Blueberries and their anthocyanins

Blueberries and Their Anthocyanins: Factors
Affecting Biosynthesis and Properties
Winny Routray and Valerie Orsat
Abstract: Blueberry is one of the most popular fruits in North America and rich in anthocyanins. Its content
in anthocyanins contributes to the health-beneficial effects of blueberry against several chronic diseases including
cardiovascular disorders, neurodegenerative diseases, diabetes, and cancer. This paper summarizes various facts presenting
blueberry as a fruit with huge potential for increased future consumption as a health-enhancing food. Factors affecting
the biosynthesis of the various anthocyanins in blueberries, including agronomic and genetic factors, and the possible
pathways of biosynthesis of the major anthocyanins present in this plant are discussed. The important health-beneficial
effects associated with blueberry anthocyanins, properties of these anthocyanins leading to the beneficial effects, and food
processing parameters leading to the depletion of the amounts of anthocyanins present in the final processed products
are also briefly discussed. Furthermore, the general methods of extraction and analyses that have been reported for being
successfully applied to blueberry anthocyanins are also reviewed.
Practical Application: Blueberries are well known for their nutritional and beneficial health effects, however,
information concerning the physiology behind the blueberry beneficial effects is still lacking. There is little or no
information on the characterization of growing conditions on anthocyanins in blueberries and research is lagging behind
on advanced methods of extracting blueberry anthocyanins.
Introduction
fruit and awareness of its beneficial effects has led to an expansion of the cultivated blueberry industry worldwide. Blueberry
and blueberry products have a wide range of uses. Blueberries are
consumed not only as fresh fruits but also as frozen fruits, in bakery goods, fruit filling, in dried form in muffin mix, or in canned
or preserved form. Processed products of blueberries are widely
popular, especially preserves, syrups, fruit juices and beverages, and
concentrates (Eck 1988). Blueberry anthocyanins are also used as
a natural food colorant (Espin and others 2000) in some parts of
the world (Bridle and Timberlake 1997). Blueberry extract can be
used as a potential prebiotic as well (Molan and others 2009). The
popularity of blueberry with yogurt, and as a part of beverages
with other berries such as cranberries, is expected to further increase the demand in the future. To meet the increasing demand,
breeding experiments and genetic modifications have been considered to obtain higher yields and other desired characteristics in
blueberries. However, blueberry is a seasonal crop for which processing and storage are important steps to maintain the availability
of blueberry benefits year-round. During these processing steps
there is a high possibility of loss of anthocyanins.
Processing studies are taking place to minimize the nutrient
MS 20110705 Submitted 6/6/2011, Accepted 7/28/2011. Authors are with
loss by optimizing and examining the working parameters of the
Bioresource Engineering Dept., Macdonald Campus, McGill Univ., Ste-Anne-deBellevue, Quebec, Canada, H9X 3V9. Direct inquiries to author Orsat (E-mail: different processing steps (Yang and Atallah 1985). In these optimization studies, chemical analysis is an important part convalerie.orsat@mcgill.ca).
sisting of preparation of the sample, extraction and isolation,
Anthocyanins are bioactive flavonoid compounds beneficial
against many chronic diseases. These are mainly consumed in the
form of foods derived from plant sources and blueberry is one of
the fruits which is popular for its taste and richness in anthocyanins.
It is among the fruits that contain high amounts of anthocyanins
(Wu and others 2006) and other polyphenolics, which have been
reported to have good antioxidant properties. Mazza and Miniati
(1993) have reported a range of 25 to 495 mg/100 g anthocyanins for highbush blueberries, but other reports have varied
with subsequent data, which account for lower and higher levels
of anthocyanins. These compounds have other beneficial effects
also, such as antidiabetic, antibacterial, and anticarcinogenic activities. A series of International Symposia on Berry Health Benefits
was started in 2005, especially to discuss the progress in research
on berry consumption and nutritional health effects, held once
in every 2 y, which is highlighting the popularity of this fruit in
North America (Seeram 2008). The popularity of blueberry as a
c 2011 Institute of Food Technologists®
doi: 10.1111/j.1541-4337.2011.00164.x
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 303
Blueberries and their anthocyanins . . .
purification, and finally analysis. This can be done for identification of the compounds at different processing steps, quantification
to detect the extent of loss, extraction from the by-products of
different processing industries to recover the compounds (Lee and
Wrolstad 2004) and adding them back as an additive into the final
processed products. Analytical determinations are also required in
the study of the distribution of the anthocyanins in different parts
of the plant, which assist scientists working in the field of breeding in improving the genetic properties of blueberries. Analytical
studies also help to better understand and modify the pathways
of production of anthocyanins in a plant system, and to study the
effects of various cultivation factors and their optimization. Analysis of different types of anthocyanins present in blueberries with
their development pathways, their relative distribution in different
parts of the plant, and variation according to the different seasons and growing conditions will be important for the continued
development of the blueberry industry.
This review thus focuses on the different aspects of the blueberry industry: varieties available, history, cultivation practices and
factors affecting accumulation of anthocyanins in different plant
parts, depletion of anthocyanins during processing, and the different anthocyanins present in blueberries. Attention is given to
the anthocyanin formation pathways in plants and their potential
health benefits to help better understand the practical importance
of these fruits and their anthocyanins in today’s scenario, and their
preservation and prospects in future research and food market developments. The different methods of analysis of anthocyanins are
discussed briefly. The methods of analysis of anthocyanins applied
in the case of other fruits and plant materials which can potentially
be used with blueberries are also discussed.
Blueberries
Blueberries are categorized under the family “Ericaceae,”
subfamily “Vacciniaceae,” genus “Vaccinium,” and subgenus
“Cyanococcus” (Gough 1994). The Ericaceae family comprises a
large group of plants which are mainly woody shrubs that grow
on acidic soils. The Vaccinium genus includes many popular berries
consumed around the world including blueberries, huckleberries,
cranberries, lingonberries, and bilberries. Vaccinium is speculated
to be derived from the Latin word “vacca” meaning cow, because
wild lingonberry otherwise known as cowberry is abundant in
Sweden, the birth place of Linnaeus (Trehane 2004).
The plants in the genus Vaccinium are dated back to the Cretaceous period, more than 100 million years ago, when they are believed to have developed and differentiated. After the Pleistocene
glaciations, the tropical forms evolved into the temperate forms,
which are now found predominantly in eastern North America.
Plants under subgenus Cyanococcus expanded into the areas cleared
after the ice sheets melted, where they hybridized and spread in
the wild (Gough 1994). The plants expanded further by dissemination of seeds by wild animals in their droppings and by spreading through rhizomes or underground runners. In today’s world,
blueberries are cultivated in North America (Canada and U.S.A.),
China (Wang and others 2010), Europe, and some countries of the
southern hemisphere, such as Chile, Argentina, Uruguay, South
Africa, New Zealand, and Australia (Lohachoompol and others
2008).
There are different kinds of blueberries and each has many
local names. Some of them are wild-growing lowbush blueberries (Vaccinium augustifolium) and cultivated highbush blueberries.
Northern highbush blueberry (Vaccinium corymbosum) is quite well
known, while the rabbiteye blueberry (Vaccinium virgatum, also
known as Vaccinium ashei) also falls under the category of highbush
variety. In many other countries of the southern hemisphere such
as Australia, the southern highbush blueberry is popular, which
is a hybrid of the northern highbush blueberry and the rabbiteye
blueberry. In European countries they have their own version of
blueberries, known as bilberries (Vaccinium myrtillus L.) which belong to same genus and are similar to North American lowbush
blueberries. In this review, we will mainly concentrate on North
American highbush and lowbush blueberries and discuss the other
varieties of blueberries in brief, wherever relevant.
Blueberries have been a part of the traditional European
food habits much before the colonies were established in North
America, and they were also a part of tradition and held with high
esteem by the natives. Blueberries were served with milk, sugar,
and spice. The fresh blueberries were also used in baking. The
method of preservation of blueberries for use in the cold winters
of North America was passed from the natives to colonists and is
still used in different versions. They were either sun-dried or dried
using smoke where sufficient sunlight was not available and also
to decrease the reliability over solar energy. These dried blueberries were used in baking breads and cakes, and the cakes available
now are the modern versions of the traditional cakes made earlier.
Powdered dried blueberries were served as a mix with parched
grain or cereal meal. Also, berries were cooked with meat to add
flavors. There are many recipes of blueberries which have been
passed through generations (Gough 1994; Trehane 2004).
Factors Affecting the Accumulation of Anthocyanins
in Different Parts of the Plant
Cultivation practices are one of the main factors which affect
the concentration level of anthocyanins in fruits and other vegetative parts when the plant is growing. The cultivation practices
of blueberries required for healthy growth of the plant have been
discussed in detail by many authors such as Trehane (2004), Gough
(1994), and Eck (1988). They have discussed different cultivars, the
important factors affecting blueberry cultivation, different types of
diseases affecting the plant, proper site selection, soil requirements,
climatic requirements, pest control, and other agronomic factors.
Sites with slopes, which encourage drainage, and less windy areas
are preferred for blueberry commercial cultivation. Well-drained
and well-aerated soil with minimal required amount of water retention, especially in summer, and proper root anchorage are basic
requirements in terms of soil characteristic, whereas optimum soil
pH is expected between 4 and 5.2 to provide an ideal nutrient
composition for proper growth and fruit-bearing (Trehane 2004).
Factors such as application of herbicides during spring, efficient
irrigation management, and proper application of fertilizers have
played a major role in increasing the production of blueberries in
the last decades. Leaf analysis is generally employed to decide on
the requirement of different minerals and the amount of fertilizers
needed. The requirement of different essential elements as nutrients and micronutrients has been reported, but the relevance of
the nutrient intake in terms of the rate of anthocyanin accumulation is not that clear, and there is no indication of the role of
these nutrients in the biosynthesis of anthocyanins (Gough 1994;
Trehane 2004). Similarly, there is no report of the correlation
between optimized cultivation practices and the anthocyanin accumulation in the different parts of the plant.
Organic farming, which is very much encouraged nowadays, has
been recently encouraged to be used with blueberry cultivation
(Drummond and others 2009). Application of pine needles and
organic manure has been suggested as a useful choice for providing
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Blueberries and their anthocyanins . . .
blueberries the proper amount of nutrients required for growth
and healthy fruit-bearing (Panicker and others 2007). The amount
of anthocyanins and total phenolics accumulated in different cultivars was either more or comparable in the case of organically
grown as compared to conventionally grown rabbiteye blueberries
(You and others 2011). A similar study focused on the effect of different cultivation practices in the case of highbush blueberries and
showed that the total anthocyanin content was significantly higher
in organically cultivated blueberries (S.Y. Wang and others 2008).
This could be a potential area of research in the future and can
contribute to the further development of commercial cultivation
of blueberries.
The season for blueberry cultivation spans May to September,
in most parts of North America. In late July the fruit reaches peak
ripeness with the entire fruit turning blue or black blue; and as the
fruit turns from green to blue, the anthocyanin content of the fruit
increases. The blue color of the fruits has been suggested as the
best criteria of fruit maturity (Hall and others 1972) and decision
making regarding fruit-picking. Fruit-picking generally takes place
during August and September in North America. The different
phases of the blueberry growth cycle vary with the varieties of
blueberry, and the time period of onset of these different phases
also differs with temperature and variation of climatic conditions.
All these factors together affect the total production which varies
from year to year.
The anthocyanins present in fruits and flowers, responsible for
their color in many cases, act as an aid in pollination and seed
dispersal (Harborne and others 1975; Steyn and others 2002).
During fall season the lowering of temperature favors the production of anthocyanins in the senescing leaves. Anthocyanins in
the leaves have been found to be a part of the defense mechanism against photoinhibition, along with other mechanisms in
photorespiration and the cycle of xanthophylls (Hoch and others
2001). Photoinhibition has been defined as “illumination of photosynthetic tissues in excess of the energy utilization potential of
carbon reduction which can lead to a marked decrease in photosynthetic capacity” (Powles 1984). As suggested by many authors,
the light screen hypothesis explains the function of foliar anthocyanins. Hoch and others (2001) extended the light screen hypothesis by proposing that “autumnal anthocyanins protect senescing
foliage from photoinhibitory irradiances, allowing the resorption
of critical foliar nutrients to occur during a period of photosynthetic instability and deteriorating photoprotective capacity.”
As observed in various plants (Nozzolillo and others 1990) and
also in the case of blueberries (Gough 1994; Trehane 2004), the
rate of accumulation of anthocyanin varies with seasonal changes
and their effects on growth patterns, and also with developmental
patterns of different species, varieties, and plant cultivars (Steyn
and others 2002). Accumulation of anthocyanins in the vegetative parts of the plants has been discussed in detail by some
authors along with the factors affecting this accumulation (Hoch
and others 2001; Steyn and others 2002). In response to many
stress factors, other than photoinhibition, such as decreased temperature, nutrient deficiency, and wounding or pathogen attack,
the synthesis and increased accumulation of anthocyanin in plants
has been observed which makes it a part of its built-in defense
mechanism.
The increase in the level of anthocyanins in the vegetative part of
the plant also subsequently affects the level of accumulation in the
fruit. Breeding experiments have either focused on the increase of
production of blueberries, sometimes by optimization of pollen
load (Dogterom and others 2000) or on the increase of the total
c 2011 Institute of Food Technologists®
amount of anthocyanins in fruits; with both approaches there is a
good likelihood of a higher rate of consumption of phytochemicals (anthocyanins) by consumers. As color is correlated to the
anthocyanins present in fruits like blueberries, during some studies with different Vaccinium species; albino fruits have been linked
with a single recessive gene not likely to be preferred (Hall and
Aalders 1963; Draper and Scott 1971; Lyrene 1988; Hancock and
others 2008). Total antioxidant capacity and total phenolic content, which includes anthocyanin content observed in blueberry
progenies, have been found to be moderately heritable (Connor
and others 2002b; Scalzo and others 2005) or in other cases from
moderately to highly heritable (Scalzo and others 2008b). Also,
the variation in anthocyanin content with different species as well
as cultivars is a well-known and established fact (Sapers and others
1984; Kalt and others 2001). During a study by Connor and others
(2002c), significant differences were found in anthocyanin content
and antioxidant activity between the same cultivars grown in different locations and different cultivars grown in the same location,
and also there was a difference in terms of year of harvest between
the same cultivars grown in the same location, proving genotypic
and environmental effects; and the effects of genotype have been
reported to be stronger than environmental effects. Even though
the direct evidence of increase of anthocyanins with progeny is not
experimentally proven in the case of blueberry breeding studies,
a significant increase is possible based on the moderate-to-high
heritability, which might be evident in terms of increased total
antioxidant capacity (Scalzo and others 2005; Scalzo and others
2008a) and careful biotechnological approach, which includes the
tools of micropropagation, genetic engineering, and genetic fingerprinting (Serres and others 1996). To constantly improve the
breeding programs, oriented toward improvement of the product (blueberries rich in anthocyanins), preservation of germplasm
with proper selection, evaluation, and dissemination would be
important steps (Debnath 2009).
To establish a strong genetic base and ensure future use of the
existing genetic resources, a thorough study of the genetic diversity could be helpful. Genes involved in anthocyanin biosynthesis
have been identified and their activities have been traced during
different developmental stages in the case of bilberries (Jaakola and
others 2002). Similar research applied to blueberries could help in
narrowing the research focused on breeding programs specifically
oriented toward the increase of anthocyanin content. Inter simple
sequence repeat markers (a polymerase chain reaction generally
used for amplification of a particular DNA sequence), based on
molecular marker assay of the genomic sequence lying between
adjacent repeating microsatellites, which are repeating sequences
of base pairs of DNA (UN-FAO 2002) for genetic diversity studies, have been developed for lowbush blueberry (Vaccinium augustifolium Ait.), and they have been found helpful in differentiating
among 43 lowbush blueberry clones (Debnath 2009). This can
also be applied in the case of other blueberry varieties and cultivars which could help to build and preserve a gene pool from
which parents with desirable characteristics such as high anthocyanin content could be selected.
Study of the different agronomic factors affecting the cultivation and influencing the level of accumulation of anthocyanins
in fruits is also helpful to provide a better product. During some
studies, the combined effect of genotype and harvest year or time
of cultivation was found to have a significant effect on total phenolic content, which includes anthocyanin. Hence, the study of
the response of germplasms over several generations was found to
play an important part in the variation in phenolic content and the
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Blueberries and their anthocyanins . . .
entire chemical composition of blueberry fruits (Howard and others 2003; Remberg and others 2004; Scalzo and others 2008b).
The variation of anthocyanin content in lowbush blueberries between 2 growing seasons was found to be up to 30% (Kalt and
others 1999b) and in highbush blueberries and interspecific hybrid
cultivars up to 35% to 40% (Connor and others 2002a).
Above-mentioned factors should be carefully considered while
selecting blueberry samples for any type of analysis, characterization, and analytical studies especially in vitro, in vivo, or ex vivo
studies. Cultivar selection and agronomic conditions influence the
uniformity or variation in anthocyanins in the sample and their
Figure 1–Skeletal structure of anthocyanins.
corresponding effects.
Effect of Processing and Preservation Practices
on Anthocyanin Concentrations
Blueberry is a seasonal crop and the harvest season of blueberries is generally in between July and September in North
America, which differs in other parts of world, while also being
cultivar-dependent. To promote the consumption of blueberries
throughout the year, a variety of storage methods (refrigeration
and freezing) are required and the effects of storage conditions
on the anthocyanin content of blueberries have been found to
be differing with different storage conditions and cultivars. In
some cases the anthocyanin content increases and in some others
it decreases with storage, depending on storage conditions (Kalt
and McDonald 1996; Kalt and others 1999a; Connor and others 2002c). Leakage from skin could be a contributing factor for
anthocyanin loss, which is low in fresh unpunctured blueberries
but is higher in soft and punctured berries, the number of which
is increasing with increased storage time. Wax content in cuticles
differs with different cultivars and lower wax content in cuticles
can increase the rate of puncture of berries, increasing the storage loss (Sapers and Phillips 1985). The production of blueberry
juice is an option to ensure the availability of blueberry benefits
throughout the year. However, fruit processing requires strict and
controlled operations to ensure quality. Loss of raw material is observed during many food processing steps and in most cases losses
are inevitable. Seven percent of raw material was lost during a series of juice processing unit operations such as enzyme maceration
and pressing in a laboratory experimental study (Skrede and others
2000), suggesting losses would be more considerable in large-scale
industrial processing. During these postharvest processing steps
there is also the possibility of losses in anthocyanins (Kalt 2005).
Enzymes like polyphenol oxidase (PPO) (Puupponen Pimiä and
others 2001), and compositional factor such as chlorogenic acid,
which enhances PPO activity, are responsible for the decrease in
anthocyanins during storage and processing (Skrede and others
2000). Anthocyanins are not the direct substrates for PPO activity (Clifford 2000). The o-quinones formed by the oxidation of
phenolic compounds by PPO trigger the degradation of anthocyanins (Kader and others 1997, 1998). Degradation depends on
the structural properties of the anthocyanins, especially the hydroxylation pattern on the B ring (Figure 1, Kader and others
1998). It is shown by several authors (Sakamura and Ohata 1963;
Sakamura and others 1965; Kader and others 1998) that PPO degrades the anthocyanins, which have a triphenolic function on
the B ring. Commercial enzymes, such as rapidase super BE depectinization enzyme and pectinases containing ß-glucosidases,
used during juice processing to improve recovery of desired fruit
components in the final product for depectinization, can contribute to the degradation (Skrede and others 2000; Smith and others 2000). Many of the processing applications using blueberries
involve cooking to some extent. Since anthocyanins are reported
to be thermosensitive, their degradation has been observed during
thermal processing (Queiroz and others 2009; Oliveira and others
2010), especially above 70 ◦ C (Mishra and others 2008). Cooking
at lower temperature can limit the damage as maintenance of a
high temperature for an elongated period of time has been observed to be the reason for degradation and it has been observed
that heating upto 40 to 60 ◦ C does not affect significantly the
total anthocyanin level (Khanal and others 2010). Oxidation and
cleavage of covalent bonds or accelerated oxidation reactions are
suggested to be the possible reasons for thermal degradation. But
the rate of degradation has been observed to be differing with
types of cultivars (Brambilla and others 2008; Oliveira and others
2010). Lowering of the moisture content by application of osmodehydration as a low-temperature process followed by hot air drying
was studied by Stojanovic and Silva (2007), but it led to a remarkable loss of anthocyanins and phenolic compounds. Application
of high-frequency ultrasound further accelerated the nonthermal
osmo-concentration process (Floros and Liang 1994), but in a later
study higher loss of anthocyanins was observed when it was applied
to rabbiteye blueberries (Stojanovic and Silva 2007). In a different
study, to increase different polyphenolic compounds in blueberry
juice, the juice was treated with Serratia vaccinii (Vuong and others
2010). Biotransformation of blueberry juice using Serratia vaccinii
bacteria, applied by Vuong and his group, is based on the logic
of prevention of cellular damage because of increased resistance
to oxidative stress due to polyphenols. The antioxidant properties
of the juice were increased and hydrogen peroxide-induced neuronal damage was prevented, because of the increase in activities
of the enzymes catalase and superoxide dismutase, and activation
of certain pathways along with blocking of some others (Vuong
and others 2010).
Some advanced processing methods like radiant zone drying,
where drying takes place in consecutive drying zones of varying
temperatures using radiant heaters (Chakraborty and others 2010),
application of ultraviolet radiation type C (Perkins-Veazie and others 2008; Wang and others 2009), pasteurization techniques, steam
blanching (Brambilla and others 2008), high-pressure application
(Buckow and others 2010), application of essential oils (C.Y. Wang
and others 2008), and a combination of multiple drying methods
(Kim and Toledo 1987; Yang and others 1987; Mejia-Meza and
others 2008), and storage methods such as modified atmosphere
packaging (Zheng and others 2003; Krupa and Tomala 2007), have
been found helpful for processing and preservation of blueberries,
which have the potential to increase shelf-life and minimize anthocyanin content loss. However, freezing is the most widely used
method of storage for blueberry and blueberry extracts during
analytical experimental studies (Lohachoompol and others 2004)
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Blueberries and their anthocyanins . . .
and freeze-drying is used as a processing measure for the longterm storage of blueberries (Yang and Atallah 1985), causing the
least deterioration. Many of the above-mentioned processes are
still under study to confirm their usefulness in the case of anthocyanin retention. Research on products such as blueberry wines
(Sánchez-Moreno and others 2003) and increase of antioxidant
activity by fermentation (Martin and Matar 2005) are relatively
new areas of study.
As today’s health-conscious society is focusing on the nutritional
quality of foods and wants good-quality processed foods with all
the required elemental factors of satisfaction, blueberry can play an
important role. It is deep-rooted in terms of traditional use and folk
knowledge about beneficial effects, which invites further research
in this field while it will remain a popular consumer product in its
fresh form. Hence, study of the composition of anthocyanins in
blueberries will give greater insight for improvement of the fruit,
improvement of the processing methods, and analytical methods
applied.
Anthocyanins Present in Blueberries and Their
Benefits
Anthocyanins are glycosidic and acyloglycosidic forms of anthocyanidins, which are polyhydroxy and polymethoxy derivatives of
2-phenylbenzopyrilium (flavilium salts). The basic structure of anthocyanin is presented in Figure 1 where it can be observed that
all anthocyanins possess the characteristic C6 C3 C6 skeletal structure, which is common for all flavonoids. The properties of the
anthocyanins are dependent on the degree and pattern of hydroxylation and methoxylation of the skeletal structure. Anthocyanins
are more stable than anthocyanidins and conversion of anthocyanins to anthocyanidins by ß-glycosidase leads to easier destruction of anthocyanidins by PPO (Buckow and others 2010). Some
anthocyanins are more vulnerable than others, hence knowledge
regarding their composition in foods can help in the selection
of proper storage and processing conditions, analytical studies for
proper estimation, and extraction studies for maximum release,
all with the least compound deterioration. The deterioration of
anthocyanins depends on factors such as pH, enzymes, other supporting substrates, and temperature, the significance of which differs according to the structural properties of the anthocyanins,
discrete from each other. The anthocyanins detected in blueberries are 3-glycosidic derivatives of cyanidin, delphinidin, malvidin,
petunidin, and peonidin (Kader and others 1996). The different
anthocyanins also have different colors, which are affected by the
pH. The structures of different anthocyanins present in blueberries are presented in Table 1 with the colors associated with them.
The most common derivatives determined are based on sugars
such as glucose, galactose, and arabinose. In lowbush blueberry
the anthocyanins were observed to be present in both nonacylated
and acetylated forms. In “Fundy” blueberries the main acetylated anthocyanins were categorized as the acetylglucoside and
3-acetylgalactoside of malvidin (Gao and Mazza 1995). In another
study (Barnes and others 2009), 25 anthocyanins, including the
(6 -acetoyl)glucoside and (6 -acetoyl)galactoside derivatives were
characterized in lowbush blueberry by high-performance liquid
chromatography (HPLC)–electrospray ionization-ion trap timeof-flight mass spectrometry. The biosynthetic pathway presented
in Figure 2 is common for these anthocyanins except for the last
few steps, and thus study of this pathway could be an essential part
of breeding programs based on quality parameters.
c 2011 Institute of Food Technologists®
Biosynthesis of anthocyanins present in blueberries
Extensive research has been done on the mechanisms of synthesis of anthocyanins in different plant species. Detailed discussions
on biosynthesis can be found in books such as “flavonoid mechanism” (Stafford 1990) and “natural food colorants” (Hendry and
Houghton 1996), which give detailed descriptions of the factors involved in the synthesis process in general. There are also
several reviews available on biosynthesis and the genetics related
to it (Holton and Cornish 1995; Weisshaar and Jenkins 1998;
Herrmann and Weaver 1999; Winkel-Shirley 2001). The knowledge base relating genetics to anthocyanin synthesis has been used
in breeding modifications to increase the production of anthocyanins in many plants (Shimada and others 2001; Katsumoto and
others 2007; Nakatsuka and others 2007). Mechanisms involved in
synthesis of the anthocyanins in different species might be the same
or very similar up to a certain extent, but there are some distinct
differences between species. Some anthocyanins are produced in
one species and not in the other, which makes their pathways
different as well (Holton and Cornish 1995). The expression of
different genes involved in anthocyanin biosynthesis during fruit
development has been reported for bilberry (Jaakola and others
2002), but according to the information collected for this review
the correlation of different anthocyanin levels and genes has not
been reported in the case of North American highbush and lowbush blueberries.
The genes involved in the anthocyanin synthesis are divided
into 2 categories, structural genes which encode anthocyanin
biosynthesis enzymes (chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3 hydroxylase, flavonoid 3 5
hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase,
methyltransferase) and regulatory genes that control structural gene
expression (Holton and Cornish 1995). Anthocyanin biosynthesis
is a process connected to many vital cellular processes, such as the
Calvin cycle that is part of photosynthesis, the pentose phosphate
pathway that is involved in the production of NADPH, and pentoses that are also part of other vital processes such as pyruvate decarboxylation forming acetyl CoA, which in turn is connected to
the Krebs cycle. The 3 main pathways which lead to the synthesis
of anthocyanins are shikimate pathway, phenyl propanoid pathway,
and flavonoid pathway; these are the basic synthesis pathways for
all flavonoids. The shikimate pathway consists of combining phosphoenol pyruvate with erythrose-4-phosphate which finally forms
chorismate, a precursor of many aromatic compounds including
amino acid phenylalanine. In some cases, this pathway ultimately
leads to the formation of phenylalanine and has also been named
“arogenate pathway” (Jensen 1986; Stafford 1990). The phenylpropanoid pathway leads to the conversion of phenylalanine to an
activated form of cinnamic acid, namely, coumaryl CoA (Stafford
1990). Acetyl CoA is converted to malonyl CoA by acetyl CoA
carboxylase reaction. The flavonoid pathway starts with a combination of 3 molecules of malonyl CoA with coumaryl CoA in the
presence of chalcone synthase. The colorful chalcone (naringenin
chalcone) is isomerized to colorless isomeric flavanones (naringenin). These flavanones get converted to dihydroflavonols (dihydroquercetin, dihydromyricetin), which act as precursors for the
formation of anthocyanidins (cyanidin and delphinidin). Dihydrokaempferol is the major dihydroflavonol from which blueberry
anthocyanins are derived. Anthocyanidins in nature are unstable
and convert to anthocyanins through glycosylation. Glycosylation
takes place in the later part of anthocyanin synthesis and is a stepwise process leading to higher glycosylated forms (Hendry and
Houghton 1996). It begins with the addition of sugars to the
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Blueberries and their anthocyanins . . .
Table 1–Anthocyanins commonly present in blueberries, their structures, sugar moieties, and color.
Anthocyanin based on
Basic structure
R (Sugar moiety)
Color
Cyanidin
Galactose,
Glucose,
Arabinose
Orange-red
Delphinidin
Galactose,
Glucose,
Arabinose
Blue-red
Malvidin
Galactose,
Glucose,
Arabinose
Blue-red
Petunidin
Galactose,
Glucose,
Arabinose
Blue-red
Peonidin
Galactose,
Glucose,
Arabinose
Orange-red
3-hydroxyl residue in the presence of UDP-glycosyl-flavonoid
3-O-glycosyltransferase. Generally, acylation occurs subsequently
with glycosylation, which leads to the formation of anthocyanins
based on peonidin, petunidin, and malvidin. It occurs in the presence of acyltransferases (for example, methyltransferase). Similarly
other anthocyanins are also formed.
The anthocyanin composition of blueberries and their content
varies with cultivars (Lohachoompol and others 2008), species, and
varieties and the amount detected varies in each study depending
on the environmental growth conditions and method of analysis (Table 2). Still, malvidin-3-galactoside has been found to be
most predominant in many cases (Skrede and others 2000; Zheng
and others 2003) and especially wild blueberry has been reported
as the best source of the petunidin- and malvidin-based anthocyanins (Wu and others 2006). Anthocyanins do not accumulate
in the cells where they are synthesized. They generally get accumulated in flowers and fruits and are partly present in the leaves
and bark. The availability of various anthocyanins in different parts
might be different, and may be a function of several factors. The
plant’s defense mechanism can increase the accumulation of anthocyanins in different parts. Anthocyanins have been reported to
be accumulated in the peels of fruits in the case of blueberry and in
both peel and pulp in the case of bilberry (V. myrtillus) (Riihinen
and others 2008). Along with the anthocyanins mentioned before,
308 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
c 2011 Institute of Food Technologists®
Blueberries and their anthocyanins . . .
Calvin
cycle
Pentose phosphate pathway
Phosphoenolpyruvate
Erythrose-4-phosphate
3-Deoxy-D-arabino-heptulosonate-7phosphate synthase
7P-2-dehydro-3-deoxy-D-arabino-heptonate
3-Dehydroquinate synthase
Dehydroquinate
Shikimate-5-dehydrogenase
Shikimate
pathway
Dehydroshikimate
3-Dehydroquinate dehydratase
Shikimate
Shikimate kinase
Shikimate-3-P
5-Enolpyruvylshikimate 3-phosphate synthase
5-O-(1-carboxyvinyl)-3-phosphoshikimate
Chorismate synthase
Chorismate
Prephanate
Pyruvate decarboxylation
Arogenate
Phenylalanine
Phenylpropanoid
pathway
Acetyl CoA
Phenylalanine ammonia lyase
Acetyl CoA carboxylase
Cinnamic acid
CO2
Flavonoid
pathway
Cinnamate 4-hydroxylase
p-Coumaric acid
4-coumaryl-coenzyme A ligase
Malonyl-CoA
Coumaryl-CoA
Chalcone synthase
Naringenin chalcone
Chalcone isomerase
Naringenin
Flavanone 3-hydroxylase
Kaempferol
Dihydrokaempferol
Flavonoid 3’ hydroxylase
F3’5’H
Dihydroquercetin
Quercetin
Dihydromyricetin
Myricetin
Dihydroflavonol 4-reductase
Dihydroflavonol 4-reductase
Catechins
Flavonoid 3’5’ hydroxylase (F3’5’H)
Leucocyanidin
Leucodelphinidin
Anthocyanidin synthase
Anthocyanidin synthase
Cyanidin
Delphinidin
Cyanidin-3-glucoside
Delphinidin-3-glucoside
UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) &
Rhamnosyl transferase (RT)
UFGT & RT
Methyltransferase
Methyltransferase
Peonidin-3-glucoside
Petunidin-3-glucoside
Malvidin-3-glucoside
Figure 2–Pathways of biosynthesis of some major anthocyanins present in blueberry.
the acetylated derivatives of malvidin and delphinidin glycosides
have been detected in blueberry juice (Skrede and others 2000).
Detailed analysis of blueberries has provided evidence of variations
with cultivars (Ehlenfeldt and Prior 2001). The mean amount of
different anthocyanins present in 34 blueberry genotypes has been
summarized by Scalzo and others (2008a). Anthocyanins such as
cyanidin and peonidin derivatives have been found in the bark of
the blueberry plant, which indicates the presence of pigments in
all parts of the plant (Hall and others 1972). In bilberry leaves,
an analysis showed the presence of anthocyanins, mainly cyanidin derivatives and hydroxycinnamoyl conjugates in the red leaves
rather than in green leaves (Jaakola and others 2004; Riihinen and
others 2008). However, a detailed study of anthocyanin composition in blueberry leaves and other vegetative parts is still required
(Naczk and others 2006).
c 2011 Institute of Food Technologists®
Benefits of blueberry anthocyanins
Some of the beneficial effects are associated with anthocyanins in
general and there exist published health reviews about the effect of
anthocyanins in general (Wang and others 1997; de Pascual-Teresa
and Sanchez-Ballesta 2008; Wang and Stoner 2008). Knowledge of
the anthocyanin composition in food is very helpful when studying their beneficial effects. The most important and commonly
reported health effect of anthocyanins is their antioxidant activity.
Antioxidant activity. Three to five percent of oxygen is expected to escape during mitochondrial electron transport without being completely reduced to water. The incomplete reduction of oxygen leads to the formation of superoxide anion (O2 − )
and eventually to hydrogen peroxide (H2 O2 ) and hydroxyl radical
(− OH) (Kalt 2005), which are together known as reactive oxygen
species (Castro and Freeman 2001). The human body produces
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 309
Blueberries and their anthocyanins . . .
Table 2–Total anthocyanin content reported in different parts of the blueberry plant.
Part of plant
Blueberry fruit
Conventionally grown
blueberry
Different cultivars of
blueberry
Anethole-treated
blueberry
Organically grown
blueberry
UV-C treated fruit
Blueberry pomace
Rabbiteye blueberry
Highbush blueberry
Different cultivars
Different cultivars
Different genotypes of
blueberries
Highbush blueberry
‘Rubel’
Amount of
anthocyanin
99.9 mg/100 g fresh
weight
111 mg/100 g fresh
weight
83.2 mg/100 g fresh
weight
20–190 mg CGE
/100 g fresh
weight
143.52–822.73
mg/100 g
82.4 mg/100 g fresh
weight
89 to 331 mg
CGE/100 g of fresh
weight
>320 mg CGE/ 100 g
fresh weight
131.2 mg/100 g
fresh weight
311 ± 9 mg/100 g
fresh weight
151.5 mg/100 g dry
weight
∼100 mg/100 g
fresh weight
790 ± 90 mg
CGE/100 g dry
weight
580 ± 30 to 1370 ±
140 mg CGE/ 100
g dry weight
62.6 ± 3.8 to 235.4 ±
6.1 mg CGE/100 g
fresh weight
73 ± 1.4 to 515 ± 3.6
mg CGE/100 g
230 mg CGE/ 100 g
of berries
Reference
Skrede and others
2000
Gao and Mazza 1994
Kalt and Dufour 1997
Kalt and others 2001
Cho and others 2004
S. Y. Wang and others
2008
Ehlenfeldt and Prior
2001
C. Y. Wang and others
2008
S. Y. Wang and others
2008
Wang and others
2009
Khanal and others
2010
Sellappan and others
2002
Wang and others
2010
Lohachoompol and
others 2008
Prior and others 1998
Moyer and others
2002
Lee and others 2004
CGE = cyanidin 3-glucoside equivalent.
The forms of presentation have been adapted from the reports themselves to show the different forms
of quantification available in the literature for anthocyanins.
another free radical species, namely nitric oxide (− NO), during
different physiological processes, which when it reacts with reactive oxygen species produces peroxynitrite (ONOO− ), which acts
as a potential oxidizing agent known as reactive nitrogen species
(Castro and Freeman 2001). Excessive production of this leads to
the imbalance of oxidants and antioxidants in the body leading
to oxidative stress (Castro and Freeman 2001), which eventually
may lead to tissue damage (Halliwell 1992), especially to DNA,
lipids, and proteins (Wang and others 1996), and may also lead to
physical disorders such as cardiovascular diseases, neurodegenerative diseases, diabetes, rheumatoid arthritis, cancer, and cataracts.
There are many published articles that correlate the reactive species
and different major diseases such as cancer, arthritis, and several
other disorders (Halliwell 1992; Castro and Freeman 2001), which
describe the way of their formation and the action of these radicals leading to these diseases (Halliwell 1991; Griveau and Lannou
1997). The body has its own built-in defense mechanism for these,
such as different categories of enzymes and other agents such as
hydrophilic radical scavengers and lipophilic radical scavengers.
Flavonoids come under the lipophilic radical scavengers’ category,
which basically helps in the retardation of chain oxidation reactions
of lipids (Castro and Freeman 2001).
There are several methods of measurement and quantification
of antioxidant property of food products. The chemically differ-
ent measurement methods and the free radical used in the assay
give an antioxidant capacity value, which may vary from method
to method (Halliwell and Gutteridge 1990; Halliwell and others
1995; Wang and others 1997). As mentioned in different reports
and considering end uses of different antioxidant components,
standardization can be done using mainly 4 assays: the oxygen
radical absorbance capacity (ORAC) assay (based on the hydrogen
atom transfer mechanism), the ferric reducing ability of plasma
(FRAP) assay, the Trolox equivalent antioxidant capacity (TEAC)
assay, and the Folin-Ciocalteu (FC) method (Joseph and others
2007). The TEAC and FRAP assays and FC method are electron transfer-based methods and give reducing capacity, whereas
the FC method is generally expressed as total phenolic contents
(Benzie and Strain 1996; Prior and others 2005). The ORAC
method has been found to be the most relevant to the human
biological system and has been recommended to be superior to
other similar methods, as “it uses an area-under-curve technique
and combines inhibition time and inhibition degree of free radical
action by an antioxidant into a single quantity” (Wang and others
1997).
According to Wang and others (1997), based on ORAC activities, among the aglycons (nonsugar part of the anthocyanin,
namely, the anthocyanidin structure [refer to Figure 1]) with the
same hydroxylation pattern on the A and C rings, increased hydroxylation on B ring leads to the increase in the antioxidant
capacity (3 , 4 di-OH as compared to 3 -OH has higher ORAC
capacity). Cyanidin was found to have higher ORAC capacity than malvidin and peonidin, but delphinidin, having 3 hydroxyl groups on the C ring, was an exception and was found
to have lower capacity (Table 1). This was expected because of
the probable decreasing effect of the 5 -OH in the presence of 3 ,
4 -OH (delphinidin) as compared to the presence of 3 , 4 -OH
only (cyanidin) (refer to Figure 1). The effect of glucosylation
varies with the type of aglycons and the type of sugar moiety
(Wang and others 1997). Also, pH has been reported to be a
factor in the antioxidant capacity of anthocyanin extracts. Anthocyanin extracts with pH 1 were reported to have higher antioxidant
capacity than extracts with pH 4 and 7 (Kalt and others 2000).
Fruit size has also been observed to be highly correlated with
the anthocyanin content within V. corymbosum L. but not in other
Vaccinium species (Moyer and others 2002). Smaller V. corymbosum
L. berries contained more anthocyanins per unit volume. Anthocyanidins have been reported to have higher radical scavenging
capacity than anthocyanins, where the radical scavenging capacity
has been reported to decrease with an increase of number of sugar
moieties (Wang and Stoner 2008).
Other methods of analysis of antioxidant activity of blueberry
extracts include tyrosine assay, galvinoxyl free radical quenching
assay, lipid oxidation assay (Smith and others 2000), and ferric reducing antioxidant power (Moyer and others 2002). Antioxidative
properties of blueberries include free radical scavenging, peroxide
decomposition, singlet oxygen quenching, synergistic effects, and
inhibition of enzymes (Wang and others 2009).
Bioavailability: a factor affecting activity of anthocyanins inside
the body. Another subject of interest is the bioavailability of an-
thocyanins. The curative effect of any phytochemical is decided
by its availability when exposed at the cellular level to an organism
through the food consumed. Bioavailability of anthocyanins has
been discussed in various reviews along with factors affecting it
(Clifford 2000; McGhie and Walton 2007), and it has been defined
as “the proportion of the nutrient that is digested, absorbed, and
metabolized” (McGhie and Walton 2007).
310 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
c 2011 Institute of Food Technologists®
Blueberries and their anthocyanins . . .
Figure 3–Different structural transformations of anthocyanins with change of pH (McGhie and Walton 2007).
Structural states play an important role in the bioactivity of
anthocyanins. In solutions, anthocyanins exist in different forms
depending on the pH of the solution and these forms are in equilibrium (Harborne and others 1975; Clifford 2000; McGhie and
Walton 2007). Initially, 3 forms were detected, which increased to
4, and recently 8 distinct structures have been recognized (McGhie
and Walton 2007). At pH 2 or lower, the hemiketal form has been
found to be the most dominant form, which, with an increase
of pH, gets converted to the blue quinoidal form. Also, through
a slow hydration process, the flavylium cation is converted to
the colorless hemiketal form, which is then tautomerized to its
chalcone form in either cis or trans configuration (Clifford 2000;
McGhie and Walton 2007). The pH in the human varies throughout the different parts of the gastrointestinal system. The pH of the
stomach is low, but human blood, small intestine, and other organs
are generally neutral (Clifford 2000; McGhie and Walton 2007).
The structural forms of anthocyanins at different pH values are
presented in Figure 3. Anthocyanins can be present in any form
in the human body, which makes the study of their assimilation
and absorption very difficult. According to the pH profile inside
the body, flavylium is the probable form of anthocyanins in the
stomach (McGhie and Walton 2007), while hemiketal seems to be
the most probable form present in most other parts of the body
(Clifford 2000). Along with pH, the microflora present inside the
body can be another factor which affects the bioavailability and
can lead to deglycosylation and demethylation of anthocyanins.
In most studies, anthocyanins are reported to be absorbed in their
c 2011 Institute of Food Technologists®
whole glycosidic form and possibly their acylated form (Mazza
and others 2002); the absorption of anthocyanins has been found
to be affected by both, the types of aglycone and the sugar moiety
(McGhie and Walton 2007).
During a study on the effects of blueberry anthocyanins on rats,
considering urine as the base of analysis for absorption, the absorption of delphinidin 3-O galactoside was found to be higher
than that of malvidin 3-O galactoside, while malvidin 3-O arabinoside was higher than malvidin 3-O glucoside (McGhie and
others 2003), which supports the dependence of absorption of anthocyanins in biological system on both the aglycone and the sugar
moiety of an individual anthocyanin molecule. Higher acylation
and presence of sugar during consumption negatively affect the
bioavailability, while consumption with alcohol has been reported
to increase the bioavailability (de Pascual-Teresa and SanchezBallesta 2008). The anthocyanins are reported to be absorbed locally from the gastrointestinal tract and through the skin (Wang
and Stoner 2008). An in vivo study on the absorption inside the
human body, especially from the gastrointestinal tract, is limited
which restricts any study of the utilization of these compounds;
the optimum amount absorbed is yet to be determined.
Consumption of blueberries by older women and the analysis of
anthocyanins in their plasma and urine showed lower absorption
and excretion of anthocyanins as compared to other flavonoids
(Wu and others 2002). The combination of blueberry extract
with other berry extracts has also been found to be better than
individual extracts in terms of antioxidant effects (Zafra-Stone and
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 311
Blueberries and their anthocyanins . . .
others 2007), and has been confirmed through in vitro analysis to be effective as antiangiogenic and anticarcinogenic (Bagchi
and others 2004). To increase bioavailability, the pharmaceutically
acceptable self-microemulsifying drug delivery system Labrasol®
was used during a study to evaluate the hypoglycemic effect of
2 pure anthocyanins, delphinidin-3-O-glucoside and malvidin3-O-glucoside, which demonstrated the more effective glycemic
modulation of malvidin-3-O-glucoside (Grace and others 2009).
Data on the bioavailability of anthocyanins are rare (Clifford
2000), and most of them do not suggest the action of any particular anthocyanin or trace down to their individual mode of action
in a biological environment. Most of the studies on the bioavailability of anthocyanins are based on flavylium cations, which is
the form present in an acid environment, and is not the most
probable form present throughout the human body (McGhie and
Walton 2007). One of the popular and useful methods of analysis
of anthocyanins used in in vivo studies is HPLC. The methods
of analysis adapted up to now are mostly based on the colorful
flavylium cation form of anthocyanins, which is also the most stable form. Established methods for analysis for other forms such as
hemiketal and chalcone do not exist and hence these forms have
not been studied in vivo. So, other forms of anthocyanins are converted to their flavylium cation form for the purpose of analysis
(McGhie and others 2003). Nonetheless, the general positive effects of consuming anthocyanins have been widely publicized. The
probable metabolic process of anthocyanins has been described in
detail by McGhie and Walton (2007). Particular discussion of the
metabolic process of anthocyanins present in blueberries has also
been reported by Wu and others (2002).
Beneficial health effects of blueberry anthocyanins. In addition
to the antioxidant effect, many other health effects are associated with blueberries. Inhibition of proteosome activity of anthocyanins is reported to be contributing to the beneficial effects
other than the antioxidant effect (Dreiseitel and others 2008). But
the effects of individual berry components and the extent of their
effects are generally variable (Zafra-Stone and others 2007) depending on concentration as well as bioavailability (McGhie and
others 2003). Many of the beneficial effects which are specifically
associated with blueberries include anticancer, cardioprotective,
and many other properties which are either confirmed by assessing the bioactivity (Smith and others 2000) through in vitro, in
vivo, and ex vivo (Kay and Holub 2002) analyses. The different effects of blueberry or blueberry anthocyanins confirmed in human
or animal studies are summarized in Table 3. The in vivo studies
can be animal or human studies. Many of the analyses are related
to blueberry-enriched diets (Ahmet and others 2009), blueberry
extracts as a whole (Paredes-López and others 2010), or on components specifically present in blueberries. The detailed discussion
of the mechanism is outside the scope of this review, but some of
the analyses are mentioned here, which are mainly based on the
animal studies conducted in recent years.
One of the major health benefits of anthocyanin consumption
is the cardiovascular protective effect. It is associated with the
other helpful effects of blueberry anthocyanins and is sometimes
discussed as the result of the other effects. Anthocyanins present
in V. myrtillus have been found to prevent cholesterol-induced
atherosclerosis in rabbits (Kadar and others 1979). By reducing the
release of inflammatory mediators, blueberry anthocyanins reduce
oxidative and inflammatory damage to microvascular endothelium (Youdim and others 2002) and are expected to reduce the
chance of occurrence of atherosclerosis (Kraft and others 2005).
Preventing atherosclerosis can lead to the prevention of cardiovascular dysfunction as this is one of the precursor reasons for the
disorders (de Pascual-Teresa and Sanchez-Ballesta 2008). Blueberry is reported to be helpful against ischemic damage of heart
(Ahmet and others 2009). This is related to the antioxidant activity of these compounds at the cellular level. The blueberry diet
increases the mitochondrial permeability transition reactive oxygen species threshold, which leads to an increase in cardiomyocyte
survival (Ahmet and others 2009). Blueberry-rich diets also affected the biomechanical properties of the aorta in rats (Norton
and others 2005). Other factors leading to cardiovascular damage
have also been found to be affected by anthocyanin consumption.
Activity against vascular endothelial growth factor, protection of
endothelial cells from CD40-induced proinflammatory signalling
(de Pascual-Teresa and others 2010), and protection of membrane
lipids from oxidation (Neto 2007) are some of the other activities
leading to cardiovascular protection. The prospect of blueberries
as one of the fruits highly helpful against cardiovascular disorders is
also explained by the presence of other polyphenolic components,
as discussed by Basu and others (2010).
The activity against cancer, of anthocyanins in general, has been
reviewed by Wang and Stoner (2008) for the different mechanisms
of their actions in animal and human studies. The different mechanisms include phase II enzyme activation, anti-cell production
(by regulating different stages of cell cycle through controlling
the cell cycle regulator protein), stimulation of apoptosis (programmed cell death), antiinflammatory effects and antiangiogenesis (angiogenesis is the procedure of formation of fresh blood
cells), antiinvasiveness, and induction of differentiation (Wang and
Stoner 2008). In vitro analysis of the blueberry extract for different
stages of carcinogenesis has also been reported (Bomser and others 1996), which has been supported by in vitro analysis showing
apoptosis of human cancer cells (Seeram and others 2006). Two
highbush blueberry cultivar extracts have been found effective in
vitro against cervical and breast cancer cells (Wedge and others
2001). Later, anthocyanins were found to be effective inhibitors of
the promotion stage of carcinogenesis using ornithine decarboxylase assay, while determining specifically the stage of carcinogenesis
for which different fractions of blueberry polyphenols are helpful
(Kraft and others 2005). Blueberry anthocyanins and their pyruvic acid adducts demonstrated anticancer potential in breast cancer
cell lines during a study of the inhibition of cancer cell proliferation and cell antiinvasive and chemoinhibitor properties (Faria and
others 2010). Recently, the modulation of the PI3K/AKT/NFκB
pathway has been explained as a reason of the antibreast-cancer
effects of blueberry extract (Adams and others 2010). Among
the different fractions of rabbiteye blueberry extract, anthocyanins
were found to be the most effective against 2 colon cancer cell
lines (HT-29 and Caco-2) in a study by Yi and others (2005).
Furthermore, the anthocyanin fraction was potentially effective
in DNA fragmentation (Yi and others 2005) and caspase-3 activity (Srivastava and others 2007), leading to apoptosis. The effect
was also correlated to the concentration of extracts (Olsson and
others 2004) and concentration of anthocyanins (Srivastava and
others 2007). Similar observations were found for cancerous cells
in other studies such as human prostate cancer cells (Matchett
and others 2005, 2006), colon cancer cells (Zhao and others 2004;
Prior and others 2008), and cervical cancer cells (Wedge and others
2001).
312 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
c 2011 Institute of Food Technologists®
Blueberries and their anthocyanins . . .
Table 3–Reports of different health benefits about blueberry anthocyanins.
Experimental substance
Blueberry
Study subject
Pig
Blueberry-enriched diet
Rat
Blueberry-rich diet
Rat
Blueberry-supplemented meal
Human
Blueberry in diet
Human
Blueberry flavonoids supplementation
Rat
Blueberry extract
Mice
Blueberry
Rat
Blueberry supplementation in feed
Blueberry-supplemented diet
Rat
Rat
Blueberry-supplemented diet
Rat
Whole blueberry powder
Mice
Purified berry anthocyanins
Mice
Conclusions
Anthocyanins get accumulated in several
parts of body including eyes and brain
Protection against ischemic damage and
prospective to avoid development of
post-myocardial infarction heart failure
“Suppressing α1-adrenergic receptor
agonist-mediated contraction” and effect on
vascular smooth muscle contractile
machinery
Increase of postprandial serum antioxidant
content in human volunteers
Diet-induced amplification of ex vivo serum
antioxidant content
Decrease in oxidative DNA damage in liver of
rats
Inhibition of growth and metastatic potential
of breast cancer cells
Effective against dextran sulfate
sodium-induced colitis
Effect on “spatial working memory”
Reversal of age-related decrease in brain’s
“heat shock protein 70 mediated”
neuroprotection
Reversal of decline of age-related neuronal
activity and behavioral aging
Reduction of adipocyte death and
inflammatory disorders leading to decrease
in whole body insulin resistance
Prevention of dyslipidemia and obesity
development
Blueberry extract has been found to be effective against different organisms leading to various diseases, such as Helicobacter pylori,
which has been identified to be the causative organism of diseases
such as duodenum ulcer and gastric cancer. It was observed that
blueberry extract at various concentrations inhibited this organism (Chatterjee and others 2004). Blueberry can have a combined
effect with probiotic bacteria in the reduction of the “severity of dextran sulfate sodium-induced colitis” and translocation
of bacteria and corresponding inflammation in rats (Osman and
others 2008). Similar prohibiting effect of blueberry extract (rich
in anthocyanins) was reported against other microorganisms including gram-negative bacteria, Enterococcus faecalis and Escherichia
coli (Ofek and others 1991) and Lactobacillus (Puupponen Pimiä
and others 2001), and Salmonella enteritidis and Listeria monocytogenes (Park and others 2011), and Citrobacter freundii (Burdulis and
others 2009). In vitro studies also showed that blueberry (V. myrtillus) extract is effective against the protozoan parasites Giardia
duodenalis and Cryptosporidium parvum, which are mainly responsible for diarrhea world-wide (Anthony and others 2007).
With increasing age, oxidative stress has been reported to be
responsible for neurodegenerative disorders such as Alzheimer’s
and Parkinson’s diseases. Oxidative stress leads to cellular disorders
and in this case damage of neurons, modification to intracellular
modulation, and apoptosis or necrosis. Anthocyanins have been
reported to be helpful against neurodegenerative disorders also for
their antioxidant properties (Prior and Wu 2006). Other antioxidants along with anthocyanins present in blueberries can also be
helpful against neurodegenerative disorders (Ramassamy 2006).
Analysis of blueberry supplementation fed to aged rats showed
that the effect of flavonoids to the brain was based on extracellular signal-related kinase, cAMP response-element-binding protein,
and brain-derived neurotrophic factor and could be correlated to
the improvement of spatial-working memory tasks after blueberry
c 2011 Institute of Food Technologists®
Reference
Kalt and others 2008
Ahmet and others 2009
Norton and others 2005
Kay and Holub 2002
Mazza and others 2002
Dulebohn and others 2008
Adams and others 2010
Osman and others 2008
Williams and others 2008
Galli and others 2006
Joseph and others 1999
DeFuria and others 2009
Prior and others 2009
consumption (Williams and others 2008). In another study, the enhancement of the ability of the brain to produce “heat shock protein 70-mediated neuroprotective response to stress” was reported
for young and old rats supplemented with blueberry (Galli and
others 2006). This shows that improvement of certain brain functions in animals and perhaps in humans is possible with blueberry
consumption. Similar results of reversal of decline of age-related
neuronal activity were reported by Joseph and others (1999).
Anthocyanins in bog blueberries have been reported to retard
the ultraviolet ray-induced skin photoaging effect as well as inhibiting collagen destruction and inflammation (Bae and others
2009). The crude extract of blueberries also showed antinociceptive and antiinflammatory effects (Torri and others 2007). In
another study, the crude extract of blueberries was found to be
effective against “nitric oxide production in LPS/IFN-activated
RAW 264.7 macrophages,” which is associated with inflammatory and cardiovascular disorders (Wang and Mazza 2002).
Blueberries were found to be effective on adipocyte physiology and gene expression for adipose tissue macrophages (movable,
large phagocytic cells derived from monocytes) (DeFuria and others 2009). In the case of high-fat-diet-fed mice, diet supplementation with blueberry powder decreased adipocyte death and the
inflammatory disorders that would lead to whole body insulin resistance. Another study concentrated on blueberry anthocyanins
and reported that when mixed with drinking water they could
bring high serum cholesterol and triglycerides levels to control
levels in the case of high-fat-diet-fed mice, better than the whole
berries (Prior and others 2009). Blueberry juice was found to be
effective for prevention of the onset of obesity in obesogenic highfat-diet-fed mice, but not as effective as blueberry anthocyanins
mixed with drinking water (Prior and others 2010). This underlines the superiority of blueberry anthocyanins, in this case, as
compared to other blueberry components; and could be explained
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 313
Blueberries and their anthocyanins . . .
by the greater and more focused activity of blueberry anthocyanins
when they are provided alone. Anthocyanins have also been reported as potentially helpful in the prevention of diabetes (Ghosh
and Konishi 2007). Usefulness of blueberry leaf extract against hyperglycemia has been reported (Allen 1927), but further research
is required in this area. Recently, the hypoglycemic activity of
anthocyanin-rich extract from lowbush blueberry was found to be
higher than for a lowbush blueberry phenolic extract showing the
higher effectiveness of blueberry anthocyanins in this particular
case (Grace and others 2009).
The possible metabolic pathway with the different forms of
anthocyanins present in the various parts of human, pig, and rat
bodies have been discussed by Prior and Wu (2006). The probable
distribution of anthocyanins in different parts of the body has
also been briefly discussed, but specific studies for blueberries are
limited. In pigs, which have been described to be similar to humans
in terms of their digestive system, anthocyanins were found in
organs beyond the blood plasma and were identified in liver, eye,
and brain of blueberry-fed pigs (Kalt and others 2008). Blueberries
are regarded as fruits which, when consumed in sufficient amounts,
can contribute to a healthier life and reduce the health problems
associated with aging (Paredes-López and others 2010).
The studies reported in the literature are mostly related to whole
blueberries or blueberry juice consumption, which offer a combination of many other beneficial and nutritional compounds.
Some reports have focused on the bioavailability and observed
final physical beneficial health effects of anthocyanin-rich blueberry extracts. But the real step-by-step mechanisms taking place
inside the human body have not been clearly defined, and will
have to be analyzed and studied in greater depth before anyone
can define a recommendation for blueberry extract or blueberry
anthocyanin dosages that will have an effect on health. Targeted
studies on blueberry anthocyanins activity in the human body will
open-up options for the utilization of blueberry products to prepare anthocyanin extracts, which would be beneficial for health
promotion.
Methods of Extraction of Anthocyanins from
Blueberries
Extraction and extract analysis are the backbones of all the studies related to anthocyanins. The extraction parameters applied
depend on the end use of the extracts, type of extracts, their
stability, reactivity, storability, and source. As these extracts contain beneficial biochemicals, there have been several bioavailabilty
studies and other health-beneficial analyses using the extracts, but
the extraction parameters used in every case were different. Anthocyanins are highly sensitive compounds and prone to fast destruction, hence the method of analysis chosen must be efficient
in terms of time and also other factors such as energy use and solvent consumption and biohazardous effects. The first documented
attempts of extraction and isolation of anthocyanins as pigments
from plants date back to 1849 by F.S. Morot. He extracted anthocyanins from Centaurea Cyanus (Onslow 1925). However, at
that time, there was not any established method of extraction and
there were a lot of variations in the reported research studies. This
uncertainty regarding the best method of extraction continues till
now. Different methods of analysis have been described in detail by
Giusti and others (2005) in the book “handbook of food analytical
chemistry-pigments, colorants, flavors, texture, and bioactive food
components.” Also, several methods have been briefly discussed
by Takeoka and Dao (2007).
The entire analytical process consists of pretreatment, extraction, concentration (if required), purification, and analysis. In the
case of blueberries, the sample can be in the form of juice, fruit
itself, fruit skin, other parts of the plant such as leaves and stem,
and so on (Garcia-Viguera and others 1997; Jaakola and others
2004; S.Y. Wang and others 2008; Buckow and others 2010). Depending on the sample, a pretreatment might be required or in
the case of fruit juice dilution might be required (Buckow and
others 2010). When the samples are fruits, other parts of plants
and some other derived products, the pretreatment might include
maceration (Chakraborty and others 2010), grinding (Wang and
others 2010), homogenization, and/or drying.
Solvent extraction is generally used for the extraction of anthocyanins. Alcohols such as methanol and ethanol mixed with acids
(Takeoka and Dao 2007) and other polar solvents such as acetone
(Giusti and others 2005; Takeoka and Dao 2007) have been used.
Methanol with a small amount of HCl is the oldest used solvent,
because of its low boiling point; but in this case the extract will
contain other compounds which act as contaminants, hence the
extract needs purification. This problem is resolved in the case
of acetone with chloroform partitioning using a separatory funnel which further isolates and partially purifies the anthocyanin
pigments (Giusti and others 2005). Other neutral solvents such as
water, n-butanol, and a combination of several others in different
proportions have been applied for the extraction of anthocyanins
from different types of samples (Jackman and others 1987; Giusti
and others 2005). Acids are used for breaking the cellular structure leading to protein release of the anthocyanin pigments and
their stabilization. But they also cause a change of the native form
of the anthocyanin and acid hydrolysis of all anthocyanins during
concentration of the extract (Giusti and others 2005). To decrease
the intensity of decomposition, weaker organic acids are used,
such as formic, citric, or tartaric acids, or small amounts of highly
volatile acids such as trifluoroacetic acid (Strack and Wray 1994).
Hydrochloric acid at low concentrations (0.01% to 0.05%) has
been suggested to decrease the decomposition (Giusti and others 2005). During a study by Nicoué and others (2007) on the
determination of anthocyanins in Quebec wild blueberries, different combinations of acids (hydrochloric, citric, tartaric, lactic,
and phosphoric acids) with ethanol were used, and the highest
extraction yield was obtained from the combination of ethanol
and phosphoric acid. For nutraceutical studies and bioavailability analyses of the extracts, ethanol might be preferred to avoid
methanol and acid toxicity (Giusti and others 2005). Sometimes
physical stirring is also used for adequate mixing of solvent and
sample to ensure proper extraction and increase in the extraction
rate (Garcia-Viguera and others 1997). Higher temperatures are
also applied to increase extraction rates (Kalt and others 2000),
and extraction temperatures ranging between 40 and 60 ◦ C have
been found to not affect anthocyanins significantly (Khanal and
others 2010); however, temperatures greater than 70 ◦ C should
be avoided, as they can lead to hydrolysis (Clifford 2000). The
sensitivity of anthocyanins has motivated researchers to use several
combinations of solvents. Some combinations of solvents used in
different studies for the extraction of anthocyanins from blueberries are summarized in Table 4.
Supercritical fluid extraction was applied for extracting polyphenols from blueberries. This method has not been specifically applied to anthocyanins, but it has potential to be applied in future.
According to the literature available, many of the modern methods
of extraction have not yet been applied for specifically extracting anthocyanins from blueberries. Most of the studies related to
314 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
c 2011 Institute of Food Technologists®
Blueberries and their anthocyanins . . .
Table 4–Different solvents and their combinations used for the extraction electric field have shown potential in terms of extraction of anof blueberry anthocyanins.
thocyanins from grape byproducts, with increases in selectivity
Solvent/
solvents
Acetone
Anthocyanins
extracted
All
Acidified methanol (about 0.1%
formic acid)
Methanol/water/acetic acid
(25:24:1)
All
Water/methanol/acetone/formic
acid
Methanol/acetic acid/distilled
water
Acetone (80%) with formic acid
(0.2%)
All
Water and formic acid
All
Acidified methanol (0.6 M HCl)
All
Methanol
All
Ethanol with 1, 3, or 5% (w/w)
citric acid
Methanol/ water/formic acid
(60:37:3)
Acetone:methanol:water
(35:35:30) with 1 cm3 36%
(w/w) HCl per l
Acetonitrile containing 4% acetic
acid
Acetone/ methanol/water/
formic acid (40:40:20:0.1,
v/v/v/v)
Methanol:water:trifluoroacetic
acid (70:30:1, v/v/v)
All
All
All
All
All
All
All
All
All
Reference
Skrede and others
2000
Chakraborty and
others 2010
Garcia-Viguera
and others 1997;
Lohachoompol
and others 2008;
Wang and others
2010
Perkins-Veazie
and others 2008
Lohachoompol
and others 2004
Wang and others
2009; Wang and
others 2008b;
Zheng and others
2003
Brambilla and
others 2008
Jaakola and others
2004
Kader and others
1996
Chen and Camire
1997
Cho and others
2004
Krupa and Tomala
2007
Prior and others
1998
Wang and others
2000
Barnes and others
2009
blueberries concentrate on the beneficial effects of anthocyanins
on increasing the antioxidant properties of the plant itself. This
has lead to reduced interest on improving the extraction methods of anthocyanins from the blueberries. The existing analytical
methods seem to work around different combinations of solvents
to improve extraction yield rather than using modern extraction
methods, which are more expensive than conventional methods.
When the objective of most of the studies is characterizing the
anthocyanins present in different varieties of blueberry and their
possible health-beneficial effects, the studies on increasing the efficiency of already existing extraction methods remain neglected.
Studies on the highest amount of extractable anthocyanins and the
optimization of extraction parameters to obtain the best extraction
method are still required.
However, for increasing the precision of analyses and application
developments for nutraceuticals, modern methods of extraction
can be very helpful. Advanced methods such as microwave-assisted
extraction (Sun and others 2007; Ghassempour and others 2008;
Yang and Zhai 2010; Liazid and others 2011), ultrasonic extraction
(Chen and others 2007; Ghassempour and others 2008), subcritical water extraction (King and others 2003; Ju and Howard 2005),
high pressure liquid extraction (Ju and Howard 2003; Mantell and
others 2003), and dynamic superheated liquid extraction (LuqueRodriguez and others 2007) have been applied for the extraction
of anthocyanins in many cases and could be applied specifically for
blueberries. Applications of high hydrostatic pressure and pulsed
c 2011 Institute of Food Technologists®
(Corrales and others 2008). These modern methods reduce the
time of extraction, reduce the amount of solvent required, and are
helpful for the extraction of sensitive phytochemicals like anthocyanins.
Purification of anthocyanins may be required depending on the
analytical process. Purification of anthocyanins by solid-phase extraction is quite common (Giusti and others 2005). According to
one study, solid-phase extraction can result in 90% to 95.6% recovery of anthocyanins (Denev and others 2010). “Mini-columns
containing C18 chains bonded on silica retain hydrophobic organic
compounds, while allowing matrix interferences such as sugars
and acids to pass through to waste” were singled out by Giusti and
others (2005). A C18 Sep Pack cartridge has been used by several
researchers for the analysis of anthocyanins (Lohachoompol and
others 2008; S.Y. Wang and others 2008; Wang and others 2009;
Chakraborty and others 2010; Wang and others 2010). For further
purification of extracts and to increase the concentration of anthocyanins, the retained pigments on the cartridges can be washed
with ethyl acetate (Giusti and others 2005). To make the extracts
suitable for HPLC, these extracts are further filtered through a
0.45-μm membrane filter (Jaakola and others 2004; S.Y. Wang
and others 2008; Wang and others 2009).
Anthocyanins in general are quite prone to destruction. Pure
anthocyanin extracts are highly prone to degradation due to the
absence of other supporting stabilizing cofactors such as flavonols
(Smith and others 2000), which makes any analysis procedure more
time-sensitive and storage of the extracts before analysis a major
issue. Usually, if the analysis of the extract follows the extraction
within 24 h, then the extract is stored at 4 ◦ C (Giusti and others
2005). However, when the analysis is done later than 24 h, the
extracts have been reported to be stored at –18 to –20 ◦ C (Giusti
and others 2005; Wang and others 2010; You and others 2011), at
–35 ◦ C (Chakraborty and others 2010), or at –80 ◦ C (S.Y. Wang
and others 2008; Wang and others 2009).
The pH differential method appears to be the most used for
the estimation of total anthocyanins in the case of blueberries invariable of the sample material (juice, fruit extract, or any other
sample) (Skrede and others 2000; Prior and others 2001; S.Y.
Wang and others 2008; Wang and others 2009; Buckow and others 2010; Khanal and others 2010). This method is based on the
reversible structural transformations of anthocyanins with change
of pH characterized by prominently different absorbance spectra;
it consists of measurement of absorbance of the extract solution
prepared in pH 1 and pH 4.5 buffers at λvis−max and at 700 nm.
The common λvis−max used in the pH differential method is 510
nm, however, there are other studies where researchers have applied different wavelengths (Skrede and others 2000; Oliviera and
others, 2010), and have reported measurements at different absorption bands (Connor and others 2002c; Lohachoompol and others
2008). This is possible because the “typical absorption band” for
anthocyanins is in the 490 to 550 nm region of the visible spectrum (Giusti and others 2005). Generally, total anthocyanins are
expressed as cyanidin-3-O-glucoside equivalents per 100 g blueberry matter (fresh or dry) (S.Y. Wang and others 2008; Oliveira
and others 2010).
Modern methods such as HPLC (Garcia-Viguera and others 1997; Riihinen and others 2008; S.Y. Wang and others
2008), vacuum chromatography (Smith and others 2000), liquid chromatography/mass spectrometry (LC/MS) (Prior and others 2001; Cho and others 2004; Taruscio and others 2004;
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 315
Blueberries and their anthocyanins . . .
Figure 4–Different steps applied for the extraction of blueberry anthocyanins.
Lohachoompol and others 2008), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Wang and others 2000), Fourier transform near-infrared spectroscopy (FT-NIR)
(Sinelli and others 2008), HPLC-electronspray ionization-mass
spectrometry (HPLC-ESI-MS) (Nakajima and others 2004; Wu
and Prior 2005), and HPLC–electrospray ionization-ion trap timeof-flight mass spectrometry (HPLC-ESI-IT-TOF-MS) (Barnes
and others 2009) have also been applied for analyzing anthocyanins
present in blueberries. These methods are helpful and very precise
in individual characterizations of the anthocyanins. The different
methods used and different analytical steps for the extraction of
anthocyanins from blueberries have been summarized in Figure 4.
HPLC appears to be the next most used method of analysis, which
differs mainly in terms of the mobile phases used (Kader and others
1996; Garcia-Viguera and others 1997; Krupa and Tomala 2007;
S.Y. Wang and others 2008; Chakraborty and others 2010). However, other methods can also be potential options for anthocyanin
analysis. Barnes and others (2009) characterized 25 anthocyanins
in lowbush blueberries, which is the highest number ever reported
for blueberries and reflects the benefit of using an advanced analytical technique. In analytical processes, in the case of unavailability
of all standards, anthocyanins are characterized in terms of one specific anthocyanin (Garcia-Viguera and others 1997; Chandra and
others 2001). Barnes and others (2009) also applied a method with
HPLC-ESI-IT-TOF-MS for the characterization of anthocyanins
without all standards available to them.
The various analytical methods are not only helpful in different basic laboratory studies but can be used for quality characterizations of products by estimation (Garcia-Viguera and others
1997) and maintenance of required level of anthocyanins among
the samples. Extraction methods can also help to find uses for
byproducts and waste products obtained from the food processing
industry (Lee and Wrolstad 2004). As anthocyanins are sensitive
compounds, better focus on modern extraction methods could
lead to optimal extraction yields and higher precision. Methanolic extracts have been reported to give high yields, but use of
other solvents, generally recognized as safe, should be singled-out
for modern extraction methods to produce clean and nontoxic
extracts.
Conclusion
Blueberry is one of the most popular berries of North
America, rich in many valuable compounds. Extensive attention
and thorough research have converted this wild plant into a widely
cultivated one. Different factors affect the concentration of anthocyanins in different parts of the plant and information regarding
the biosynthesis can help in improving and providing the optimal
conditions for highest concentration of anthocyanins. The healthbeneficial effects are now well known but in many cases not well
proven in terms of scientific studies. With the use of modern
and efficient extraction procedures and high-precision analytical
methods, clean blueberry anthocyanin extracts can be prepared
and tested in clinical trials for their beneficial effects. Agricultural
and food processing wastes from the blueberry industry are a
potential source of anthocyanins and can be a source of extra
income for farmers and processing industries. This fruit has served
as a beneficial food as well as a food ingredient, and with modern
extraction methods it has the potential of contributing even more.
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