,dMv-3-glu.

Stability of anthocyanins is affected by several environmental factors, particularly by thermal treatment. Interestingly, some acylated anthocyanins have an unusual stability in neutral or weakly acidic solutions. Packaging can also speed up the non-enzymatic browning and reduce the concentration of anthocyanins. Especially packaging in atmosphere of high CO2 free amino acids are released due to tissue damage of fresh fruits and react with anthocyanins. Data from the analysis of solid forms (dry extracts) lead to the conclusion that they are degraded through an increase in ambient temperature (Patras et al., 2010; Yamamoto et al., 2013a, 2013b).

Flavonoids

The average amount contained in 100 g of fruits is 14 mg of flavonoid glycosides (Hansel et al., 1994). Since June flavonoids concentration decreases as fruits ripen. The following flavonoids were reported from bilberry: apigenin, luteolin, chrysoriol, kaempferol, hyperoside, quercetin, quercitrin, isorhamnetin, myricetin, laricitrin, syringetin, quercetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-glucuronide, avicularine, quercetin-3-O-rhamnoside, quertine-3-O-galactoside, quercetin-3-O-arabinoside, quercetin-3-O-xyloside, 3-[[4-O-(4-carboxy-3-hydroxy-3-methyl-1- oxobutyl)-6-deoxy-α-L-mannopyranosyl]oxy]-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1- Benzopyran-4-one, 3-[[4-O-(4-carboxy-3-hydroxy-3-methyl-1-oxobutyl)-6-deoxy-α-L- mannopyranosyl]oxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-Benzopyran-4-one, isorhamnetin-3-O galactoside, myricetin-3-O-glucuronide, myricetin-3-O-glucoside, myricetin-3-O-xyloside, laricitrin-3- O-glucuronide, isorhamnetin-3-O-glucoside, syringetin-3-O-glucoside, kaempferol-3-O-glucoside, myricetin-3-O-galactoside, isorhamnetin-3-O-xyloside, quercetin-3-O-rutinoside and astragalin (Laaksonen et al., 2010; Su 2012; Bilberry Fruit Extract. Summary of data for chemical selection).

Alkaloids

The quinolizidine alkaloid myrtine was found. However it is not explained precisely by the authors of the publication, whether it comes from the fruit or the leaves (Slosse and Hootelé, 1978).

Iridoides

Asperuloside and monotropeine are found in immature fruits, but in ripe fruits they are no longer detectable (Friedrich and Schonert 1973).

Tannins

Condensed and hydrolyzable tannins. Dried ripe fruit contains minimum 1.0% of tannins, expressed as pyrogallol (Ph. Eur. 8.0, 2008:1588).

Triterpenes

Oleanolic acid, ursolic acid (0.25%) (Szakiel et al., 2012)

Organic acids

Chlorogenic, ferulic, syringic, caffeic, p-coumaric acids (Clifford 2000; Brenneisen and Steinegger 1981a, 1981b; Määttä-Riihinen et al., 2004).

Vitamins

In fresh fruits: Vitamin C, B1, panthotenic acid, nicotinamide (Hansel et al., 1994).

Other substances

Aliphatic alcohols, aldehydes, ketones, terpene derivatives. For the distinctive aroma of the fruits trans-hexenal, ethyl 3-methyl butyrate and ethyl 2-methylbutyrate may be responsible (Garcia 2008).

Herbal preparation(s)

V. myrtillus (L.), fructus dry extract prepared from fresh bilberry fruit; DER:153-76:1; extraction solvent methanol 70% V/V containing 36% anthocyanosides, corresponding to 25% anthocyanidins (Herein after referred to as BEM – see section 2.1.1. Information about products on the market in the EU/EEA Member States).

Extracts from bilberry are usually refined to the range of 34 to 36% anthocyanosides, which corresponds to a content of 25% anthocyanidins (aglycons). The amount of anthocyanin in many commercial preparations substantially varies fluctuating between 2.0 – 200 mg/g (Prior and Cao 2000).

Extracts are mainly prepared from the fresh bilberry fruits by a suitable procedure using ethanol (96% V/V) or methanol (minimum 60% V/V) at 10 – 60 °C, diluted with water, filtered and afterwards concentrated and refined usually by means of ion-exchange chromatographic techniques (Upton et al., 2001; Ph. Eur. 8.0, 2008:2394).

According to the European Pharmacopoeia 8.0 the refined and standardized dry extract of fresh bilberry fruit contains 32.4% to 39.6% of anthocyanins, expressed as cyanidin 3-O-glucoside chloride. The appropriate chromatogram for the assay of refined and standardized fresh bilberry fruit dry extracts contains: 1. Delphinidin 3-O-galactoside chloride, 2. Myrtillin (delphinidin 3-O-glucoside chloride), 3. Cyanidin 3-O-galactoside chloride, 4. Delphinidin 3-O-arabinoside chloride, 5. Chrysanthemin (cyanidin 3-O-arabinoside chloride, 6. Petunidin 3-O-galactoside chloride, 7. Cyanidin 3-O-arabinoside chloride, petunidin 3-O-glucoside chloride, 9. Delphinidin chloride, 10. Peonidin 3-O galactoside chloride, 11. Petunidin 3-O-arabinoside chloride, 12. Peonidin 3-O-glucoside chloride, 13.

Malvidin 3-O-galactoside chloride, 14. Peonidin 3-O-arabinoside chloride, 15. Malvidin 3-O-glucoside chloride, 16. Cyanidin chloride, 17. Malvidin 3-O-arabinoside chloride, 18. Petunidin chloride, 19. Peonidin chloride, 20. Malvidin chloride.

Combinations of herbal substance(s) and/or herbal preparation(s) including a description of vitamin(s) and/or mineral(s) as ingredients of traditional combination herbal medicinal products assessed, where applicable.

Not applicable.

1.2. Search and assessment methodology

Databases assessed up to September 2013:

Science Direct, PubMed, Embase, Medline, Academic Search Complete, Toxnet

Search terms: Vaccinium myrtillus, bilberry, anthocyanins

2. Data on medicinal use

2.1.Information about products on the market

2.1.1.

Information about products on the market in the EU/EEA Member

States

Dry bilberry fruit has been present as single active ingredient in 115 herbal teas on the German market for more than 30 years, traditionally used for unspecific acute diarrhoea, mild inflammation of the oropharyngeal mucosa.

Comminuted dry bilberry fruit has been on the Polish market as single active ingredient for more than 30 years and it is also registered as a herbal tea for unspecific acute diarrhoea in Austria.

A bilberry methanolic dry extract prepared from fresh fruit (DER 153-76:1; extraction solvent methanol 70% V/V) containing 36% anthocyanosides, corresponding to 25% anthocyanidins has been on the Italian market for more than 30 years, at least since 1984, being the active substance of three medicinal products, in soft and hard capsules and as a granulate for oral solution.

Information on medicinal products marketed in the EU/EEA

Table 2: Overview of data obtained from marketed medicinal products

This overview is not exhaustive. It is provided for information only and reflects the situation at the time when it was established.

Information on relevant combination medicinal products marketed in the EU/EEA

Belgium

Pharmaceutical form> containing 100 mg V. myrtillus L., anthocyanosidic extract (no further detail available) + 5 mg beta-carotene (not authorized).

Indication: capillary fragility, CVI, visual problems related to circulatory problems

Posology: 3 to 6 times 100 mg a day

On the market from 1965 to 1991.

Italy

Soft capsules containing 70 mg V. myrtillus L, fructus recens dry extract; DER 153-76:1; extraction solvent methanol 70% V/V containing 36% anthocyanosides, corresponding to 25% anthocyanidins + 40 mg d,l-alfa-Tocoferil-acetate + 10 mg beta-carotene

Indication: conditions of capillary fragility

Posology: 4 to 6 capsules a day or according to medical prescription

On the market since 1993.

Information on other products marketed in the EU/EEA (where relevant)

Five single active ingredient herbal teas on the Polish market as food supplements (for unspecific acute diarrhoea).

2.1.2. Information on products on the market outside the EU/EEA

Not applicable

2.2. Information on documented medicinal use and historical data from literature

Bilberry fruits are traditionally used for diarrhoea and in the conditions of increased fragility of blood vessels and chronic venous insufficiency. Anecdotal explanations start back to World War 2, when British pilots supposedly ate bilberry jam before the night flights in order to improve night vision (Kramer 2004).

The use of V. myrtillus L., fructus has been included in the following handbooks: Table 3: Overview of historical data

2.3. Overall conclusions on medicinal use

Table 4: Overview of evidence on period of medicinal use

Long-standing medicinal use for at least 30 years within the European Union therefore demonstrated for the following preparations and indications:

1)V. myrtillus L., fructus siccus (dry bilberry fruit), whole or comminuted, as herbal tea for oral use as an adjuvant in unspecific acute diarrhoea. Traditional medicinal use of this preparation is substantiated by extensive bibliography and the presence on the German and Polish market for more than 30 years. The daily dose in adults and adolescents over 12 years ranges from 15 to 60 g, divided in 3-4 single dose of 5 to 15 g in 250 ml as a 10 minutes decoction. (In Poland it is used as a decoction (for 10 -20 minutes under cover): 4 g in 200 ml of boiling water, 2 – 3 times daily)

2)V. myrtillus L., fructus siccus (dry bilberry fruit), whole or comminuted, as a decoction for oromocosal use for the topical treatment of mild inflammation of the mucous membranes of the mouth and throat. Traditional medicinal use of this preparation is substantiated by extensive bibliography and the presence on the German and Polish market for more than 30 years. It is used as a 10% decoction to rinse the mouth several times daily.

2) V myrtillus L., fructus recens dry extract; DER 153-76:1; extraction solvent methanol 70% V/V containing 36% anthocyanosides, corresponding to 25% anthocyanidins (BEM), in solid dosage forms for oral use for the treatment of symptoms of venous insufficiency and conditions of capillary fragility. Traditional medicinal use of this preparation is substantiated by the presence of medicinal products since 1984 in Italy. Single dose: 80-160 mg; Daily dose: 160-540 mg

3. Non-Clinical Data

3.1. Overview of available pharmacological data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof

3.1.1. Primary pharmacodynamics

Vasoactive properties

Bilberry extract

In vitro experiments

Effect of the V. myrtillus fresh fruits extract, DER 153-76:1, extraction solvent methanol 70% V/V containing 36% anthocyanosides (corresponding to 25% of anthocyanidines) (BEM) on the venous smooth muscles to contraction (response to the 5-HT) was investigated in vitro by Bettini et al., (1984a). The study was performed on isolated thoracic vein calf preparations. V. myrtillus extract (25% of anthocyanosides) (25–100 g/ml) alone caused a moderate decrease in tension as the response to the contractions induced by 5-HT (0.5–1 g/ml). The effect was more pronounced after addition of ascorbic acid (1–4 g/ml). The relaxation effect was nullified or highly decreased by the pre-treatment with indomethacin (1 g/ml) or lysine acetylsalicylate (1 g/ml).

Contractility of the segments of internal thoracic vein calf preparations induced by barium chloride (50g/ml) was reduced by V. myrtillus extract concentration dependently (25, 50, 75, 100 g/ml). Indomethacin (1–30 g/ml) and lysine acetylsalicylate (1–30 g/ml) reduced or completely suppressed the reduction of the venous muscle tone produced by BEM (Bettini et al., 1984c).

In other experiment the influence of BEM on contractility of the smooth muscles of the calf splenic arteries segments induced by 5-HT was investigated. V. myrtillus extract (25, 50, 75, 100 g/ml) alone

caused a concentration dependent decrease in tension of the arterial muscles as the response to the contractions induced by 5-HT (0.2 g/ml). The effect was potentiated after addition of ascorbic acid (1–4 g/ml). Indomethacin (1-30 g/ml) and lysine acetylsalicylate (1–30 g/ml) decreased the reduction of the arterial muscle tone produced by BEM. The results obtained by the authors indicate that the mechanism of the vasodilatating effect of BEM on vascular muscles is based on the local synthesis of prostacyclin (Bettini et al., 1984 b).

Effect of the extract of BEM on contractility of calf isolated coronary artery preparations was tested in vitro by Bettini et al., (1985a). Contractions induced by barium chloride (50 g/ml) were concentration dependently mildly suppressed by BEM (25, 50, 75, 100 g/ml). The effect was more pronounced after addition of ascorbic acid (1–4 g/ml). Relaxation induced by BEM was concentration dependently reduced with indomethacin (1–30 g/ml) or lysine acetylsalicylate (1–30 g/ml) (Bettini et al., 1985a).

Bettini et al., (1985b) reported the influence of BEM on potentiation of activity of adrenaline on the isolated calf coronary vessels. Adrenaline (0.2 g/ml) vasodilating activity was concentration dependently increased by BEM (25, 50, 75, 100 g/ml) (Table 5). The potentiating effect was completely abolished in the presence of pyrogallol (50 g/ml) a catechol-o-methyl transferase (COMT) inhibitor. The authors conclude that the vasodilating mechanism of BEM results from COMT inhibition.

Table 5: Mean percentage increase (± S.D.) in the response of the preparation of the calf coronary artery to adrenalin (0.2 g/ml) in the presence of BEM (afterBettini et al., 1985b)

In another study Bettini et al., (1991) investigated contractile responses of the isolated calf coronary vessels to acetylcholine (ACh) and methylene blue. Experiments were carried out without removal of the endothelium. BEM (50–200 g/ml) decreased both the tone and in higher concentrations the contractile response of the preparations to ACh (0.001–1 g/ml). A small decrease of the tone was observed with BEM alone (50–200 g/ml) and the effect was more marked with addition of the ascorbic acid (100–300 g/ml). Indomethacin (40 g/ml) and lysine acetylsalicylate (40 g/ml) reduced or completely suppressed the reduction of the venous muscle tone produced by BEM. Pre-treatment with methylene blue (24 g/ml) resulted in the partial reduction of the vasodilatatory effect of BEM. The authors concluded, that the vasodilator effect of BEM is related to the release of prostaglandins and to a facilitation of the endothelium-derived relaxing factor (EDRF) release, as the methylene blue is known to block the release of EDRF.

Continuing research of Bettini et al., (1993) found after use of BEM (5-100 g/ml) a significant increase in contractility of endothelium-deprived isolated calf coronary arteries induced by ACh administration (0.001–1 g/ml). The potentiating effect was completely suppressed by methylene blue (24 g/ml) or haemoglobin (0.015–0.020 g/ml) which block the release of EDRF.

The direct vasorelaxating activity of a lyophilized dry bilberry extract (BE) (no further details are available) was tested in vitro by Bell and Gochenaur (2006) on coronary arterial rings isolated from pigs. BE contained 15 different anthocyanins including cyanidin, peonidin, delphinidin, petunidin, and malvidin. The total anthocyanin composition was 12.1 g/100 g and total phenolics 35.7 g/100 g. BE produced dose- and endothelium-dependent vasorelaxation in isolated rings with endothelium (% maximal relaxation at 5 mg/l total anthocyanins: 59±10). The authors tested the role of nitric oxide (NO) in these relaxations and found that such relaxation could be abolished by the application of 100

M NO2-L-arginine (Nitric Oxide Complex with L-arginine). These observations suggest that the endothelial NO system may be involved in the relaxation response of coronary arteries to the BE. At a concentration too low to directly alter coronary vascular tone, they did not alter coronary responses to endogenous or exogenous NO. However, this same low concentration ( 100 nM) had a considerable potential to prevent loss of endothelial-dependent relaxation caused by exposure of arteries to exogenous ROS (Reactive Oxygen Species) as pyrogallol. The authors concluded that the tested anthocyanins concentration roughly reflects that seen in several studies to exist even in human plasma after oral consumption of these products, hence they may have vasoprotective properties.

Mechanism of vasodilatation induced by bilberry anthocyanins was investigated by Ziberna et al., (2013). Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. The endothelium was preserved in the rings. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the endothelial plasma membrane carrier, that transports flavonoids, resulted in decrease of vasodilatation induced by cyanidin 3-glucoside and bilberry anthocyanins. For experiments a purified methanolic BE (no further detail) was used. Anthocyanins composition was analyzed by HPLC-DAD method: 14.3% of delphinidin 3-galactoside, 14.0% of delphinidin 3-glucoside, 9.2% of cyanidin 3-galactoside, 12.1% of delphinidin 3-arabinoside, 10.1% of cyanidin 3-glucoside, 4.0% of petunidin 3-galactoside, 7.7% of cyanidin 3-arabinoside, 8.8% of petunidin 3-glucoside, 1.1% of peonidin 3-galactoside, 2.6% of petunidin 3-arabinoside, 3.7% of peonidin 3-glucoside, 2.5% of malvidin 3-galactoside; 0.5% of peonidin 3-arabinoside, 7.9% of malvidin 3-glucoside, 1.5% of malvidin 3-arabinoside. Concentration of total anthocyanins in the BE was expressed in mg/l as equivalents of cyanidin 3-glucoside (no further detail given). In vascular reactivity experiments concentration-relaxation curves to cyanidin 3-glucoside (1 nmol/l-10 mmol/l) and bilberry anthocyanins (0.01-20 mg/l expressed as equivalents of cyanidin 3-glucoside) were constructed. Vasorelaxations were examined before the 30 minutes incubation in each of the tested solutions, and after incubation followed by 30 minutes equilibration periods to avoid ACh tolerance. Anthocyanins are especially rich in bilberries, consisting on the average 80-90% of total phenolic compounds. Anthocyanins are substrates of bilitranslocase, suggesting that they greatly contribute to the endothelium-dependent vasodilatory effect. In presented experiments, observed vasodilatation activity was in the range from 10 nM on, thereby in the range of reported pharmacokinetic post- absorption plasma concentration.

In vivo experiments

Influence of fresh fruits BEM on the capillary fragility has been studied in the model of rats deprived of dietary flavonoids (Cristoni and Magistretti 1987). Wistar rats were fed for 3 weeks on the diet devoid of flavonoids. On depilated skin the capillary resistance – the lowest negative pressure on the skin that induces petechiae was defined by use of the vacuum gauge. Immediately after the BEM corresponding to 25% anthocyanidins was administered by intraperitoneal injection and the capillary fragility was estimated again after 2, 4 and 6 hours (Table 6).

Table 6: Activity of BEM on the capillary resistance of rats fed on a flavonoid-devoid diet (after Cristoni and Magistretti 1987)

Anti-inflammatory activity

Bilberry extract

In vitro experiments

Triebel et al., (2012) studied the influence of a lyophilized BE (no further details are available) and comprising anthocyanins on pro-inflammatory genes in IFN-γ/IL-1β/TNF-stimulated human colon epithelial cells (T84) by real-time polymerase chain reaction (qRT-PCR) and cytokine activity. Analysis of the extract is displayed in Table 7 which also shows the concentrations (μM) of the substances in 25 μg/ml BE extract (used in incubations with the cultured cells).. Fifteen anthocyanins were detected in the BE by HPLC-DAD analysis, the most numerous being del-3-gal, del-3-glc, del-3-ara, cy-3-gal, and cy-3-glc.

Table 7: Anthocyanin concentrations in the BE and corresponding initial concentrations in in vitro incubations with 25 μg/ml extract (afterTriebel et al., 2012)

aAccording to the manufacturer’s specifications. bFor an in vitro incubation with 25 μg/ml extract.

The authors studied the expression of inflammatory bowel diseases-associated pro-inflammatory marker genes (TNF-α, IP-10, IL-8) in the cultures of the human colon epithelial cells (T84) by quantitative real-time PCR. The cytotoxic effects of BE and the singular anthocyanins/anthocyanidins on T84 cells were determined using a resazurin reduction assay. Selected cytokines and chemokines were analyzed using the “Human Cytokine Array Panel A” antibody array.After 4 and 24 hours, 250 μg/ml BE reduced viability to 80±5 and 60±3% and 200 μM cyanidin reduced it to 83±2 and 78±3%, whereas 200 μM delphinidin reduced it to 79±1 and 63±2%, respectively. All investigated anthocyanins had only slight cytotoxic effects at 200 μM (cell viability > 95%) after 4 and 24 hours of incubation. Cytotoxic effects of the anthocyanidins were stronger than the corresponding anthocyanins declining in the order delphinidin > cyanidin > pelargonidin >peonidin > malvidin so that increasing with increases in hydroxylation. BE significantly and dose-dependently inhibited expression of the pro- inflammatory marker genes TNF-α and IP-10 in CM stimulated T84 cells, at concentrations of 2.5 and 25 μg/ml, respectively.

Influence of chosen anthocyanins on expression of TNF-α, IL-8, IP-10 genes in T84 stimulated cells depended on both the aglycone and of sugar residues. IP-10 expression was significantly inhibited by cyanidin-3-ara, the most potent inhibitor, cyanidin-3-glc at 25 μM (the lowest concentration tested), and cyanidin-3-gal at 50 μM. Peonidin-3-O-glycosides were active concentration dependently only as glucose conjugates in the tested concentrations (25, 50, 100 μM). The investigation of activity of the corresponding anthocyanidins have shown that cyanidin, delphinidin, and petunidin significantly down- regulated IP-10 mRNA expression, but peonidin or malvidin did not (even at 100 μM). Cyanidin significantly reduced TNF-α transcript levels at ≥50 μM, but peonidin or malvidin did not have any effect. Pre-treatment with cyanidin (25 μM) and BE (25 μg/ml) completely inhibited synthesis of interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell alpha chemo-attractant (I- TAC), and soluble intercellular adhesion molecule 1 (sICAM-1). Authors concluded that single anthocyanins from BE modulates inflammatory genes and protein secretion in vitro and thus may act as transcription-based inhibitors of the pro-inflammatory gene expression associated with inflammatory bowel diseases. Moreover, the anti-inflammatory activity of the investigated anthocyanins is strongly dependent on their aglycone structure and the attached sugar moieties (Triebel et al., 2012).

In the study of Song et al. (2010), a newly established human corneal limbal epithelial cell line (HCLEC) was investigated to study the effects of a BE on the cell growth, cell cycle and the expression of hyaluronic acid (HA) and glycosaminoglycans (GAGs) in corneal epithelial cells. A commercially available BE containing 25% total anthocyanins (no further details are available) was used. The content of anthocyanins present in BE was quantified by HPLC using cyanidin-3-O-glucoside as external standard. The cells were incubated with different concentrations of BE for 24 hours and 48 hours. BE (10−5 M) promoted cell growth to about 120% compared with the control group (p<0.05) after 24 hours incubation while three concentrations (10−6, 10−5and 10−4 M) were effective in increasing cell viability to 112.9%, 130.1% and 113.8%, respectively, (p<0.05) after 48 hours incubation. For the HA and GAGs assay, HCLEC cells were incubated with different concentrations of BE. The GAGs content in the supernatant of the cells increased significantly after incubation with BE for 48 hours, but the increase was not dose-dependent. Two separate concentrations (10−7 and 10−4 M) significantly induced the secretion of GAGs (p<0.05), while no significant changes were observed for the expression of hyaluronic acid. According to the authors the results showed that BE may be advantageous for the physiological recovery and homeostasis of corneal epithelial cells.

Isolated compounds

Hou et al. (2005) investigated cyclooxygenase-2 (COX-2) inhibiting activity of five anthocyanidins. Only delphinidin and cyanidin inhibited lipopolysaccharide (LPS)-induced COX-2 expression in the culture of macrophage RAW264 cells. The significant dose dependent inhibition was present at the concentrations of 50, 75 and 100 µM, but peonidin and malvidin did not. Delphinidin was the most potent on mRNA and protein level. Delphinidin suppressed LPS-mediated COX-2 expression by blocking mitogen-activated protein kinase (MAPK) pathways with the attendant activation of nuclear factor-kB (NF-κB), CCAAT/enhancer-binding protein (C/EBPδ) and activator protein-1 (AP-1).

Microcirculation injury

Bilberry extract

In vivo experiments

In another study Bertuglia et al., (1995) tested activity of BEM from fresh fruits (100 mg per day/kg p.o. for 2 and 4 weeks) in the microcirculation ischemia model. Ischemia was induced by clamping the

hamster cheek pouch for 30 minutes with subsequent reperfusion also lasting 30 minutes. Changes in the microcirculation were visualized by fluorescence method. Ischemia and reperfusion were associated with increased number of leukocytes sticking to venules, decreased number of perfused capillaries and increased permeability. After treatment there was a significant reduction in ischemic symptoms (p<0.01, Table 8).

Table 8: Number of sticking leukocytes (l per 100 μm venules), % of perfused capillary length (capillary perfusion), permeability increase (normalized grey levels) in Control (Con) and in hamsters treated with BEM for 2 (A) and 4 (B) weeks after ischemia reperfusion (Bertuglia et al., 1995).

*p<0.01, compared to controls, †p<0.05 relative to bilberry A group.

Table 9: Overview of the main non-clinical data/conclusions

A large amount of data from preclinical studies on the beneficial effects of anthocyanins on the regeneration of rhodopsin exist. Such studies, among others, were carried out by Matsumoto et al., 2003; Tirupula et al., 2009; Yanamala et al., 2009. Circadian clock regulation of the pH in the retina of vertebrates showed the pH increased upon exposure to light. It appeared that the pH of the retina is more alkaline during the day. According to the authors this may be important because the pH of the environment plays an important role in the various activities of anthocyanins. Their concentration in the tissues of the eye is very low (Manach et al., 2005; Ichiyanagi et al., 2004a, 2004b; Matsumoto et al., 2003).

As believed by Kalt et al., (2010) other phenolic flavonoids may play an important role in the survival of the cultured human retinal pigment epithelial cells and regeneration of rhodopsin. These could be the flavanone eriodictyol (Johnson et al., 2009, Maher and Haneken 2005, 2008) baicalein, luteolin, galangin, fisetin or quercetin.

3.1.2. Secondary pharmacodynamics

Impact on lipid peroxidation

Bilberry extract

In vitro experiments

The comprehensive and extensive monograph by Upton et al., (2001) showed that an extract of bilberries protected microsomes from rat liver against oxidative damage and apolipoprotein B before brought about by UV radiation.

An anthocyanoside complex extract from V. myrtillus was tested for its ability to inhibit lipid peroxidation and to scavenge hydroxyl and superoxide radicals (Martín-Aragón et al., 1998; 1999). An antiperoxidative action of this V. myrtillus extract was assayed by the Fe3+ -ADP/NADPH method in rat liver microsomes. Superoxide anions were generated by preparing a mixture of hypoxanthine and xanthine oxidase. The results were expressed as percentage inhibition of cytochrome C reduction (Upton et al., 2001).

Laplaud et al., (1997) tested in vitro an aqueous extract of V. myrtillus on human low density lipoproteins. They found, that the extract, which contained 74.2±4.9 mg/g of total polyphenols with a proportion of catechin of 17.3±3.3% caused potent protective action on LDL particles during in vitro copper-mediated oxidation. Trace amounts of V. myrtillus aqueous extract (15 to 20 μg/ml) induced statistically significant changes in the oxidation behaviour of low density lipoproteins (LDL). Observed effects included: 1) Prolongation of the lag-phase of conjugated diene production (p<0.01); 2) Reduction in the formation of lipoperoxides and of thiobarbituric acid-reactive substances up to 7 hours and especially between 1 and 5 hours (p<0.01); 3) Inhibition of modification in the net negative charge of LDL. The antioxidant effect of the extract could be compared to the corresponding action of ascorbic acid or butylated hydroxytoluene in the protection of LDL particles from oxidative stress.

Antioxidant activity

Bilberry extract

In vitro experiments

In in vitro studies conducted by Cluzel et al. (1969; 1970) it was found that anthocyanins of

V. myrtillus affected the activity of various enzymes of retina in pig and in rabbit (inhibiting the activity of phosphoglucomutase, and increasing the activity of lactate dehydrogenase, α-hydroxybutyrate

dehydrogenase, 6-phosphogluconate dehydrogenase and α-glycerophosphate dehydrogenase). However, in these studies, the authors used a complex formulation consisting, beyond of an extract of bilberry, of other components, including beta-carotene. Therefore the significant effect of beta- carotene contained in the preparation in large quantities cannot be excluded.

It was found, that the extract scavenged superoxide anion and inhibited microsomal lipid peroxidation at all concentrations (25, 50 75 and 100 μg/ml) (p<0.01) and a 50% inhibition of rate of reaction was observed with a final concentration of 25 μg/ml. The anthocyanoside complex extract was able to inhibit lipid peroxidation (IC50=50.28 mg/ml) and to scavenge superoxide anion (IC50<25 mg/ml). The ability to remove hydroxyl radical exerted by this extract was detectable from 50 mg/ml of extract in the reaction mixture (Martín-Aragón 1998).

According to Prior et al. (1998), comparison of the antioxidant capacity in a variety of Vaccinium species have shown high activity of V. myrtillus (Table 10). Bilberries were extracted with acetonitrile/acetic acid for the analysis of ORAC, total anthocyanins, and total phenolics. In conclusion the increased maturity at harvest increased the ORAC, the anthocyanin, and the total phenolic content. A linear relationship existed between ORAC and anthocyanin (rxy=0.77) or total phenolic (rxy=0.92) content.

Table 10: Antioxidant activity of V. myrtillus L. (after Prior et al., 1998).

V. myrtillus L.

aExpressed as micromole Trolox equivalents per gram of fresh fruit. Oxygen radical absorbance capacity (ORAC ROO). Data in parentheses expressed per gram of dry matter. Bilberry was harvested on 7/2/97.

bConcentration based upon cyanidin-3-glucoside as standard. cConcentration based upon gallic acid as standard. dAnthocyanin/phenolics.

Direct in vitro influence of the bilberry fruit extracts on the oxidative phosphorylation of isolated rat heart mitochondria was tested by Trumbeckaitè et al. (2013). For testing, two types of extracts were used: the hydroethanolic extract (BEE) of the crushed plant material was prepared by maceration with 50% ethanol at room temperature (1:10, V/V), initially for 48 hours and thereafter until exhaustion; the aqueous extract (BAE) was prepared using repercolation method (1:10, V/V). The obtained hydroethanolic extract was filtered and concentrated under vacuum (at 50oC) and then subjected to freeze drying. Freeze dried bilberry powder was packed into a glass jar and dissolved before experiments. The levels of anthocyanidins, measured by use of HPLC, varied in the two extracts (see Table 11)

Table 11: Anthocyanidins in the two extracts tested by Trumbeckaitè et al. (2013)

Amount of anthocyanidins (ng/ml) in 1 μl) of bilberry fruit extracts

BAE, bilberry aqueous fruit extract; BEE, bilberry ethanolic fruit extract.

When measured the effects of BEs on complex I-dependent substrate pyruvate plus malate oxidation, mitochondrial respiratory rates only in the presence of 5–30 ml/1.5 ml of the BAE extract the mitochondrial state 3 respiration rate decreased from 33% to 61% (p<0.05). BEE induced the decrease in state 3 respiration rate also starting from 5 ml/1.5 ml. High doses of BEE (15–30 ml/1.5 ml) induced a decrease in the state 3 respiration rate by 35–56% that is similar to BAE. In effect at higher concentrations, BAE and BEE induced significant uncoupling of oxidative phosphorylation and decrease in the state 3 respiration rate. The true mechanism of the diminishing of the state 3

respiration rate by BEs may be the inhibition of mitochondrial respiratory chain at complexes I and II. Pure anthocyanins, the main components of used extracts, malvidin-3-glucoside, malvidin-3- galactoside, and cyanidin-3-galactoside, had no effect on oxidation of pyruvate plus malate. A statistically significant decrease in H2O2 production by mitochondria was found in the presence of bilberry fruit extracts. BAE at concentrations of 1.5 and 15 μl/1.5 ml clearly suppressed this process and caused a 46% and 62% reduction, respectively, in the H2O2 generation as compared with that in the absence of BAE. Similar effects (reduction by 50%) were obtained by BEE (15 ml/1.5 ml), whereas lower amounts of BEE (1.5 ml/1.5 ml) suppressed H2O2 generation by 16%, that is, less than BAE. The results revealed that the effect of BAE and BEE on mitochondrial function is bivalent: lower concentrations (they correspond to 6–9 mg/l of total anthocyanins) had no effect on mitochondria, whereas at high concentrations (they would correspond to 18–52 mg/l of total anthocyanins), the extracts caused an obvious decrease in the state 3 respiration, but the radical scavenging activity remained increased. The effects of BAE and BEE on mitochondria were dose dependent (Trumbeckaitè et al., 2013).

A BE from dried fruits containing anthocyanins (25.0%, w/w) (no further details are available) reduced UVA-induced oxidative stress in keratinocytes (Svobodová et al., 2008). In the first experiment keratinocytes grown in culture medium were pre-treated with the BE (5–100 mg/l) in serum free medium at 37oC for 1 hour, irradiated and incubated in serum-free medium at 37oC for another 4 hours. In the second experiment keratinocytes were irradiated and after UVA exposure the BE (5–100 mg/l) in the serum-free medium was added to the cells for 4 hours. The effect of extract in the concentration range of 1–250 mg/l, various UVA doses (10–40 J/cm2) or combinations of the extract and UVA on keratinocytes cell viability was assessed after 4/24 hours. Pre-treatment (1 hour) or post- treatment (4 hours) of keratinocytes with the BE resulted in attenuation of UVA-caused damage. Viability of the cells was determined photometrically. BE at the concentrations tested (1–250 mg/l) did not affect incorporation of water-soluble dyes into lysosomes, but decreased lactate dehydrogenase (LDH) activity in medium samples containing 100 and 250 mg/l of the extract after 24 hours. The last finding evaluated the activity of a cytosolic enzyme, which reflects cell membrane integrity. Moreover, application of the extract significantly reduced UVA-stimulated ROS (Reactive Oxygen Species) formation in keratinocytes: the maximal decline in ROS generation was at concentrations of 50 and 100 mg/l of the extract. Administration of BE also prevented/reduced UVA-caused peroxidation of membrane lipids: the maximal protection was observed in pre-treatment at a concentration of 50 mg/l (over 90%), post-treatment with the extract also markedly inhibited membrane lipid damage with maximum at concentrations of 25 and 50 mg/l (75–80%). The extract also induced depletion of intracellular GSH: pre-treatment with the extract significantly protected against UVA caused GSH depletion, especially at concentrations of 25 and 50 mg/l (55%). Post-treatment was the most effective in the concentration range of 50–100 mg/l (50%).

In the other experiments of the same group HaCaT keratinocytes were used to assess the effects of pre-and post-treatment with BE phenolic fractions (5–50 mg/l) on keratinocyte damage induced UV radiation by a solar simulator (295–315 nm) (Svobodová et al., 2009). For the assessment of UVB (photo) protective potency of phenolic fractions non-toxic concentrations (5, 10, 25 and 50 mg/l) of BE were used. BE efficiently reduced the extent of DNA breakage (especially at concentrations of 25 and 10 mg/l) together with caspase-3 and -9 activity. The effect of post-treatment on caspase-3 activity was similar for all concentrations tested. Pre-treatment of keratinocytes with BE also reduced caspase- 9 activity. BE effect was concentration-dependent (maximal protection of 87%). BE was the most potent at a concentration of 5–10 mg/l (around 80%), which slightly decreased at higher concentrations. Application of the extract before UVB exposure significantly prevented DNA fragmentation. BE effectiveness culminated at a concentration of 10 mg/l (70%) and at higher

concentrations the protection decreased to 50% and 40% at a concentration of 25 mg/l. The phenolic fraction of Vaccinium myrtilli berries significantly decreased generation of reactive oxygen and nitrogen species (RONS), of oxidizing lipids, proteins and DNA. Application of the BE (4 hours) to non-irradiated HaCaT slightly reduced RONS generation compared to untreated non irradiated cells. The effectiveness of the extract showed 40% protection at the highest concentration. Supreme RONS elimination was found in post-treated cells. At concentrations of 25 and 50 mg/l phenolic fractions reduced RONS amount to control level. The extract decreased IL-6 production in irradiated cells, when it was applied before UVB exposure. The effect of BE was concentration-dependent with maximal protection 35%. The maximal potency was found at the highest concentration approximately 33%.

Antioxidant activity of bilberry (Vaccinium myrtillus L.) and blueberry (Vaccinium corymbosum L.) was examined at the cellular level in different cell lines: human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and rat vascular smooth muscle (A7r5). The bilberry crude methanolic extract was further purified in order to obtain the anthocyanin fraction: [(crude BE: phenolic acids, proanthocyanidins, flavanols, flavonols) (purified BE: anthocyanin fraction)]. Anthocyanins had intracellular antioxidant activity if applied at very low concentrations (<1 μg/l; nM range) (Table 12). Delphinidin and cyanidin glycosides were the predominant anthocyanins in BEs, whereas malvidin glycosides dominated in the blueberry extract (Bornsek et al., 2012).

Table 12: Half maximal effective concentrations (EC 50) of the extracts in 4 cell lines (afterBornsek et al., 2012)

Data were expressed as mean±SEM, number of independent measurements was n=6. Statistical analysis was performed using one- way ANOVA with post-Bonferroni test. Statistically significant differences (p<0.05) are marked with letters (a,b,c) in the same row.

The effective concentrations achievable after oral administration (Mazza et al., 2002; Felgines et al., 2008) are in the range of plasma anthocyanin concentrations in the presented experiments, showing cellular antioxidant activity at very low concentrations in different human cell lines. Such values in the range of 1nM are attained after consumption of ordinary servings of berries (McGhie and Walton 2007).

Cytoprotective effect of a BE from fresh fruits against oxidative damage in primary cultures of rat hepatocytes was studied by Valentová et al., (2007). The BE analysed by HPLC contained 25% of total anthocyanins. Activity of BE against oxidative cell damage induced by tert-butyl hydroperoxide and allyl alcohol in primary cultures of rat hepatocytes was investigated. The hepatocyte monolayers were incubated with the tested extract for 4, 24 and 48 hours and the viability of the cells was assessed by the MTT test. In the concentrations tested (100 and 500 μg/ml), no significant toxicity was registered. The extract showed significant dose-dependent protective activity against oxidative damage in rat hepatocytes primary cultures induced by tert-butyl hydroperoxide and allyl alcohol. Maximum cytoprotection (58.16%) was noted in the culture pre-incubated with 500 μg/ml of the extract (Table 13).

Table 13: Protective effect of the bilberry extract on tert-butyl hydro peroxide induced damage of rat hepatocytes primary cultures (afterValentová et al., 2007)

After 30 minutes of pre-incubation with the BE, the cell monolayers were treated with tert-butyl hydroperoxide (tBH, 0.5 mmol/l) during 1.5 hours. Results are expressed as mean±SD, n=9. a p<0.01 vs. non-treated cells. b p<0.01 vs. tBH-treated cells.

Antiradical activity was evaluated spectrophotometrically like the ability of the tested substances to reduce 1.1-diphenyl-2-picrylhydrazyl (DPPH) radical. The extract has shown scavenging activity; 50% inhibition was achieved at 3.99±0.14 μg/ml (IC50). In the same experimental condition, IC50 of the synthetic analogue of vitamin E, trolox, was 2.15±0.06 μg/ml (8.57±0.25 μmol/l). Estimation of the antioxidant activity of the BE was also investigated in the xanthine/XOD superoxide generating system. The BE scavenged the superoxide radical and its activity was equivalent to 108±7.2 units of SOD per mg of extract. In the same system, trolox had an activity equivalent to 16.4±0.19 units of SOD/mg (Valentová et al., 2007).

Ogawa et al., (2011) studied the lipid peroxidation and free radical scavenging activity of a BE (containing more than 25% anthocyanosides–no futher details are available) in murine stomach tissue homogenates. BE in the concentration dependent range significantly induced decrease of activity of the lipid peroxide levels and revealed strong scavenging activity against superoxide and hydroxyl radicals (Table 14; Table 15).

Table 14: Superoxide anion radical scavenging activity of the bilberry extract and its main anthocyanidins (delphinidin, cyanidin and malvidin) (afterOgawa et al., 2011)

IC50, 50% inhibitory concentration. The parentheses show 95% confidence limits

Table 15: Hydroxyl radical scavenging activity of the bilberry extract and its main anthocyanidins (delphinidin, cyaniding and malvidin) (afterOgawa et al., 2011)

IC50, 50% inhibitory concentration. The parentheses show 95% confidence limits.

Isolated compounds

Different berry phenolics, V. myrtillus included, and their antioxidant activity were investigated by Kähkönen et al., (2001). Extraction methods for berries and apples were examined to create phenolic extracts with high antioxidant activity. Evaluation of antioxidant activity was performed by auto- oxidazing methyl linoleate (40°C, in the dark). The extraction method affected prominently both the phenolic composition and the antioxidant activity (Table 16). However, from the observation of the results obtained the effects cannot be clearly related with the content of phenolic of the individual subgroups.

Table 16: Anthocyanin, flavonol, hydroxycinnamic acid (HCA), hydroxybenzoic acid (HBA), ellagitannin, flavanol and procyanidin, and total phenolic contents (data expressed as mg per 100 g of weight), and antioxidant activity (data expressed as inhibition percentage) of bilberry extracts produced using different extraction methodsa) (afterKähkönen et al., (2001).

aMeans (SD of duplicate assays. Values in the same column for each berry having the same letter are not significantly different at p<0.05. ND, not detected. NA, not analyzed. bConcentration based upon cyanidin-3-glucoside as standard. cConcentration based upon rutin as standard. dConcentration based upon chlorogenic acid as standard. eConcentration based upon gallic acid as standard. Concentration based as ellagic acid as standard. gConcentration based as (+)-catechin as standard. hConcentration based upon gallic acid as standard. iInhibition of methyl linoleate hydroperoxide formation after 72 hours of incubation at the concentration of 500 ppm of dry raw extract. jInhibition of methyl linoleate hydroperoxide formation after 72 hours of incubation at the concentration of 500 ppm of dry extract after sugar removal with SPE.

The antioxidant activity of phenolics (at concentrations of 1.4, 4.2, and 8.4 μg of purified extracts/ml of liposome sample) such as anthocyanins, ellagitannins, and proanthocyanidins from bilberry was studied by Viljanen et al. (2004) in a lactalbumin-liposome system. Phenolic profile of BE determined using an analytical HPLC method. The extent of protein oxidation was measured by determining the loss of tryptophan fluorescence and formation of protein carbonyl compounds and that of lipid oxidation by conjugated diene hydroperoxides and hexanal analyses (Table 17).

Table 17: Inhibition of lipid and protein oxidation (after 6 days of oxidation) by bilberry phenolics incorporated into lactalbumin-lecithin liposomes (% inhibition, Mean±SD) (after Viljanen et al., 2004)

aSD, standard deviation. Negative values indicate pro-oxidant activity. Values in the same column at the same concentration followed by different letters are significantly different (p<0.05).

Bilberry phenolics exhibited good overall antioxidant activity toward protein oxidation. The antioxidant effect toward lipid oxidation was more pronounced than the effect on protein oxidation (Table 18).

Table 18: Phenolic profiles of bilberry extract (expressed as % of total phenolics measured using HPLC; ND, not detected) (afterViljanen et al., 2004)

Anthocyanins; amount based upon cyanidin-3-glucoside as standard. b(+)-Catechin as standard. cChlorogenic acid as standard. dGallic acid as standard. eProAs, proanthocyanidins; flavan-3-ol as standard. ET, ellagitannins; ellagic acid as standard. gEA, ellagic acid.

Antimicrobial activity

It has long been known that several phenolic substances such as flavonoids, phenolic acids, tannins and lignans have antimicrobial activity (Heinonen 2007). It is believed that it is the flavonoid anthocyanins component in V. myrtillus that exerts such an effect. The mechanism of antimicrobial activity may include antiadhesion activity, destruction of the cytoplasmic phospholipid bilayer of the cell wall in microbes, damage of the outer membrane with disintegration of the liposaccharide (LPS) layer by phenolics, tannins complexation of metal ions and inhibition of plasma coagulation by bacteria. Another mechanism is the inhibition of antibacterial multidrug resistance (MDR) and impairment of the efflux pump activity in bacteria (Puupponen-Pimiä et al., 2005a, 2005b, 2005c).

Bilberry extract

In vitro experiments

Rauha et al. (2000) evaluated the antimicrobial activity of a number of plants, including bilberry. To the in vitro studies, an aqueous solution of the dry extract prepared from the dry plant material (acetone/methanol 70% V/V–no further detail) was used to determine the diameter of the inhibition zones in the agar cultures of bacteria. Clear antimicrobial effect has been found for the BE (500 µg samples) against the Micrococcus luteus (inhibition zone (i.z.) of sample=3-4 mm>i.z. of methanol and slight antimicrobial activity against Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli and

Candida albicans (i. z. of sample=1-3 mm)>i.z. of methanol.

The antimicrobial activity of many plants, including V. myrtillus extract (acetone-water 70:30 V/V; elution with MeOH) prepared from fresh frozen berries was screened against the human pathogenic microbial strains on agar plates to estimate their growth and adherence of the bacterial cells to a berry material. The BE (1 mg/ml) revealed the death of the culture of Helicobacter pylori, very strong inhibition of growth of Bacillus cereus and strong inhibition of growth of Clostridium perfringens and

Staphylococcus aureus (Nohynek et al., 2006).

Binding of Neisseria meningitidis pili to V. myrtillus berries and juice polyphenolic fractions containing anthocyanins, proanthocyanidins and flavonols have been identified by Toivanen et al. (2009; 2011). Prevention of adhesion of pathogenic bacteria to host cell surfaces may constitute the protection from the activity of bacteria that use adhesins to colonize the host cells.

Activity of bilberry against Gram positive and Gram negative intestinal pathogens was examined in in vitro cultures of Salmonella, Staphylococcus, Listeria and Lactobacillus bacteria (Puupponen-Pimiä et al., 2005a; 2005b). The BEs (water/ethyl acetate/methanol) prepared from fresh frozen berries containing phenolic acids and fractions eluted with methanol (ellagitannins and anthocyanins) were tested. BE (2 mg/ml) inhibited the growth of Staphylococcus aureus for 12 and 24 hours (5×101- 5×102) and Salmonella enterica Typhimurium (10-5×101). BE fractions (10 mg/ml) exhibited stronger inhibition against Staphylococcus aureus (>5×104) for 12 and 24 hours compared with control.

Stronger inhibition of growth was also seen against Salmonella enterica Typhimurium (5×102-5×103) compared with control.

Influence of various preparations of BE prepared from fresh berries, on trophozoites of Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts was examined in in vitro experiments by Anthony et al., (2007, 2011). The water soluble extracts of bilberry containing polyphenols (167 µg/ml of gallic acid equivalents) killed 90.4+2.8% of Giardia duodenalis trophozoites. Increase of the spontaneous excystation of Cryptosporidium parvum oocysts observed after administration of the BE (equivalent to 213 µg/ml of the gallic acid). Because anthocyanins represent more than 70% of the polyphenols, it was concluded that they could be responsible for antiprotozoan activity of bilberry.

Bilberry juice

In vitro experiments

Similar experiments were conducted by Huttunen et al. (2011) who found through in vitro studies that a 10-100 kDa molecular size fraction (9 mg/g) of the bilberry juice inhibited the binding of Streptococcus pneumoniae to human bronchial cells (Calu-3) in the adhesion model. The adhesion inhibition by bilberry juice, consisting essentially of small amounts of phenolic compounds with a low molecular weight, was 52%. The test fractions were devoid of solvents and are water-soluble.

In contrary, juice fractions of V. myrtillus of the higher molecular weight with the dominance of anthocyanins, proanthocyanidins and flavonol glycosides exerted antiaggregation effect on the pairs of bacteria common in the pathology of the dental plaque in the oral cavity: Streptococcus mutans with

Fusobacterium nucleatum or Actinomyces naeslundi (Riihinen et al., 2011).

Antineoplastic activity

Bilberry extract

In vitro experiments

Bilberry fruit extracts were screened to antineoplastic activity by in vitro tests to determine the ability to induce phase II detoxification of quinone reductase and inhibition of the induction of ornithine decarboxylase. Raw extracts containing anthocyanin and proanthocyanidin fractions showed little activity (Bomser et al., 1996).

Esselen et al., (2011) investigated the influence of BE on topoisomerases activity in a cell-free system and in human HT29 colon carcinoma cells. Topoisomerases I and II are targets of clinically used anti- cancer drugs. A BE containing 36% w/w anthocyanin was used. The major anthocyanins concentrations were established by HPLC mass spectrometric analysis. About 1.2 million HT29 cells were spread into Petri dishes (two Petri dishes for one concentration) and allowed to grow for 48 hours. Topoisomerase protein was detected using rabbit polyclonal antibodies specific for either topoisomerase I, topoisomerase II a, or topoisomerase II b. The effect of BE on the growth of the cell line HT29 was determined using the sulforhodamine B assay. Incubation of HT29 cells with the extract up to 500 μg/ml for 72 hours led to an inhibition of cell growth but without reaching an IC50 value. However, the extract potently inhibited the catalytic activity of topoisomerase I at a concentration of 25 μg/ml. Activity of topoisomerase I was completely suppressed by the extract at concentrations 50 mg/ml. The BE was found to inhibit the catalytic activity of topoisomerase II at concentrations exceeding 1 μg/ml. The catalytic activity of topoisomerase IIa was fully blocked at 5 μg/ml. Similar effects were obtained with recombinant topoisomerase IIb. Pre and co-incubation of HT29 cells with BE (1 g/ml) significantly suppressed (p<0.001) the strand-breaking effects of camptothecin. The extract was found

to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations 1 μg/ml (p<0.001). Authors concluded that anthocyanins show a preference for inhibition of topoisomerase II (Esselen et al., 2011).

Isolated compounds

In vitro experiments

Lamy et al., (2007) studied the activity of anthocyanidins (aglycons of anthocyanins) in prevention of migration of glioblastoma cells. Because the full resection of malignant glioblastomas is not possible due to their diffuse structure, the development of new projects for cancer therapy and prevention is very important. It was found that aglycons of the anthocyanins: cyanidin, delphinidin, and petunidin acts as potent glioma (U-87) cell migration inhibitors. The most potent was found delphinidin 3-O glucoside (5, 10, 20 µM, (p<0.05, p<0.01) and 50 µM (p<0.001)), with significant differences versus control alone. Since the antocyanins cross the blood brain barrier and pass to the CNS in the concentration range of 192.2±57.5 ng/g after administration of the single dose (Passamonti et al., 2003), it was noted that the chronic intake can significantly inhibit the migration of glioblastoma cells and may affect the results of cancer treatment (Lamy et al., 2007).

Antiulcer activity

Bilberry extract

In vivo experiments

Antiulcer activity of a BE (corresponding to 25% anthocyanidins) was tested in vivo in Wistar rats in experimental models of pyloric ligature induced ulcers, ulcers induced with the use of reserpine, phenylbutazone, ulcers caused by restraint and a local application of acetic acid to the gastric mucosa (Cristoni and Magistretti 1987). The results of experiments were compared with the control groups and groups of rats receiving carbenoxolone and cimetidine. The results of the performed experiments were analyzed by the Mann-Whitney U test or the Dunnett t test (Table 19; Table 20; Table 21; Table 22; Table 23).

Table 19: Activity of V. myrtillus extract in pyloric ligature ulcer model in rats (afterCristoni and Magistretti 1987)

Substances were given 50, 30, 25, 6 hours before and immediately after pyloric ligature (*) p<0.05; (**) p<0.01; Mann Whitney U test.

Table 20: Activity of V. myrtillus extract in reserpine ulcer model in rats (afterCristoni and Magistretti 1987)

Substances were given once a day for 8 days; (**) p<0.01 (Mann-Whitney U test).

Table 21: Activity of V. myrtillus extract in phenylbutazone ulcer model in rats (Cristoni and Magistretti 1987)

Substances were given twice a day for 4 days (*) p<0.05; (**) p<0.01; Mann Whitney U test.

Table 22: Activity of V. myrtillus extract in restraint ulcer model in rats (Cristoni and Magistretti 1987)

Substances were given once a day. (*) p<0.05; (**) p<0.01; Mann Whitney U test.

Table 23: Activity of V. myrtillus extract in acetic acid ulcer model in rats (Cristoni and Magistretti 1987)

Smooth muscles contractility

Bilberry extract

In vivo experiments

Influence of a BE (corresponding to 25% of anthocyanidins on rat stomach muscles to stimulation of post-ganglionic fibres was studied in vitro by Bettini et al. (1986). The extract in concentration of 1- 4 μg/ml enhanced contractility of the rat stomach muscles preparations stimulated intramurally. The effect was potentiated by addition of an ascorbic acid (500 μg/ml). Ganglionic blockade with use of

hexamethonium (20 μg/ml) partially decreased the facilitatory response (Table 24). Authors concluded that the BE enhances the liberation of ACh at the level of the postganglionic nerve endings.

Table 24: Mean percentage increase in the response of the preparation to transmural stimulation (after Bettini et al., 1986)

A) in standard Krebs solution; B) with hexamethonium (20 g/ml); note that the percentage increase is greater in the absence of hexamethonium.

3.1.3. Safety pharmacology

Platelet aggregation

Effects of BE (containing 36% of anthocyanosides) on platelet aggregation in humans was studied by initially by Bottecchia et al., (1987), later by Pulliero et al., (1989) and by Morazzoni and Magistretti (1990) in rabbit and rats.

Bilberry extract

In preliminary in vitro studies Bottecchia et al. (1987) showed 50% inhibition of the clot retraction at concentration of 75 µg/ml of the BE. Moreover, the platelet aggregation induced by ADP, collagen and arachidonic acid was inhibited in a concentration dependent manner (50, 100 and 150 µg/ml). The researchers believed that BE stimulates the release of prostacyclin (PGI2), which has the effect of increasing the concentration of the intracellular cAMP or reducing the level of thromboxane A2 in platelets.

In experiments conducted by Morazzoni and Magistretti (1990) in rabbits and rats in vitro and in vivo, not only the activity of the BEM was tested, but also three principal anthocyanosides occurring in the extract. BE, as dipyridamole and aspirin inhibited platelet aggregation (Table 25). Both cyanidin 3-O

glucoside, delphinidin 3-O-glucoside and malvidin 3-O-glucoside added to rabbit plasma inhibited platelet aggregation induced by ADP, collagen and sodium arachidonate (Table 26).

Table 25: Inhibition of rabbit platelet aggregation by BEM, dipyridamole and aspirin (after Morazzoni and Magistretti 1990)

PRP (Platelet-rich plasma) *IC50 values were determined after 3 minutes incubation of test compound (30 sec for dipyridamole) withy PRP or GFP at 370C. Each IC50 value and its confidential limits (in brackets) were calculated on four to eleven experiments performed with four to six concentrations of inhibitor. **Gel-filtered platelets (GFP) were prepared using Tyrode’s albumin buffer according to Tangen et al., 1971.

Tab 26: Inhibition of rabbit platelet aggregation by cyanide 3-O-glucosoide, malvidin 3-O-glucoside and delphinidin 3-O-glucoside (after Morazzoni and Magistretti 1990).

PRP (Platelet-rich plasma) *IC50 values were determined after 3 minutes incubation of test compound with PRP at 37oC Each IC50 value and its confidential limits (in brackets were calculated on four to eleven experiments performed with four to six concentrations of inhibitor. **Reconstituted extract: a mixture containing 38% of cyaniding 3-O-glucoside, malvidin 3-O-glucoside and delphinidin 3- O-glucoside in the same ratio as in the V. myrtillus extract

BEM significantly and dose dependently (5–400 mg/kg) induced in rats in vivo lengthening of bleeding time. The effect was independent from the influence on the coagulation system. Indeed hematocrit (44.9±0.3; Control 44.7±0.4), Cephotest (18.6±0.7; Control 19.4±0.9) and Normotest (26.7±1.2; control 25.8±1.1) values were normal after 2 hours after a single oral dose of the extract (400 mg/kg) (Table 27).

Table 27: Time course of the effects of single oral doses of BEM (100 mg/kg) on bleeding time in rats (after Morazzoni and Magistretti 1990)

Bleeding time (sec) (Mean±S.E.)

*p≤0.05; **p≤0.01 with Student’s test for paired data. The experiment was performed on groups of 10 rats for each time.

Similar to ticlopidine change in platelet adhesiveness was found in male mouse after single oral administration of BEM at the dose 400 mg/kg 2 hours before the test. In the treated group marked decrease in the number of the adhesive platelets was found compared to control values (Table 28)

Table 28: Effect of single oral dose of BEM and ticlopidine hydrochloride on male mouse platelet adhesiveness (after Morazzoni and Magistretti 1990).

BEM and ticlopidine hydrochloride were administered orally 2 hours before the test (**p≤0.01 with Dunnett’s t test).

3.1.4. Pharmacodynamic interactions

Fuchikami et al. (2006) studied in vitro the influence of the water/ethanol bilberry extract on the uptake estrone-3-sulfate by the organic anion-transporting polypeptide B (OATP-B) in the culture of human embryonic kidney (HEK293) cells. The bilberry extract potently inhibited estrone-3-sulfate uptake (about 75%) by the transporter protein OATP-B expressed in the intestine. This transporter plays a role in absorption of several drugs as glibenclamide, fexofenadine and pravastatin. Hence, the authors concluded, that there is a possibility that the extract could reduce the absorption of these drugs and their therapeutic effectiveness.

Except this single report no animal studies of the bilberry fruit or extracts interactions were identified (Gardner and McGuffin 2013).

3.1.5. Conclusions

Available non-clinical data underline the plausibiliy of the traditional use of bilberry fruits and extracts for the treatment of diarrhoea and peripheral vascular disorders. The therapeutic effects observed in clinical trials may be explained by the results of preclinical pharmacological studies.

As dried ripe fruits of bilberry contain at least 1% of tannins, the traditional use of the dry fruit preparations in diarrhea and topically in mild inflammation of the mucosa is justified on the basis of their astringent properties.

Bilberry is also recommended for the treatment of vascular disorders. Many therapeutic proprieties of bilberry is ascribed to anthocyanin activity. This concerns both the relaxing action on blood vessels like arteries and veins, an improvement of capillaries after microcirculation injury and protective effects of the BE and anthocyanins against oxidative damage. Preclinical studies have also reported their antimicrobial, antinflammatory, antiplatelet and antineoplastic activity.

3.2. Overview of available pharmacokinetic data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof

Absorption

Bilberry extract

In the study of Talavéra et al., (2003) anthocyanins from BEM (88 µM/l) were injected directly into the stomach of anaesthetized rats for 30 minutes and their appearance in plasma was a measure of absorption from the stomach. The absorption of anthocyanins was very diverse (19–37%) and depended on their structure. Of bilberrry anthocyanins the greatest absorption underwent with delphinidin glycosides. After absorption from the stomach glycosides were rapidly excreted into the bile.

Other bioavailability studies in rats demonstrated that after oral administration of a single dose of 400 mg/kg of BEM (mixture of 15 anthocyanosides) they were quickly absorbed with a Cmax=2.5 µg/ml at the plasma peak Tmax value of 15 minutes (absorption rate constant=0.13 /min). After 2 hours after administration no detectable concentrations were observed. According to the authors, despite the low bioavailability of BEM the concentration of 2–3 µg/ml in plasma obtained after administration of a single dose can guarantee sufficient biological effects. There is a lot of evidence that anthocyanidins are enabled in vitro at concentrations of about 2-7 µg/ml (Morazzoni et al., 1991, Morazzoni and Bombardelli 1996).

Isolated compounds

Anthocyanins have a low bioavailability as evaluated by urinary excretion, appear very quickly after ingestion in systemic circulation (peak plasma concentrations appear after 1.5 hours). Several data showed that only less than 1% of the consumed anthocyanins can be detected in the plasma and urine (Felgines et al., 2005; Kay et al., 2005; Nielsen et al., 2003). Intact anthocyanidin glycosides have been detected in plasma indicating that cyanidin-glycosides are absorbed from the digestive tract into the blood stream in their intact glycosylated forms, as the only flavonoids which occur as glycosides in plasma. Indeed, anthocyanins are mainly absorbed as intact glycosides from the stomach and also from the upper part of the small intestine (McGhie et al., 2007; Matuschek et al., 2006; Passamonti et al., 2003; Nurmi et al., 2009). Anthocyanins absorption through the mucosa of the gastrointestinal tract was evaluated in tests carried out in vivo (in situ perfusion model). It was found that 10-22% the anthocyanins administered is absorbed in the small intestine.

This high level of anthocyanins absorption was comparable to the studies of Felgines et al., (2006) (about 20% in the stomach and 7% in the small intestine). It can generally be assumed on the basis of these results that about 10% total the anthocyanins administered can be absorbed from the stomach and small intestine (Talavéra et al., 2004).

Anthocyanin specific transporter is the bilitranslocase, a membrane carrier in the liver responsible for the transport of the dye bromosulphophtalein. This transporter is located on luminal side in the apical domain of epithelial cells of the stomach and intestines. Experimental simulation of antocyanins absorption with the use of intestinal epithelial Caco-2 cells demonstrated high retention of anthocyanins in the cells (up to 60%) but complete lack of their diffusion across the basolateral (serosal) side (Steinert et al., 2008).

This phenomenon may explain why the bioavailability of anthocyanins is so low. When the anthocyanins come into contact with the bacterial microflora of the colon, rapid deglycosylation occurs with conversion to phenolic metabolites (Fleschhut et al., 2006; Keppler and Humpf 2005; Kemperman et al., 2010; Nurmi et al., 2009; Selma et al., 2009). Most anthocyanins do not appear to undergo extensive metabolism of the parent glycosides to glucuronic, sulfo- or methyl- derivatives (Mc Ghie and Walton 2007; Ichiyanagi et al., 2004a, 2004b).

Distribution

Bilberry extract

Lietti and Forni (1976) studied the tissue distribution of anthocyanidins after the administration to rats of the extract of V. myrtillus equivalent to 25% of anthocyanidins dissolved in saline in doses of 25 mg/kg i.p. or 20 – 40 mg/kg i.v. Bilberry anthocyanidins were rapidly distributed in the tissues especially to skin and kidney as compared to plasma (Table 29). The calculated estimated volume of distribution in the rat as extrapolated from the plots was 22 ml, which corresponds approximately to the sum of the plasma plus the interstitial fluid volume.

Table 29: Tissue distribution of anthocyanidins in rat 1 hour after i.p. administration of 200 mg of V. myrtillus anthocyanosides (equivalent to 25% of anthocyanidins). Mean±SE of five animals per group (after Lietti and Forni 1976).

Anthocyanidins seem to have longer persistence in the skin compared with the plasma (Table 29). This long-term persistence of anthocyanidin in the skin may be associated with the observed increase in capillary resistance, once they are not found in detectable concentrations in the blood. (Table 30 adapted from Lietti and Forni 1976).

Table 30: Relationship between plasma and skin level of anthocyanidins and pharmacological activity of V. myrtillus anthocyanosides (200 mg/kg i.p.) on rat capillary resistance. Mean±SE of five rats per group (after Lietti and Forni 1976)

The distribution of bilberry anthocyanidins was tested in mice (Sakakibara et al., 2009). After single oral administration of 100 mg/kg of BE (obtained by use of 80% ethanol acidified with hydrochloric acid) with the total concentration of anthocyanins 67.3 µmol/100 mg of the extract the total plasma concentration has attained a maximum of 1.18±0.3 µM after 15 minutes and afterwards rapidly decreased almost to basal levels after 120 minutes (Table 31).

Table 31: Time-dependent changes of plasma anthocyanins after administration of bilberry extracts (after Sakakibara et al., 2009)

aValues are indicated as the mean (min-max) of plasma anthocyanins, n.d., under the detection limit in this study (<15 nM).

When mice received a diet containing 0.5% BE for 2 weeks, anthocyanins were detected in the liver, kidney, testes and lung. Malvidin-3-glucoside and -3-galactoside were the prevailing anthocyanins (Table 32). Anthocyanins were not detectable in spleen, thymus, heart, muscle, brain, white fat and eye balls. After the long intake of BEs, the levels of anthocyanins are maintained in the liver, kidney, testes, and the lung (Sakakibara et al., 2009).

Table 32: Anthocyanin concentrations in tissues of mice fed a 0.5% bilberry diet for 2 weeks (after Sakakibara et al., 2009)

aValues are indicated as the mean (min-max) of 10 mice, n.d., under the detection limit in this study as follows: <15 nM in plasma, <10 pmol/g tissues. bDel-G, delphinidin glycosides (peak no. 1 + 2 + 4); Cy-G, cyaniding glycosides (peak no. 3 + 5 + 7); Pet-G, petunidin glycosides (peak no. 6 + 8 + 10); Mal-G/Peo-G, malvidin glycosides + peonidin glycosides (peak no. 9 + 11 + 12 + 13 + 14 + 15). Peak numbers refer to Table 26.

Binding of anthocyanins to plasma albumin

Isolated compounds

Cahyana and Gordon (2013) have found that the effect of the structure of anthocyanins (pelargonidin, cyanidin, delphinidin, malvidin) on the affinity to human plasma albumins was pH-dependent. Electrostatic binding of anthocyanins to albumins is favoured at pH 7.4. The more polar glucosides showed stronger binding, while methylation, which reduced the polarity of the anthocyanins, decreased the association. Hydroxyl substituents and glycosylation of anthocyanins decreased the affinity for binding to at lower pH (especially pH 4), but increased the strength of binding at pH 7.4. In contrast, methylation of a hydroxyl group enhanced the binding at acidic pH, whilst this substitution reduced the strength of binding at pH 7.4 (Fossen et al., 1998).

The above experiments have shown that the most common anthocyanins in plasma and tissues were malvidin glycosides, and in second place followed by peonidin glycosides. These and other anthocyanins are converted to methoxyl or glucosyl substituents with subsequent result on the interaction with serum albumin. Since the pH at inflammatory sites is acidic it can determine their beneficial health effects (Cahyana and Gordon 2013).

Elimination

Isolated compounds

Elimination of anthocyanidins proceeds quite fast regardless of the route of administration (Lietti and Forni 1976). After 4 hours, approximately 20% of the administered dose was eliminated in the urine.

After 24 hours, 15% of the intravenous dose and 18% of the administered via the intraperitoneal route was excreted in the bile.

Anthocyanins are readily reactive compounds and therefore are easily deteriorating or reacting with the other ingredients in the mixtures to form colourless or brown compounds. After the passage through the gastrointestinal tract after oral administration they are exposed to different pH and temperature conditions and to different chemical substances. Their different molecular forms are in dynamic equilibrium. Most of the anthocyanins are transformed in the colon to phenolic acids by bacteria (Aura et al., 2005; Keppler & Humpf, 2005). The enzymatic reactions include ring cleavage, hydrolysis of glycosides, glucuronides, amides and esters, and reduction, decarboxylation, demethylation and hydroxylation (Aura 2008; Dall’Asta et al., 2012; Kay et al., 2005; Kay 2006; Manach et al., 2005; Williamson and Manach 2005). On the other hand, anthocyanins given to patients with an ileostomy are accumulating mostly unchanged in the bag (85%), giving little evidence of metabolism after gastrointestinal transfer up to ileum (Kahle et al., 2006). Known mammalian metabolites of anthocyanidins are presented in the Table 33 (after Williamson and Clifford 2010).

Table 33: Known mammalian metabolites of anthocyanidins (after Williamson and Clifford 2010).

The expected B-ring fragments for the common anthocyanidins and their known mammalian metabolites

The metabolites of anthocyanins most likely are the C6, C1-dihydro acids. Microbial metabolites may be much more efficiently absorbed than the parent compounds, because they are there in high concentrations (similar to mM) and the surface of absorption from the colon mucosa is large.

In vitro studies with anaerobic human microflora demonstrated that protocatechuic acid is the most probable main degradation product of anthocyanins (Galvano et al., 2008). Also, studies in humans with the administration of orange juice and administration of cyanidin-3-O-glucoside confirmed that protocatechuic acid is the predominant unconjugated metabolite of anthocyanins (Vitaglione et al., 2007).

The oral intake by volunteers of oats added to a purée of bilberries (glycosides of delphinidin, accompanied by small amounts of malvidin, peonidin and petunidin glycosides) and lingonberries (cyanidin glycosides) (Ek et al., 2006) resulted in urinary excretion of 3-methoxy-4- hydroxyphenylacetic (homovanilic) and vanilic acid and a low amounts of syringic acid (Ichyanagi et al., 2004a, 2004b; Katsube et al., 2003). Urinary excretion of these acids was maximal at 4–6 hours (Nurmi et al., 2009).

3.3. Overview of available toxicological data regarding the herbal substance(s)/herbal preparation(s) and constituents thereof

3.3.1. Single dose toxicity

Herbal preparations

The acute oral toxicity of BE was studied in mice and rats and no signs of toxicity were observed at doses higher than 2000 mg/kg (Eandi, Data on file 1987; quoted after Morazzoni and Bombardelli 1996).

Oral administration of a single dose of BEM (no further details) (3000 mg/kg, equivalent to 1.08 g/kg anthocyanins) to dogs, besides a clear darkening stool and urine, did not result in adverse reactions (Eandi, Data on file 1987; quoted after Morazzoni and Bombardelli 1996). This symptom gave evidence that the absorption and elimination of the preparation are via the kidney.

Dosage used, however, broadly exceeds the human exposure (usually around 5-10 mg/kg). The above studies suggest that the extract used is practically non-toxic.

3.3.2. Repeated dose toxicity

Subacute

No toxic effects were noted in guinea pigs received daily BEM for 2 weeks and rats for 6 weeks at doses up to 43 mg/kg (Pourat et al., 1967, quoted in Upton 2001).

Similarly no abnormalities were found in rats receiving BEM for 4 weeks up to 36 mg/kg i.v. daily or in dogs treated for 13 weeks 12 mg/kg daily. There was, however, a dark blue colour of the urine, skin, eyes, and, in certain cases, liver, kidney and the ovaries (Eandi, Data on file 1987, quoted in Morazzoni and Bombardelli 1996).

Both oral daily administration of the BEM to rats for 6 months (125–500 mg/kg) and to dogs (80-320 mg/kg) did not have a toxic effect or resulted in adverse reactions (Eandi, Data on file 1987, quoted by Morazzoni and Bombardelli 1996). There were no variations of haematological or biochemical parameters. The autopsy also showed no morphological changes.

Chronic Toxicity

No 2 years toxicity studies of BE were identified in the available literature.

Toxicological studies of anthocyanins are limited and are made using extracts from the various species. Human population is naturally affected by exposure to anthocyanins as regularly eating fruits and vegetables.

3.3.3. Genotoxicity

Mutagenicity

Weak mutagenic activity in the Ames test was observed after application of an extract of V. myrtillus (Schimmer et al., 1994). However, the results refer to the extract of the leaves of the plant and not to fruit preparations.

Antimutagenicity

In the study of Malaveille et al., (1996) cyanidin chloride inhibited hepatic S-9-mediated mutagenicity in the Salmonella typhimurium TA98 heterocyclic amines, (for 10 and 45% with 2 and 6 µg respectively) by 2-amino-3 ,8-dimethyl-imidazo [4,5-f] quinoxaline (MeIQx) and (for 5, 10, 20 and 50% with 0.2, 0.6, 2 and 6 µg respectively) 2-amino-1-methyl-6-phenyl-[4,5-b] pyridine (PhIP).

On the contrary, cyanidin, malvidin, and delphinidin were inactive in prevention of the S-9 mediated mutagenicity in S. typhimurium TA98 of the heterocyclic amines, 2-amino-3-methylimidazo [4,5-f] Quinoline (IQ) and 3-amino-1-methyl-5H-pyrido [4,3-b] indole (Trp-P72) from cooked food (Edenharder et al., 1993).

3.3.4. Carcinogenicity

Laboratory and clinical studies provide strong evidence that increased intake of berries which have among others a high content of anthocyanins, may contribute to a reduced risk of certain cancers, especially colorectal cancer (Brown et al., 2012; Stoner et al., 2008; Wang and Stoner 2008).

3.3.5. Reproductive and developmental toxicity

In the study of Pourrat et al., (1967) anthocyanin glycosides from the currants, blueberries and elderberries, when given in doses of 1.5, 3, or 9 g/kg for 3 successive generations did not induce teratogenic activity in rats, mice and rabbits (http://www.inchem.org/documents/jecfa/jecmono/v17je05.htm).

Bhargava (1990) reported that malvidin chloride inhibited the spermatogenesis in langur monkeys receiving 50 mg/kg for 60 days. Testicular and epididymal mass was diminished and the disappearance of Leydig cells was seen. The level of total RNA protein, sialic acid, the acid/alkaline phosphatase in the testes was reduced as the amount of cholesterol in epididymides.

3.3.6. Local tolerance

No data available.

3.3.7. Other special studies

No data available

3.3.8. Conclusions

Toxicological studies are limited.

Tests on reproductive toxicity and genotoxicity were performed almost 50 years ago and are not in accordance with the current standards.

Adequate tests on toxicity, genotoxicity and carcinogenicity have not been performed. No reason of concern is arisen from data of human consumption.

3.4. Overall conclusions on non-clinical data

Non-clinical data on vasoactive influence, microcirculation, anti-inflammatory and antioxidant activity of BE support the traditional use to relieve symptoms of discomfort and heaviness of legs related to minor venous circulatory disturbances.

Results from relevant experimental studies are very limited, but the antimicrobial, astringent and antihadesive properties of the tannins present in the herbal substance can explain the traditional uses of the dried fruit as a decoction for symptomatic treatment of mild diarrhoea and for symptomatic treatment of minor inflammations of the oral mucosa.

Pharmacokinetic studies showed that anthocyanin glycosides are rapidly absorbed from the stomach after ingestion and they enter the central compartment after first pass through the liver. In the liver they undergo methylation and glucuronidation reactions and some of the metabolites are transported to the bile. Anthocyanin glycosides which are not absorbed from the stomach move to the jejunum, and are absorbed to systemic circulation. Anthocyanins that reach the colon are exposed to a microbial transformation with production of phenolic compounds, later they are degraded to aldehydes and phenolic acids.

As there is no valid information on reproductive and developmental toxicity the use during pregnancy and lactation cannot be recommended.

Toxicological studies are limited and were performed using extracts from the various species.

Adequate tests on reproductive toxicity, genotoxicity and carcinogenicity have not been performed.

Oral administration and oromucosal use of decoction of dried bilberry fruit can be regarded as safe at traditionally used doses and adequate duration of use.

4. Clinical Data

4.1. Clinical pharmacology

4.1.1. Overview of pharmacodynamic data regarding the herbal substance(s)/preparation(s) including data on relevant constituents

Platelet aggregation

Bilberry extract

In clinical study Puliero et al., (1989) 30 volunteers of both sexes (mean age 45 years) were involved. They were divided into three groups, of which received orally for 60 days : A) BEM 480 mg/day in 3 divided doses; B) Ascorbic acid 3 g in three divided doses, C) BEM 480 mg/day in 3 divided doses + ascorbic acid 3 g in three divided doses. Blood samples were taken before treatment and after 30, 60 and 120 days after the beginning of treatment. The combination of BEM and ascorbic acid concentration dependently reduced the platelet aggregation induced by either collagen (2-4 µ/ml) or ADP (0.5, 1, 2, and 3 µM), the combination was more active than each product separately. The values of platelet aggregation returned to baseline in the treated groups in 120 days after beginning of the treatment.

4.1.2. Overview of pharmacokinetic data regarding the herbal substance(s)/preparation(s) including data on relevant constituents

Elimination

Isolated compounds

Clinical Studies

In vitro studies with anaerobic human microflora demonstrated that protocatechuic acid is one of the most probable main degradation products of anthocyanins (Galvano et al., 2008). Also, studies in humans with the administration of orange juice and administration of cyanidin-3-O-glucoside confirmed that protocatechuic acid is the predominant unconjugated metabolite of anthocyanins (Vitaglione et al., 2007).

The oral intake of oats added to a purée of bilberries by volunteers (glycosides of delphinidin, accompanied by small amounts of malvidin, peonidin and petunidin glycosides) and lingonberries (cyanidin glycosides) (Ek et al., 2006) resulted in urinary excretion of 3-methoxy-4- hydroxyphenylacetic (homovanilic) and vanilic acid and a low amounts of syringic acid (Ichyanagi et al., 2004a, 2004 b; Katsube et al., 2003). Urinary excretion of these acids was maximal at 4–6 hours (Nurmi et al., 2009).

4.2. Clinical efficacy

4.2.1. Dose response studies

No data available.

4.2.2. Clinical studies (case studies and clinical trials)

Table 34: Clinical studies on humans

Treatment of disorders of the circulatory system (e.g. altered microcirculation and peripheral venous insufficiency) Non Randomised Studies

Dry eye syndrome (DES) (keratoconjunctivitis sicca) – Controlled study

Bilberry OSDI group showed a statistically significant improvement (p<0.01)

4.3. Clinical studies in special populations (e.g. elderly and children)

No data available.

4.4. Overall conclusions on clinical pharmacology and efficacy

Bilberry fruits are traditionally used for diarrhoea and in the conditions of increased fragility of blood vessels and chronic venous insufficiency. Bilberry intake is claimed to produce improvement of microcirculation.

Numerous clinical trials have been carried out since the early 1960s, involving the treatment of vascular fragility and improvement of vision. Modern-controlled, randomized clinical trials have not yet confirmed the benefits of using products containing Vaccinium myrtillus in people with good vision. Such studies have been carried out just 50 years ago, with well-known methodological limitations (Camire 2002).

The systematic review of clinical studies dedicated to improving vision in conditions of reduced light (Canter and Ernst 2004) does not provide clear conclusions. Of the 12 studies with placebo, five studies were randomized. Unfortunately, four of these recent studies gave negative results. The only positive study (Jayle and Aubert 1964) showed an increase in the area of the visual field. In this study patients received, however, a complex product containing BE and beta-carotene.

In most discussed publications the age of the patients varied greatly from young to advanced age. It is known, however, that normal night vision begins to decline in middle age. In addition, it has not been mentioned anywhere at what time of day research was carried out. Rigorous clinical trials are needed to prove the effects of BE on subjects suffering from impaired night vision (Canter and Ernst 2004).

The problem of stability of anthocyanins is essential, as improper storage leads to their degradation. It is especially relevant when carrying out long, ongoing clinical trials.

5. Clinical Safety/Pharmacovigilance

5.1. Overview of toxicological/safety data from clinical trials in humans

No data available.

5.2. Patient exposure

Aside from market presence and data from studies, an epidemiological study has been published concening patient exposure. Bilberry is often used in self-treatment. Recent epidemiological data from Scandinavia estimated that it is the most commonly used herbal product co-used every day with conventional drugs in Norwegian general practice. Almost 40% of patients on anticoagulants used garlic and bilberry. The patients were mostly female, above of 70 years with chronic diseases. Only 50% of patients informed physician on using herbal products together with other treatment (Djuv et al., 2013).

5.3. Adverse events, serious adverse events and deaths

In the clinical trials no serious adverse events were reported, in Biedermann et al (2013) 33% of patients reported mild to moderate flatulence. No cases of death were reported.

5.4. Laboratory findings

Very limited data are available.

5.5. Safety in special populations and situations

5.5.1. Use in children and adolescents

Particular use in children has not been reported. Therefore, the use in children up to 6 years is not recommended.

According to the instruction of the package leaflet of the products marketed in Poland and in Germany the use in children of comminuted dry bilberry fruit is not recommended.

In Germany the use of in children up to 2 years is contraindicated.

5.5.2. Contraindications

None reported

5.5.3. Special Warnings and precautions for use

When used to relieve symptoms of discomfort and heaviness of legs related to minor venous circulatory disturbances, if there is inflammation of the skin, thrombophlebitis or subcutaneous induration, severe pain, ulcers, sudden swelling of one or both legs, cardiac or renal insufficiency, a doctor should be consulted.

5.5.4. Drug interactions and other forms of interaction

Peris et al., (2008) described the case of the patient (age and sex not stated) after the 6-month follow-up. They observed intensification of the oral anticoagulant action expressed by an increased value of the international normalized ratio (INR 16.6, the correct values in patients not treated 0.8 – 1.2). Unfortunately, dosages, duration of treatments, reaction onset and therapeutic indication were not stated.

Paoletti et al., (2011) reported a case about the reduction to 1.6 of INR (time to onset 4 days) in 80 years old woman, who was receiving long-term oral anticoagulant therapy with warfarin (27.5 mg/week for 1.5 years) and received bilberry juice (200 ml/day, indication not reported). Herbal drug was withdrawn, warfarin dosage increased to (32.5 mg/w) and patient completely recovered. According to the Naranjo adverse drug reaction (ADR) probability scale, authors classified the probability that an adverse event is related to drug therapy (possible value of 4). In this case, the reduction activity of warfarin is surprising, since bilberry anthocyanosides, having antiplatelet properties should increase the risk of bleeding with the concomitant use of an anticoagulant therapy. The mechanism of such a reciprocal interaction is unclear.

Aktaş et al., (2011) published a case history of the 77 years old man with hypertension, who consumed for 5 years large amounts of bilberries. The patient for 6 years suffered from hypertension, and because it was found recently that he has an atrial fibrillation and a year before he had a stroke, he received an anticoagulant treatment. In emergency he sought medical assistance because of rectal bleeding and dizziness, which occurred 16 days after introduction of warfarin therapy. His prothrombin time (PT) was 110.5 seconds, INR=15.0, and the activated partial thromboplastin time (APTT) 76.4

seconds. He was given intravenous plasma, but the next day he returned with abundant haematuria and dizziness. His INR was 6.24, and the prothrombin time (PT) was 55.7 seconds. So again he was the subject of further hospitalization.

Despite the presented anecdotal cases it has not been possible to prove a genuine threat of interactions with anticoagulants and antiplatelet agents at the recommended dose of bilberry preparations.

5.5.5. Fertility, pregnancy and lactation

No adverse effects were registered in more than 200 women receiving BEM (no further details) equivalent to 57–320 mg/kg/day anthocyanins for 60 to 102 days during pregnancy for venous complaints, including haemorrhoids in several non controlled studies (quoted after review of Morazzoni and Bombardelli 1996).

However the safe use of BE and BEM in pregnancy and lactation has not been adequately investigated and established. In the absence of sufficient data, the use of the dry extract of fresh bilberry fruit during pregnancy and lactation is not recommended.

No concern has arisen about any malformation in humans, following the consumption of dried bilberry fruit.

No fertility data available.

5.5.6. Overdose

No cases of overdose have been reported.

5.5.7. Effects on ability to drive or operate machinery or impairment of mental ability

No data available.

5.5.8. Safety in other special situations

Not applicable.

5.6. Overall conclusions on clinical safety

Some reports presented in section 5.4.4. do not provide real threat to confirm interactions with anticoagulants and antiplatelet drugs at the recommended doses of bilberry. However, some attention is needed in patients treated with antiplatelet agents or anticoagulants.

6. Overall conclusions (benefit-risk assessment)

The results of clinical data available for bilberry fruit preparations are not considered sufficient to support a well-established use indication.

The systematic review, conducted in 2004 by Canter and Ernst, of clinical trials designed to improve vision in conditions of poor light with preparations of bilberry fruits did not provide clear conclusions. Out of 12 studies with placebo, only five studies were randomized. Unfortunately, four of these recent studies gave negative results. The only positive study (Jayle and Aubert 1964) showed an increase in

the area of the visual field. In this study patients received, however, a complex product containing BE and beta-carotene.

Several clinical studies on disorders of the circulatory system (e.g. altered microcirculation and peripheral venous insufficiency) were performed about 30 – 40 years ago and were not randomized. Hence, despite the conviction of the beneficial effects of the anthocyanins therapy in cardiovascular diseases, the medicinal use of bilberry preparations is based on tradition.

All the requirements for TU (self-medication character, specified strength/posology, appropriate route of administration, period of traditional use, plausibility and safety) are met.

The traditional medicinal use of bilberry dried fruit and bilberry fruit extract has been documented in several medicinal handbooks with indications consistent with the existing pertinent pharmacological experiments performed in vitro and in vivo and it is substantiated by the presence of medicinal products on the European market.

The experimental toxicological data are limited, but given the history of long-term and present use in humans, also in food, there are no safety concerns for the oral or oromucosal use of decoctions from dried bilberry fruit and the oral use of the bilberry dry extract from fresh fruit.

Long-standing traditional medicinal use within the European Union for at least 30 years according to Directive 2004/24/EC is therefore considered fulfilled for the following preparations and indications:

1)V. myrtillus L., fructus siccus (dry bilberry fruit), whole or comminuted

a)Traditional herbal medicinal product for symptomatic treatment of mild diarrhoea. Daily dose in adults and adolescents over 12 years as an herbal tea for oral use: 15 to 60 g, divided in 3-4 single dose of 5 to 15 g in 250 ml as a 10 minutes decoction. Therapeutic area for browse search with TU indications: gastrointestinal disorders.

b)Traditional herbal medicinal product for symptomatic treatment of minor inflammations of the oral mucosa. It is used as a 10% decoction for oromocosal use to rinse the mouth several times daily. Therapeutic area for browse search with TU indications: mouth and throat disorders.

2)V. myrtillus L., fructus recens dry extract; DER 153-76:1; extraction solvent methanol 70% V/V containing 36% anthocyanosides, corresponding to 25% anthocyanidins, in solid dosage forms for oral use the following indications:

a)Traditional herbal medicinal product to relieve symptoms of discomfort and heaviness of legs related to minor venous circulatory disturbances.

b)Traditional herbal medicinal product to relieve symptoms of cutaneous capillary fragility. Single dose: 80 – 180 mg; Daily dose: up to 160 – 540 mg.

Therapeutic area for browse search with TU indications: venous circulatory disorders.

Whole or comminuted dried bilberry fruit

As a general precaution related to the therapeutic indication “for symptomatic treatment of mild diarrhoea”, the product information should include a warning text advising the patient to consult a doctor or a qualified health care practitioner if the symptoms worsen or persist longer than 3 days during the use of the product. In case of the oromucosal use “for symptomatic treatment of minor inflammations of the oral mucosa” the warning should refer to 1 week.

Bilberry fruit cannot be recommended for oral use in children under 12 years of age due to lack of adequate data.

No concern has arisen about any malformation in humans, following the consumption of dried bilberry fruit. They can be used during pregnancy and lactation if clinically needed. No data on fertility is available.

Fresh bilberry fruit dry extract; DER 153-76:1; extraction solvent methanol 70% V/V containing 36% anthocyanosides, corresponding to 25% anthocyanidins

The recommended duration of use of the fresh bilberry fruit dry extract “to relieve symptoms of discomfort and heaviness of legs related to minor venous circulatory disturbances” or “to relieve symptoms of cutaneous capillary fragility” is 4 weeks. However, if the symptoms persist for more than 2 weeks during the use of the medicinal product, a doctor or a qualified health care practitioner should be consulted.

The following warnings are included in section 4.4. of the monograph on bilberry dry extract: “If there is inflammation of the skin, thrombophlebitis or subcutaneous induration, severe pain, ulcers, sudden swelling of one or both legs, cardiac or renal insufficiency, a doctor should be consulted.”

Bilberry dry extract cannot be recommended for oral use in children and adolescents under 18 years of age due to lack of sufficient safety data.

Safety during pregnancy and lactation has not been established. In the absence of sufficient data, the use during pregnancy and lactation is not recommended. No fertility data is available.

On the basis of the available information anthocyanins are considered by the HMPC as contributing to the activity of the V. myrtillus L., fructus recens dry extract (DER 153-76:1; extraction solvent methanol 70% V/V containing 36% anthocyanosides, corresponding to 25% anthocyanidins) and therefore they might be used as active markers. Improper storage leads to their degradation.

A European Union list entry is not supported due to lack of adequate data on genotoxicity.

Annex

List of references