Fraochan

“That Which Grows Amongst the Heather”

DEVELOPING A SUSTAINABLE BLAEBERRY INDUSTRY FOR

SCOTLAND

Heather J GriggsDissertation, BSc (Hons)

Herbal MedicineNapier University, Edinburgh

May 2007

2

Acknowledgements:

Richard Constanduros (Highland Natural Products)Douglas Hardie (Highland Natural Products)

Graham Strachan (Highland Council)Fiona Sinclair (Project Blaeberry)Sarah Allen (Highland Council)Anne Thompson (Ella Drinks)

And

With grateful thanks to Liz Craigen without whose support and generosity thisresearch would have been impossible.

3

Dedicated to my dear friend and classmate,

Arthur Clibury.

You will be profoundly missed.

4

Contents

Summary of Work 1

Chapter 1.1: Vacciniummyrtillus in European History

2

Chapter 1.2: Vacciniummyrtillus in Scottish History

5

Chapter 2: Vacciniummyrtillus (L.) Botany

7

Chapter 3.1.1: ChemicalComposition of Vacciniummyrtillus

11

Chapter 3.1.2: AntioxidantProfiles and Recent Research

15

Chapter 3.2: BiomedicalResearch: Recent Trends andNew Horizons

18

Chapter 4.1: The Role ofVaccinium myrtillus in the WildFood Industry

22

Chapter 4.2: Vacciniummyrtillus Crop Trials

26

Chapter 4.3: Harvesting andProcessing

30

Chapter 5: Discussion andConclusion

34

References 40

Appendix One:Bibliography

51

Appendix Two:Reproduction and MycrorrhizalDependence

65

5

Appendix Three: FlavonoidAnthocyanin Chemistry

68

Appendix Four: BlaeberryLeaflets

72

6

Summary of Work

Background: Used for millennia worldwide as a food and medicinal plant, the

berries of Vaccinium myrtillus L., commonly known as bilberries, or blaeberries in

Scotland, have drawn increasing interest from the medical and health industries

since scientific studies were first performed on the extracts in the 1950s. More

recent studies on the antioxidant properties of bilberry anthocyanins have shown

potential for medical applications ranging from rheumatoid disorders to vascular

disease, cancer to diabetes. Whist the bulk of the world’s blaeberries are now

harvested in Scandinavia and Eastern Europe, recent investigations show that

France, Germany, Portugal, Russia, America, Canada, Japan, and Australia are

investigating sustainable sources of this valuable crop. Most Scottish fruit

producers have shunned any attempt to grow or harvest native blaeberries, while

Finnish companies harvest millions of tons per annum. Purpose and Methods:

This research will take a multi-disciplinary approach to the question of developing

a blaeberry industry in Scotland, covering such diverse areas of study as

ethnobotany, botany, biochemistry, medical research, funding and legislation,

non-timber forestry products, sustainability, industrial research, and harvest and

production methods already in place in other countries. Using a thorough

literature review, information on industrial processes, government reports, and

information gathered from those involved in recent blaeberry trials, this paper will

attempt to outline the possibilities and problems with industrial-scale harvesting

and processing of blaeberries in Scotland. Finally, this work will attempt to

provide a solid basis for future research into blaeberry production in Scotland and

the study of Scottish blaeberry antioxidants.

7

Chapter 1: Introduction

1.1 Vaccinium myrtillus in European History

Humans have been using Vaccinium species as food and medicinal plants

for many millennia. In Europe, wild cranberry (Vaccinium oxycoccus) residues

were found at a Bronze Age archaeological site in Denmark (Trehane 2004, 15),

and Vaccinium spp. pollen was found in the digestive tract of the Tyrolean

Iceman discovered in the Alps in 1991 (Dickson et al. 2000, 1845). Vaccinium

myrtillus (L.), widely known as bilberry, or blaeberry in Scotland, is the most

commonly found Vaccinium species in the wild in Europe (Trehane 2000, 18). In

the13th century, abbess and herbalist Hildegaard of Bingen (Figure One) claimed

in her herbals that blaeberry fruits induced

menstruation, and the 16th century German herbalist

Hieronymous Bock asserted that blaeberries could

be used to treat bladder stones, lung, and liver

disorders (Trehane 2004, 20). The first Amsterdam

Pharmacopoeia (1636) also includes an entry for

Fructus Myrtilli and a recipe for Oleum Myrtillorum

(Koning and Arnold 1961). Curiously, Morazzoni

and Bombardelli (1996, 4) claim that it was not

named in many European herbals before the 16th

century, but they do note, however, that the berries

were used traditionally in many European countries for diarrhoea, dysentery,

haemorrhoids, gastrointestinal inflammation, scurvy, and urinary complaints.

Decoctions of the leaves were also used internally for diabetes and externally for

inflammation of the eyes and mouth, infections, and burns (ibid.).

In Britain, blaeberries have also been in use for thousands of years.

Found on upland moors and in woods in areas as diverse as Somerset,

Derbyshire, Pembrokeshire, and throughout Scotland, the berries of Vaccinium

8

myrtillus are known by a variety of names across the British mainland (Figure

Two).

Common Name Region/County

Blaeberry Scotland, Cumbria, Lancastershire, Northumberland, Shropshire, Yorkshire

Blueberry Cumbria, Yorkshire

Brylocks Scotland

Hartberry Dorset, Somerset

Hurtleberry Devon, Somerset

Hurts Cornwall, Devon, Gloucestershire, Hampshire, Surrey, Sussex, Pembrokeshire

Whortleberry Somerset, Wiltshire

Whinberry/Wimberry Cheshire, Derbyshire, Gloucestershire, Herefordshire, Shropshire

Figure Two: Common names for Vaccinium myrtillus in Britain(After Trehane 2004)

Blaeberry remains have been found at diverse archaeological sites in

Britain including a Neolithic burial cairn in Sutherland (Dickson and Dickson

2000, 63) and near a Roman fort in Carlisle (ibid, 123). In British historical

literature, Culpepper makes mention of blaeberries in his famous herbal of 1653

(1981, 19):

It is a pity they are used no more in physic than they are. The blackbilberries are good in hot agues, and to cool the heat of the liver andstomach; they do somewhat bind the belly, and stay vomitings andloathings; the juice of the berries made in a syrup, or the pulp made into aconserve with sugar, is good for the purposes aforesaid, as also for an oldcough, or an ulcer in the lungs, or other diseases therein.

Maud Grieve (1971), in whose early 20th century classic A Modern Herbal

blaeberry holds an entry, notes that the leaves can be used for urinary tract

disorders and for diabetes, whilst the fruits are good for diarrhoea and

‘discharges’. However, in all of the 19th century materia medicae (Murray 1825;

Duncan 1829; Craig 1879; and Leonard 1892) available for examination by the

author, blaeberries seem to have disappeared completely. C. Pierpont Johnson

(1862, 163), who includes a section on blaeberries in his mid-19th century ‘A

9

Treatise Upon the Principle Native Vegetables Capable of Application as Food,

Medicine, or in the Arts, and Manufactures’, comments that, at the time,

blaeberries were picked and consumed in large quantities in the rural areas of

Britain where they grew most abundantly, but were ‘less esteemed’, especially in

London, after the introduction of the cranberry from the Balkan states.

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1.2 Vaccinium myrtillus in Scottish History

Being a country made up largely of heath land and open mixed forest, it

seems likely that Scotland would have always produced an abundance of

blaeberries for use by its animal and human populations. The Gaelic name for

Vaccinium myrtillus is fraochan from the word fraoch, or heather. It is listed in

MacBain’s Etymological Gaelic Dictionary ([online] MacBain1982) as meaning

‘heather-protector’, after its association with the top part of a walking shoe. Also

known as fraoch nan curra-bhiteag and fraochan caora-mhiteag (Comhairle nan

Eileen Siar 2007), fraochan can be loosely translated as ‘that which grows

amongst the heather’. Camilla and James Dickson’s Plants and People in

Ancient Scotland (2000) catalogues archaeological remains of blaeberry found

various sites around Scotland including a Neolithic site in Embo, Sutherland that

yielded two carbonized berries (Dickson and Dickson 2000, 63), a 12th century

site in Perth (ibid. 180), 13th century sites in Aberdeen and Paisley (ibid, 189,

196), and a 14th century site in Elgin, which contained a vessel with blaeberry

seeds in it (ibid, 190). Examination of the initial archaeological reports from the

Soutra Hospital Project (SHARP) excavations in the Scottish Borders (Moffatt et.

al. 1988; 1989) revealed no Vaccinium remains that could be strictly attributed to

human usage, but more recent excavations have revealed remains which

principle archaeologist Dr. Brian Moffatt attributes to usage by locals for

treatment of intestinal parasites (Vickers 2005).

Despite the ubiquitous presence of blaeberries in the Scottish landscape,

there are not as many historical or cultural references as one might expect in

Scottish literature (Sinclair 2000, 3). In her Millennium Award funded Blaeberry

Project (2000), Fiona Sinclair lists only a handful of references to blaeberries in

Scottish literature from the 15th through the 19th centuries. The Flora Celtica

database ([online] RGBE 2001), compiled by botanists at the Royal Botanic

Garden Edinburgh, also contains a small number of entries with reference to

11

historical blaeberry use in Scotland including uses as a dye, and for food and

medicine. In her book Healing Threads: Traditional Medicines of the Highlands

and Islands, Mary Beith (1995, 206) catalogues some common medicinal uses

for blaeberries including to ease dysentery, diarrhoea, and as a tea for dissolving

kidney stones. Allan and Hatfield (2004, 123) reiterate these uses, and add that

the berries were also used for cold symptoms and sore throats. Likewise,

Sinclair (2000, 4-5) quotes a number of historical sources from the 18th to the 20th

centuries that proclaimed the usefulness of Vaccinium myrtillus for ‘fluxes’, or

bleeding from the orifices, diarrhoea, and kidney stones. Perhaps the dearth of

historical references to blaeberry use can be partly attributed to the fact that they

were such a part of the landscape that their use was taken for granted (Sinclair

2000, 3). The author, however, suspects multiple forces at play, including the

economically, politically, socially, and linguistically fractured landscape of

Scotland. Although blaeberry jam was reportedly first imported to the court of

James V of Scotland in 1513 by the cooks of his French bride (Trehane 2004,

18), it seems more than likely that the peoples of Scotland have used blaeberries

in syrups and tinctures, honeys and preserves for at least as long as they have

been settled here.

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Chapter Two: Vaccinium mytillus (L.) Botany

2.1 Botany and Ecology

The genus Vaccinium, belonging to the family Ericaceae, contains 450

species that are found across the globe. Of these, six can be found in the wild in

Europe. In his definitive book The Genus Vaccinium in North America (1988,

130), Vaccinium expert S.P. Vanderkloet identifies Europe’s native low-bush

blueberry, Vaccinium myrtillus (Linneus) as follows:

Shrubs (10) 17 – 45 (60) cm high, rhizomatous, forming open colonies.Twigs green, conspicuously 3-angled, glabrous. Leaves broadly ellipticalor ornate, 7 – 11 mm wide, 19 – 27 mm long, green, laxly glandularbeneath; margin serrate. Calyx continuous with pedicel, green andglabrous; lobes very small or absent; margin of the calyx tube merelysinuate. Corolla globose, pink, cream, or greenish – white, 5 – 7 mmwide, 3 – 5 mm long; lobes very small. Filaments glabrous, anther sacsawned; pollen tetrads 34 – 38 µm in diameter. Berry purple, black, usuallynot glaucous, 7 – 9 mm in diameter; nutlet approximately 1 mm long.Chromosome number 2n = 24.

Figure Twohttp://www.ag.uidaho.edu/sandpoint/images/Bilberry%20(Vaccinium%20myrtillus).jpg

13

Figure Threehttp://www.treesforlife.org.uk/images/blaeberry2_200.jpg

Lang (1987, 148) provides a detailed description of the habitat of

Vaccinium myrtillus as it is found across the British Isles. For northern areas, he

explains that blaeberry is:

…abundant on heaths, moors, and oak, birch, and pinewoods on acidsoils throughout northwest Britain at altitudes up to 1200m. The dwarfshrub zone composed of bilberry and cowberry, is one of the mainfeatures on the mountains above the heather zone and below the opensummits, and it may form isolated island communities in sheltered, well-drained places on otherwise exposed bare tops especially where the rocksbreak the wind. They are a characteristic feature of the Cairngorms above1000m although they are absent from patches where the snow lives late inthe season.

He continues (1987, 149):Bilberry can tolerate very dry conditions and extremely acid peat, oftengrowing with cowberry, and it can tolerate shade better than heather, so itpersists on wood margins. It probably reaches maximum growth andreproductive performance in open pinewoods such as those in the SpeyValley and it always fruits best with a little shelter.

Although blaeberry is able to grow at altitudes up to 1000m, it prefers drier, acid,

and shady environs, like those found in the open birch and pine woodlands of the

Caledonian Forest (Featherstone 2002) (See Figure Four).

14

Figure Four: Blaeberries growing in Clashwood, Ross and CromartyTaken by the author, July/August 2006

It likes to grow on the forest floor over old tree trunks and rocks and on some

moors and heaths it can be an indicator of woodlands having been there

previously (ibid.). Whilst blaeberry is plentiful in the north and west of Scotland, it

has declined over the past 300 years due to the destruction of Scotland’s native

woodlands and over-grazing by large-scale sheep husbandry (ibid.).

Vaccinium myrtillus plants are usually pollinated by bumblebees (Bombus

ssp.) or wasps, but may also self-pollinate to some degree (Featherstone 2002).

In the field of horticultural science, there are ongoing, international studies of

pollination by various insects within the Vaccinium genus, and these are easily

accessed via the internet from websites such as the International Society for

Horticultural Science Acta Horticulturae site (http://www.ishs.org/acta/index.htm).

The berries themselves develop soon after pollination in May (the author has just

seen the first berries of 2007 on May 1st) and usually ripen by the end of July or

15

early August. Vaccinium myrtillus, like some other fruiting trees and shrubs, are

subject to masting1 cycles of 3 to 4 years (Selas 2000, 424), a process that may

have some impact of blaeberry cultivation.

Reproduction methods are the subject of a good deal of research on

Vaccinium myrtillus and many of these studies address the matter of sexual

versus clonal reproduction. Sources (Featherstone 2002; Nuortila et. al. 2002;

Vanderkloet and Hill 1994) suggest that Vaccinium myrtillus does not have a

successful rate of sexual reproduction because seed germination can vary

considerably depending on the environment, the time in the season, and whether

the seeds have passed through the digestive system of a foraging bird or

mammal. A 1994 study of Canadian soil samples by Vanderkloet found that

seeds from this genus were underrepresented in the seed banks of soils where

they are found in North America (Vandekloet and Hill 1994, 56) even where the

plants were growing abundantly. He hypothesized that loss of seeds may be

taking place through dispersal by birds, between seed deposition and

establishment in the soil, through germination, or because of fungal

decomposition (ibid.). Contrary to earlier studies (as cited in Vanderkloet and Hill

1994, 57), a recently published study by Honkavaara et. al. (2007) found an

increase in rates of germination for Vaccinium myrtillus seeds when they were

passed through the digestive systems of thrushes (Turdus spp.). They found that

germination rates increased for ‘passed’ seeds except those from berries picked

very early in the season (Honkavaara et. al. 2007, 15). Variation in seed

germination rates also occurred between sample-years, leading them to

hypothesize that that there is potential variation in germination success within the

berry season itself, between crop years, and when seeds are eaten by birds or

foragers (ibid., 15 – 16). Clearly there is scope for continued research in this

area. (See Appendix Two for more information on blaeberry reproduction and

ecology).

1 In some plant species, individuals store up energy for two or more years, inorder to produce a large seed crop in one season. When all of the plants in apopulation are synchronised to perform this mass seed production in the sameyear, it is called masting (Selas 2000, 423).

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Chapter Three: Biochemistry and Pharmacological Applications

3.1.1 Chemical Composition of Vaccinium myrtillus (L.)

Jean Bruneton (1999, 361 - 362) lists the chemical composition of bilberry

fruits and leaves in his comprehensive treatise on plant chemistry,

Pharmacognosy, Phytochemistry, Medicinal Plants:

[Blaeberries] are rich in water…, sugars…, and organic acids. Phenolicacids, flavonoids (hyperin…,quercitrin), proanthocyanidins…,andmonomeric flavan-3-ols (catechin and epicatechin) have been identified.The anthocyanin level in the fresh fruits is about 0.5%. These glycosides,about fifteen of them, are C-3 O-glucosides, O-galactosides, and O-arabinosides of cyanidin, peonidin, delphinidin, malvidin, and petunidin.

The bilberry leaf contains phenolic acids, flavonoids…, and tracesof quinolizidine alkaloids… . It is rich in proanthcyanidins and catechin (upto 10%) and in the 1950s, hydroquinone and arbutin were isolated. Lateron, it was not possible to characterize them again.

In fact, knowledge of Vaccinium myrtillus berry and plant chemistry has improved

much in the last two decades due to economic interest and advancing chemical

assay techniques. Morazzoni and Bombardelli (1996, 6) report the presence of

glucosides, galactosides and arabinosides of delphinidin, cyanidin, petunidin,

peonidin, and malvidin in a purified Vaccinium myrtillus berry extract called

Myrtocyan®, manufactured by Italian phytochemical giant Indena. They also cite

an earlier German study, which found 109 constituents in the berries including

aliphatic alcohols, aldehydes, ketones, terpene derivatives, and aromatic

compounds (Morazzoni and Bombardelli 1996, 5). According to various

European studies (as cited in Morazzoni and Bombardelli 1996, 5) the leaves

have been shown to contain quercetin and its glycosides, (+)-catechin, (-)-

epicatechin and their gallates, catechic tannins, iridoids, derivatives of cinnamic,

chlorogenic, and caffeic acids, and trace amounts of arbutin and hydroquinone

(ibid.).

17

Isolation and identification of flavonoid anthocyanins2 from blaeberry fruits

has been a subject of considerable research since the 1990s because they are

well known to be highly antioxidant. Blaeberries are also known to contain the

highest quantity of anthocyanins of all berry varieties, with a ratio of 30:36:13

parts for cyanidin, delphinidin, and malvidin 3-O-glycosides, respectively (Du et.

al. 2004, 60). Two papers from issue 49 of the 2001 Journal of Agricultural and

Food Chemistry examined the identification of anthocyanins in Vaccinium

myrtillus by two different methods of high-performance liquid chromatography

(HPLC)3, the most common method for identifying anthocyanins. Dugo et. al.

(2001) used a technique called electrospray ionization mass spectrometry (ESI-

MS) combined with the HPLC method to examine anthocyanin extracts from

various berries to elucidate differences between them. This technique confirmed

the identification of 14 anthocyanins in Vaccinium myrtillus including 3-O-

arabinosides, 3-O-glucosides, and 3-O-galactosides of cyanidin, delphinidin,

peonidin, petunidin, and malvidin (Dugo et al. 2001, 3989), as reported by

Bruneton (1999) (See Figure Seven).

Figure Seven: Anthocyanins Identified in Blaeberry

delphinidin-3-galactoside

delphindin-3-glucoside

cyanidin-3-galactoside

delphidin-3-arabinoside

cyanidin-3-glucoside

cyanidin-3-arabinoside

petunidin-3-glucoside

petunidin-3-galactoside

peonidin-3-galactoside

petunidin-3-arabinoside

peonidin-3-glucoside

malvidin-3-galactoside

malvidin-3-glucoside

malvidin-3-arabinoside

After Dugo et. al (2001), 3990.

2 For a detailed explanation of flavonoid anthocyanin chemistry, see AppendixThree.3 High performance liquid chromatography (HPLC) is a commonly usedtechnique for separating, identifying, and purifying many chemical compounds.See HPLC: A User’s Guide at http://www.pharm.uky.edu/ASRG/HPLC/hplcmytry.html.

18

This research is significant because it helps with the formation of a library of

anthocyanin fractions for production as commercial standards and with the

identification of anthocyanins in other matrices (ibid.).

Nyman and Kumpulainen (2001, 4183) used HPLC in combination with

diode array detection (DAD) to discern the anthocyanidin content of several

berries. They assert that the antioxidant behaviour of anthocyanins is related to

hydroxyl substitutions made on the B-ring, which leads to increased antioxidant

capacity. Thus methods for identifying the various types and quantities of

anthocyanidins present in berries may play a significant role in understanding the

benefits of these foods and the pharmacological agents that might be produced

from them. They make several important points in their discussion. Firstly, they

observe that the quality of anthocyanin standards available for purchase is poor

because they are unstable (Nyman and Kumpulainen 2001, 4185). This may

lead to possible biases in research due to selection and use of a single standard

reference glycoside for calibration, for instance (ibid.). Secondly, as mentioned

earlier, the structure of an anthocyanin is pH dependent and solvent systems for

samples may affect absorption characteristics (ibid.). With their HPLC-DAD

method, Nyman and Kumpulainen elicited an improvement in the sensitivity of

the identification of anthocyanidin peaks on a chromatograph and they

reconfirmed the method over 14 days to insure its repeatability without excess

variation. This kind of pure chemistry is imperative to finding better methods of

identifying and quantifying anthocyanins for use in other kinds of research.

A number of other recent papers have examined the various compounds

found in blaeberry. Zhang et. al. (2004) compared different HPLC methods

(HPLC-DAD and HPLC-MS) in an attempt to develop better methods for

distinguishing between blaeberry anthocyanins. Their conclusion is that HPLC

analysis before hydrolysis is very useful for controlling product quality in

identification and consistency of raw materials, but hydrolysis is better for

quantifying the individual anthocyanidins (Zhang et. al. 691). Du et. al. (2004)

also isolated two anthocyanin sambubiosides using another technique, high

speed counter-current chromatography (HSCCC), which has the benefit over

19

HPLC of high sample loading capacity, no adsorption of the materials to solid-

phase column material, and complete sample recovery, making it an effective

method for producing pure anthocyanins on a laboratory scale (Du et. al. 2004,

63). Witzell et. al. (2003) examined variation in phenolic compounds for

Vaccinium myrtillus leaves collected from an unfertilized Picea abies forest in

northern Sweden. They established that the concentration of certain compounds

(e.g. p-coumeric acid) varied throughout the growing season (Witzell et. al. 2003,

125). Such temporal variation has obvious ramifications for harvest times,

should these phenolics prove to have properties applicable to industry or

medicine. Zadernowski et.al. (2005) classified phenolics from Vaccinium

myrtillus picked in northeastern Poland. They identified 17 phenolic acids in

Vaccinium myrtillus berries, as compared to five or less in cultivated high-bush

varieties (Zadernowski et. al. 2005, 2120). This has implications because

phenolics are known to be antioxidant, and the quantity of phenolics present in

berries is affected by a number of factors including maturity of the fruit, storage

conditions, and processing methods, amongst others (ibid., 2118). There is

clearly scope for more research on this subject using blaeberries from Scotland.

20

3.1.2 Antioxidant Profiles and Recent Research

Free radicals and reactive oxygen species (ROS) are oxidants that arise in

the human body from various sources including mitochondrial respiration, contact

with environmental toxins, drug metabolism, and cell necrosis. They are

cytotoxic and have been shown to create oxidative stress and inflammation in the

tissues when they accumulate (Donaldson et al. 2003; Brown et. al. 2004; Emerit

et. al. 2001; Stringer and Kobzik 1998). In addition to leading to the natural

process of aging, free radicals/ROS can be implicated in a number of

pathological processes including atherosclerosis, nerve damage, Parkinson’s

disease, cancer, and rheumatoid arthritis. Therefore, encouraging the presence

of natural anti-oxidants, like anthocyanins, in the body to help mop up excess

ROS may help to lower risk factors for these diseases.

Over the last 10 years, research has focused on identifying and

quantifying the antioxidant capacity of anthocyanins. This study found at least

ten scholarly articles on anthocyanin anti-oxidant capacity from extracts of

Vaccinium myrtillus berries in that period. Wang et. al. (1997) used an oxygen

radical absorbing capacity (ORAC)4 assay to determine the antioxidant capacity

of 14 available anthocyanin extracts when compared to anti-oxidant standard and

vitamin E analogue, Trolox. They were able to demonstrate that the five

anthocyanidins commonly present in fruits (dephinidin, cyanidin, malvidin,

peonidin, and pelargonidin) have varying rates of ORAC capacity based on the

hydroxylation of their B-rings, whether they were glycosylated, and the type of

glycosylation (e.g. glucose v. rhamnose) for each molecule. The cyanidin-3-

glucoside had the highest anti-oxidant capacity, and pelargonin had the lowest of

all the anthocyanins tested (See Figure Eight).

4 Oxygen radical absorbing capacity (ORAC) is a method for determining theROS inhibition capacity for a number of compounds varying from animal tissue tofruits and vegetables.

21

Figure Eight: Order Ranking of Anthocyanins by ORAC Capacity

Compound Common Name Present in Vaccinium

myrtillus

Percent of Total

Anthocyanins in

Blaeberry

1 cyanidin-3-glucoside kuromanin cyanidin-3-glucoside 8.1%

2 cyanidin-3-rhamnoglucoside keracyanin

3 cyanidin

4 cyanidin-3-galactoside ideain cyanidin-3-galactoside 8.9%

5 malvidin

6 delphinidin

7 peonidin-3-glucoside peonidin-3-glucoside 2.7%

8 cyanidin-3,5-diglucoside cyanin

9 peonidin

10 pelargonidin-3-glucoside callistephin

11 malvidin-3,5-diglucoside malvin

12 pelargonidin

13 malvidin-3-glucoside oenin malvidin-3-glucoside 8.2%

14 pelargonidin-3,5,diglucoside pelargonin

After Wang et. al. (1997), 306; Dugo et.al. 2001, 3990.; Kahkonen et. al. 2003, 1409.

Similarly, Kahkonen et. al. (2003) and Nakajima et. al. (2004) profiled

anthocyanins from Vaccinium myrtillus and other berries and measured their

antioxidant properties. Nakajima et. al. (2004) measured anti-oxidant activity by

DPPH5 radical-scavenging activity. They found that blaeberries have more

dephinidin and cyanidin glycosides than commercially produced high-bush

blueberries (Nakajima et. al. 2004, 244). Kahkonen et. al. (2003) used fresh,

purchased fruits from Finland. They note that there are many variables that can

affect anthocyanin content of berries including varietal and regional diversity,

growing conditions, humidity, temperature, fertilizer use, and stress factors like

disease (Kahkonen et. al. 2003, 1408). They note in their results that the total

amount of anthocyanins present in blaeberries was similar to that found in other

studies, but that amounts of malvidin-3-glucosides varied (Kahkonen et. al. 2003,

1409). This makes it all the more important that berries from different regions

5 2,2-Diphenyl-1-picrihydrazyl EPR spectroscopy is another method for effectivelydetermining the ROS-scavenging capacity of a compound.

22

and environments in Scotland be tested for variation in anthocyanin content and

quantity, and the results compared with other studies.

Likewise, a number of other studies have examined antioxidant and anti-

lipid peroxidation by the anthocyanins and phenolics in Vaccinium myrtillus

berries. Faria et. al. (2005) used field-picked blaeberries (origin unspecified) and

free-radical scavenging ability was measured using DPPH and the ferric-

reducing/antioxidant power (FRAP)6 assay. Significantly, in their study

chemically modified extracts were found to have higher radical scavenging and

anti lipid-peroxidation capacities than pure blaeberry extracts. These results

have potential for maintaining stability in pigments used in the food industry, but

their ramifications for human consumption or health are unknown. Prior et. al.

(1998) and Ehala et. al. (2005) examined antoxidant capacity by phenolic content

(and anthocyanin content in the case of Prior et. al.) in Vaccinium myrtillus

berries from Germany and Estonia. Prior et. al. sampled various Vaccinium ssp.

berries from America and Maine, but ordered their blaeberries from Germany.

They found that the blaeberries and their close relatives the low-bush blueberries

from Canada had the highest antioxidant capacity, but they also concluded that

there was considerable variability amongst all of the Vaccinium spp. samples

(Prior et. al. 1998, 2690) underscoring the necessity to sample regionally in

Scotland and compare these results to other studies. Ehala et. al. (2005), using

fresh berries purchased in Talinn, Estonia, showed that blaeberries were found to

have the highest total phenolic content and antioxidant capacity of all of the

berries they tested. However, all of the studies agreed that much more work

needs to done in order to understand the variability in berry anthocyanins and

phenolics, as this will affect their value as antioxidants in both the food and

phytochemical markets.

6 The ferric reducing antioxidant power (FRAP) assays are a group of assays fortesting the ferric reducing capacity, therefore antioxidant ability, of a number ofbiological fluids.

23

3.2 Biomedical Research: Current Trends and New Horizons

Although the earliest medical studies on bilberry extracts were concerned

with whether they affected eyesight and ocular vasculature, more recent work

has focused on possible applications for the antioxidant properties of Vaccinium

myrtillus berries. Medical and pharmacological research seems to fall into a

number of categories amongst which are pharmacokinetic studies, studies on the

cytoprotective qualities of anthocyanins, and studies on the antidiabetic

properties of anthocyanins. Some of the earliest studies focused on

pharmacokinetics, because bio-availability is crucial for blaeberry antioxidants to

be useful in human health and medicine. Lietti and Forni (1976, 834) found that

blaeberry anthocyanins administered to rats moved rapidly from the blood to the

tissues, particularly the kidneys and skin, where they had a long-acting activity on

the capillaries that lasted beyond when the chemicals were no longer detectable

in the blood. However, low bio-availability in rats has been reported due to

hepatic extraction (Lietti and Forni 1976, 835; Morazzoni and Bombardelli 1996,

17), so the mechanisms for this are not understood. More recently, research has

shown that anthocyanins are well absorbed in the stomachs and small intestines

of rats, (Talavera et. al. 2003, 4181; Talavera et. al. 2004). They also found that

the delphinidin glycosides, which make up approximately 40% of the total

blaeberry anthocyanin content, were better absorbed into the animal tissues than

other anthocyanins (Talevera et. al. 2003, 4181). The bio-availability of

anthocyanins in humans is also unknown (Guohua et. al. 2001; He et. al. 2005).

Guohua et. al. (2001) gave elderberry anthocyanins to a small group of elderly

women and were able to measure the unchanged anthocyanins in blood and

urine by HPLC, with a peak at 71 minutes after ingestion. They found that most

of the anthocyanins had passed out of the body after about 4 hours (Guohua et.

al. 2001, 924). More recent studies of adult males (Kay et. al. 2004) have

concluded that anthocyanins are metabolised into other molecules in the human

24

body, which may contribute to or detract from their bioactivity. Research on

anthocyanin metabolism in the human body is not extensive (Kay 2006;

Morazzoni and Bombardelli 1996, 14), sample groups have been very small, and

methodological problems in previous studies have been raised (Kay 2006, 141),

so while it is still thought that anthocyanins have health benefits, more research

on metabolism and pharmacokinetics needs to be done before the health

benefits of anthocyanin ingestion can be confirmed.

Whilst anthocyanin bio-availability is, in general, unknown, their bio-

activity is the subject of much current research. Bilberry extracts have been

shown to be protective against oxidative stress in rat hepatocytes (Valentova et.

al. 2007), in aged rats with cognitive deficits related to oxidative stress (Kolosova

et. al. 2005), and in human subjects fed a high-fat diet (Kay and Holub 2002).

One recent paper suggested that the antioxidant activity of blaeberry extract

could be the reason for the improvement of fibromyalgia symptoms in sufferers

participating in a small double-blind, placebo-controlled trial (Logan and Wong

2001, 453). Blaeberry extracts have also been shown to have anti-inflammatory

(Lietti et. al. 1976; Morazzoni and Bombardelli 1996) and wound-healing

activities (Morazzoni and Bombardelli 1996), vasoprotective activity (Boulakia et.

al. 2000; Morazzoni and Bombardelli 1996; Lietti et. al. 1976), Anti-ulcer activity

(Morazzoni and Bombardelli 1996; Chatterjee et. al. 2004), insulin-regulatory

ability (Jayaprakasam et. al. 2004), and lipid-lowering activity (Morazzoni and

Bombardelli 1996; Cignarella et. al. 1996). Ironically, the traditional use of

blaeberry to improve dark-adaptation and night vision has never been clinically

proven. Muth et. al. (2000) performed a double-blind, placebo-controlled trial of

blaeberry anthocyanins on the improvement of night vision in young male

subjects with no significant effect. Canter and Ernst (2004) also reviewed a

number of placebo-controlled trials on night vision improvement in healthy

subjects, and found that there was no evidence to support this hypothesis. On

the other hand, other studies (as cited in Morazzoni and Bombardelli 1996; Mills

and Bone 2000) have shown improvements in diabetic retinopathy and retinal

damage in those with compromised eyesight. All of these applications have

25

potential for development as more research is completed on anthocyanins and

the other phytochemicals present in Vaccinium myrtillus.

One particular field of study remains the ‘holy grail’ of medical research,

and that is the effect of phytochemicals, here anthocyanins, on cancer cell

proliferation. Two recent papers on the general subject of anthocyanins and

cancer prevention (Cooke et. al. 2005; Kang et. al. 2003) suggest that

anthocyanins are able to inhibit the growth of cancer cells in vivo, although

Cooke et. al. (2005, 1938) caution that pharmacological potency differences

between different anthocyanins should be established, and tissues in which their

protective ability may be applied should be explored. Olsson et. al. (2004)

examined the inhibition of breast cancer cells by anthocyanins from various fruits,

including the commercial high-bush blueberry, and found that blueberries had

among the highest inhibition effects of breast cancer cells HT-29, although mixed

rates of proliferation were reported. Wisely, they also suggest that the effects on

proliferation may be influenced by the synergistic activity of more than one

compound found in an extract (Olsson et. al. 2003, 7269), an observance that is

not often made in pharmacological studies on plant chemicals. Similarly,

experiments have been performed on a variety of cancer cells to establish

whether Vaccinium myrtillus extracts and other blueberry extracts can induce

apoptosis in these cells. Katsube et. al. (2003) found that delphinidin and its

glycoside extracted from Vaccinium myrtillus inhibited the growth of HCT116

colon cancer cells in vitro by apoptosis and that dephinidin and malvidin and their

glycosides inhibited growth of HL60 colon cancer cells by apoptosis. Yi et. al.

(2005) found that anthocyanin extracts from American commercially grown

blueberries showed the highest anti-proliferation activity in HT-29 and Caco-2

colon cancer cells when compared to other fractions like phenolic acids,

flavonols, and tannins. This is significant because wild blueberries have been

shown to have significantly higher amounts of anthocyanins than high-bush

blueberries (Moyer et. al.2002; Beccaro et. al. 2006), and therefore may prove to

have more effective anti-proliferation activities on the these cells lines. Finally,

Zhao et. al. (2004) found that anthocyanin rich extracts (AREs) from several

26

berries including Vaccinium myrtillus inhibited growth of colon cancer cells HT-

29, but did not inhibit non-tumorigenic cells. This is significant in an environment

like the colon, where high cell turnover plays a role in tissue health.

As a final point, two studies from 2005 focused specifically on how

anthocyanidins and anthocyanins work on a molecular level in COX-27

production and colon cancer (Hou et al. 2005; Lala 2005). Hou et al. (2005)

used anthocyanidins on LPS-induced RAW264 murine macrophages to observe

COX-2 expression in vitro. Their study showed that delphinidin and cyanidin,

had dose-dependent COX-2 inhibition at mRNA and protein levels. This

experiment also showed that this was due to blocking the MAPK-mediated

pathway including activation of inflammation mediator proteins NF-κB and AP-1.

Geeta Lala (2005) studied the bio-availability and chemoprotective activity of

anthocyanin rich extracts (AREs) on an Azoxymethane-induced rat colon cancer

model. She found that, in confirmation of in vitro experiments on colon cancer

cell line HT-29, COX-2 mRNA was significantly down-regulated in rats fed AREs.

The importance of Lala’s research is two-fold: it is the first known study in which

inhibition of colon cancer cell proliferation by bilberry AREs has been shown in

vivo (Lala 2005, 56), and by using AREs, she demonstrates that the ingested

anthocyanins are, in part, responsible for the chemoprotective effect. It remains

unknown whether the anthocyanin aglycones used by Hou et al. (2005) are bio-

available in animals after berry consumption, in the lab they must be produced

chemically. As chemical assays and equipment become more complex and the

molecular mechanisms of disease are better understood, it seems likely that

there will be more novel applications for bilberry extracts within the occidental

and herbal medical establishments.

7 Cyclooxygenase-2 is an enzyme mediator of prostaglandin synthesis in thebody. Prostaglandins perform a number of important duties in the body includingregulating inflammation and protection and repair of tissues.

27

Chapter Four: Vaccinium myrtillus as an ‘Industry’

4.1 The Role of Vaccinium myrtillus in the Wild Food Industry

The Non-Timber Forestry Products (NTFP) industry is a developing

industry in many countries, including the Scandinavian countries and Eastern

Europe, although many would argue that people have been using the forests to

supplement their needs for millennia. This categorization is a catch-all label for a

large number of fungi, wild plants, and plant-related products that are the subject

of growing interest from academics, rural developers, and industries across the

world (Emery et. al. 2006, 1). NTFPs are also occasionally referred to as special

forest products (Molina et. al. 1997) or non-wood forest products (NWFPs)

(Olmos 1999; Saastamoinen 1999), and include the berries, flowers, stems,

twigs, seeds, nuts, or bark (and products made from these) of many plants that

grow in a forested or wild environment. Mushrooms and lichens are also included

in this category. Interest in NTFPs has generated many recent studies by a

variety of international, national, and local groups including the United Nations

and other international development agencies like the NTFP Exchange

Programme for South and Southeast Asia, the United States Department of

Agriculture Forestry Service, the Royal Botanic Gardens at Kew and Edinburgh,

the Forestry Commission, Reforesting Scotland, and other quangos and interest

groups. The current surge in research about NTFPs in the UK is being driven by

government and Forestry Commission interest, even though the NTFPs,

themselves, may have no direct link to forestry (Douglas Hardie, Highland

Natural Products, pers. comm. 24/4/07). For instance, new work funded by

Forestry Commission Scotland and Scottish Enterprise and carried out by

Reforesting Scotland, is currently assessing the possibility of creating a trade

body for the support of NTFP businesses in Scotland8. There is currently too

8 Links to this and a number of other research projects on NTFPs in Scotland areavailable from http://www.forestharvest.org.uk/projects.htm.

28

much research taking place in this subject area for it to be covered in detail in

this work, but it is important to discuss recent NTFP research on blaeberries here

and abroad.

In many European countries, NTFPs are free to the general public

provided people harvest and process them without damaging the forest

ecosystem (Saastamoinen 1999), but increasing use of NTFPs on an industrial

scale has been cause for conflict and discussion in more than one European

country. Sustainability, along with access rights and ownership, are among the

topics that NTFP interest groups are now debating. Recently, a number of

studies have been published about NTFPs across the globe, and many of these

include reference to blaeberry collection. A study for the Russian Far East

(Khaborovsk Region) funded by the Canadian International Development Agency

found blaeberries to be one of the most popular berries harvested in the region,

and that numerous processing companies exist for turning them into jam that is

mainly used for Russian consumption (Sprieszl 2001). Similarly, the USDA

Forestry Service (1999) commissioned a study to investigate the potential for

marketable NTFP products from Alaskan forests. Although this was largely a

paper examining NTFP policies in America and Canada, 70% of the businesses

interviewed for the study were seeking new sources of blaeberry leaf and berry at

the time. Demand quotes from these businesses for Vaccinium myrtillus were

approximated at 10 to 20 tonnes per annum for dried leaf, and 5 to 10 tonnes per

annum dried berry with quoted prices of $3/kg for berry and $3.20/kg for leaf. It

was claimed that the demand for dried leaf was particularly high, which has the

potential of adding value to the crop if harvesting and processing methods for

leaf can be established in Scotland. In Project Blaeberry, Fiona Sinclair (2000)

reports on European and Scandinavian markets, where Sweden and Finland

remain the top producers of wild berries for the export market. Sweden averages

a harvest of 485 million kg of wild berries (including blaeberries, lingonberries,

and cloudberries) per annum, and Finland averages 450 million kg, but can

produce 730 million kg in a good year (Sinclair 2000, 7). It appears that, like in

29

Russia, most of the berries are harvested and individually quick frozen (IQF) for

the jam industry, but that berry concentrates for drinks are also produced (ibid.).

Here in the UK, Vaccinium myrtillus berries as NTFPs are also the subject

of much research. Sanderson and Prendergrast (2004) were commissioned by

Kew Gardens and The Countryside Agency to look at the harvest of ‘wild foods’

in England and Scotland. They report the annual harvesting of ‘whinberries’ in

Shropshire, sales of which are known to top £10,000 per annum (Sanderson and

Prendergrast, 56). They quote a market price of £5.50/kg paid for blaeberries in

Shropshire in the year 2000. They also make some important observances

about the costs and difficulties of harvesting wild foods in the UK, including

problems with an infrastructure, which is largely non-existent in most places, and

the seasonality of crops, which affects collectors and supply to industry. In

Scotland, blaeberries as wild foods are also very much on the research agenda.

Over a decade ago, Agros Associates produced a report for Highlands and

Islands Enterprise outlining the possibilities for harvesting a number of wild plant

products in Scotland. They project the total market outlet for Vaccinium myrtillus

to be 50 tonnes annually for berries and 6 tonnes annually for leaf, for Europe

alone. Notably, since this report was written, the healthcare applications for

bilberry anthocyanins have grown exponentially based on new research into anti-

oxidants, and it would be interesting to recalculate these figures for the current

market.

More recently, Fiona Sinclair’s (2000) Project Blaeberry, funded by the

Millennium Forest for Scotland Trust, had the purpose of studying the

possibilities of inter-cropping blaeberries in a forest environment, where they fruit

best. It remains a very important monograph of Vaccinium spp. research until

the year 2000, although there is little information within on the applications of

such research to the actualities of blaeberry management, harvesting, and

processing in Scotland. The Scottish Executive also funded a 2001 study on the

sustainable development of Scottish plants for industry. Whilst berries in general

are discussed as a possible development area with regards to the food and

drinks industry, the report contains no new information on Vaccinium myrtillus

30

development for any industry, especially herbal medicine. Finally, the Non-

Timber Forest Products Scotland group and their associates continue to sponsor

research9 and seminars on developments in this growing area of production, and

future work on a ‘blaeberry industry’ will certainly require regular communication

with them as projects develop.

9 For recent NTFP research projects seehttp://www.forestharvest.org.uk/projects.htm.

31

4.2 Vaccinium myrtillus Crop Trials

High-bush blueberries have been cultivated in Europe since the

early part of the 20th century, but even today there is very little information on

cultivation of Vaccinium myrtillus from anywhere in the world. In his overview of

Vaccinium ssp. cultivation in Europe, Pliszka (1997) noted that work on blaeberry

cultivation has taken place in Slovenia, but unfortunately no further details about

this research were given. A Ph.D. student at the Swedish University of

Agricultural Sciences, Andreas Akerström, is currently running crop trials on

Vaccinium myrtillus. He is interested in the cultivation of blaeberries because,

amongst other reasons, Polarica AB10 has decided that there is no longer a

future in wild-harvesting berries while the market continues to grow (Andreas

Akerström Profile: http://www.hfon.org/fon/engelska/eng-doktorander.htm). The

author did attempt to contact Mr Akerström about his research, but he has yet to

return any emails, so the outcomes of his study are currently unknown.

The Australian Government recently published a study on blaeberry trials

that they have been running in Tasmania (Australian Government RIRDC 2006).

They had interesting results that may benefit future crop trials here in Scotland.

As there is no Vaccinium myrtillus native to Tasmania, and quarantine rules and

fees are prohibitive for imported plants, they had to germinate seed themselves.

Initially, they had a low germination success rate, even with stratification, so they

instead propagated by cultivating leaf cuttings, buds, and stems, which they

found was not practical for large-scale plantings. Subsequent germinations with

viable seed produced 1000 seedlings for trial at two sites, one in a mild coastal

climate, and one in a cooler area at an altitude of 615m. The soil was acidified

and fertilised, and plants were monitored for 3 years. The plants flowered in the

10 Polarica AB is Europe’s largest supplier of berries and has been in the berrybusiness for 25 years. They have considerable knowledge and expertise in thearea of purchasing and processing of wild berries.

32

third year, when the plants were 32 months old. There was fruit-set at altitude,

but none set in the milder climate. Additionally, the plants at the higher altitude

grew 50% larger than those in the lower, more temperate field. Additional

cultivation information arose from the author of the RIRDC study travelling to

Poland to research the growth and harvest of blaeberry in the forests there. He

discovered from his Polish colleagues that blaeberry was sparse in forests where

deciduous species dominated, which may be the case in Scotland, as well. In

Polish forests, the best blaeberry stands were found in 50% canopy cover and

blaeberry grows relatively slowly there, gaining only 14% cover after 50 years

(Australian Government RIRDC 2006, 26), information that may prove useful for

the cultivation of blaeberry in Scotland.

Another recent study of Vaccinium myrtillus use in the Ukraine

(Konovalchuk and Konovalchuk 2006, 56) found that, there, blaeberries grow

best in pinewoods, mixed birch and pinewoods, and mixed oak and pinewoods.

The Ukranian study estimated crop yields of around 80 kg per hectare, but they

report that other studies have found this to be 110 kg per hectare with roughly

half of that being suitable for harvesting11 (ibid.). They also recommend tree

felling in the winter, when blaeberry plants are dormant, and they advise the use

of organic fertilisers, herbicides, and pesticides for improved husbandry. Parlane

et. al. (2005) studied growth of Vaccinium myrtillus in Pinus sylvestris woodland

in Strathspey in Scotland, with relationship to light levels and herbivorous

browsing. They found that blaeberry cover was best in open stands, but rare in

dense plantings, and that browsing reduced cover suggesting the need to control

herbivores to encourage blaeberry growth (Parlane et. al. 2005, 276). They

determined that blaeberry growth was optimal at an irradiance of 0.35, and they

recommend controlling tree height to encourage establishment (ibid.).

11 There are other studies on crop estimation for Vaccinium myrtillus berries.Ihalainen et. al. (2002) created mathematical models of yield based on currentforestry methods, site, and stand characteristics in Finland which might proveuseful in creating similar predictions for Scottish forests.

33

The author could find no literature to suggest ongoing trials or cultivation

of Vaccinium myrtillus in North America. However, the native wild blueberry

Vaccinium angustifolium is grown extensively in Maine and along the east coast

of Canada into Nova Scotia, providing up to 50% of the total blueberry crop for

North America (Yarborough 2002, 409). Yarborough (2002, 410) surveyed fields

of wild North American blueberries (Vaccinium angustifolium) that had been

cultivated from wild stands, with the intention of creating a better model for

estimating crop yield. He found considerable clonal variation in all of his sampled

locations. He also discovered that by current methods, all estimates of berry

yield were considerably higher than the average yields by nearly 40%. He

attributes this to issues with estimating field cover. Fiona Sinclair (2000) visited

wild blueberry growers in Canada for Project Blaeberry. She found that the

farmers maintained their wild blueberry fields by mowing or burning in a two-year

cycle. This apparently has the knock-on effect of producing more fruit. She also

learned that wild blueberry fields often took up to 10 years to establish by

rhizomatous spread, and that they were generally burnt every 6 years to keep

down weed growth. In 1997, Yarborough noted that 52,800 hectares of wild

blueberries were cultivated in North America. He, like Sinclair, found that adding

bee hives to the fields, reducing weed competition, and developing appropriate

fertilising methods greatly increased wild blueberry yield (Yarborough 1997, 34).

Yet, because of clonal differences within crops, wide variations in fruiting do exist

in wild species, and continuing research into blossoming, fruit set, and berry size

amongst clones is necessary to improving the yield in these crops (Smagula

1997, 113). Certainly, continued study of the cultivation techniques of these

blaeberry cousins growing in America and Canada may provide some insight into

better ways to manage blaeberry cultivation in Scotland.

In 2002 Highland Council and the local enterprise companies in

association with Colin Stirling of HortiCS began a project to determine if certain

berry crops could be established and grown intensively (Stirling 2006, 2) at a

number of sites in the highlands. A three-year crop trial was established at 14

sites. These included Skye, Arisaig, Halkirk, Bonar Bridge, Borgie Forest, and

34

Helmsdale (Sarah Allen, pers. comm. 24/04/07) for cultivation of Vaccinium

myrtillus. Site holders were expected to prepare the site for cultivation and the

plants came from four sources: the Clashnadarroch Forest (Aberdeenshire),

North Sutherland, Alba Trees Ltd. (Borders), and from Finland where they had

been produced by micropropagation. Some blaeberries were planted in an

exposed site, and suffered badly from wind damage throughout the trial. As this

is a forest species, it would make more sense to cultivate them in a forest

environment, and this was noted in the report (Stirling 2006, 4). The report

concluded that blaeberries would take at least 5 years to establish as a crop, but

this author feels that if a comparison is made to Vaccinium angustifolium crops in

Canada, this could, in actuality take as long as 10 years (Sinclair 2000).

Unfortunately, the official report on these trials was very brief and contained no

actual data. Any extended blaeberry project would need to see further

investigation into the trial data. The project coordinator did compile some short

leaflets (See Appendix Four) with cultivation instructions for the Highland Berry

Growers Group, but only extended trials will determine the optimum environment

necessary for effective blaeberry cultivation. The author does not know what has

happened to the sites since the trial was finished in 2005, the contact addresses

the author was given are no longer functioning, and the author has been unable

to reach any of the people involved with the trials. A brief chat via email with Ella

Drinks managing director Anne Thompson revealed that the project had gone

quiet, simply because there was no one currently willing to coordinate the

ongoing research (Pers. comm. 11/04/07). There is plenty of scope for further

investigation, here, however. Extension of this research will allow more time to

find and communicate with the project participants and, perhaps, examine the

data from the trials and visit the sites to see if any valuable information might be

gained from the crop trial study.

35

4.3 Harvesting and Processing

The blaeberry industry continues to grow worldwide because of demand

for the berries for food, beverages, condiments, and medicines. Yet there are

many logistical issues that dominate potential crop development, including

harvesting and processing methods, extract production, and shipping and

marketing, to name a few. Harvesting is an almost universal problem for

industrial scale production of Vaccinium myrtillus berries. It is the greatest

expense in producing the wild blueberry crop in America (Yarborough 2002,

329), where most of it is still collected by hand (Sinclair 2000, 12) using the hand

rake or a similar tool (See Figure Nine).

Figure Nine: Collecting Blaeberries in Clashwood, Ross and CromartyTaken by the author July/August 2006

It is the author’s supposition that this is one reason that berry purchasers and

processors like Polarica AB are no longer interested in wild-harvesting berries;

the cost of buying through agents that have collected berries from individuals at

collection stations (Leven and Segerstedt 2004) has most likely become

prohibitive in a rapidly expanding market. If Scotland is to finally develop

36

Vaccinium myrtillus as an agricultural crop then considerable resources will have

to be put into developing a mechanical harvester that does not crush the fruit,

and can work in a woodland environment. Recent development of a harvester

uniquely designed for the harvest of Bog Myrtle (Myrica gale) in a collaboration

between Cranfield University, Highland Natural Products, and LogLogic has

shown that cooperation between industry and academia can produce new

technology specifically designed for unique harvesting situations. Yarborough

(2002) discusses the progress towards developing a wild blueberry harvester for

North American crops. He reports that the most commonly used mechanical

harvester for wild blueberries is the Bragg harvester (See Figure Ten), but that in

trials this only recovered between 59% and 69% of berries as compared to hand

harvest (Yarborough 2002, 330).

Figure Ten: A Bragg Blueberry Harvester on a Tractorhttp://www.wildblueberries.net/harv.html

However, during recent trials, use of a prototype harvester by Nimco with canoe-

shaped teeth that strip the berries without damaging the plant, eliminated the

problem of crushing of berries that happens with reel-type harvesters. The

picking head was also monitored by a set of sensors that allowed it to move over

hummocks and obstacles, preventing damage to the head, which can often

happen on rocky terrain. This harvester performed as efficiently as hand-

harvesting in most cases, but it was slower than other harvesters because it was

a prototype and not suitable to be monitored for harvesting efficiency

37

(Yarborough 2002, 331). As with the other research areas discussed in this

work, it seems clear that under the right circumstances there is plenty of scope

for successful development of new technology for large-scale harvesting of

blaeberry, even if it is being cropped in a wooded environment.

Post-harvest processing of blaeberries is another area where there has

been significant research. Vaccinium myrtillus berries, and their constituents, are

fragile and must be properly preserved by freezing, drying, or cooking within 48

hours of picking (Leven and Segerstedt 2004). Polarica AB, one of the world’s

largest processors of blaeberries, uses freezing tunnels to freeze the berries that

can then be packed into 500 kg boxes and stored (ibid.) for future sorting or

shipment. This practice is only limited by freezer space and by how rapidly

berries arrive at the processing plant during the short picking season. Berries

can also be individually quick frozen (IQF), a technology also used for seafood

and other foods, spray-dried, heat-dried or cooked, but most of these will require

the processor to provide space for expensive equipment, and a food-standard

production environment. Research in this area is very much focused on

treatments that will improve the quality and ripeness of the fruit, reduce waste

during processing, reduce infection by biological contaminants, and preserve the

quality and efficacy of the juice or extract. Please see the bibliography

(Appendix One) for a more resources on this subject.

Preparing anthocyanin extracts from blaeberry is a complicated and

expensive process that requires several steps and expensive laboratory

equipment:

The first step in the manufacturing process is the biological elimination ofthe sugars by Saccaromyces spp.; the medium recovered from thefermentors is clarified by on-line centrifugation and concentrated undervacuum at low temperature. The purification of this crude extract entailsrepeated treatment with boiling ethanol. Upon cooling of the ethanolsolution, the insoluable anthocyanins precipitate and are recovered bycentrifugation (the free anthocyanidins, organic acids, and other impuritiesremain in solution (Bruneton 1999, 362).

However, more recent techniques like sub-critical water extraction (SWE) are

providing cheaper and more efficient methods for producing anthocyanin

38

extracts. King et. al. (date unknown)12 working through the Los Alamos National

Laboratory in New Mexico have developed a method for extracting anthocyanins

without ethanol or other solvents by using the SWE technique, which alters the

boiling point of the fluid (water) under pressure, changing its properties and

allowing it to extract polyphenolic molecules from berries (King et. al., 3).

Anthocyanin extraction from elderberries (Sambucus nigra) and chokeberries

(Aronia ssp.) by SWE produced equivalent or better extraction of anthocyanins

than by ethanol (King et. al, 4). They conclude that SWE would be a good

technique for extracting anthocyanins cheaply and efficiently, and it would prove

very interesting to see how well the technique worked for blaeberry

anthocyanins. Another recently developed technique for extraction of plant

polyphenolics and antioxidants is supercritical fluid extraction (SFE) particularly

by supercritical CO2. A brief survey of internet-based businesses selling

blaeberry extracts, seemed to indicate that this was a common way to produce

pharmaceutical-grade blaeberry anthocyanins, but a similar survey of scholarly

articles on the Ingenta Sciencedirect website did not being up any relevant

articles (search terms: supercritical fluid extraction + bilberry; supercritical fluid

extraction + anthocyanin). SFE-CO2 has until very recently been a very

expensive way of producing extracts (Douglas Hardie, pers. comm. 25/04/07).

More research into these techniques and other current research will be

necessary as a blaeberry project develops, but it is not possible within the current

scope of study to develop this topic any further.

12 This paper was found on the website of the Los Alamos National Laboratorywebsite, http://scrub.lanl.gov/index2.htm, but its publication date is unknown. Thewebsite was first published in 2004.

39

Chapter Five: Discussion and Conclusion

5.1 Discussion

When the preliminary research plan for this study was undertaken in May

of 2006, it was to encompass a thorough literature review of the subject of

Vaccinium myrtillus cultivation for use in food, drinks, and healthcare, and an

extended monograph of the plant including current research from the last 10

years on its industrial use and cultivation in Europe and Scotland. A year later, it

has become clear that such a project was naïvely well intentioned, but

completely impossible within the time frame and scope established for this

dissertation. The literature alone has taken many hundreds of hours to locate

and sift through, much less to read and analyse. However, the benefits of taking

on such an unwieldy project have also become apparent. Blaeberry research is

on the rise, and so much groundwork has already been done, that it seems

possible that work on formalizing blaeberry production in Scotland could really

begin in earnest if the proper infrastructure were put into place. After much

consideration of the literature presented here, it is possible to create a catalogue

of subjects that should be addressed in order to make blaeberry viable as a

commercial crop.

1. Botany and Ecology

a. Continuing research into several aspects of Vaccinium myrtillusecology will be necessary to effective crop production. This willinclude pollination research for Scottish plants in their naturalhabitat and in test ‘fields’, even if these are wooded. The researchshould examine which insect species pollinate blaeberry in Scottishwoodlands, its levels of self-pollination, and fruiting success ratesbetween kinds of pollination. Studies of the environmental impactblaeberry pollination and fruiting would also prove useful. Thesecould include the effects of temperature, wind, light, soil moistureand acidity, fertilisers and pesticides, weed growth, and evengenetics on successful reproduction.

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b. Continuing research into mycorrhizal associations between Scottishblaeberries and other forest species may help to elucidate therelationship between these species and its effect on successfulgrowth and nutrient uptake. If Vaccinium myrtillus has optimalgrowth in Pinus sylvestris woodlands, is this related to their sharedmycorrhizal association (MacKay 2004)? Would mycorrhizalassociations be necessary to improve nutrient uptake in cultivatedblaeberry? How would crop maintenance (e.g. burning) affect themycorrhiza? How do they regenerate? Perhaps a mycorrhizalspecialist at RBGE or an agricultural college would be interested inpursuing this line of study.

c. Continuing research into reproduction, both sexual and clonal,would be very important to establishing blaeberry as a fruiting cropin Scotland. Recent reports (e.g. Stirling 2006) did not includecoordinated data to show which clones or seedlings wereestablished at which trial site, and of these, which clones orseedlings did best in growth and flower. On the scant evidenceavailable, clonal reproduction seems to be the best candidate forlarge-scale production of plants. Growers would need to know ifclones are more likely than seedlings to successfully establish inlarge scale planting. It would be helpful to know, as well, ifrhizomatous propagation might reduce cost of planting on a largescale. Some of the issues surrounding using a single clone for cropestablishment have been presented in Appendix Two, andmasting behaviour may also be a very big problem with regularannual harvests. Using multiple clones may be necessary. Ifcompanies like Alba Trees have stocks of plants, they may havedeveloped efficient ways of germinating or propagating them.Investigations into which companies have developed methods forpropagating Vaccinium myrtillus will be necessary, as willdetermination of potential problems with genetic inbreeding andmasting behaviours. Finding a nursery willing to take on this areaof the project would be ideal.

2. Chemistry

a. Most of the chemistry research that has taken place on Vacciniummyrtillus extracts has taken place outside of Scotland, in Japan,America, Italy, Sweden, and Finland. Although chemicalexperiments have used a variety of samples from different sources,very little research has been done chemically on Vacciniummyrtillus in Scotland. The Berry Scotland website lists recentresearch into anthocyanin anti-oxidants in Scotland(http://www.berryscotland.com/Recent%20research.htm), and thismight offer a route to finding university departments interested inanalysing Scottish blaeberries for their constituents. Ideally, plants

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and berries should be sampled at different times in the season andtested for their various constituents. One could even comparesamples from different regions of Scotland with samples from othercountries to see if constituents are present in similar quantities.

b. Anti-oxidant research would need to take place on the Scottishblaeberry extracts, because, as the above literature review shows(Chapter 3.1.3), there is considerable spacial and temporalvariation in the antioxidant content of Vaccinium myrtillus berries.The ORAC and FRAP assays are not expensive and mostlaboratories will be able to undertake these assays.

c. It would also be worthwhile to create links with bio-medicalresearchers and perform in vitro and in vivo experiments onpossible health applications for Scottish blaeberry extracts. Thiskind of research is taking place abroad, particularly in Finland,Japan, and America, with increasing frequency. Without this kindof study, Scotland runs the risk of falling behind in developing newapplications for blaeberry extracts and the techniques necessary toproduce them. Napier University School of Life Sciences has anumber of professors and postgraduate students whose expertiselies in antioxidant research and molecular mechanisms ofpathology induced by oxidative stress. There is scope here forlinking with Napier or other universities to undertake some of thisresearch for the benefit of a blaeberry development project.

3. Non-Timber Forest Products

a. Full advantage should be taken of the current trend of fundingresearch into NTFPs. Since Vaccinium myrtillus is likely to growand fruit best as a woodland crop, creating links with forestry andforest owners will be beneficial to running trials, taking samples fortesting, or even wild-harvesting. As many forest owners are nowtrying to add value to their woodlands, using timber extraction,rather than clear-felling, and encouraging blaeberry growth at theoptimum canopy density has the potential to increase the value ofwoodlands. Olmos (1999) noted that, at the time of his research,blaeberries picked in one season from one hectare of Polish forestwere worth nearly the same amount in money as the timberextracted from one hectare of woodland in a whole year.Obviously, market values would need to be established forblaeberry plant products, but this may prove and excellent incentivefor forest owners to work in partnership with a blaeberry cropproject.

b. Legislation and ownership rights for NTFPs need to be examined,as these are not clear at the moment (see

42

http://www.forestharvest.org.uk/owners.htm). It is likely that asystem for regulating the collection of forest ‘crops’ would need tobe implemented, as has been done in England. Decisions shouldalso be made about the mapping out of blaeberry resources inScotland. Surveys of blaeberry yields over a number of years willbe also be necessary to determine mast cycles, optimal fruitingenvironments, and ecological relationships. Sustainable harvestingpractices will also need to be established. Any ongoing blaeberryproject should maintain close links with the NTFP Scotland groupestablished recently.

4. Blaeberry Crop Development

a. Creating a LINK-type project between academia, agriculture, andindustry would be the ideal way to manage development ofblaeberry as an agricultural crop. Some feasibility studies (Sinclair2000) and crop trials (Stirling 2006) have already taken place, butthe data for the trials need to be examined more closely. A numberof areas for study have arisen from past crop trials, including theappropriateness of field sites, propagative techniques, tree cover infields, temperatures during the flowering and fruiting season, soilwater levels, light levels, the use of fertilisers and pesticides oncrops, mycorrhizal interactions, and pollination methods, to name afew. From the analysis of data from recent trials in Scotland, anddata from ongoing crop trials in Sweden and Tasmania, it wouldbecome possible to consider how to move forward with blaeberryas a crop. A mechanical harvester prototype would need to bedesigned, especially if blaeberry were grown as a woodland crop.Processing infrastructure would also need to be developed. Berriesdo not keep more than 48 hours, and during the harvesting seasonpotentially thousands of tonnes of berries per day would need to beshipped from field to processing plant. Industry, including food,drinks, and healthcare might be persuaded to invest in helping withthis research.

b. The author contacted several Scottish businesses via email thatsell or use wild foods in their products, and only one responded.They were also unwilling to share any information about their use ofblaeberries or potential volume needs for Scottish berries in theirbusiness. Unless industry can be persuaded to participate inblaeberry research, it will be very difficult to calculate market valuesfor the crop and weigh those against development costs. It is alsolikely to take five to ten years to develop blaeberry as a crop, sodiscussions about wild-harvesting potential should be taken in themeantime. Those involved in previous research, like Fiona Sinclair,should also be interviewed about their work. The author contactedFiona Sinclair by phone, but has yet to be able to organise an

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interview. Interviews with the Highland Berry Growers Group wouldalso prove very interesting, as would an interview with Colin Stirlingof HortiCS who coordinated the most recent crop trials. To date,the author has been unable to contact Mr. Stirling via email or byphone. Emails to professor Ulla Bäng and Ph.D. student AndreasAkerström of the Swedish University of Agricultural Sciences alsoremain unanswered. Extension of this project will allow time tomake contact with them and possibly find out about their research.

c. Finally, market research will need to be undertaken to identifypotential buyers for Scottish blaeberries. It was originally theintention of the author to include this subject area in thisdissertation but time and space constraints have prevented it fromhappening. Some Scottish food and drink businesses wereapproached, but there are likely to be other types of businesseswho might also be interested including herbal tea producers,healthcare products producers, herbal tincture makers, etc. Itwould also be useful to trace supply chains from field to processorto producer for a number of industries, if permissible. This wouldallow a blaeberry project to establish the range of products thatblaeberries could be used for, and which of these would be themost lucrative.

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5.2 Conclusion

Despite the overwhelming scale of this project, it has been a most

interesting task to examine the state of research on Vaccinium myrtillus in so

many fields of study. There is scope for development in every area, and the right

individual, in cooperation with business, government, and academia will have

every chance of moving forward with a blaeberry development programme for

Scotland. One thing has become very clear from this study: the project needs a

leader, or coordinator. This should be someone who has the right links, and who

is willing to put in the hard work that will be required to follow up the many

strands of evidence uncovered here. Additionally, much more research needs to

be done, especially on the industrial aspects of harvesting, processing, and

extractions. There will need to be consultations with freezer and processor

suppliers and engineers, experts in developing infrastucture, funding

programmes like the Northern Peripheries Project, and perhaps SEERAD or

other governmental departments and quangos responsible for developing

programmes and protocols in agriculture and the countryside. The author

recommends that surveys of Vaccinium myrtillus cropping and ecology start

immediately, even if mapping and collecting are performed on small scale for a

couple of years. One Scottish business that the author spoke to has expressed

an interest in supporting such a venture this summer. Most importantly, the

momentum building behind this project should be sustained. There is growing

media interest, in the form of national radio and print, on blaeberries as a

medicinal and food plant in Scotland. The author has had one interview about

this work, and has had interest from radio for another possible spot this summer.

This interest should be used to push forward a blaeberry project, rather than

letting it fall silent again, as it has since 2005 crop trials finished. It is a project

full of promise, but the work and investment have only just begun.

45

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Appendix Two: Blaeberry Reproduction and Mycorrhizal Dependence

A 1994 study of Canadian soil samples by Vaccinium expert S.P.

Vanderkloet found that seeds from this genus were underrepresented in the seed

banks of soils where they are found in North America (Vandekloet and Hill 1994,

56) even where the plants were growing abundantly. He hypothesized that loss

of seeds may be taking place through dispersal by birds, between seed

deposition and establishment in the soil, through germination, or because of

fungal decomposition (ibid.). Contrary to earlier studies (as cited in Vanderkloet

and Hill 1994, 57), a recently published study by Honkavaara et. al. (2007) found

an increase in rates of germination for Vaccinium myrtillus seeds when they were

passed through the digestive systems of thrushes (Turdus spp.). They found that

germination rates increased for ‘passed’ seeds except those from berries picked

very early in the season (Honkavaara et.al. 2007, 15). Variation in seed

germination rates also occurred between sample-years, leading them to

hypothesize that that there is potential variation in germination success within the

berry season itself, between crop years, and when seeds are eaten by birds or

foragers (ibid., 15 – 16).

Since sexual reproduction is known to not be particularly successful in

Vaccinium myrtillus, research is also focusing on clonal reproduction. Blaeberry

plants have a rhizome system from which new shoots may arise for up to 15

years (Featherstone 2002). This system also allows the plants to survive burning

episodes, as may be the case in forest fires or crop-maintenance practices.

Rhizomatous propagation may also be used commercially as a means of rapidly

generating fruit-bearing plants. However, as with all clonal plants, genetic

diversity and plant reproductive health must be considered. A recent study of

clonal diversity in Belgium (Albert et. al. 2004) took samples of blaeberry from 3 x

3 m plots across a population and sampled them using RAPD procedure. They

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found that genetic diversity within a local population was highly variable and that

Vaccinium myrtillus seems to use a phalanx growth structure in which ramets

belonging to a clone are grouped closely together to form clumps (Albert et. al.

2004, 158). They attribute the genetic variation to a number of factors including

the ‘architectural parameters’ of rhizome formation (e.g. internode length and

branching angle) and ‘environmental heterogeneity’ and they hypothesize that

sexual reproduction within a population can only take place within windows of

opportunity, explaining why seedlings and seed banks are often missing from

Vaccinium populations (ibid. 160).

Nuortila et. al. (2002) also examined reproductive success in clonal

Vaccinium myrtillus plants by examining the pollination patterns of closely

situated plants both by hand and through pollination by Bombus spp. Their

hypothesis, that proximal plants would be closely genetically related and that this

might lower seed production due to inbreeding or self-incompatibility, was found

to have foundation. They found that in northern and southern boreal forests of

Finland pollination was entirely done by bees, whereas in other experiments in

Belgium found a 10% fruit set through self-pollination alone (Nuortila et. al. 881).

Additionally, they found that blaeberries set four times more seed when cross-

pollinated at a 10m distance, and that cross-pollinations produced more fruit than

self-pollinations, as well (ibid., 882). Since studies like the one mentioned earlier

have determined that proximal clonal structures can show high genetic diversity,

this cannot be the only reason for smaller fruit set within short pollination

distances. Furthermore, since reproductive success and genetic diversity will

play a role in the development of Vaccinium myrtillus as a fruit crop in Scotland, a

more detailed study of this area of research is necessary, although not time-

permissible for this particular work. Observation of pollination, genetics, and fruit

sets within local populations and between populations in Scotland, with regard to

the environment and other variables will be a place to start.

It is necessary to mention that Vaccinium species are known to have

mycorrhizal associations with fungi such as Pezizella ericaea and Clavaria ssp.

that infect the roots and increase the uptake of nitrogen and phosphorus from

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acidic soils (Vanderkloet 1988, 25). Vaccinium myrtillus are among the species

that share a mycorrhizal relationship, and some scholars have suggested that

this is the reason that Vaccinium species, in general, are tolerant of heavy metals

(ibid.). MacKay (2004) investigated the effects on local mycorrhizae of managed

burning on a Pinus sylvestris plantation in Inverness-shire. He found that

although he was unable to specifically identify most mycorrhizae morphotypes in

association with the pines, one, Piceirhiza bicolorata, was found associated with

the roots of both Pinus sylvestris and Vaccinium myrtillus. Previously, it was

thought that Ericoid mycorrhizae and the ectomycorhizae associated with Pinus

syvlestris were unrelated (MacKay 2004, 21). This relationship may be one

reason why blaeberries are more successful in the pine woodlands of Scotland

than on the moors. Finally, an earlier study by Turnau et. al. (1992) found that N-

P-K fertilization of oak and pine woodlands in southern Poland reduced the

frequency of root cells colonized with mycorrhizae. This had the effect of causing

the Vaccinium myrtillus population to disappear whilst rapidly expanding other

species like grasses Festuca gigantea and Milium effusum. Although this is a

single study from two decades ago, it underlines the importance of observing and

investigating the mycorrhizal relationship with blaeberries as part of an ongoing

research plan. The most recent issues of the International Symposium on

Vaccinium Culture (Issues 574 and 715) from the ISHS Acta Horticulturae

(http://www.ishs.org/acta/index.htm) also contain new and ongoing research into

Ericoid mycorrhizae and their relationship to Vaccinium culture.

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Appendix Three: The Flavonoid Anthocyanins

Anthocyanins are part of a much larger class of natural chemicals called

flavonoids, a vast group of aromatic plant products whose many sub-categories

include phenylpropanoids, tannins, and quinones (Brielmann 1999, 20).

Flavonoids are a product of the shikimate and acetate pathways and provide

plants with pigments that serve a variety of functions including UV ray protection,

antioxidant activity, and enzyme inhibition (Pengelly 2004, 34). The basic

flavonoid structure is two benzene rings (A and B) separated by a propane unit

(See Figure One). The three-carbon chain is often closed to form a heterocyclic

third ring (C-ring).

Figure One: Flavonoid Structurehttp://www.uky.edu/~dhild/biochem/17/skeleton.gif

Flavonoids are water-soluble and mostly occur in nature as glycosides (Dewick

2001, 150). Research has shown them to be ‘good scavengers of free radicals

due to high reactivities of their hydroxyl substituents in a hydrogen atom

abstraction reaction’ (Korkina and Afanas’ev 1997, 152). Furthermore, they have

been shown to have superoxide inhibition activity through a variety of

mechanisms incuding xanthine oxidase and superoxide dismutase (ibid). They

have also been shown to inhibit prostaglandin up-regulation, inhibit cell

proliferation in tumour cell lines, and have antimutagenic properties in human

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lymphocytes (Korkhina and Afanas’ev 1997, 159). For these reasons, several

classes of flavonoids are important to human health maintenance including the

flavonols, flavones, flavonones, and anthocyanidins (Lila 2004, 306).

Since the second World War, when British RAF pilots claimed that eating

bilberry jam helped improve their night vision (Muth et al. 2000, 164), biochemists

and medical researchers have become increasingly interested in the compounds

contained in bilberries and other red, purple, and blue fruits. Much of this interest

lies in the antioxidant qualities of some of the phytochemicals bilberries contain,

namely the flavonoid anthocyanins. Anthocyanins, whose name is derived from

the Greek words for flower (anthos) and blue (kyanos), are water-soluble

pigments that provide flowers, leaves, and fruits with a wide range of colours

ranging from pink, red, and purple to blue and black. The bright colour and low

toxicity of anthocyanins plays an important role in aiding plant pollination by

insects and seed dispersal, thus reproduction, thorough foraging mammals and

birds (Bruneton 1999, 356). Anthocyanins have a basic flavonoid inner structure

of 3 carbon rings to which a number of sugar molecules may be attached,

particularly at hydroxylated positions 3, 5, and 7 on the A and heterocyclic rings

(See Figure Two).

Figure Twohttp://wwwchem.uwimona.edu.jm:1104/spectra/chime/anthocy.gif

In strong acidic media (pH<3), anthocyanins are stable (and red), but they

become less stable (and blue) in weak acids (pH 4 – 6), and the maintenance of

their colour in living media may be due to a number of other mechanisms

including intermolecular co-pigmentation with other molecules like flavonoids,

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proteins, and pectins (Bruneton 1999, 358). Anthocyanins are also unstable in

oxygen, heat, light, and in reaction with sulfur dioxide (ibid.).

The inner structure of the anthocyanin is called the anthocyanin aglycon or

anthocyanidin. Cyanidin, the most abundant anthocyandin found in nature, is

present in 69% of fruits and 50% of flowers (Evans 2002, 250). Pelargonidin and

delphinidin (See Figure Three) are also very common, ‘virtually ubiquitous’

(Bruneton 1999, 356), but peonidin, petunidin, and malvidin are also known.

Figure Three: Structures of some common anthocyanidinshttp://www.shieldsgardens.com/DLPlace/anthocyanidins.jpg

The anthocyanidins themselves are unstable because their 3-hydroxyl group

makes them very reactive, therefore they are never found in nature

unglycosylated (Bruneton 1999, 357). Anthocyandins may be attached to a

number of different sugars, including glucose, galactose, rhamnose, and

arabinose, and are often known by the number of sugar molecules in their

structure (e.g. monosides, biosides, triosides) (Lala 2005, 16). Their diversity

can be attributed to the varying number of sugar molecules in their structure and

by chemical combination of these sugars with organic acids like phenylpropanoic

or benzoic acid (ibid., 14). It is important to take notice of the differences

between anthocyanins and anthocyanidins and when they occur because this is

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a matter of considerable importance when considering their pharmacological

applications. Similarly, it should be noted that anthocyanin solutions are very

unstable, and can only be kept in nitrogen, at low temperature and in the dark

(Bruneton 1999, 359.). This has obvious ramifications for both their experimental

usage and their pharmacological and food industry applications.