Blueberries and Their Anthocyanins: Factors Affecting Biosynthesis and Properties

Authors


Abstract

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

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 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 loss by optimizing and examining the working parameters of the different processing steps (Yang and Atallah 1985). In these optimization studies, chemical analysis is an important part consisting of preparation of the sample, extraction and isolation, 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 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 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 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 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).

Figure 1–.

Skeletal structure of anthocyanins.

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) and freeze-drying is used as a processing measure for the long-term 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 C6C3C6 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 time-of-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.

Table 1–.  Anthocyanins commonly present in blueberries, their structures, sugar moieties, and color.Thumbnail image of
Figure 2–.

Pathways of biosynthesis of some major anthocyanins present in blueberry.

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 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, 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).

Table 2–.  Total anthocyanin content reported in different parts of the blueberry plant.
Part of plantAmount of anthocyaninReference
  1. CGE = cyanidin 3-glucoside equivalent.

  2. The forms of presentation have been adapted from the reports themselves to show the different forms of quantification available in the literature for anthocyanins.

Blueberry fruit99.9 mg/100 g fresh weightSkrede and others 2000
 111 mg/100 g fresh weightGao and Mazza 1994
 83.2 mg/100 g fresh weightKalt and Dufour 1997
 20–190 mg CGE /100 g fresh weightKalt and others 2001
 143.52–822.73 mg/100 gCho and others 2004
Conventionally grown blueberry82.4 mg/100 g fresh weightS. Y. Wang and others 2008
Different cultivars of blueberry89 to 331 mg CGE/100 g of fresh weightEhlenfeldt and Prior 2001
Anethole-treated blueberry>320 mg CGE/ 100 g fresh weightC. Y. Wang and others 2008
Organically grown blueberry131.2 mg/100 g fresh weightS. Y. Wang and others 2008
UV-C treated fruit311 ± 9 mg/100 g fresh weightWang and others 2009
Blueberry pomace151.5 mg/100 g dry weightKhanal and others 2010
Rabbiteye blueberry∼100 mg/100 g fresh weightSellappan and others 2002
Highbush blueberry790 ± 90 mg CGE/100 g dry weightWang and others 2010
Different cultivars580 ± 30 to 1370 ± 140 mg CGE/ 100 g dry weightLohachoompol and others 2008
Different cultivars62.6 ± 3.8 to 235.4 ± 6.1 mg CGE/100 g fresh weightPrior and others 1998
Different genotypes of blueberries73 ± 1.4 to 515 ± 3.6 mg CGE/100 gMoyer and others 2002
Highbush blueberry ‘Rubel’230 mg CGE/ 100 g of berriesLee and others 2004

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 (H2O2) and hydroxyl radical (OH) (Kalt 2005), which are together known as reactive oxygen species (Castro and Freeman 2001). The human body produces 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 different 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 anthocyanins. 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).

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 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).

Figure 3–.

Different structural transformations of anthocyanins with change of pH (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 Sanchez-Ballesta 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 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 malvidin-3-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.

Table 3–.  Reports of different health benefits about blueberry anthocyanins.
Experimental substanceStudy subjectConclusionsReference
BlueberryPigAnthocyanins get accumulated in several parts of body including eyes and brainKalt and others 2008
Blueberry-enriched dietRatProtection against ischemic damage and prospective to avoid development of post-myocardial infarction heart failureAhmet and others 2009
Blueberry-rich dietRat“Suppressing α1-adrenergic receptor agonist-mediated contraction” and effect on vascular smooth muscle contractile machineryNorton and others 2005
Blueberry-supplemented mealHumanIncrease of postprandial serum antioxidant content in human volunteersKay and Holub 2002
Blueberry in dietHumanDiet-induced amplification of ex vivo serum antioxidant contentMazza and others 2002
Blueberry flavonoids supplementationRatDecrease in oxidative DNA damage in liver of ratsDulebohn and others 2008
Blueberry extractMiceInhibition of growth and metastatic potential of breast cancer cellsAdams and others 2010
BlueberryRatEffective against dextran sulfate sodium-induced colitisOsman and others 2008
Blueberry supplementation in feedRatEffect on “spatial working memory”Williams and others 2008
Blueberry-supplemented dietRatReversal of age-related decrease in brain's “heat shock protein 70 mediated” neuroprotectionGalli and others 2006
Blueberry-supplemented dietRatReversal of decline of age-related neuronal activity and behavioral agingJoseph and others 1999
Whole blueberry powderMiceReduction of adipocyte death and inflammatory disorders leading to decrease in whole body insulin resistanceDeFuria and others 2009
Purified berry anthocyaninsMicePrevention of dyslipidemia and obesity developmentPrior and others 2009

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).

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 Salmonellaenteritidis 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 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 high-fat-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 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.

Table 4–.  Different solvents and their combinations used for the extraction of blueberry anthocyanins.
Solvent/ solventsAnthocyanins extractedReference
AcetoneAllSkrede and others 2000
Acidified methanol (about 0.1% formic acid)AllChakraborty and others 2010
Methanol/water/acetic acid (25:24:1)AllGarcia-Viguera and others 1997; Lohachoompol and others 2008; Wang and others 2010
Water/methanol/acetone/formic acidAllPerkins-Veazie and others 2008
Methanol/acetic acid/distilled waterAllLohachoompol and others 2004
Acetone (80%) with formic acid (0.2%)AllWang and others 2009; Wang and others 2008b; Zheng and others 2003
Water and formic acidAllBrambilla and others 2008
Acidified methanol (0.6 M HCl)AllJaakola and others 2004
MethanolAllKader and others 1996
Ethanol with 1, 3, or 5% (w/w) citric acidAllChen and Camire 1997
Methanol/ water/formic acid (60:37:3)AllCho and others 2004
Acetone:methanol:water (35:35:30) with 1 cm3 36% (w/w) HCl per lAllKrupa and Tomala 2007
Acetonitrile containing 4% acetic acidAllPrior and others 1998
Acetone/ methanol/water/ formic acid (40:40:20:0.1, v/v/v/v)AllWang and others 2000
Methanol:water:trifluoroacetic acid (70:30:1, v/v/v)AllBarnes and others 2009

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 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 (Luque-Rodriguez 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 electric field have shown potential in terms of extraction of anthocyanins from grape byproducts, with increases in selectivity (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; 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 time-of-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.

Figure 4–.

Different steps applied for the extraction of blueberry anthocyanins.

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 health-beneficial 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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