Anthocyanins and Human Health: Biomolecular and therapeutic

SPRINGER BRIEFS IN FOOD, HEALTH,

AND NUTRITION

123

Muhammad Riaz

Muhammad Zia-Ul-Haq

Bashar Saad

Anthocyanins and

Human Health:

Biomolecular

and therapeutic

aspects

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Muhammad Riaz • Muhammad Zia-Ul-Haq

Bashar Saad

Anthocyanins and Human

Health: Biomolecular

and therapeutic aspects

ISSN 2197-571X ISSN 2197-5728 (electronic)

SpringerBriefs in Food, Health, and Nutrition

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DOI 10.1007/978-3-319-26456-1

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Muhammad Riaz , Ph.D.

Shaheed Benazir Bhutto University

Sheringal , Pakistan

Bashar Saad , Ph.D.

Al-Qasemi Academic College

Baga Algharbiya, Israel

Arab American University Jenin

Jenin, Palestine

Muhammad Zia-Ul-Haq , Ph.D.

The Patent Ofﬁ ce

Karachi , Pakistan

Pref ace

During the last ﬁ ve decades, epidemiological studies as well as basic and clinical

studies have consistently shown that there is a signiﬁ cant positive relationship

between intake of herbs, fruits, and vegetables and reduced rate of chronic diseases

in humans, such as cardiovascular diseases, diabetes, common cancers, and other

degenerative diseases as well as aging. This is attributed to the fact that these prod-

ucts provide an optimal mix of basic macromolecules (carbohydrates, proteins,

lipids, and nucleic acids) as well as dietary ﬁ ber, antioxidants, vitamins, and miner-

als. Various molecules in the diet can control the physiological functions of the

body and supporting immune responses. Immune functions are indispensable for

defending the body against attack by pathogens or cancer cells and thus play a

pivotal role in the maintenance of health. However, the immune functions are dis-

turbed by malnutrition, aging, physical and mental stress, or undesirable lifestyle.

Therefore, the uptake of diets with immune-modulating activities is considered an

efﬁ cient way to prevent immune functions from declining and reduce the risk of

infection or cancer.

During the last years increasing consideration has been placed in plants and

foods which can contain antioxidant substances. The chemical compounds present

in plants that are related to health-promoting beneﬁ ts considered several antioxi-

dants, as vitamins C and E, carotenoids, and ﬂ avonoids. The chemical variety,

molecular weight, three-dimensional conformation, and biochemical and physical

properties of these ﬂ avonoids allow them to interact with different targets in many

live organisms. Pigmented ﬂ avonoids, mainly anthocyanins, are considered the

most important group of ﬂ avonoids in plants having more than 600 compounds

identiﬁ ed in nature. Anthocyanins are water-soluble compounds that provide color

to plant tissues (leaves, stems, roots, ﬂ owers, and fruits) ranging from red, purple,

to blue according to the environmental pH and their structural composition.

Regarding the human consumption, the high intake of foods rich in anthocyanins

offers potential health beneﬁ cial effects on various disorders associated with cancer,

aging diseases, obesity, neurological diseases, inﬂ ammation, diabetes, as well as

bacterial infections.

vi
 This book explores and introduces information available concerning the struc-
ture, composition, and abundance of anthocyanins in fruits and its bioavailability 
and biological activity related to health-promoting effects. This book includes nine 
chapters, embracing particularly historical aspects and present uses of traditional 
Arab-Islamic herbal medicine. Chapter   1     focuses on botanical medicines or herbal 
medicines. These therapies are still utilized as the primary form of medicine by 
about 80 % of the world’s population. Over 80,000 species of plants are in use 
throughout the world. Usually, leaves, fruits, ﬂ owers, seeds, and roots are formu-
lated into tablets or pills, teas, extracts, tinctures, ointments, or creams. Currently, 
about 25 % of the commonly used modern pharmaceutical drugs are of herbal origin 
or contain at least one herbal-derived active compound. Indeed, some are extracted 
from herbal crude extracts; others are chemically modiﬁ ed to produce a pharmaceu-
tically active drug that agonists plant active molecule. The therapeutic effects of 
medicinal plants are generally labeled as antidiabetic, anti-inﬂ ammatory, laxative, 
carminative, demulcent, antiseptic, or antitussive. Chapter   2     provides a general 
introduction on anthocyanins including their general chemical structure, their thera-
peutic efﬁ cacy, and their safety. Chapter   3     focuses on the chemical structures of 
anthocyanins. It provides understanding of anthocyanins’ chemistry including their 
occurrence in nature, e.g., plants and the recent discovered anthocyanins. Chapter   4     
describes the utilization of anthocyanins as natural color in food and beverages. 
Chapter   5     is subdivided into ten subsections, which describe intake, metabolism, 
and secretions of anthocyanins in the human body. Chapter   6     provides knowledge 
about the biosynthetic pathways through which these compounds are synthesized in 
natural system. Chapters   7    –  9     describe the state-of-the-art knowledge of in vitro, 
in vivo, and clinical literature regarding the efﬁ cacy and safety of anthocyanins, 
including anti-inﬂ ammatory, antioxidant, antidiabetic, and anticancer effects and 
prevention and treatment of degenerative diseases.  
  Sheringal,  Pakistan      Muhammad      Riaz  ,          Ph.D.    
 Karachi,  Pakistan      Muhammad      Zia-Ul-Haq ,   Ph.D.    
 Jenin,  Palestine      Bashar      Saad ,   Ph.D.      
Preface

vii

Abbreviations

AC Anthocyanidins

ACN Anthocyanin

ACNs Anthocyanins

ACN-3-gly Anthocyanins-3-glycoside

AMPK Adenosine monophosphate protein kinase

AN-gluc Anthocyanins glucuronide

BHA Butylated hydroxyl anisole

BHT Butylated hydroxyl toluene

CBG Cytosolic B-glucosidase

CD Cluster of differentiation

CH Chalcone

max

Maximum concentration

CNS Central nervous system

COMT Catechol- O -methyltransferase

COX Cyclooxygenase

Cy Cyanidin

Cyd-3-glu Cyanidin-3-glucoside

Cyd-3-rut Cyanidin-3-rutinoside

Dp Delphinidin

EMIQ Enzymatically modiﬁ ed isoquercitrin

FC Flavylium cation

FRAP Ferric reducing antioxidant potential

Gal Galactoside

Glu Glucoside

HDL High-density lipoprotein

ICAM Intracellular adhesion molecule

IL Interleukin

iNOS Inducible nitric acid synthase

LDL Low-density lipoprotein

LPH Lactase phlorizin hydrolase

LPS Lipopolysaccharide

viii

MAPK Mitogen-activated protein kinase

MCP Monocyte chemotactic protein

MCP-1 Monocyte chemotactic protein-1

MDA Malondialdehyde

MI Myocardial infarctions

Mv Malvidin

NO Nitric oxide

ORAC Oxygen radical absorbance capacity

PACNs Pyranoanthocyanins

PB Pseudo-base

PC Protocatechuic acid

PDE Phosphodiesterase

PEDF Pigment epithelial-derived factor

Pg Pelargonidin

Pn Peonidin

PPAR γ Peroxisome proliferator-activated receptor gamma

Pt Petunidin

QB Quinoidal base

RDI Recommended daily intake

max

Maximal rate of excretion

RNS Reactive nitrogen species

ROS Reactive oxygen species

RPE Retinal pigment epithelial

Rut Rutinoside

SAOC Serum antioxidant capacity

SGLT Sodium-glucose co-transporter

SREBP1c Sterol regulatory element-binding protein 1c

STZ Streptozocin

SULT Sulfotransferase

TAS Total antioxidant status

TBARS Thiobarbituric acid reactive substances

TEAC Trolox equivalent antioxidant capacity

TNF-α Tumor necrosis factor-α

TRAP Total reactive antioxidant potentials

UDP-GT UDP-glucuronosyltransferase

UV Ultraviolet

VEGF Vascular endothelial growth factor

VLA Very late antigen

WHO World Health Organization

Abbreviations

Contents

1 Diet and Herbal-Derived Medicines ........................................................ 1

1.1 Introduction ........................................................................................ 1

1.2 Current Status of Food and Herbal-Based Medicine ......................... 3

1.3 Medicinal Plants from Tradition to Evidence-Based Application ..... 4

1.3.1 Nigella sativa ........................................................................ 5

1.3.2 Olea europaea ....................................................................... 5

1.3.3 Punica granatum ................................................................... 6

1.3.4 Trigonella foenumgraecum .................................................. 6

1.3.5 Salvia ofﬁ cinalis ................................................................... 6

1.3.6 Ammi visnaga ...................................................................... 7

1.3.7 Silybum marianum ............................................................... 7

1.3.8 Inula viscose ......................................................................... 7

1.3.9 Portulaca oleracea ................................................................ 7

1.3.10 Eruca sativa .......................................................................... 8

1.3.11 Cichorium intybus ................................................................ 8

1.3.12 Allium sativum, Garlic, and Onion (Allium cepa L.) .......... 8

1.3.13 Urtica dioica ......................................................................... 9

1.3.14 Melissa ofﬁ cinalis ................................................................ 9

1.3.15 Pimpinella anisum ................................................................ 9

1.3.16 Chamomilla recutita ............................................................. 10

1.3.17 Zingiber ofﬁ cinale ................................................................ 10

1.3.18 Rosmarinus ofﬁ cinalis ......................................................... 10

1.4 Administering Herbal-Based Treatment ............................................ 11

1.5 Herbal Active Compounds ................................................................. 11

1.6 Synergistic Actions of Foods and Phytomedicines ............................ 13

1.7 Therapeutic Properties of Herbal-Based Active Compounds ............ 14

1.8 Examples of Herbal Compounds and Pharmacological Properties ... 15

1.9 Conclusions ........................................................................................ 16

References ................................................................................................... 17

2 Introduction to Anthocyanins .................................................................. 21

2.1 Introduction ........................................................................................ 21

2.2 Chemical Structure ............................................................................. 23

2.2.1 Glycone Moiety and Acylating Acids .................................... 24

2.3 Pyranoanthocyanins ........................................................................... 28

2.4 Conclusions ........................................................................................ 28

References ................................................................................................... 31

3 Occurrence of Anthocyanins in Plants .................................................... 35

3.1 Introduction ........................................................................................ 35

3.2 Concentration of Anthocyanins in Fruits, Vegetable and Nuts .......... 37

3.2.1 Variation in Anthocyanins Content ........................................ 38

3.3 Typical New Anthocyanins Found in the Past Years ......................... 40

3.4 Conclusions ........................................................................................ 42

References ................................................................................................... 42

4 Anthocyanins as Natural Colors .............................................................. 47

4.1 Introduction ........................................................................................ 47

4.2 Use of Anthocyanin-Based Colorants ................................................ 51

4.3 Acylated Anthocyanins as Colorants for the Food Industry .............. 52

4.4 Conclusions ........................................................................................ 54

References ................................................................................................... 54

5 Anthocyanins Absorption and Metabolism ............................................ 57

5.1 Introduction ........................................................................................ 57

5.2 Daily Intake ........................................................................................ 58

5.3 Anthocyanins Absorption .................................................................. 59

5.3.1 Gastric Absorption ................................................................. 60

5.3.2 Absorption in the Small Intestine .......................................... 60

5.3.3 Pharmacokinetics ................................................................... 62

5.4 Carbohydrates Moieties Deconjugation ............................................. 64

5.5 The Inﬂ uence of Colonic Microﬂ ora ................................................. 64

5.6 Metabolism in Intestinal Mucosa and Tissues ................................... 65

5.7 Tissue Distribution ............................................................................. 65

5.8 Excretion ............................................................................................ 66

5.9 Conclusions ........................................................................................ 67

References ................................................................................................... 67

6 Biosynthesis and Stability of Anthocyanins ............................................ 71

6.1 Introduction ........................................................................................ 71

6.2 Stability of Anthocyanins .................................................................. 73

6.3 Relationships Between Structure and Stability .................................. 74

6.4 Factor Affecting Stability of Anthocyanins ....................................... 75

6.4.1 pH ........................................................................................... 75

6.4.2 Co-pigmentation Effect .......................................................... 75

6.4.3 Solvent Effects ....................................................................... 77

6.4.4 Temperature ........................................................................... 78

Contents

6.4.5 Concentration Effects ........................................................... 78

6.4.6 Oxygen ................................................................................. 78

6.4.7 Light ..................................................................................... 78

6.4.8 Enzymes ............................................................................... 79

6.4.9 Ascorbic Acid ...................................................................... 79

6.4.10 Sugars ................................................................................... 79

6.4.11 Sulﬁ tes.................................................................................. 80

6.5 Stability of Anthocyanins in Food Products ...................................... 80

6.6 Anthocyanins Degradation in Plants .................................................. 80

6.7 Drawback and Derivatives of Anthocyanins ...................................... 81

6.8 Anthocyanins Stabilization Mechanisms ........................................... 81

6.9 Conclusions ........................................................................................ 83

References ................................................................................................... 83

7 The Role of Anthocyanins in Health as Antioxidant,

in Bone Health and as Heart Protecting Agents..................................... 87

7.1 Introduction ........................................................................................ 87

7.2 Presumed Health-Promoting Effects of Anthocyanins ...................... 88

7.3 Toxicity of Anthocyanins ................................................................... 91

7.4 Reproductive and Developmental Toxicity ........................................ 92

7.5 Anthocyanins Biological and Pharmacological Activities ................. 92

7.6 Antioxidant Activity .......................................................................... 93

7.7 Protection Against Cardiovascular Diseases ...................................... 95

7.8 Anthocyanins and Bone Health ......................................................... 98

7.9 Conclusions ........................................................................................ 100

References ................................................................................................... 101

8 The Role of Anthocyanins in Obesity and Diabetes ............................... 109

8.1 Introduction ........................................................................................ 109

8.2 Anthocyanins and Obesity ................................................................. 110

8.3 Anthocyanins and Diabetes ................................................................ 111

8.3.1 Oxidative Stress ..................................................................... 112

8.3.2 β Cells .................................................................................... 113

8.3.3 Insulin Secretion .................................................................... 114

8.3.4 Insulin Resistance .................................................................. 115

8.3.5 α-Glucosidase Inhibitory Action ............................................ 115

8.3.6 Obesity and Diabetes ............................................................. 116

8.4 Improvement of Eye Vision ............................................................... 117

8.5 Conclusions ........................................................................................ 119

References ................................................................................................... 119

9 Anthocyanins Effects on Carcinogenesis, Immune System

and the Central Nervous System ............................................................. 125

9.1 Introduction ........................................................................................ 125

9.2 Anti-Inﬂ ammatory Activity ............................................................... 125

9.3 Anthocyanins and Cancer .................................................................. 126

Contents

xii

9.4 Anthocyanins, Alzheimer Diseases and Brain Function .................... 131

9.5 Miscellaneous Activities .................................................................... 131

9.6 Pharmaceutical Products .................................................................... 133

9.7 Conclusions ........................................................................................ 133

References ................................................................................................... 134

Contents

M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_1

Chapter 1

Diet and Herbal-Derived Medicines

1.1 Introduction

Health and food and their inter-relationship are one of most-debated topics by

people of all age and income groups and are one of the most sought after and refer-

enced topic in E-world. A peek at the magazine rack of nearby library or websites

of newspapers conﬁ rms this as there are speciﬁ c sections for healthy-eating there.

Healthy-diet is affordable and does not include the side effects and the metabolic

and physiologic burden that medication-packages impose on human body-systems.

Diets rich in diversiﬁ ed eating pattern of plant-based foods are among the recom-

mended lifestyle modiﬁ cations to decrease the risk of diseases. Fruits, nuts, herbs,

spices, grains and legumes and their products or by-products are an integral part of

the cultural, socio-economic and health systems of all countries due to their estab-

lished health-promoting effects and veriﬁ ed immunity-boosting claims.

Botanical medicines, or herbal medicines herbal medicines also known as phyto-

therapies, are the ancient healthcare remedies known to mankind. Hundreds of wild

edible herbs and animal-derived preparations (e.g., milk, blood serum, urine, bones,

and feathers) are utilized by traditional healers to prepare remedies for the treat-

ment/prevention of all types of known illnesses as well as in maintaining healthy

body, soul, and spirit. These therapies are still utilized as the main form of drugs by

about 80 % of the world’s population. Over 80,000 species of herbs are utilized for

their medicinal properties throughout the world. Usually, leaves, fruit, ﬂ owers,

seeds, and root are formulated into tablets or pills, teas, extracts, tinctures, ointments,

or creams [ 1 – 4 ].

The last three decades have witnessed a tremendous growth in utilization of

herbal-based diet and medicines as well as a signiﬁ cant progress in studying risks

and beneﬁ ts of these products at cellular and molecular levels. Herbs build an

increasingly important source of new drugs. Currently, about 25 % of the commonly

used modern pharmaceutical drugs are of herbal origin or contain at least one

herbal-derived active compound. Indeed, some are extracted from herbal crude

extracts; others are chemically modiﬁ ed to produce a pharmaceutically active drug

that agonists plant active molecule. The therapeutic effects of medicinal plants are

generally labeled as anti-diabetic, anti-inﬂ ammatory, laxative (induces bowel move-

ments or to loosen the stool), carminative (blocks the gas formation in the gastroin-

testinal tract or facilitates the expulsion of gas), demulcent (cover the mucous

membrane with soothing ﬁ lm, healing pain and inﬂ ammation of the membrane),

antiseptic (Healing from infections) or antitussive (cough suppressants). In contrast

to pharmaceutical medicines, which are often synthetic and usually consist of a

single compound), phytomedicines contain multiple constituents (Fig. 1.1 ) [ 4 – 6 ].

The popularity in utilization of the major traditional medicines has increased

worldwide over the last half century. One of the main reason for the currently

witnessed popularity is probably the belief that these medical systems have been

used for hundreds of years and that natural product-based diet and herbal-based

remedies are safe. The resurgence of interest in medicinal plant-based therapies at

the global level has been so drastic that sales of these preparations in the world are

estimated at more than 100 billion dollars per annum. Germany and France are the

principal countries in Europe in the sale of plant-based preparation. The majority of

German physicians (about 80 %) prescribe herb-based preparations [ 5 – 8 ].

Herbal medicines in the United States are sold as dietary supplements and in

many European countries are classiﬁ ed as drugs, whereas in China and India as well

Persian, Mesopotamia

Medicine, Ancient Arabic Medicine

Innovations

introduced by Arab

and Muslim

physicians

Western medical

system

Greco-Arab

and Islamic

Persian, Chinese

Indian medicine

Pre-Islamic Arabic

medicine

Arab-Islamic medical

system

Traditional Arabic and

Islamic medicine/ Unani-

Tibb

Greekmedical system

Ayurveda and

Chinese traditional

medicines

Western traditional

medicine

Fig. 1.1 The development of various types of medical systems

1 Diet and Herbal-Derived Medicines

3
as in the Arab-Islamic world they are mostly sold over the counter without clear 
regulations. One of the problems with herbal drugs is that the concentrations of 
active compound(s) varies according to the soil, the weather conditions, and other 
environmental factors. Safety assessments of herbal products used in these tradi-
tional medicines have often been neglected due to their prolonged and apparently 
safe utilization. Nevertheless, a scientiﬁ c evidence of the toxicity of such products 
has accumulated. This is not surprising, since herbal extracts consist of mixtures of 
tens of secondary metabolites, many of which are potentially toxic (e.g., hepatotox-
icity, mutagenicity, and carcinogenicity). Therefore, the extensive consumption and 
fame of herbal products brought apprehensions and doubts over professionalism of 
healers and quality, efﬁ cacy, and safety of these products. Safety, contaminations, 
inappropriate preparation, or lack of knowledge regarding plant and drug interac-
tions [ 6 – 9 ]. 
 This chapter will provide a brief introduction to medicinal plants, including their 
therapeutic aspects and safety. In addition, the various major types of active com-
pounds will be discussed. In the course of the following chapters, we intend to 
reveal the complexities, encourage comparisons various form of anthocyanins, and 
highlight relationship between their structure and functions. To keep within the 
scope of this introductory chapter, we will give a brief overview of the main topics 
of this book. The following chapters will comprehensively discuss the chemistry, 
metabolism, in vitro and in vivo scientiﬁ c literature as well as the clinical signiﬁ -
cance of anthocyanins. We have organized this book around nine major topics, 
reﬂ ected by the titles of these chapters: (1) diet and herbal-derived medicines, (2) 
introduction to anthocyanins, (3) occurrence of anthocyanins in plants, (4) antho-
cyanins as natural color, (5) anthocyanins absorption and metabolism, (6) biosyn-
thesis and stability of Anthocyanins, (7) the role of anthocyanins in health, (8) the 
role of anthocyanins in obesity and diabetes, and (9) anthocyanins effects on carci-
nogenesis, immune system and the central nervous system.  
1.2      Current Status of Food and Herbal-Based Medicine 
 In parallel with the revival of interest in the traditional foods and medicinal plants, 
there is also an intensive research activity dealing with their safety and efﬁ cacy as 
well as with their action mechanisms at cell biological, biochemical and molecular 
biological levels. The modern clinical medicine is now beginning to accept the 
 utilization of foods and herbal-based remedies once their efﬁ cacy and safety are 
scientiﬁ cally investigated and validated. As a result, there is an increasing trend in 
Europe as well as in the USA and Canada to incorporate herbal-derived prepara-
tions as an obligatory course in the medical curriculum. Herbalists do not isolate a 
particular herbal active compound. In generally, they use the whole plant or water 
extracts from parts of plants, e.g., the leaves or roots (Fig.  1.2 ). They argue that the 
large number of active molecules present in herbs amplify therapeutic beneﬁ ts and 
reduce possible side effects [ 6 – 13 ].
1.2   Current Status of Food and Herbal-Based Medicine

Currently, products from biological sources, mainly from plants, animals, fungi,

and algae represent a main source of new medicines. The World Health Organization

(WHO) estimates that about 80 % of the world population presently utilizes plant-

based preparations for prevention or treatment of all known diseases. Although

ﬁ nancial funding for natural product–based drug development in the pharmaceutical

industry has been reduced from 1984 to 2003, the percentage of natural product–

based substances has remained relatively at relatively high level. Approximately

70 % of anticancer drugs developed since 1940s (155 drugs) have their origin from

natural products [ 5 – 8 , 13 ].

1.3 Medicinal Plants from Tradition to

Evidence-Based Application

Medicinal plants are classiﬁ ed as wild-grown or as cultivated. As a result of natural

selection, wild medicinal plants are grown in places with optimal soil and environ-

mental conditions. These herbs may have been exposed to environmental pollution

and pesticides. Hence, cultivated organic herbs grown under well controlled

Fig. 1.2 Commonly used preparation procedure

1 Diet and Herbal-Derived Medicines

5
conditions represent a better alternative to wild-grown herbs. Organic vegetables 
and fruits are becoming increasingly available, as more and more farmers start to 
adapt the organic system. With careful management, organic food farms can  provide 
high quality organic products to the market. In the case of medicinal plants, it is 
important that medicinal plant as well as fruits and vegetables have been harvested 
at the optimum season in order to achieve optimal therapeutic effects. For instance, 
many edible wild plants are collected in the spring, winter, and fall, but not in 
 summer, when energy of plants is utilized for the purposes of ﬂ owering and growth. 
 To keep within the scope of this chapter we will give a brief overview of the most 
commonly used medicinal herbs in the Mediterranean region where more than 2600 
plant species are found and about 450–700 plants are mentioned in various scripts 
for their uses as medicinal herbs. Plant parts used include seeds, roots, ﬂ owers, 
leaves, stems and fruits [ 
1 – 5 ,   12 – 14 ]. 
1.3.1       Nigella  sativa  
 The seeds of  Nigella sativa  (black seeds) are commonly used medicinal herbs 
throughout the Arab counties. These seeds have been used since dawn of civiliza-
tion as a spice and food preservative and for the treatment and prevention of a wide 
range of diseases. Thymoquinone, dithymquinone, thymohydroquinone, and  thymol 
are the main bioactive constituents responsible for the pharmacological properties 
of black seeds [ 
14 ,   15 ]. Pharmacological and toxicological effects of the seeds have 
been extensively studied. In recent years, scientiﬁ c journals have published 
 numerous articles pointing to potential therapeutic properties  Nigella sativa , 
 including antimicrobial, hypotensive, antidiabetic, anticancer, antihistaminic, 
immunomodulatory and anti-fertility effects [ 8 ,   14 – 17 ].  
1.3.2       Olea  europaea  
 The olive, like  Nigella sativa , is one of the most commonly utilized medicinal herbs 
throughout the Mediterranean. Both, olive oil and olive leaf are well known for their 
health beneﬁ ts and have been for these properties for thousands of years. The 
 primary medical constituents of olive leaf are the antioxidants oleuropein, hydroxy-
tyrosol, hydroxytyrosol acetate, and the ﬂ avonoids luteolin, and luteolin- glucosides. 
Oleuropein and its hydrolysis products are those of the greatest therapeutic  potential. 
Oleuropein has a vasodilator effect, increases blood ﬂ ow in the coronary arteries 
and improves arrhythmia. It has proven to be a potent anti- inﬂ ammatory  and 
 antioxidant compound. Various scientiﬁ c reports show that oleuropein also exhibits 
in antimicrobial activity against viruses, retroviruses, bacteria, yeasts, fungus, 
molds and other parasites [ 
8 ,   14 ,   18 ,   19 ].  
1.3   Medicinal Plants from Tradition to Evidence-Based Application

6
1.3.3       Punica  granatum  
 The pomegranate has been used since long in Arab and Islamic traditional medicine 
to treat a wide range of diseases. These include anticancer, anti-inﬂ ammatory and 
anti-rheumatic activities. In Ayurvedic medicine the fruits as well as other parts of 
the plant are regarded as “remedy for every disease”. For example, the bark and 
roots are used to manage vermifuge and helminthic conditions and the juice is 
 considered as “refrigerant” and “blood tonic”. Pomegranate ﬂ owers serve as a 
 remedy for diabetes mellitus. The hydrolyzable tannins known as punicalagins 
show anti- oxidant effects in in vitro experiments. A Medline search using therapeu-
tic potential and toxicological effects  Punica granatum  discloses greater than 350 
citations, including antioxidant, cardiovascular protection, oral hygiene, as well as 
anti- inﬂ ammatory properties [ 8 ,   14 ,   20 ].  
1.3.4       Trigonella  foenumgraecum  
 Locally known as fenugreek, it is one of the most used herb in the Mediterranean. 
Most of the medicinal beneﬁ ts of  Trigonella foenum - graecum  are found in the 
seeds, which have been used for thousands of years in Chinese and Ayurvedic 
 medical systems. The anti-diabetic effects of fenugreek are mediated through the 
active compound, 4-hydroxy isoleucine. The action mechanisms include 
 translocation of glucose transporter-4 (GLUT4) to the plasma membrane, delay of 
gastric emptying, slowing glucose absorption, and transport from the ﬁ ber content, 
as well as increased erythrocyte insulin receptors and modulation of peripheral 
 glucose   utilization  [ 8 ,   14 ,   21 ,   22 ].  
1.3.5       Salvia  ofﬁ cinalis  
 Commonly known as sage, pharmacological and toxicological properties of  Salvia 
ofﬁ cinalis  leaf have been extensively investigated using in vitro and in vivo tests. The 
main active compounds of sage are present in its essential oil, which contains thu-
jone, borneol, and cineole. In addition, sage leaf contains ursonic acid, tannic acid, 
ursolic acid, oleic acid, chlorogenic acid, fumaric acid, cornsolic acid, caffeic acid, 
niacin, nicotinamide, ﬂ avonoid glycosides, and estrogenic compounds.  Salvia ofﬁ ci-
nalis  exhibits a wide spectrum of pharmacological effects. These includes  antioxidant, 
anti-inﬂ ammatory, antimicrobial, carminative, weakly spasmolytic, astringent, and 
antihidrotic (inhibits perspiration).  Salvia ofﬁ cinalis  is also a well-known tonic and 
stimulant for the nervous and digestive systems. Clinical studies have demonstrated 
beneﬁ cial therapeutic properties of  Salvia ofﬁ cinalis  leaves in elderly patients 
 suffering from mild to moderate Alzheimer’s disease [ 8 ,   14 ,   23 – 25 ].  
1  Diet and Herbal-Derived Medicines

7
1.3.6       Ammi  visnaga  
 Khella is used to treat breathing system disorders such as asthma, bronchitis, whoop-
ing cough, cardiac diseases and liver and gall bladder disorders and is believed to help 
discharge of kidney stones and gallstones. Khella contains coumarins and furocouma-
rins the most signiﬁ cant being khellin, visnadin and visnagin. Visnadin exhibits 
 coronary and peripheral vasodilatory activities in isolated vascular smooth muscle of 
rat model. The coronary, urological and respiratory clinical and therapeutic effective-
ness of khellin is well-established. Furthermore, khella has been found effective in the 
treatment of mild angina complaints, postoperative treatment of urinary calculus and 
supportive treatment of mild forms of obstructive pulmonary diseases [ 8 ,   14 ,   26 – 28 ].  
1.3.7       Silybum  marianum  
 Milk thistle is used in liver supporting functions, treatment of chronic and acute 
liver disease, as well as promoting detoxifying functions of the liver. The active 
compounds of  Silybum marianum  are ﬂ avonolignans including silidianin, silybin 
and silichristine, jointly recognized as silymarin. Silybin is the molecule with the 
greatest degree of pharmacological effects. Silymarin protects against several types 
of xenobiotic including alcohol. Medical studies have veriﬁ ed the pharmacological 
effects of standardized milk thistle extracts in cases of cirrhosis, toxic liver and 
allied liver conditions [ 8 ,   14 ,   29 ].  
1.3.8       Inula  viscose  
 Tayun, has been used traditionally as one of the most effective medicinal herb for 
the treatment of infections, inﬂ ammations, various skin diseases and wound  healing. 
The roots have been utilized to treat cough, phlegm and sepsis. The leaves contain 
essential oils, ﬂ avonoids such as rhamnocitrin, glycosyl analogue of diacylglycerol, 
sakuranetin, methylaromadendrin, acetyl-methylaromadendrin, and sesquiterpene 
lactones.  Inula viscosais  well documented to have anti-ulcerogenic effects and to 
cause abortion in mammals. Further effects include antimicrobial and fungi effects, 
anti-inﬂ ammatory and anti-diabetic properties. Due to these wide range of medical 
effects, this plant is appreciated by pharmaceutical industries [ 8 ,   30 – 33 ].  
1.3.9       Portulaca  oleracea  
 Purslane, is used traditionally in the treatment of a wide range illnesses, such as 
include headache, stomach ache, enteritis, painful urination, mastitis. It is also used 
to increase milk production in nursing women and in treating postpartum bleeding. 
1.3   Medicinal Plants from Tradition to Evidence-Based Application

8
Arial parts of this plan have therapeutic effects in healing burns, earache, ulcers, and 
pruritis. They are also used to treat inﬂ ammations, skin sores, insect stings, eczema, 
and wound healing. Purslane contains relatively high levels of a neurohormone 
( L -norepinephrine) that was found to exhibit vasopressor and anti-hypotensive and 
anti-haemorrhage effects. Various in vitro and in vivo studies indicate that aqueous 
extracts of this plant exhibit skeletal muscle relaxant effects [ 3 ,   4 ,   8 ].  
1.3.10       Eruca  sativa  
 Rucola, is considered traditionally as a general tonic and potent aphrodisiac. 
Additional traditional uses of  Eruca sativa  include stimulation of spermatogenesis 
and fertility, antibacterial effects, and promoting kidney function and digestion. 
 Eruca sativa  extract was found to exhibit signiﬁ cant antioxidant properties. 
Glucoerucin and ﬂ avonoids are the major antioxidants present in  Eruca sativa . 
Feeding of  Eruca sativa  extract to rats induced a signiﬁ cant protection against HgCl 
induced renal toxicity. In addition, there are several scientiﬁ c reports that indicate 
that  Eruca sativa  exhibit antimicrobial effect [ 8 ,   14 ,   34 ,   35 ].  
1.3.11       Cichorium  intybus  
 Wild chicory, ﬁ nds a widespread use both in the inhibition and treatment of a wide 
spectrum of illnesses. It is useful in promoting liver functions (detoxifying  functions) 
as well as encouraging the eliminative pathways both through the intestine and the 
kidneys. Arabic traditional healers recommend chicory as part of a combined treat-
ment of metabolic problems, colds, and ﬂ u. The roots of chicory contain inulin and 
oligofructose polysaccharides. Chicory, like many plants that support liver function 
and immunity, has strong antioxidant effects in vitro, but the clinical signiﬁ cance of 
this has not been tested. However there have been several studies in humans on the 
therapeutic effects of the inulin and oligofructan polysaccharides. They have been 
found to undergo fermentation in the colon and to selectively stimulate of the growth 
of healthy biﬁ dobacteria population that results in the decrease of colonic diseases 
and diabetes, as well as support for the immune system [ 14 ,   36 ,   37 ].  
1.3.12       Allium  sativum ,  Garlic,  and  Onion  ( Allium  cepa   L.) 
 These are one of the most used plants for their well-known health beneﬁ ts. Garlic 
has been used for centuries for prevention and treatment of a large number of 
 illnesses. Chinese as well as Greeks and Romans utilized onion and garlic-based 
preparations for the treatment and prevention of diseases. The two plants are rich 
1  Diet and Herbal-Derived Medicines

9
sources of large number of active compounds. Onion and garlic is important ingre-
dient of the Mediterranean diet. Various scientiﬁ c reports indicate their efﬁ cacy in 
the treatment and prevention of a wide range of pathological conditions, such as 
cancer, cardiovascular diseases and obesity [ 3 ,   4 ,   8 ,   14 ].  
1.3.13       Urtica  dioica  
 Stinging nettle is highly appreciated in Greco-Arab medicine for its beneﬁ cial 
effects. These include anti-rheumatic effects, anti-colds and anti-cough, and 
 promoting liver functions, anti-hypotensive and anti-inﬂ ammatory  effects. 
Evidence- based therapeutic application of this plant includes anti-diabetes antioxi-
dant, anti-inﬂ ammatory, increasing cell growth lymphocytes in humans, anti- 
prostatic hyperplasia and anti-hypertension [ 8 ,   14 ,   38 ,   39 ].  
1.3.14       Melissa  ofﬁ cinalis  
 Lemon balm, the therapeutic uses of this plant dates back into ancient times. Greco- 
Arab and Islamic physicians used the herb to treat heart disorders.  Melissa ofﬁ cina-
lis  leaves contain about 0.1 % of essential oil, consisting of a highly variable mixture 
of constituents. These include and polyphenolic compounds (mainly rosmarinic 
acid and monoterpene glycosides), monoterpenoid aldehydes, and ﬂ avonoids. 
Currently,  Melissa ofﬁ cinalis  is highly appreciated by traditional healers. The herb 
ﬁ nds a widespread use in the treatment of skin diseases (mainly acne). Additional 
therapeutic effects include sedative effect on the central nervous system of mice, 
antimicrobial and antiviral effects, anti-hyperthyroidism and anti-depression effects. 
Lemon palm has also positive effects on the nervous system. In addition to anti- 
depression effects on patients, the plant is effective in decreasing symptoms of 
Alzheimer’s and dementia such as memory loss [ 14 ].  
1.3.15       Pimpinella  anisum  
 Anise, as cumin, fennel, carrots, cilantro, and dill belong to the Apiaceae family. 
The seeds (“fruits”) are used traditionally to treat of a wide range of illnesses, 
 particularly for their beneﬁ cial effects in reduction of problems related to digestion. 
Seed-based therapies are commonly used with babies and children to heal from 
baby colic. Furthermore, these seeds are also recommended by traditional healers to 
treat symptoms associated with indigestion and nausea. In additional, their antispas-
modic effect is one of most known therapeutic property of anise. The seeds 
 commonly used to treat menstrual pain, asthma attacks, whooping cough and other 
1.3   Medicinal Plants from Tradition to Evidence-Based Application

10
spasmodic coughs. Furthermore, anise seed-based preparations are rationally used 
for their ability to increase the production of milk in nursing mothers. Anise-derived 
essential have the same therapeutical properties as the whole seeds. Women in the 
ﬁ rst term of pregnancy must should avoid taking anise [ 8 ,   14 ].  
1.3.16       Chamomilla  recutita  
 Chamomile is appreciated for the medicinal beneﬁ ts of the essential oils and 
 infusions prepared from ﬂ ower heads. Due to their aromatic, ﬂ avoring and coloring 
properties they ﬁ nd a widespread use in commercial products including liniments, 
balms, hair products, soaps, detergents, perfumes, bakery and confectionary 
 products, and herbal teas. Phenolic compounds, primarily the ﬂ avonoids quercetin, 
patuletin, apigenin and luteolin are the main active compounds of the ﬂ owers. 
Medicinal beneﬁ ts of Chamomile-based remedies include antioxidant, antimicro-
bial activities and antiplatelet activity. Animal model investigations verify 
 anti-inﬂ ammatory effects, anti-mutagenic and cholesterol-lowering effects, as well 
as anti-spasmotic and anxiolytic properties [ 8 ,   14 ].  
1.3.17       Zingiber  ofﬁ cinale  
 The rhizome (the underground stem) of the ginger is appreciated globally both as 
spice and for its medicinal properties to treat arthritis, colic, diarrhea, painful men-
strual pains, as well as common cold and ﬂ u. The rhizomes have been utilized since 
ancient times as a one of the effective herbal remedies in various systems of medi-
cine. Currently, traditional healers recommend ginger for the treatment/prevention 
of nausea and vomiting related with pregnancy, cancer and motion sickness. In 
addition, rhizome-based extracts ﬁ nd a wide spread use as a gastrointestinal utility 
for minor stomach troubles and to cure inﬂ ammations like arthritis [ 8 ,   14 ].  
1.3.18       Rosmarinus  ofﬁ cinalis  
 Rosemary, the areal parts of this highly aromatic plant, known for its bitter and 
astringent taste, are customarily utilized all over the Mediterranean region, both as 
cooking spice and for their medicinal properties. Rosemary contains a number of 
bioactive phytochemicals, such as the antioxidants carnosic acid and rosmarinic 
acid. Rosemary is known for its effects on muscle relaxation. Because of this prop-
erty it is conventionally used to relieve digestive problems and to ease menstrual 
pains. A tea made from the leaves is also taken as a tonic for calming nerves and 
used as an antiseptic. Several studies showed that carnosic acid, found in rosemary, 
exhibit a strong antioxidant and antimicrobial properties [ 
14 ,   40 ].   
1  Diet and Herbal-Derived Medicines

1.4 Administering Herbal-Based Treatment

Several preparation methods were developed in major traditional medicines are still

practiced by traditional herbalists to prepare herbal-based medicines. The majority

of herbal preparations are used as tea or water diluted extracts. Heating fresh or

dried plant parts in a solvent result in the extraction of bioactive phytochemical. In

addition, this procedure helps to reduce or even to eliminate impurities and poisons

and prior to application (Table 1.1 ). The chemical composition and concentration of

an extract is largely affected by the solvent used in the extraction. Water extracts

will be rich in hydrophilic phytochemicals, oil on the other hand will absorb hydro-

phobic substances. Alcohol will help in extracting, both polar and un-polar

compounds. Other extraction methods include the inhalation of aerosols, essential

oils (Essential oils are volatile, complex, natural compounds formed by aromatic

plants), tinctures (tinctures are preparations containing alcohol), capsules and

tablets and vaporized plant juices or teas [ 1 – 5 , 41 ].

1.5 Herbal Active Compounds

Plants produce metabolites as part of their normal cellular metabolic functions.

These are classiﬁ ed as primary metabolites, present in all plants, and secondary

metabolites eliciting pharmacological effects in man and animals (Table 1.2 ). Basic

metabolism comprises all primary metabolites essential for the survival of the plant

which are involved in the primary anabolic and catabolic cellular processes respon-

sible for types of cellular activities (e.g., cell growth and differentiation). In con-

trast, secondary metabolites are those that found usually only in special, differentiated

cells/tissues and are not necessary for the cells/tissue themselves but are important

for the plant as a whole. The number of known secondary metabolites that have

been discovered to date is increasing at a constant rate. Yet, it is not only plants that

produce these bioactive compounds; rather, other organisms such as fungi, bacteria,

Table 1.1 Preparations methods used for oral administrations

Administration form Preparation methods

Whole plant Fresh juice; fresh/dried areal parts and other underground parts

Tinctures Preparations of plant extract with varying ratios of water and alcohol

Tisanes Hot water extracts of plants

Decoctions and teas Made by steeping and soaking herb (leaves, ﬂ owers, stems, roots,

and bark) in water for a few minutes

Vinegars Prepared as tinctures

Syrups Extracts of herbs made with syrup or honey

Extracts Extracts are liquids with a lower alcohol level than tinctures

Essential oils Essential oil extracts are usually diluted in carrier oil

1.5 Herbal Active Compounds

sponges, as well as animals, are also capable of synthesizing a large number of these

compounds. In general, secondary metabolites often possess interesting therapeutic

properties in humans and animals, and therefore their investigation is very important.

It should not be forgotten that plants synthesize these compounds as part of their

own survival strategies. For example, some secondary products are pheromones

used to attract insects for pollination, while others are toxins used to deter predation.

Phytoalexins protect the plant against fungal or bacterial infections. Flavonoids acts

as antioxidants to neutralize free radicals generated during photosynthesis.

Anthocyanins may attract pollinators or seed dispersers. Alkaloids can protects

against herbivore animals or insect attacks. Plants regulate their cellular metabolism

in response to the present herbivores, pollinators, microorganisms, and other

environmental stresses. In addition, recent evidence has pointed to additional roles

for secondary metabolites in plant development. Although the term “ secondary

metabolites” perhaps infers a less important role for these compounds than those

involved in primary metabolism, this is not the case. In fact, many essential and

nonessential compounds in this group are found in plants, and even so-called

“ nonessential materials” can play a role in a plant’s responses against abiotic and

biotic stress.

Table 1.2 Secondary metabolites and their properties

Metabolites Examples

Primary metabolites :

Organic compounds produced in

plants

Polypeptides, cellulose, amino acids, nucleic acids,

mono-saccharides, and lipids

Essential for basic cell growth and

differentiation

Produced all plant tissues

Secondary metabolites :

Organic compounds produced in

plants

Generally grouped into classes:

Do not have essential role involved

in growth and differentiation

Polyphenols (Widely distributed in the plant

kingdom, responsible for the colors of many

ﬂ owers, others are present in bark, roots and leaves

that play an important role in tanning hides and

skins to give leather. Yet others are simpler

compounds found in most fresh fruit and vegetables

Produced in different plant

families, in speciﬁ c groups of plant

families or in speciﬁ c tissues, cell/

tissue speciﬁ c, produce at different

developmental stages and in

response to environmental stresses

Terpenoids and steroids are derived

biosynthetically from isopentenyldiphosphate).

Over 35,000 compounds are known

Fatty acid - derived substances and polyketides are

biosynthesized from simple acyl precursors such as

acetyl CoA. More than 10,000 molecules are known

Alkaloids are derived biosynthetically from amino

acids. More than 12,000 compounds are known

Nonribosomal polypeptides are biosynthesized

from amino acids

Enzyme cofactors are coenzymes such as pyridoxal

phosphate

1 Diet and Herbal-Derived Medicines

13
   In general, secondary metabolites occur as complex mixtures. The chemical com-
position and concentration of same plant can vary over time in response to variation 
in environmental conditions. Their biosynthesis can also be inﬂ uenced by a variety 
of factors during development, in addition to stress, which makes the determination 
of their complete pattern essentially very difﬁ cult. Whilst secondary metabolites can 
occur in the tissues as active compounds, they can also be synthesized as inactive 
compounds that must be transformed into active products. Compounds that are bio-
synthesized under stress conditions are typically not detectable in unstressed tissues; 
when they are synthesized after the invasion of plants by various pests. The patterns 
of secondary metabolites will differ depending on the species. The synthesis of 
 secondary metabolites can occur in all plant organs, including the roots, shoots, 
leaves, ﬂ owers, fruit, and seeds. Some metabolites are stored in speciﬁ c compart-
ments, which may be either whole organs or specialized cell types. Within these 
compartments the concentration of toxic secondary metabolites may be very high, so 
that they can exert an efﬁ cient defense against herbivores. 
 In order to identify or quantify a compound of interest, the metabolite must ﬁ rst 
be extracted from the plant tissues. However, the chemical properties of a material 
under investigation isof great importance in the development of a relevant puriﬁ ca-
tion scheme (Fig.  1.1 ). The most important issues to be taken into account include: 
It must be deﬁ ned whether a compound or a broad range of already known should 
be extracted and quantiﬁ ed. In addition, for individual compounds, it must be 
 determined which properties are already known, and which solvents can be used for 
their extraction. And ﬁ nally, the purity of the compound might be important for 
 identiﬁ cation and also for bioactivity assays; in this situation the metabolite must be 
further puriﬁ ed using chromatographic methods [ 5 ,   7 ,   40 ,   42 ].  
1.6      Synergistic  Actions  of  Foods  and  Phytomedicines 
 In contrast to synthetic drugs based upon one pure active molecule, the majority of 
herbal-derived medicines exert their pharmacological action via synergistic or 
 additive pathway of a mixture active biomolecules acting at single or multiple target 
tissues associated with a pathophysiological pathway. In addition to desired thera-
peutical action these synergistic and additive effects can be advantageous by 
 reducing negative side effects allied with the use of drugs consisting of a single 
pharmaceutical molecule. Additive and synergistic effects likely have their origin in 
the physiological and metabolic roles of secondary products in stimulating plant 
survival, regeneration and growth. For instance, a combination of secondary metab-
olites having additive or synergistic action at multiple target cell/tissue would not 
only guarantee efﬁ cacy in ﬁ ghting wide range of pathogens and herbivores but 
would also reduce or even eliminate the probabilities of these pathogens developing 
adaptive responses or resistance [ 11 – 14 ].  
1.6   Synergistic Actions of Foods and Phytomedicines

1.7 Therapeutic Properties of Herbal-Based Active

Compounds

Plants synthesize a wide range of secondary metabolites but most are derived from

a few chemical motifs. These phytochemicals can have pharmacologic properties in

humans and can be chemically modiﬁ ed to produce new medicines. Numerous

herbal-derived substances have been investigated for their pharmacologic potential

as new drugs. These include ﬂ avonoids, coumarins, saponins and alkaloids.

Flavonoids, in particular anthrocyanins, are probably the best elucidated phyto-

chemicals of these biomolecules due to their potent antioxidant activity. The medical

beneﬁ t of numerous plant herbal-based remedies used by traditional healers, at least

in part, is attributed to their effective antioxidant effects.

As above discussed, Black seed has been used for centuries in Greco-Arab and

Islamic medicine for its magic healing properties as well as its disease prevention

effects. Avicenna (980–1037 AC) highlighted the medical beneﬁ ts of black seeds

that they act as energy-booster of human body and serves to recover from fatigue

and dispiritedness. Thymoquinone presents the main active molecule responsible

for the biological and pharmacological properties of black seed. It was found to

inhibit a wide range of pathogenic processes. For example, antioxidant, immun-

emodulatory, anti-cancer, hypolipidemic, and vasoconstrictive properties in cell

culture systems and animal models. Additional therapeutical properties of black

seeds include, inhibition of iron-dependent microsomal lipid peroxidation, cardio-

toxicity induced by doxorubin in rats, drug-induced toxicity and ameliorates the

anticancer effects. One of the very important pharmacological properties of thymo-

quinone is its high cytotoxic effects as assessed in canine osteosarcoma, colon

cancer, skin cancer and prostate cancer. In contrast thymoquinone showed low

cytotoxicity to normal cells. Thymoquinone also cures many multidrug-resistant

types of pancreatic adenocarcinoma, human leukemia and uterine sarcoma.

Furthermore, many in vitro and in vivo mechanistic studies indicate that thymoqui-

none induces apoptosis through affecting multiple cellular and biochemical targets.

Therefore, this compound present a promising example of phytochemical that is

helpful for the prevention and treatment of many types of cancer cells. This antican-

cer property was also supported by studies in prostate and other cancer cells.

Thymoquinone was found to inhibit angiogenesis in vivo, prohibited tumor angio-

genesis in a xenograft human prostate cancer model in mouse and blocked human

prostate tumor growth without any side effects. Thymoquinone also exhibits

anti- proliferative activity in colon and prostate tumors implanted in nude mice.

Taken together, these ﬁ nding show that the anticancer and cytostatic properties are

due to the effect of thymoquinone on cell cycle [ 18 , 19 ]. In addition, these results

indicate a great potential for the development of new synthetic derivatives of thymo-

quinone as anticancer drugs [ 43 – 45 ].

Another example of potential group phytochemical is anthocyanins. This phyto-

chemicals are one of the most abundant ﬂ avonoid compounds and one of the most

widespread families of natural pigments in the plant kingdom. These pigments,

1 Diet and Herbal-Derived Medicines

present in fruits and vegetables, provide color and promote health beneﬁ ts to con-

sumers due to their antioxidant capacity. To date, more than 600 anthocyanins have

been identiﬁ ed in the plant kingdom. The different anthocyanin absorb light at about

500 nm and are responsible for the red, blue and purple color of fruits and vegeta-

bles. All known anthocyanins conjugates are based on six anthocyanidin aglycones

derived from ﬂ avylium backbone with different glycosylations and acylations. As

discussed in the coming chapters of this book, many studies in cell lines, animal

models and human clinical trials suggest that anthocyanins have anti-carcinogenic

and anti-inﬂ ammatory activities, provides cardiovascular disease prevention,

promote obesity and diabetes control beneﬁ ts, and also improve visual and brain

functions. Those health beneﬁ ts are mainly associated with their antioxidant effects,

which clearly are inﬂ uenced by the molecular mechanism related to the expression

and modulation of key genes.

1.8 Examples of Herbal Compounds and Pharmacological

Properties

Many drugs currently listed as conventional medications are prepared from herbal-

derived active compounds. The majority of these herbal-derived medicines were

discovered by the study of the old traditional medical systems, namely, the Chinese,

Ayurvadic, and Greco-Arab medicine. For instance, the cardiac glycoside obtained

from the foxglove ( Digitalis purpurea ) are the most cited molecules of herbal-

derived drugs for treatments of cardiovascular diseases. They are matchless by any

synthetic or semi-synthetic medicines and have exceptional efﬁ cacy with selective

cardiotonic activity. Another example is the study of cardiovascular properties of

herbs that led to the discovery of reserpine over 65 years ago. Reserpine is derived

from the roots of Rauwolﬁ a serpentine and Vakil in 1949 reported it as a hyperten-

sive drug. About 10 years later, reserpine was isolated said plant, its structure was

elucidated and it was synthesized in labs. Later on, reserpine was found to be a

potent agent in treating Parkinson disease and depression. These results stimulated

further scientiﬁ c research and it was observed that reserpine decreased brain

nor- epinephrine, dopamine as well as serotonin. This was a break-through in

research on transmitter amine defects in depression and Parkinson’s disease. This

was a milestone for the development of many psychoactive drugs and led to a

substantial interaction between scientists and pharmacological industry.

Other examples of herbs as a source of pharmaceutical active compounds

include: Vincristine is obtained from Periwinkle and used as an anti-cancer remedy.

Cinchona bark is the source of malaria-ﬁ ghting quinine. For centuries, herbalists

prescribed echinacea obtained from purple coneﬂ ower to ﬁ ght infection. This herb

was one of the most extensively recommended medicines in the US before the

discovery and synthesis of antibiotics. Now it has been conﬁ rmed that echinacea

improves the immune system by increasing the generation of lymphocytes. Willow

bark-derived salicylic acid (Aspirin) is a key anti-inﬂ ammatory, antipyretic and

1.8 Examples of Herbal Compounds and Pharmacological Properties

analgesic molecule frequently used in clinical medicine. Another example of herbal-

derived medicines is opium poppy ( Papaver somniferum )-derived morphine which

is one of the early compounds used in conventional medicine systems and is a

premium painkiller. The isolation of morphine from crude opium by Serturner in

1806 stirred so much research on herabal drugs that Megendie published a medical

formulary in 1821, containing only pure chemical entities, hence laid paving the

pathway for the use of single and pure compounds instead of medicinal plants and

their extracts [ 43 – 45 ].

1.9 Conclusions

In parallel with the increasing in utilization of nutraceuticals and herbal-based

medicines, there is also an intensive scientiﬁ c research activity dealing with their

safety and health beneﬁ cial effects. These include management of wide range of

diseases as well as in elucidation of their action mechanisms in vitro, in vivo and

in clinical studies. Plants metabolites are classiﬁ ed as primary metabolites, like

proteins and lipids which are found in all plants, and secondary metabolites elicit-

ing pharmacological effects in man and animals. Basic metabolism comprises all

primary metabolites necessary for the survival of the cells, which are found in all

plants and are responsible for the primary metabolic functions of building and

sustaining plant cells. In contrast, secondary plant metabolites are those that occur

usually only in special, differentiated tissues and are not necessary for the cells

themselves but are important for the plant as a whole. The number of known sec-

ondary metabolites that have been discovered to date is increasing at a constant

rate. Herbal-derived medicines exercise their pharmacological actions through the

synergistic or additive pathway of numerous active molecules that act at single or

many target tissue linked with a physiological pathway. Many drugs currently

listed as conventional medications are prepared from herbal-derived active com-

pounds. These herbal-derived drugs were discovered through the study of tradi-

tional medical systems, namely, the Chinese, Ayurvadic, and Greco-Arab medicine.

Anthocyanins are one of the most abundant ﬂ avonoid compounds and one of the

most widespread families of natural pigments in the plant kingdom. The following

chapters will comprehensively discuss the chemistry, metabolism, in vitro and

in vivo scientiﬁ c literature as well as the clinical signiﬁ cance of anthocyanins.

Regarding pharmacological properties of anthocyanins, a lot needs to be eluci-

dated. Understand their action mechanisms in the prevention of chronic diseases,

cancer, neurodegenerative diseases, and aging are still to be unveiled. As we will

see in following chapters of this book, investigations regarding absorption and

distribution anthocyanins are still needed. Furthermore, the effect of long-term

exposure to anthocyanins is still largely uninvestigated and more in depth in vivo

and clinical studies are needed in order to elucidate implications of anthocyanins

in the before mentioned health-promoting effects.

1 Diet and Herbal-Derived Medicines

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1 Diet and Herbal-Derived Medicines

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M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_2

Chapter 2

Introduction to Anthocyanins

2.1 Introduction

Medicinal plants and diet therapies are the oldest methods of prevention and treat-

ment of all types of known diseases and built a substantial part of the main tradi-

tional medical systems in maintaining healthy body, soul, and spirit. The past three

decades have witnessed signiﬁ cant increase in utilization as well as a big progress

in studying the cellular and molecular interactions between intake of healthy foods

(e.g., edible wild plants, nuts, seeds, fruits, vegetables as well as olive oil) and

reduced rate of diabetes, common cancers, cardiovascular diseases, ageing and

degenerative diseases. The terms functional foods and nutraceuticals refer to phy-

tochemical substances that have long-term health beneﬁ cial effects. Although there

is very thin difference between medicinal plants and functional foods, the latter also

known as super-foods provide health-promoting and immunity boosting effects

besides providing optimum nutrition with minimum amounts. The use of medicinal

plants however leads to speciﬁ c biological actions without any nutritional aspect.

These pharmacological effects of medicinal plants, their extracts or their formula-

tions are due to secondary metabolites also known as bioactive compounds or phy-

tochemicals present in them. These secondary metabolites are speciﬁ c for plant

species and families and also serve a role in taxonomic identity of their respective

species and families. The secondary metabolites differ from primary metabolites

such as lipids, proteins, carbohydrates and nucleic acids which are commonly

found in all plants and are responsible for primary metabolism and cell growth and

regeneration. Various plant secondary metabolites, also known as active compounds

can regulate the metabolism and physiological functions of the human body, and

support immune responses. As discussed in Chaps. 7 , 8 , and 9 , both arms of the

immune system, namely the innate and the adaptive systems are indispensable for

defending the body against attack by pathogens or cancer cells, and thus play a

pivotal role in the maintenance of health. Hence, the intake of herbs and foods with

22
immunity-modulating effects are an effective way to inhibit deterioration of immu-
nity potential and decrease the chances of allied disorders like cancer, inﬂ ammation 
or infection [ 1 – 3 ]. 
 The term anthocyanin is coined from the Greek  anthos , meaning ﬂ ower, and 
 kyanos , meaning blue in 1835 by a German scientist Ludwig Marquart as previ-
ously they were known as colored cell sap, cyanophylls, chrythophylls or cyanins. 
Earlier it was believed that these colors arise from the degradation of chlorophyll 
during autumn. Boyle in 1664 in his research paper entitled, “Experiments and 
considerations touching colors” for the ﬁ rst time noticed that color change of the 
“syrup of violet” from intense purple to green is due to “acid liquor”. The ﬁ rst book 
ever written was, “The anthocyanin pigments of plants” by Muriel Wheldale in 
1916. Willstatter and Everest (1913), Willstatter and Nolan (1915) and Shibata 
et al. (1919) performed ﬁ rst time experiments on pH dependent changes of antho-
cyanin colors of  Centaurea cyanus   ﬂ owers. This group reported for the ﬁ rst time 
that development of  C. cyanus  petal color is due to conjugation of anthocyanins 
with metal ions [ 4 – 9 ]. 
 The molecular structure of anthocyanins was discovered by Richard Willstatter 
and his colleagues from 1912 to 1916 shortly after the rediscovery of Mendel’s 
laws of inheritance. In this era, anthocyanins were subject of molecular genetics 
since Mendel’s pea had distinct color due to anthocyanins [ 10 ]. The color dis-
played by these constituents was described by Pauling for the ﬁ rst time in 1939 
who suggested that the resonate structure of the ﬂ avylium cation causes intensity 
of their color. These are the biggest group of vacuolar natural colors that are water 
soluble [ 11 ] and are the most important pigments followed by chlorophylls that 
are visible to human eye. Their color is due to their resonating structure which is 
also the basis of their instability. Being phytochemicals, these molecules cannot 
be synthesized by humans and animals and they have to rely on plants for their 
requirements. Anthocyanins are found in many plant tissues predominantly in the 
epidermal tissues, palisade, spongy mesophyll of leaves, ﬂ esh of fruits/stem and 
underground storage organs [ 12 ]. but most commonly accumulated in ﬂ owers and 
fruits [ 13 ]. Anthocyanins provide color to plant tissues (ﬂ owers, stems, leaves, 
roots) ranging from red, purple to blue according to the pH and their structural 
composition. 
 The anthocyanins are recently the focus of substantial basic and pharmacological 
research because of their sparkling color, high water solubility and valuable biologi-
cal properties. They are less potent as compared to corresponding medications, but 
since they are consumed in substantial amounts in regular diet, they exert clearly 
visible long-term biological effects. The French paradox revealed that French popu-
lation has less chances of coronary heart disease because of more consumption of 
red wine. The plethora of available ﬂ ora in region and their possible combinations 
used in making this wine provides a diverse and rich pool of anthocyanins. These 
compounds play an important role in human in prevention and treatment of a wide 
range of diseases. Now-a-days, anthocyanins are becoming an integral part of human 
diet. Nutritionists and diet-counselors are increasingly recommending the use of 
2  Introduction to Anthocyanins

anthocyanin-rich foods as well as pure anthocyanins to treat many diseases, due to

their proven ability to simulate speciﬁ c hormones and neurotransmitters, to inhibit

some enzymes, and to act as antioxidant. They also act as a secondary antioxidant

defense system in plant tissues exposed to different abiotic and biotic environmental

stresses like fungal pathogens, UV light, cold temperature and dry weather [ 14 ].

2.2 Chemical Structure

The understanding of the chemistry of anthocyanins is of vital importance in assess-

ment their biological and pharmacological effects. Although hundreds of anthocya-

nins have been identiﬁ ed, only a small fraction of these molecules has been studied

in depth. Each anthocyanin has its speciﬁ c and precise three dimensional structures

which ensures optimal molecular ﬁ tting into speciﬁ c cell and sub-cellular binding

sites leading to biological effects. The biological properties of anthocyanins depend

on their chemical structure, substitutions, conjugations and polymerization. In

nature, these exist as glycosides of ﬂ avylium salts and 90 % of identiﬁ ed anthocya-

nins are based on six major anthocyanidins (aglycone). They differ from each other

depending upon sugar moiety attached to the ﬂ avyliumn cation and to the acylation,

hydroxylation and methoxilation pattern. Spectroscopic, molecular and functional

genomic studies have exhibited remarkable similarity among all anthocyanins in

sense as they all share a common skeleton. Basic skeleton structure of anthocyanins

is shown in Fig. 2.1 .

Chemically, anthocyanins belong to the ﬂ avonoid group that are glycosylated

polyhydroxy and polymethoxy derivatives (3,5,7,39-tetrahydroxyﬂ avylium cation)

of ﬂ avilium salts, possessing a characteristic C-6 (A ring)-C-3 (C ring)-C-6 (B ring)

carbon structure [ 14 ]. Structurally, these are heterosides of an aglycone unit (antho-

cyanidin) which is a derivative of the ﬂ avylium ion. The general formula of antho-

cyanins is

Fig. 2.1 Basic skeleton

structure of anthocyanins

2.2 Chemical Structure

24
  
Anthocyanidins sugars Anthocyanins+Þ
   
  The aglycone (anthocyanidins) part of the anthocyanins are quite reactive due 
to electron deﬁ cient ﬂ avylium cation and has lower solubility so they occur 
always as glycosides in nature i.e. bonded to glycosyl moiety to make it more 
stable than the aglycones. The most common glycone moiety are  D -glucose, 
 L -rhamnose,   D - galactose,   D -xylose and arabinose, these moieties are usually 
located at carbons 3, 5, 7, 3′, and 5′ [ 15 ,   16 ]. Glycosylation at C-3 position is 
most common as compared to other position [ 17 ]. All anthocyanins are type 
 O -glycosides i.e. the sugar substituent is attached through O linkage [ 15 ].  Various 
factors affect their stability in food including processing and storage temperature, 
pH, content and identity in food matrix, oxygen, enzymes and metallic ions. 
Anthocyanins are different from each other by number of hydroxylated groups in 
the anthocyanidin, the nature and the number of bonded sugars in their structure, 
the aliphatic or aromatic carboxylates bonded to the sugar in the molecule, and 
the position of these bonds [ 18 ]. The variation of methoxylation and hydroxyl-
ation patterns in these structures produces hues from orange-red (pelargonidin) to 
blue-violet (delphinidin) at pH ≈ 1. Remarkably, the anthocaynins behave in same 
way  in vivo : nasturtium ﬂ owers ( Tropaeolum majus  L.), radish ( Raphanus sativus  
L.) and strawberry ( Fragaria ananassa  Duch.) are orange-red due to pelargonidin 
derivatives, while larkspur petals ( Delphinium consolida  L.), blueberries 
( Vaccinium  sp.) and grapes ( Vitis  sp.) are blue-violet due to delphinidin-type 
anthocyanins [ 19 ]. Due to their water solubility, they are incorporated into aque-
ous food systems. Anthocyanins occurring in nature contain several anthocyani-
dins or aglycones, but only six are common in foods e.g. cyanidin, peonidin, 
pelargonidin, malvidin, delphinidin, and petunidin [ 20 ]. The structures of some 
common anthocyanins are given in Fig.  2.2 .
2.2.1        Glycone  Moiety  and  Acylating  Acids 
 Glucose, arabinose, rhamnose, galactose, xylose or glucuronic acid are the reported 
monosaccharaides chains that are linked to C-3/5/7/3′/4′/5′, which are often acyl-
ated by aliphatic acids such as acetic, malonic, malic, oxalic, tartaric and succinic 
acids and also by cyclic acids like caffeic, sinapic, ferulic, gallic, p-hydroxybenzoic 
and p-coumaric acids, concurrently up to three acylating acids may be present [ 21 ]. 
Pendant sugars increase stabilization, and absorption of these small molecules 
(±500 g/mol) into human cells and their small size helps in penetrating the blood- 
brain barrier and cell walls. Some common monosaccharides that are linked to agly-
cone moiety are shown in Fig.  2.3  while structures of some acylated cyclic and 
aliphatic acids are depicted in Figs.  2.4  and  2.5 ,  respectively.
2  Introduction to Anthocyanins

Fig. 2.2 Various anthocyanins isolated from natural sources

Apigeninidin (Ap)

Arrabidin (Ab)

Aurantinidin (Au)

Capensinidin (Cp)

Carajurin (Cj)

Cyanidin (Cy)

Delphinidin (Dp)

Europinidin (Eu)

Hirsutidin (Hs)

Luteolin (Lt)

Malvidin (Mv)

5-MethylCyanidin (5-MCY)

Pelargonidin (Pg)

Peonidin (Pn)

Petunidin (Pt)

(continued)

2.2 Chemical Structure

lucose Ar

binose

OH OH

Rhamnose

Galactose

HO OH

Xylose

Glucuronic acid

Fig. 2.3 Monosaccharides

that are linked to aglycone

Pulchellidin (Pl)

Riccionidin A (RiA)

Rosinidin (Rs)

Tricetinidin (Tr)

30-HydroxyAb (3'OHAb)

6-HydroxyCy (6OHCy)

6-HydroxyDp (6OHDp)

6-HydroxyPg (6OHPg)

Fig. 2.2 (continued)

Fig. 2.4 Acylated aliphatic acids

ffi

cac

Gallic acid

p-hydroxybenzoic acid

p-coumaric acid

Fig. 2.5 Acylated cyclic acids

2.2 Chemical Structure

28
2.3            Pyranoanthocyanins 
 Pyranoanthocyanins are the products of anthocyanins and low molecular weight 
such as pyruvic acid, 4-vinylphenol and ﬂ avonols. They are stable derivatives of 
anthocyanins and considered to contribute the age-related color changes in plants, 
and the ﬁ rst pyranoanthocyanins was reported in 1996 in red wines ﬁ ltrates [ 22 ]. 
Some pyranoanthocyanins are also detected in black carrot juice and in blood 
orange juices [ 22 ,   23 ]. Pyranoanthocyanins are stable anthocyanins derivatives that 
were identiﬁ ed in red grape wines [ 24 ]. The cyclo-addition of anthocyanins and 
ethylenic bond of another molecule at C-4 and C-5 results in the formation of bleach 
and pH change resistant, pyranoanthocyanins [ 25 ,   26 ]. Rosacyanin B violet was the 
ﬁ rst pyranoanthocyanidin isolated from plant petals  Rosa hybrida . 
 These derivatives of anthocyanins have important qualities in terms of stability 
to gastro environment may results in more pharmacological active molecule and 
also results more stable color to change of temperature, pH or other chemical reac-
tion like bleaching with SO 
2
 . Figure  2.6  indicates structure of various reported 
pyranoanthocyanins.
   For example, Vistins, a group of pyranoanthocyanins in wine with orange-red 
color, exhibit 11 and 14 times greater color at wine pH, than malvidin-3- O - glucoside 
[ 27 ]. Almost all reported pyranoanthocyanins have orange color except portisins 
which has blue color. Lu et al. (2002) isolated four pyranoanthocyanins, namely 
pyranodelphinin C and D and pyranocyanin C and D, possessing the pyrano[4,3,2- 
 de ]-1-benzopyrylium core structure from an extract of blackcurrant seeds [ 28 ]. 
Table  2.1  shows various reported anthocyanins.
   Pyranoanthocyanins have also been observed in small quantity in extracts of red 
onions [ 21 ] and strawberries [ 38 ]. Four reported methyl pyranoanthocyanins iso-
lated from black currant seeds were identiﬁ ed as the oxidative cyclo-addition prod-
ucts of the anthocyanins and extraction solvent i.e. acetone [ 39 ]. It is conﬁ rmed 
from studies that pyaranoanthocyanins are more stable than their parent anthocya-
nins, so a new strategy would be to increase the stability of anthocyanins, the impor-
tance of this group has already been discussed in previous chapters.  
2.4      Conclusions 
 Most living matter in nature has color and a major percentage of these colors are 
plant-derived. Anthocyanins are incorporated in food chains because of their pres-
ence in fruits and vegetables and their corresponding beverage products like juices, 
jams, jellies, wines, confectionary and bakery products as food color, functional 
food or as a dietary supplement. Anthocyanins have aromatic rings containing polar 
substituent groups (–OH, –OCH 
3
 ) and glycosyl residues making them a polar mol-
ecule. Structurally these are benzopyran derivatives; i.e. oxygenated heterocyclic 
compounds while chemically these are chromophores having conjugated double 
2  Introduction to Anthocyanins

Fig. 2.6 Structure of various reported pyranoanthocyanins

COOH

OGlu

Vitisins A

OGlu

Vitisins B

OGlu

Methyl pyranoanthocyanins

OGlu

10-acetylpyranoanthocyanins

OGlu

Pinotins

Flavanylpyranoanthcyanins

(continued)

2.4 Conclusions

OGlu

Portisins A

OGlu

Portisins B

OGlu

Oxovitisins

OGlu

Pyranoanthocyanin dimer

Pinotin A

Fig. 2.6 (continued)

2 Introduction to Anthocyanins

31
bonds. Due to the technological advancements in instruments and chemical deter-
minations, considerable information has been gained on the identiﬁ cation and char-
acterization of anthocyanins. Despite so much progress, still hundreds of 
anthocyanins remain unidentiﬁ ed and so full potential of many plant species has not 
been explored yet. More work is required to isolate and characterize remaining 
anthocyanins and to decipher their biological activities as it will provide invaluable 
insights into mechanism underlying their health-promoting and immunity boosting 
effects.   
     References 
    1.    Saad, B., & Said, O. (Eds.). (2011).  Greco-Arab and Islamic herbal medicine: Traditional 
system, ethics, safety, efﬁ cacy, and regulatory issues . Hoboken: Wiley.  
    2.     Saad,  B.  (2015).  Integrating  traditional  Greco‐Arab and Islamic diet and herbal medicines in 
research and clinical practice. In I. Ramzan (Ed.),  Phytotherapies: Efﬁ cacy, safety, and regula-
tion  (p. 142). Hoboken: Wiley.  
    3.    Saad, B. (2015). Greco-Arab and Islamic diet therapy: Tradition, research and practice. 
 Arabian Journal of Medicinal and Aromatic Plants, 1 ,  1–24.  
     4.     Boyle,  R.  (2004).   Experiments and considerations touching colours (1664) . London: Henry 
Herringman.  
    5.     Wheldale,  M.  (1916).   Anthocyanic pigments of plants . Cambridge: Cambridge University 
Press.  
   6.    Willstätter, R., & Everest, A. E. (1913). Untersuchungen über die Anthocyane. I. Über den 
Farbstoff der Kornblume.  Justus Liebigs Annalen der Chemie, 401 (2),  189–232.  
   7.    Willstätter, R., & Nolan, T. J. (1915). Untersuchungen über die Anthocyane. II. Über den 
Farbstoff der Rose.  Justus Liebigs Annalen der Chemie, 408 (1),  1–14.  
   Table  2.1     Various types of reported pyranoanthocyanins   
 Cyclo-addition   Type  of  pyaranoanthocyanin   References 
 Anthocyanin + pyruvic acid   Carboxypyranoanthocyanins (Vitisin A)   [ 
29 ] 
 Anthocyanin + acetaldehyde   Pyranoanthocyanins  (Vitisin  B)   [ 
30 ] 
 Anthocyanins + pyruvic 
acid + ﬂ avanols in the presence of 
acetaldehyde 
 Portisins   [ 
31 ] 
 Anthocyanins + pyruvic 
acid + vinylphenol + phloroglucinol 
 Portisins   [ 
32 ] 
 Anthocyanins + pyruvic 
acid + phenol 
 Portisins, oxovitisins, and 
pyranoanthocyanin dimmers 
 [ 
33 ] 
 Anthocyanins + pyruvic 
acid + acetone 
 Methyl-pyranoanthocyanin   [ 
34 ] 
 Anthocyanins + diacetyl   10-acetylpyranoanthocyanins   [ 
35 ] 
 Anthocyanins +  p -coumaric  or 
sinapic acids 
 Pinotins 
(hydroxyphenylpyranoanthocyanins) 
 [ 
36 ] 
 Pyranoanthocyanin + ﬂ  avanols   Flavanylpyranoanthocyanins   [ 
32 ] 
 Anthocyanins + 4-vinylcatechol   Pinotin  A   [ 
37 ] 
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859–871.

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References

35
© The Author(s) 2016 
M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food, 
Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_3
    Chapter  3   
 Occurrence  of  Anthocyanins  in  Plants                     
3.1                  Introduction 
 Currently, there are plenty of scientiﬁ c studies dealing with biological and pharma-
cological properties of anthocyanins as well as with their structure, composition, 
and abundance in the plant kingdom. Anthocyanins, are considered the most impor-
tant group of ﬂ avonoids in plants having more than 600 compounds identiﬁ ed in 
nature. These water-soluble compounds are widely distributed in plant tissues and 
provide color to leaves, stems, roots, ﬂ owers and fruits ranging from red, purple to 
blue according to the environmental pH and their chemical structure. Due to their 
chemical structure anthocyanins are soluble in water solutions and frequently found 
in the vacuoles of epidermal cells, and in some species, they are bound to membrane 
of the main cell-vacuole, called anthocyanoplasts. The relative abundance, compo-
sition, and chemical structure of anthocyanins may vary according to the species or 
from fruit to fruit of the same species depending on external and internal dynamics. 
Genetic factors and agronomic practices, intensity and type of light, temperature, 
processing season, cultivation and horticultural practices, stage of maturity at har-
vest and post-harvest storage conditions. Thus identifying absolute anthocyanins 
identity and content of speciﬁ c plant-derived diet is very difﬁ cult and therefore 
range of values is used usually. Figure  3.1  shows anthocyanins-based colors of 
 various storage conditions inﬂ uence the level of anthocyanins. Although, anthocya-
nins are found in almost all members of the plant kingdom, plants belonging to 27 
families are rich in anthocyanins [ 1 ].
   Rich edible sources of anthocyanins are colored fruits such as berries, cherries, 
peaches, grapes, pomegranates, and plums as well as many dark-colored vegetables 
such as black currant, red onion, red radish, black bean, eggplant, purple corn, red 
cabbage, and purple sweet potato [ 2 ]. All these fruits and vegetables are regularly 
consumed either in diets or in juices, soft drinks, alcoholic beverages, and similar 
other products [ 3 ]. Figure  3.2  summarizes the main known biological functions of 
anthocyanins.

Almost all highly pigmented fruits and their products as well as byproducts are

potential sources of anthocyanins colorants, such as cranberry press cake, red- grapes

extracts and their by-products [ 4 ], blueberries [ 5 ], elderberries, Hibiscus calyces ,

black choke berries, purple corn, and black current berries etc. However, the antho-

cyanins composition of all of these comprises chieﬂ y mono and di- glucosides which

stabilize against pH changes and hydration in a limited manner [ 6 ].

Fig. 3.1 Examples of anthocyanins colored fruits, vegetable, leaves and ﬂ owers

3 Occurrence of Anthocyanins in Plants

As discussed in Chap. 1 , anthocyanins impart shiny blue, orange, violet, pink and

red colors (Fig. 3.1 ) to the ﬂ owers, vegetables, beverages and fruits of some plants

thus these less stable and more water soluble metabolites than carotenoids, can be

extracted from tulips, roses and orchids [ 7 ]. In addition, Heliophila coronopifolia

from cape areas has blue ﬂ owers [ 8 ] and ‘Dalicha’ ( Tali camellia ) from Yunnan has

red ﬂ owers because of anthocyanins [ 9 ] similarly Rosa hybrida , ( Rosaceae ), has

colored appearance because of anthocyanins and carotenoids [ 10 , 11 ]. Saito et al.

(2011) reported 44 ﬂ oral anthocyanidin glycosides of pelargonidin, cyanidin and

peonidin from the Japanese morning glory ( Ipomoea nil or Pharbitis nil ) [ 8 ].

3.2 Concentration of Anthocyanins in Fruits,

Vegetable and Nuts

As mentioned above, contents and composition of anthocyanins in plant tissue are

affected by genetics (variety and cultivar), geographic origin, growing seasons,

agronomic practices utilized during farming, collection time, climate (temperature,

light, altitude) and geological conditions of area from where that food commodity

or plant part (vegetable, fruit or ﬂ ower) is collected, processing method and the

Functions of

anthocyanins

Protection

against UVB

radiations

Osmotic

adjusters

during

drought or ↓

temperature

Attracting

pollinators

and seed

dispensers

Color in

plants

Light

attenuators

Transport

vehicles for

monosacc-

haride

Active

defense

against fungi

and

herbivores

Fig. 3.2 Functions of anthocyanins in plants

3.2 Concentration of Anthocyanins in Fruits, Vegetable and Nuts

method used for determination. Knowledge on individual and population level

anthocyanins intake relies on precise information about their level in foods.

Therefore source and content of anthocyanins in food is of vital importance for

researcher, nutrition-counselors, food processors, manufacturers and consumers.

Several tables and databases of anthocyanins content of foods have been compiled

to facilitate anthocyanins classiﬁ cation and quantiﬁ cation of intake (Tables 3.1 and

3.2 ). Due to the important role in food industry and human nutrition, the common

food of the United States, fruit, vegetable and nuts are assayed for total anthocya-

nins content per 100 g of fresh weight or form consumed (Table 3.1 ).

3.2.1 Variation in Anthocyanins Content

Anthocyanins are present at cellular level in vacuoles, at organ level in ﬂ owers epi-

dermal cells and occasionally mesophylls, however in certain leaves they are

restricted to mesophylls only e.g. leaves of Secale cereal [ 40 ]. Different food matri-

ces require different methods for complete release of anthocyanins without degrada-

tion and therefore accurate determination of anthocyanins contents requires

optimum and validated procedures both for isolation and quantiﬁ cation. Even for

same food matrix, various factor like varietal differences, time of harvest, farming

practices and raw versus processed food inﬂ uence the anthocyanin contents.

Sometimes many of above mentioned variables affect anthocyanin contents and

Table 3.1 Anthocyanins content of commonly consumed fruits and vegetables in United States [ 12 ]

Fruit

Total

anthocyanins (mg) Fruit

Total

anthocyanins (mg)

Apple (Fuji, Gala, Red

delicious)

1.8, 3.2 and 17 Marion blackberry 433

Blackberry 353 Blueberry cultivated 529

Gooseberry (average of

various cultivars)

6.43 Elderberry 1993

Chokeberry 2147 Cranberry 133

Peach 4.7 Concord grape 192

Nectarine 9.2 Red grape 42.7

Currant black, 533 Currant red 14.3

Cherry (sweet) 177 Blueberry wild 705

Strawberry (average) 52.1 Raspberry black,

red

845, 116

Black plum 82 Plum 12.5

Blackbeans 23.1 Red cabbage 113

Eggplant 35.1 Red leaf lettuce 1.5

Red onion 38.8 Small red bean 6.2

Red radish 116 Pistachio 2.1

mg per 100 g fresh weight

3 Occurrence of Anthocyanins in Plants

39
while it may be possible to make generalization about one factor, in reality numer-
ous factors exert their inﬂ uence during synthesis and accumulation of anthocyanins. 
Further their post-harvest stability is affected by processing, cooking, heat- 
treatment, exposure to light and oxygen and storage which not only affects content 
but also form of anthocyanins. The concentration of anthocyanins is variable, it was 
reported to be 0.1–1 % per dry weight for most of the fruits and vegetable, it may be 
present as single main type e.g. Dianthus, apple, cherry, two main type e.g.  cranberry 
and many/mixture of anthocyanins e.g.  Rosa ,   Tulipa , grapes [ 40 ]. Table  3.3   shows 
various rich sources of anthocyanins.
   Andersen and Jordheim (2006) reported that there are more than 500 different 
anthocyanins [ 47 ] and 23 anthocyanidins [ 47 ,   48 ]; of which only six are the most 
common in vascular plants, pelargonidin, peonidin, cyanidin, malvidin, petunidin 
and dephinidin [ 49 ]. The glycoside derivatives of the cyanidin, dephinidin and 
 pelargonidin, i.e. three non-methylated anthocyanidins, are mostly found in nature, 
with 50 %,69 % and 80 % in ﬂ owers, fruits and leaves respectively [ 50 ]. 
 The distribution of the six common anthocyanidins in fruits and vegetables in the 
abundance order as: cyanidin (50 %), pelargonidin (12 %), peonidin (12 %), delph-
inidin (12 %), petunidin (7 %), and malvinidin (7 %), while publishing another 
report he included more anthocyanins sources and the abundance order was likely 
to be cyanidin (30 %), delphinidin (22 %), pelargonidin (18 %), peonidin (7.5 %), 
malvidin (7.5 %), and peunidin (5 %). In both reports, the three non-methylated 
anthocyanidins (cyanidin, delphinidin, and pelargonidin) were exposed to be more 
prevalent than the three methylated anthocyanins (peonidin, malvidin, and petuni-
din). So it was conﬁ rmed that cyanidin-3-glucoside is the most common anthocya-
   Table  3.2     Plants as sources of anthocyanins   
 Anthocyanins  sources   Reference   Anthocyanins  sources   Reference 
  Vegetables    [  
13 ]   Elderberries   [ 14 ] 
 Potato   [ 
15 ]    Hibiscus calyces    [  16 ] 
 Tomato   [ 
17 ]   Black  currents   [ 18 ] 
 Spinach   [ 
19 ]   Purple  corn   [ 20 ] 
 Paprika   [ 
21 ]   Berries   [ 22 ] 
 Purple  corn   [ 
23 ]   Strawberry   [ 24 ] 
 Red  radish   [ 
25 ]   Mulberry   [ 26 ] 
 Red and green cabbages   [ 
27 ,   28 ]   Tamarillo  fruit  ( Solanum 
betaceum   Cav.) 
 [ 29 ] 
  Fruits    Garcinia  indica  Choisy   [ 
30 ] 
 Cherries   [ 
31 ]   Litchi   [ 32 ] 
 Extracts of red grapes and its 
by-products 
 [ 
33 ]   Apple  skin   [ 34 ] 
 Cranberry press cake   [ 
35 ,   36 ]   Pomegranate 
( Punica granatum   L.) 
 [ 37 ] 
 Blueberries   [ 
5 ]   Muscadine  grapes 
( Vitis rotundifolia ) 
 [ 38 ] 
 Black  chokeberries   [ 
39 ] 
3.2   Concentration of Anthocyanins in Fruits, Vegetable and Nuts

40
nins in nature and more than 90 % of anthocyanins contain glucose as a glycosylating 
sugar [ 7 ,   40 ].   
3.3      Typical New Anthocyanins Found in the Past Years 
 Four anthocyanins with furanose sugar (apiose) were obtained from leaves of 
 Synadenium grantii   ( Euphorbiaceae )  [ 51 ], which is very unusual in anthocyanin 
family. In 2003 and 2004, Honda and Tatsuzawa et al. (2004) discovered two novel 
C-glycosyl anthocyanins, which were isolated from the ﬂ owers of  Tricyrtis formo-
sana  cultivar Fujimusume [ 52 ]. Pyranoanthocyanins are a newer member with a 
pyran ring in the anthocyanins family, which were reported in 1996 as the pigments 
detected in red wines ﬁ ltrates [ 53 ]. Furthermore, there are some unique anthocyanin–
ﬂ avanol condensed pigments (polymeric anthocyanins) reported in existing studies 
[ 54 ,   55 ]. Table  3.4  shows major and minor anthocyanins in various food while 
Table  3.5  shows the occurrence of anthocyanins in common Mediterranean diet.
   Table  3.3     Anthocyanins content in vegetables and fruits   
 Sources   Cultivar 
 Total  anthocyanin 
content (mg/100 g)   Reference 
 Blackberries,  raw    Rubus fruticosus    100.61   [ 
41 ] 
 Blueberries,  raw    Vaccinium myrtillus    163.30   [ 
41 ] 
 Cranberry,  raw    Vaccinium oxycoccos    103.70   [ 
41 ] 
 Gooseberry,  raw    Ribesuva - crispa    9.51   [ 
41 ] 
 Raspberry,  raw    Rubus idaeus    48.63   [ 
41 ] 
 Strawberry,  raw    Fragaria xananassa    27.01   [ 
41 ] 
 Grape,  raw    Vitis vinifera    48.04   [ 
41 ] 
 Grape,  dried    Sunbelt    107.60   [ 
42 ] 
 Grape, raw, skin  Black     Olympia    97.50   [ 
43 ] 
 Pomegranate,  raw    Mollar de Elche    56.09   [ 
43 ] 
 Purple tomato, dried    V118    72.31   [ 
44 ] 
 Purple carrot, dried    Rain    44.27   [ 
45 ] 
 Purple carrot, dried    Haze    57.22   [ 
45 ] 
 Red cabbage, dried    Gario    198.61   [ 
45 ] 
 Purple  cauliﬂ ower, dried    Grafﬁ tti    201.11   [ 
45 ] 
 Purple potato, dried    Majesty    97.71   [ 
45 ] 
 Purple potato, dried    Mackintosh    48.74   [ 
45 ] 
 Red potato, dried    Y38    85.23   [ 
45 ] 
 Red potato, dried    Thumb    43.09   [ 
45 ] 
 Red onion, dried    Pier - c    7.77   [ 
45 ] 
 Red onion, dried    Pearl    18.95   [ 
45 ] 
 Red onion, raw    Morada de Amposta    23.30   [ 
46 ] 
 Eggplant,  dried    Black Beauty    29.55   [ 
45 ] 
3  Occurrence of Anthocyanins in Plants

41
   Table  3.4     Occurrence pattern of anthocyanins in common vegetables and fruits [ 
56 ]   
 Name 
 Major 
anthocyanins  Minor anthocyanins 
 Strawberry   Pelargonidin-
3- glucoside 
 Cyanidin-3-glucoside,  Pelargonidin-3-rutinoside 
 Blackberry   Cyanidin-
3- glucoside 
 Cyanidin-3-rutinoside,  malvidin-3-glucoside 
 Raspberry   Cyanidin-
3- glucoside 
 Pelargonidin-3-glucosides,  Pelargonindin-3-rutinoside 
 Sweet  cherries   Cyanidin-
3- rutinoside 
 Cyanidin-3-glucoside,  Peonidin-3-rutinoside 
 Blackcurrant   Cyanindin-
3- rutinoside 
 Cyanindin-3-glucoside,  Delphinidin-3-glucoside 
 Bilberry   Delphinidin-
3- galactoside 
 Peonindin-3-glucoside,  Peonindin-3-galactoside 
 Red  onions   Cyanidin 
3-glucoside 
 Delphinidin-3-glucoside,  Petunidinglucoside 
 Blood  orange   Cyanidin 
3-glucoside 
 Delphinidin 3-glucoside, cyanidin 3,5-diglucoside, 
cyanidin 3-sophoroside, delphinidin 
3-(6″-malonylglucoside), peonidin 
3-(6″-malonylglucoside), cyanidin 
3-(6″-dioxalylglucoside) 
   Table  3.5     The occurrence of anthocyanins in common Mediterranean diet   
 Plant   Anthocyanins   References 
 Blackberries  ( Rubus 
ulmifolius Schott ) 
 Cyanidin-3-glucoside (major) and cyanidin-3-
rutinoside (minor) 
 [ 
57 ] 
 Eggplant  ( Solanumm 
elongena L .) 
 Nasunin   [ 
58 ] 
 Fig  ( Ficus carica L .)   Cyanidin-3- O -rhamnoglucoside  (cyanidin-3- O - 
rutinoside) (major), cyanidin 3-glucoside, cyanidin 
3,5-diglucoside and pelargonidin 3-rutinoside 
 [ 
59 ,   60 ] 
 Grape  ( Vitis vinifera L .) 
and Wine 
 3- O -monoglucoside of pelargodin-3- O -glucoside, 
cyanidin-3- O -glucoside,  delphinidin-3- O -glucoside, 
peonidin-3- O -glucoside,  petunidin-3- O -glucosideand 
malvidin-3- O -glucoside 
 [ 
61 ] 
 Lentil  ( Lens culinaris 
Medik .) 
 Delphinidin-3- O -(2- O - β- 
D -glucopyranosyl-α- L -
arabino- pyranoside 
 [ 62 ] 
 Myrtle  ( Myrtus 
communis L .) 
 Anthocyanin  arabinosides   [ 
63 ] 
 Olive  ( Olea europea L .)   Cyanidin  3- O -glucoside and cyanidin 3- O -rutinoside   [ 
64 ] 
 Sweet Cherry ( Prunu 
savium L .) 
 Cyanidin 3-rutinoside, cyanidin 3-glucoside, peonidin 
3-rutinoside, cyanidin 3-sophoroside, pelargonidin 
3-glucoside, pelargonidin 3-rutinoside, 3-glucoside 
 [ 
65 ,   66 ] 
 Plum  ( Prunus 
domestica L .) 
 Cyanidin-3-glucoside,  cyanidin-3-rutinoside, 
peonidin- 3- glucoside, and peonidin-3-rutinoside 
 [ 
67 ,   68 ] 
(continued)
3.3   Typical New Anthocyanins Found in the Past Years

3.4 Conclusions

Various fruits, vegetables, nuts, herbs, spices and horticultural crops are good source

of anthocyanins. Diverse factors like packaging, freezing, lipid composition and

other bioactive constituents present in fruits and vegetables affect anthocyanin

contents. Release of organic acids that can lead to isomerization during slicing and

juicing of fruits, surface area, porosity and enzymatic oxidation during slicing,

peeling, pulping or juicing all affect anthocyanins contents in fruits. Therefore

marketing minimally processed fruits and vegetables is continuously increasing due

to consumer demand for high quality and nutritive products. The development of

novel and more ample sources of anthocyanins in horticultural crops, vegetables

and fruits and improvement of their shelf-life and consumer acceptance can improve

human health signiﬁ cantly. The ongoing research on horticulture and crops is

expected to bring more verities on grocery shelves and in hands of consumers

having more anthocyanins contents than traditional varieties.

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Plant Anthocyanins References

Onion ( Allium cepa L .) 3-(3-Glucosyl-6-malonyl-glucoside), 3-(6-malonyl-

glucoside), 3-(3-glucosylglucoside) and cyanidin-3-

glucoside, respectively 3-(3,6-dimalonylglucoside),

3-(3-malonylglucoside), and 3,5-diglucoside of

cyanidin, 3-glucoside, 3,5-diglucoside and

peonidin-3-malonyl-glucoside

[

69 , 70 ]

Red Radish ( Raphanus

sativus L .)

Cyanidin and pelargonidin derivatives pelargonidin-3-

sophoroside-5-glucoside derivative compounds were

found

[

71 ]

Red chicory ( Cichorium

intybus L. var. Silvestre

Bisch .)

Cyanidin-3- O -(6-malonyl-β-glucopyranoside)

delphinidin derivative has been recently identiﬁ ed

[

72 , 73 ]

Pomegranate ( Punica

granatum L .)

3-Glucosides and 3,5-diglucoside such as

delphinidin-3- glucoside, cyanidin-3,5-diglucoside,

delphinidin-3- glucoside and pelargonidin-3-glucoside

[

74 , 75 ]

Table 3.5 (continued)

3 Occurrence of Anthocyanins in Plants

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3 Occurrence of Anthocyanins in Plants

M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_4

Chapter 4

Anthocyanins as Natural Colors

4.1 Introduction

Colors play a very important but often under-estimated role in our lives. Since

ancient times natural colors derived from plants, lichens and insects have been used

as sources for natural colors, suitable for multiple applications including textiles,

paints, works of art, food, clothing and cosmetics. Colors fascinate human and ani-

mals as they are one of the ﬁ rst characteristics observed by senses and helps in

determining the acceptability of food and play a crucial role in the plant pollination

process by animals.

Since, a decent outlook is crucial to fetch high market price of food item, natural

or synthetic color is added to the food. Natural pigments from plant or animal origin

are believed to be more safe and cost-effective than synthetic ones. Synthetic dyes

have been suspected to affect adversely central nervous system both behaviorally

and neurologically. Colors from biological origin are called ‘biocolors’. The suc-

cess of any color, natural or synthetic, depends upon its consumer-acceptance,

approval by regulatory bodies and the size of the investment required in bringing the

color to store-shelves. The use of natural colors is an inﬂ uential tool for selling a

product in market. The food as such or in packed form is judged by its color or even

by color of package. It has led to claims appearing on colored food products and

commodities like, “no synthetic added” or “all natural”.

There are many pigmented classes of phytochemicals, such as carotenoids, ﬂ avo-

noids and anthocyanins. The later class has been focus of attention as these are safe

compared to artiﬁ cial colors like Sudan red III and other synthetic colors.

Anthocyanins like other bio-colors have a high market value with prices variations

from US $1250 to $2000 per kg. No permission is required for work on these pig-

ments as these occur naturally.

Chlorophyll, carotenoids, and anthocyanins are the most abundant natural occur-

ring pigments in plant tissues. We recognize anthocyanins indirectly because of the

splendid red to red-brown to purple autumn colors of the leaves of trees. In combi-

48
nation with the yellow red carotenoids, they give leaves, ﬂ owers and fruits a wide 
spectrum of colors. The anthocyanins are now exclusively used as food colors but in 
older times they were used in paints for precious manuscripts. They are now pre-
pared from the peels of grapes (enocyanin) as a side product of the grape harvest 
and winemaking. Red cabbage is also a good source of anthocyanins. The rainbow 
of colors of anthocyanins is due to conjugated bonds present in their structures. 
Most of the ﬂ ower colors are ﬂ avonoids in nature and red or purple is associated 
with anthocyanins alone or as co-pigment responsible for other related colors. The 
pigmentation of ﬂ ower attract insects and contribute in its propagation, another 
important role of anthocyanins for plants is protection from external plant enemies 
e.g. cyanidin and peonidin glucosides inhibited the growth of  Xanthomonas oryzae  
[ 1 ]. Their importance may not be overlooked because they have been sub-grouped 
as color of current EU approved additives with E163, E-Number by EU food stan-
dard agency [ 2 ]. Safety and water solubility of anthocyanins allow their integration 
into aqueous foodstuff without unwanted negative effects [ 3 ]. 
 Like synthetic color, pure single natural color is hard to obtain thus extract from 
the concerned natural source is used as coloring agent for example the most com-
monly anthocyanins enriched extract used are grape peel and black currant. This 
natural color varies with the stage and season of the plant, leaves when young have 
different color than aged leaves and same is for fruit and ﬂ owers. The reason for 
color variation is thought to be the intra and intermolecular bioconversion of antho-
cyanins or derivatives with age and environment to its variant analog moieties that 
have speciﬁ c colors. For example the interconversion of aromatic polyacylated 
anthocyanins is responsible for bluing effect and stability of ﬂ ower color. Taking the 
advantage of the advancement in plant biotechnology the cultivar of desired color 
may be produced by incorporating the gene of interest. Investigation on this side is 
required to produce natural color in greater in quantity and quality. 
 As mentioned above, the color of anthocyanins is due to the conjugated double 
bonds carrying a positive charge on the heterocyclic oxygen ring under acidic con-
ditions. Various factors like pH, co-pigmentation and interactions with metal ions 
lead to variation of colors in anthocyanins by affecting the conjugated double bond 
system present in basic skeleton of anthocyanins. At pH near to 1 the free ﬂ avylium 
cation is red while at higher pH, the color changes to purple and blue as it goes in 
transition to the quinoidal base and subsequently to ionized quinoidal base. At 
higher pH, the colorless carbinol pseudo-base is formed which ultimately changes 
to chalcone molecule having a pale yellow color. Substitution of hydroxyl or methyl 
group also affect the color of anthocyanins (Figs.  4.1  and  4.2 ) for example increas-
ing the number of hydroxyl increase bluishness, while increasing the number of 
methyl group intensify redness [ 1 ].
    Anthocynanin color depends upon number of OH groups attached especially on 
B ring. Addition of OH groups shifts their color from orange to violet. Similarly 
glycosylation of anthocyanidin leads to extra red color of anthocyanins while pres-
ence of aromatic or aliphatic acyl moieties does not change color but affects antho-
cyanin solubility as well as stability. Literature indicates that more than 650 
anthocyanins exist in nature. In 1993, over 250 different anthocyanins have been 
4  Anthocyanins as Natural Colors

Apigeninidin

(Orange)

Delphinidin

(Purple, mauve and blue)

Aurantinidin

(Orange)

Cyanidin

(Magenta and Crimson)

6-Hydroxycyanidin

(Red)

Luteolinidin

(Orange)

Pelargonidin

(Orange, salmon)

Triacetidin

(Red)

Fig. 4.1 Natural OH substituent’s anthocyanidins and change in color [ 1 ]

4.1 Introduction

Peonidin

(Magenta)

Capensinidin

(Bluish red)

Europenidin

(Bluish red)

Hirsutidin

(Bluish red)

Malvidin

(Purple)

5-Methylcyanidin

(Orange red)

Petunidin

(Purple)

Pulchellidin

(Bluish red)

Rosinidin

(Red)

Fig. 4.2 Natural methyl substituent’s anthocyanidins and change in color [ 1 ]

4 Anthocyanins as Natural Colors

51
isolated from plants [ 4 – 7 ] and 500 anthocyanin structures reported by the year 2000 
[ 8 ]. The six common anthocyanidins yield greater than 540 anthocyanins in nature 
[ 9 ], due to acylation of sugar residues with organic-acids and structural differences 
of glycosidic substitution at the 3 and 5 positions [ 10 ]. Andersen and Jordheim, in 
2006 reported the number 500 [ 10 ] and the number increased to 635 in 2008 [ 11 ]. 
 Anthocynains are exclusively used as red color for food stuff although sometime 
betalains are also used. The chief reason of this use in food matrixes is requirement 
of marinating the acidic pH (less than 3.5) to obtain the required red color. Grape 
extracts are more stable towards pH variations so they are widely used as commer-
cial sources of anthocyanins. Further grapes are available globally since they are a 
major fruit in many countries. 
 The cheap source of anthocyanin is by-products of some industrial practices like 
grape skin extracts. Therefore more focus is being given to anthocyanin-rich waste 
by-products like purple corncobs and banana bracts. The use of these by-products is 
however very challenging. Other ingredients can be added. Designing an accurate 
recipe of different reagents to increase their color stability as well suitable process-
ing and storage time can lead to stable colors. 
 Although being obtained from plants, there are many challenges. For example 
the supply is subjected to long cultivation times, climatic and seasonal variations, 
disease risks, increasing cost of agriculture as well as declining availability of culti-
vable land all making their availability highly challenging. New feasible candidate 
to be investigated as new natural pigment must be readily available, economical and 
high yield plant and with satisfactory tinctorial strength. The great activity in this 
ﬁ eld is supported by data from a survey by Frost and Sullivan in 2002, who predict 
an expansion of the European color market by 1 % per year till 2008, whereas color-
ing foodstuff is estimated to grow even by 10–15 % in the same time range [ 12 ].  
4.2      Use  of  Anthocyanin-Based  Colorants 
 Anthocyanins are being used by humans since beginning of civilization for decora-
tive purposes. Egyptians used wine as source of food color to enhance the color of 
candies approximately 1500 BC. The oldest anthocyanin used and marketed is enocy-
nain obtained from pomace of red grape in Italy since 1879. According to the USA 
regulations, “color additives are deﬁ ned as any dye, pigment or substance capable of 
imparting color when added or applied to a food, drug or cosmetic (21CFR70.3). ANs 
are permitted as food colorants in the USA under the category of fruit (21CFR73.250) 
or vegetable (21CFR73.260) juice color.” Under these categories only suitable for 
eating part of plants and their water extracts are permitted for use. 
 Anthocyanins themselves and any of anthocyanin-derived colorant are recog-
nized as a natural colorant (labeled as E163) by the European Union in  Codex 
Alimentarius  system. In USA, the Food and Drug Administration Authority (FDA) 
has cataloged anthocyanin under list of natural colors that do not require certiﬁ ca-
tions, i.e. without FD&C numbers. Out of these exempted 26 colorants approved by 
4.2   Use of Anthocyanin-Based Colorants

52
FDA, 4 are based on anthocyanin pigments. Anthocyanins are a potent alternative to 
FD&C Red No. 40 which is a synthetic dye with highest consumption in US. Various 
food plants have been considered as marketable source of anthocyanin-based colo-
rants, however their use has been limited by pigment stability, availability of raw 
material and economic considerations [ 3 ].  
4.3      Acylated  Anthocyanins  as  Colorants 
for the Food Industry 
 Anthocyanins are potential food color substitute however the major demerit is poor 
stability, however acylation perk up color and pigment stability. These acylated 
anthocyanins have variety of shades that are dependent on pH of food matrix and 
structure changes in anthocyanins. For example acylated pelargonidin derivatives 
obtained from radish and potato at acidic pH match allura red and acylated cyanidin 
from black carrots or cabbage pH dependent hues ranging from deep red to purple. 
More than 65 % of reported anthocyanins whose structures have been properly 
identiﬁ ed are acylated. Anthocyanins diversity greatly depends on number, nature 
and linkage position of acyl groups. 
 Acyalted derivatives of anthocyanins are stable and have wide color range so 
scientists are in search of acylated anthocyanin-rich material (Table  4.1 ) for exam-
ple red cabbage in Polish food industry [ 13 ,   14 ] has color like blueberries at pH 4.2–
4.5 and usually the extract of red cabbage is available in a liquid or sprayed-dried 
water-soluble form [ 3 ]. Another exceptional source of acylated anthocyanins is 
black carrot ( Daucus carota  L) consumed in Afghanistan, Egypt, Pakistan, Spain, 
and Turkey [ 3 ]. It is well-known now that poly acylated anthocyanins are more 
stable while simple anthocyanins quickly drop their colors by hydration at 2- position 
of anthocyanidin nucleus.
   Another acceptable source of stable acylated anthocyanins is red potatoes [ 15 , 
 16 ]. Extracts of acylated anthocyanins can be applied for foods having a low pH 
level, including jellies, sauces, conserves, confectionery and bakery items and soft 
drinks [ 17 ]. Some other potential applications of acylated anthocyanins are neutral 
or slightly alkaline products, such as powdered and ready-to-eat desserts, milk- 
drinks, ice cream and panned products [ 3 ,   17 ]. Table  4.1  shows sources of some 
acylated anthocyanins, their properties and their use in food industry. 
 Anthocyanins react very rapidly with metals forming stable complexes with iron, 
copper and tin, these complexes can be used as potential coloring agents due to their 
higher stability. Acylation decrease water solubility while glycosylation increases 
it. Hydroxylation (with more –OH groups) produces blue colors while methoxyl-
ation (with more methoxyl groups) produce red colors. The number and type of 
glycosidic units at C-3 don’t change color signiﬁ cantly while additional glycosyl-
ation at C-5 induces a slight shift to red-purple.  
4  Anthocyanins as Natural Colors

Table 4.1 Sources of acylated anthocyanins, their properties and their uses in food industry

Source Properties Types of anthocyanins Trade name Company

Maqui berry ( Aristotelia

chilensis L)

Excellent ROS/RNS scavenger, ↓

oxidative stress, blood glucoe, platelet

activity

Derivatives of delphinidin

3,5-O-diglucoside, delphinidin

3-O-samb, 5-O-glucoside, delphinidin

3-O-glucoside

Delphinol

Maqui New Life

Red cabbage ( Brassica

oleracea L)

Expensive, produces different shades at

different pH, have pleasant taste,

stability to heat and light, low in

polyphenols reducing the risk of hazing

with proteins, available all year round

and have potential health beneﬁ ts when

included into the diet

15 Anthocyanins (mainly diacylated)

being derivatives of cyanidin-3-

diglucoside, -5-glucoside acylated with

sinapic, ferulic and/or p -coumaric acids

Magento™ Overseal Foods Ltd

Black carrot ( Daucus

carota L)

Provide an excellent bright strawberry

red shade in acidic products, exhibit

mauve to blue tones under neutral pH,

vegetarian and kosher alternative to

carmine, has low levels of polyphenols

and improved stability to heat, light and

Derivatives of cyanidin-3- rutynoside-

-glucoside- galactoside acylated with one

cinnamic acid ( p - -coumaric, ferulic or

sinapic)

Exberry

Carantho

GNT Group,

Overseal Foods Ltd

Red radish ( Raphanus

sativus L).

Acylated pelargonidin derivatives from

red radish give red color to maraschino

cherries very close to that of synthetic

colorant FD&C Red # 40 at pH 3.5

This vegetable contains 12 acylated

anthocyanins (8 diacylated)

ColorPure™ RFI

Red potatoes ( Solanum

tuberosum )

Possible natural alternative pigment

source to synthetic colorant FD&C Red

# 40

Monoacylated anthocyanins Not available Nil

Purple corn ( Zea mays )

and oxalis ( Oxalis

triangularis )

Under consideration as potential food

color sources

Not yet known Not available Nil

4.3 Acylated Anthocyanins as Colorants for the Food Industry

4.4 Conclusions

Pigments act as ﬁ ngerprint of a food commodity determining its overall quality.

Anthocyanin-rich extracts obtained from purple corn, blackcurrant, grape skin and

red cabbage are now approved as natural colorants in New Zealand, Australia and

Europe and are commercially used in food industry. However, anthocyanins as nat-

ural colors have their own limitations. Difﬁ culties in collection of plant materials,

lack of standardization, extraction procedure from plants material and low color

value makes them less focused as substituent to synthetic colors. If somehow

obtained and used, they face problem of instability due to temperature, oxygen,

light, pH and enzymes. Further the color imparted is fugitive sometimes and a mor-

dant needs to be added to increase colors. However it should not be a disappointing.

Their stability can be increased by adding stabilizers like dextrin additives obtained

from tart cherries. Further research should be conducted for identiﬁ cation, charac-

terization, documentation, assessment and potential utilization from un-prospected

plant species. The large scale cultivation from these species and increasing anthocy-

anin contents in existing species by genetic engineering will lead to availability of

sufﬁ cient amounts for industrial extraction, improvement of quality and quantity of

anthocyanins and enhance their stability during processing and storage.

References

1. Delgado-Vargas, F., Jiménez, A., & Paredes-López, O. (2000). Natural pigments: Carotenoids,

anthocyanins, and betalains—Characteristics, biosynthesis, processing, and stability. Critical

Reviews in Food Science and Nutrition, 40 (3), 173–289.

2. Current EU approved additives and their E numbers. (2014). EU Food Standard Agency .

http://

www.food.gov.uk/science/additives/enumberlist

3. Giusti, M. M., & Wrolstad, R. E. (2003). Acylated anthocyanins from edible sources and their

applications in food systems. Biochemical Engineering Journal, 14 (3), 217–225.

4. Mazza, G., & Miniati, E. (1993). Anthocyanins in fruits, vegetables, and grains . Boca Raton:

CRC.

5. Harborne, J. B., & Williams, C. A. (2001). Anthocyanins and other ﬂ avonoids. Natural

Product Reports, 18 (3), 310–333.

6. Kong, J. M., Chia, L. S., Goh, N. K., Chia, T. F., & Brouillard, R. (2003). Analysis and biologi-

cal activities of anthocyanins. Phytochemistry, 64 (5), 923–933.

7. Strack, D., Steglich, W., & Wray, V. (1993). Betalains. Methods in Plant Biochemistry, 8 ,

421–450.

8. Pietta, P.-G. (2000). Flavonoids as antioxidants. Journal of Natural Products, 63 (7),

1035–1042.

9. Andersen, Ø. M., Fossen, T., Torskangerpoll, K., Fossen, A., & Hauge, U. (2004). Anthocyanin

from strawberry ( Fragaria ananassa ) with the novel aglycone, 5-carboxypyranopelargonidin.

Phytochemistry, 65 (4), 405–410.

10. Wrolstad, R. E., Durst, R. W., & Lee, J. (2005). Tracking color and pigment changes in antho-

cyanin products. Trends in Food Science and Technology, 16 (9), 423–428.

4 Anthocyanins as Natural Colors

11. Anderson, Ø., & Jordheim, M. (2008). Anthocyanins: Food applications . In Proceedings of 5th

International Congress Pigments in Food: For Quality and Health, University Helsinki

Helsinki, Finland .

12. Stintzing, F. C., & Carle, R. (2004). Functional properties of anthocyanins and betalains in

plants, food, and in human nutrition. Trends in Food Science and Technology, 15 (1), 19–38.

13. Baublis, A., Spomer, A., & Berber Jiménez, M. D. (1994). Anthocyanin pigments: Comparison

of extract stability. Journal of Food Science, 59 (6), 1219–1221.

14. Giusti, M. M., Luis, E. R., Donald, G., & Ronald, E. W. (1999). Electrospray and tandem mass

spectroscopy as tools for anthocyanin characterization. Journal of Agricultural and Food

Chemistry, 47 (11), 4657–4664.

15. Fossen, T., & Andersen, Ø. M. (2003). Anthocyanins from red onion, (Allium cepa) with novel

aglycone. Phytochemistry, 62 (8), 1217–1220.

16. Terahara, N., Konczak, I., Ono, H., Yoshimoto, M., & Yamakawa, O. (2004). Characterization

of acylated anthocyanins in callus induced from storage root of purple-ﬂ eshed sweet potato,

Ipomoea batatas L. BioMed Research International, 2004 (5), 279–286.

17. Naturex . (2014). Ultimate botanical beniﬁ ts . Cited September 2014.

References

M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_5

Chapter 5

Anthocyanins Absorption and Metabolism

5.1 Introduction

Biologically-active compounds, after ingestion, interact during metabolism to mod-

ify biological responses that can either strengthen or restrict their efﬁ cacy and

potency. Regarding anthocyanins, as discussed before in Chaps. 2 and 3 , no member

of animal kingdom can synthesize these molecules and they have to rely on plants.

Although humans are not colored by anthocyanins, low to signiﬁ cant contents of

anthocyanins are observed in human blood, tissues and organs. Demographical

studies indicate that most of the world population lives in poverty and do not have

luxury to afford such dietary recommendations. The efﬁ ciency of food in digestion

and bioavailability is the chief factor, i.e. the amount of anthocyanins released, solu-

bilized, absorbed, transported and metabolized. It is once the anthocyanins are in

human body that the major uncertainty begins. The ratio of total digested anthocya-

nins to bioavailable anthocyanins must always be considered. The outcomes are

highly inconsistent and sometimes contradictory. Due to variations of experimental-

designs, analytical procedures and species analyzed, the statistical ﬁ gures reported

and arithmetical comparisons between studies are not precise.

Stability and functionality of anthocyanins in human body depends on their con-

tent, type, and location in food matrix and presence of other bioactive compounds in

fruits and vegetables. Their instability depends directly on number of –OH groups

and indirectly on number of –OCH

groups. Further glycosylation also affects stabil-

ity as diglucoides are more stable than monoglucosides. Being polar in nature, antho-

cyanins are more soluble in polar solvents as compared to non-polar solvents. Further

food processing can lead to qualitative and quantitative changes since anthocyanins

are susceptible to oxidation and isomerization. Their bioefﬁ cacy depends upon their

bioavailability and bioaccessibility. Studies indicate that bioavailability is variable

depending upon various diverse parameters. Anthocyanidins and anthocyanins are

the least absorbed phenolics. Therefore, their abundance in term of dietary intake

does not necessarily mean their intact and relative metabolites in human body.

58
 The main criterion for absorption and resultant exerted bioactivity is liberation 
from food matrix, i.e. bioaccessibility. Bioaccessibility indicates the amount of 
anthocyanins available for absorption in gut. Bioaccessibility and bioavailability 
depends on the physicochemical characteristics of food matrix which affects the 
efﬁ cacy of the enzymatic and physicochemical digestion processes. Actually food 
micro-structure determines release, transformation and absorption of anthocyanins 
in digestive tract. Complexation with other food components, attachment to speciﬁ c 
organelles and restriction within intact cell walls or entrenchment in food matrix 
may affect the release of anthocyanins. Consequently anthocyanins bioavailability 
and bioactivity depends upon food matrix behavior, the presence of promoters and 
inhibitors of anthocyanins absorption. Food processing disrupts the food matrix 
which can affect their bioavailability. While the analytical methods themselves are 
subject to various sources of errors, the main problem with anthocyanins analysis 
lies in their inherent instability.  
5.2      Daily  Intake 
 Currently, we are watching a worldwide sharp increase in research activity as well 
as in dietary supplement containing anthocyanins. Despite the fact that the need for 
anthocyanins is different and depends on income level, information of anthocyanins 
content and composition is necessary so that guidelines can be provided on food 
sources that can provide ample to rich supplies of preferred anthocyanins. 
Anthocyanins are quickly being incorporated into the mainstream channel of func-
tional foods, dietary supplements and nutraceuticals and progressively being recog-
nized by the public due to the demand for health-promoting diets. Depending upon 
the dietary habits and socioeconomic conditions, the daily intake of anthocyanins 
varies from micrograms to hundreds of grams per person. Although individuals in 
developed countries use more anthocyanins due to awareness of their health- 
promoting effects created by television commercials and all types of media- 
campaigns, intake of diets containing anthocyanins is gradually increasing in 
low-income countries also because of easy commercial and market availability of 
juices, drinks, slushes, concentrates and extracts of fruits and vegetables containing 
greater anthocyanins contents. Since anthocyanins are not classiﬁ ed as essential 
nutrients so their recommended daily intake (RDI) has not been established yet. 
Daily intake of anthocyanins in USA had previously (1976) been estimated to be 
180–215 mg per day per person [ 1 ], but a recent study by USDA, based on the 
evaluation of more than 100 common foods in the USA, daily intake was found to 
be 12.5 mg per day per person [ 2 ]. Interestingly 100 g of berries offer up to 500 mg 
of anthocyanins [ 3 ]. This intake of anthocyanins is higher compared with dietary 
ﬂ avonoids such as genistein and quercetin (estimated at 20–25 mg/day) [ 4 ].  The 
daily intake of anthocyaninsis higher compared than in the USA. According to mul-
tinational study across ten European countries report (36,037 individuals in the age 
of 35–74 years), the mean daily intake of anthocyanidins [ 5 ] ranged in men from 
5  Anthocyanins Absorption and Metabolism

59
19.8 to 64.9 mg, while in women from 18.7 to 44.1 mg. In addition, this study 
showed high anthocyanidins intake by southern European, non-obese and non-
smoker older women [ 6 ]. The different values of daily intake are mainly due to 
different food intake data. Authentic and exact food intake data along with complete 
food anthocyanin concentration data is necessary for estimation of daily intake. 
 Proanthocyanidins content of food ﬂ uctuates depending upon distribution of 
monomers (ﬂ avan-3-ols), oligomers and polymers (usually N10, ﬂ avan-3-ol mono-
mers), and their inter-ﬂ avan bonds [ 7 ]. The mean daily intake of oligomeric and 
polymeric proanthocyanidins was estimated to be higher than that of monomeric 
ﬂ avan-3-ols, and twice as high as the combined overall intake of other ﬂ avonoids 
[ 8 ]. The total proanthocyanidin intake by American adults range from 96 to 137 mg 
per day (mg/day) [ 9 ],whereas those reported for Spanish 189 mg/day and for Finnish 
adults was [ 10 ] 128 mg/day [ 11 ]. Wang et al. (2011) recorded the mean daily intake 
in American adults to be 95 mg/day [ 12 ], the value of intake lies at lower end of 
amount that was previously seen by Gu et al. [ 9 ,   13 ].  
5.3      Anthocyanins  Absorption 
 The preventive and therapeutic actions of anthocyanins are directly proportional to 
their absorption and metabolism in human body. Many diverse challenges exist in 
conducting kinetic studies in humans mainly due to their low levels in circulation. 
Comprehensive information of qualitative and quantitative distribution of anthocya-
nins in food chain, human blood, tissues and organs is crucial in order to establish a 
statistically signiﬁ cant correlation between consumption of anthocyanins and their 
preventive and pharmacological effects. The absorption of anthocyanins depends 
upon many factors including chemical structure, nature of food matrices, interaction 
with other micro, macro and phytonutrients, type and extent of food processing and 
preparation, as well as the nutritional, pathophysiological and genetic factors of 
individual. The change in absorption of anthocyanins is also associated with its 
biological action, in general the absorption of the anthocyanins are less, so this point 
may be used for increasing the biological actions by playing with anthocyanins 
pharmacokinitics via different ways. However the structural and functional differ-
ences should be considered with respect to the source of anthocyanins for example 
the vegetable anthocyanins are more complex than fruit and thus their hydrolysis 
rate, bioavailability and metabolism is different. 
 Various factors can inﬂ uence the ability to absorb, convert and metabolize the 
anthocyanins. Due to their nutritive and health promoting effects, interest in factors 
that inﬂ uence their bioavailability has intensiﬁ ed. Biological activities of anthocya-
nins are dependent on its bioavailability, i.e. the amount of anthocyanins reach to 
systemic circulation and it is usually based on absorption and metabolism. Despite 
much information, still our knowledge about anthocyanins distribution and 
 availability is limited. Usually acylated anthocyanins are less absorbed than non- 
acylated ones. The type and numbers of backbone and acyl group are believed to 
5.3  Anthocyanins Absorption

60
affect their bioavailability in humans. Bioavailability from supplements is usually 
much better. In mouth, chewing releases anthocyanins from the food matrix. The 
bioavailability of anthocyanins is very low, with <1 % of the ingested amount reach-
ing the plasma, glucuronidated and methylated anthocyanin metabolites were found 
twice than their parent (intact) compounds [ 14 ]. In human body anthocyanins inter-
act physically and chemically with other phytochemicals which may lead to syner-
gism or interference. 
5.3.1      Gastric  Absorption 
 Anthocyanins absorption may start in the stomach and appear in the blood immedi-
ately after ingestion. Passamonti et al. (2003) and Talavera et al. (2003) used analo-
gous methods to establish that anthocyanins were proﬁ ciently absorbed in the 
stomach [ 15 ,   16 ]. Passamonti et al. injected grape anthocyanins into the stomach of 
19 Wistar male rats and collected blood from both the portal vein and the heart at 
6 min. Malvidin-3-glucoside was present in both portal and systemic plasma. 
Importantly, malvidin-3-glucoside appeared in the plasma within 6 min, presenting 
an evidence of stomach absorption. Peonidin-3-glucoside, petunidin-3-glucoside, 
and malvidin-3-acetyl glucoside derivatives were inconsistently detected, perhaps 
owing to animal variability. Neither anthocyanins aglycones nor conjugated deriva-
tives were detected in the plasma. Talavera et al. [ 16 ] (2003) infused anthocyanin 
standards as well as bilberry and blackberry extract into the stomach of pylorus- and 
sphincter-ligated rats. Gastric contents sample from gastric vein and blood sample 
from abdominal aorta were taken 30 min after the administration. HPLC analysis 
revealed that a high proportion (∼25 %) of anthocyanin mono-glycosides, including 
glucoside and galactoside, was absorbed from the stomach, whereas the rutinoside 
was poorly absorbed. It was suggested that gastric absorption of anthocyanins 
involves bilitranslocase (TC 2.A.65.1.1), an organic anion membrane carrier in the 
gastric mucosa [ 17 ].  
5.3.2      Absorption  in  the  Small  Intestine 
 The small intestine is the major site for anthocyanins absorption. Absorption of 
anthocyanins in small intestine of anesthetized rats was estimated by an in situ per-
fusion method [ 18 ] supplemented in physiological buffers. The amount of antho-
cyanins remaining in the efﬂ uent was used to estimate the rate of anthocyanins 
absorption in the small intestine. Depending on their structures, the absorption rate 
of supplemented anthocyanins ranged from 22.4 ± 2.0 % (cyanidin-3-glucoside) to 
10.7 ± 1.1 % (malvidin-3-glucoside). Such high absorption rates seemed to contra-
dict the very low levels of anthocyanins observed in the blood [ 
19 ]. However, it has 
to be noted that these absorption rates were calculated based on the disappeared 
5  Anthocyanins Absorption and Metabolism

amount in the efﬂ uent, thus they could indicate the portion of anthocyanins being

taken up into the small intestine tissue but not necessarily transferred into the blood.

Recently, our research group also demonstrated that as high as 7.5 % of the admin-

istered black raspberry anthocyanins could be taken up by rat small intestinal tissue,

yet very limited amount can be detected in urine [ 20 ].

Endogenic β-glucosidases are involved at this stage to release aglycones from

anthocyadin-glucosides. Since anthocyanin are highly water soluble and are very

large molecules, their absorption cannot take place by passive diffusion. Therefore

absorption of anthocyanins should be either hydrolyzed to the aglycone in small

intestine by α-rhamnosidase, β-glucuronidase or β-glucosidase or they should uti-

lize an active transport mechanism to transport glycosides across the intestinal wall.

Aglycones in free status are more hydrophobic and are smaller than the glycosides

and can easily inﬁ ltrate the epithelial layer passively. However intact glycosides are

absorbed by the small intestine, either by sodium-dependent glucose transporter

(SGLT1) or by the inefﬁ cient passive diffusion. Recent evidence have suggested

that acylated anthocyanins are somewhat bioavailable in the intact form [ 21 ],

although, likely owing to their increased molecular size, acylated ANs are much

less efﬁ ciently absorbed than their counterparts without the acylation [ 22 ].

Phase II enzymes convert anthocyanins to glucuronids, methylates and sulfates

in liver and kidney [ 23 , 24 ]. These conjugated forms of anthocyanins may be

excreted via bile to jejunum and recycled by enterohepatic circulation system in

intestine/colon. Figure 5.1 indicates the potential mechanism of anthocyanins (ANs)

absorption

Fig. 5.1 Potential mechanism of anthocyanins (ANs) absorption, sodium-glucose co-transporter,

AN-3-gly anthocyanins-3-glycoside, CBG cystolic B-glucosidase, LPH lactate pholorizin hydro-

lase, UDP-GT UDP-glucuronosyl-transferase, AN-gluc anthocyanins glucuronide, COMT

catechol- O-methyl = transferase, SULT sulfotransferase [

25 ]

5.3 Anthocyanins Absorption

62
   Many  studies  have  conﬁ rmed the blood circulation of anthocyanins after fast- 
track absorption in intact form from stomach and small intestine and their excretion 
as methylated, sulfo-conjugated or glucueonide derivatives via urinary system [ 15 , 
 16 ,   23 ,   26 ]. These are one of few anthocyanins that are found in intact forms in 
blood plasma [ 27 ]. In a recent study when anthocynain-rich diet was fed to rats for 
almost 17 days, their presence was observed in stomach, small intestine, liver, kid-
ney and brain. Total anthocyanin content in brain (blackberry anthocyanins and 
peonidin 3- O -glucoside) was found 0.25 ± 0.05 nmol/g of tissue [ 14 ,   28 ].  
5.3.3      Pharmacokinetics 
 The knowledge of pharmacokinetics is vital to comprehend the effect of daily intake 
of anthocyanins on health-improvement. There is limited information on complete 
pharmacokinetic data of anthocyanins. Challenges exist in understanding kinetics 
of anthocyanins in biological systems due to their low levels in human circulation 
making it hard to observe their absorption and metabolism. Despite this limitation, 
considerable work has been carried out in last decade in assessing the dynamics and 
kinetics of anthocyanins in humans. Figure  5.2  shows an overview of anthocyanins 
(ANs) absorption and excretion.
   Previous studies indicate that average quantity of anthocyanins in blood plasma 
vary from 1.4 and 592 nmol/l followed by post-consumption of dose from 68 and 
1300 mg between 0.5–4 h. Further, the average urinary excretion of anthocyanins 
varies from 0.03 and 4 % of the total ingested anthocyanins, and maximum excre-
tion occurs between 1–4 h and having a t 
1/2
  of 1.5–3 h. Although quercetin are 
excreted slowly (t 
1/2
  11–28 h), which indicates the probability of their bioaccumula-
tion [ 29 ], the excretion of anthocyanins is pretty quick (t 
1/2
  1.5–3 h) and no signiﬁ -
cant bio-accumulation occurs after a diet containing normal amount of anthocyanins. 
However the importance of increase of colonic metabolites and aglycone metabolic 
products should be explored. 
 Pharmacokinetic investigations indicate the presence of anthocyanins and their 
glucuronide derivatives in blood up to 5 h after intake while transformation into 
methyl derivatives increases over time (6–24 h). Another study also conﬁ rmed the 
presence of metabolites with their basic anthocyanin skeleton in the urine for up to 
24 h [ 26 ] thus biological activity changes with metabolic transformation e.g. glyco-
sylation and acylation patterns decrease the bioavailability of an anthocyanin; how-
ever, glycosidases present in the GIT may hydrolyze anthocyanins into 
anthocyanidins, thereby increasing their biological potential but decreasing their 
stability. The presence of a glucose moiety compared with a galactose or arabinose 
on the cyanidin and peonidin anthocyanidins present in cranberry juice seems to 
make them more bioavailable as a percentage of the delivered dose [ 30 ]. 
 The absorption of anthocyanins from food is insufﬁ cient and the contents detected 
in plasma are from nM to low μM [ 26 ,   28 ]. Kay et al. [ 26 ], reported the total cumula-
tive concentration 172.96 ± 7.44 μg h/mL of anthocyanins (parent and metabolites) 
5  Anthocyanins Absorption and Metabolism

detected in the serum over a 7 h sampling, peak plasma level 44.86 ± 2.82 μg/mL

was achieved (C

max

) within 2.8 h (t

max

), cyanidin 3-glycosides was found the parent

of the total anthocyanins in the serum. Further, the total urinary excretion of metabo-

lites and parent compounds over 24 h was 1071.54 ± 375.46 μg, reaching a maximal

rate of excretion (R

max

) of 202.74 ± 85.06 μg/h at 3.72 ± 0.83 h (t

max

) and having an

elimination half-life (t

1/2

) of 4.12 ± 0.4 h. Correspondingly, only 32.5 % of the antho-

cyanins excreted in the urine were the parent compounds with an average of 67.5 %

occurring as conjugated metabolites.

To exert any physiological effect, bioactive molecules should be bioavailable.

The percentage of anthocyanins, both native and metabolites, absorbed and evacu-

ated in urine is extremely small in-comparison to consumed amount. The bioavail-

ability of anthocyanins is very low, with <1 % of the ingested amount reaching the

Fig. 5.2 An overview of anthocyanins (ANs) absorption and excretion

5.3 Anthocyanins Absorption

64
plasma, glucuronited and methylated anthocyanin metabolites were found twice 
than their parent (intact) compounds [ 14 ]. 
 The reason for low bioavailability of anthocyanins can be underestimation that 
may occur because concentration of some metabolites, such as protocatechuic acid, 
might be below the detection limit of the analytical methods employed and the pre-
dominance of the colorless carbinol (75–80 %) and chalcone (15–20 %) forms of 
anthocyanins present in blood and urine at neutral pHs, it is highly likely that these 
chemical forms may escape detection, and/or be chemically bound to other compo-
nents in the plasma or urine, and therefore not included in the analyzed fraction. 
Labeling anthocyanins for identiﬁ cation of all generated metabolites may overcome 
these shortcomings [ 14 ,   27 ].   
5.4      Carbohydrates  Moieties  Deconjugation 
 Glycosylated anthocynains are better hydrophilic as compared to aglycosylated 
(aglycones) ones and passively diffuse the cell membrane due to their more lipid 
solubility and small molecular size. During this passage, ingested anthocyanins are 
deglycosylated. Despite the strong acidic conditions of stomach, there are very little 
chances of acid hydrolysis (non-enzymatic deglycosylation). Deglycosylation of 
glycosylated anthocyanins [ 31 ] was not observed after pepsin-HCl digestion even at 
stronger conditions (pH 2.0, 37 °C, 2 h). There are chances of in vivo enzymatic 
deglycosylation as observed by using animal models although very little evidence is 
available to support this path. GIT of rats and pigs showed selective degradation of 
anthocyanin-glucosides in small intestine [ 2 ,   20 ], however further characterization 
of deglycosylation patterns of anthocyanins under the effect of isolated small intes-
tinal β-glucosidases is needed. Even in rat small intestine in situ perfusion model, 
the disappearance of cynadin-3-glucoside was signiﬁ cantly higher than other glyco-
sides of cynadin [ 18 ]. Limited information available suggests that anthocyanins 
containing xylose are better retained in the fecal content and feces as opposed to 
anthocyanins containing galactose and glucose [ 21 ]. Detailed studies are required to 
interpret the destiny of such glycosides. 
 Various factors inﬂ uence the bioavailability of anthocyanins, like quantity of gut 
microﬂ ora, food matrix [ 32 ], analytical detection problems (e.g. carbinol and chal-
cone) and differences in xenobiotic metabolism in GIT, liver, and other tissues.  
5.5      The  Inﬂ uence of Colonic Microﬂ ora 
 Gut  microﬂ ora converts anthocyanins into less stable anthocyanidins at neutral pH 
leading to degradation within 20 min thus Cyanidin-3-rutinoside was ﬁ rst hydro-
lyzed into cyanidin-3-glucoside and then into cyaniding aglycon, which rapidly 
degraded into protocatechuic acid (3,4-dihydroxybenzoic acid) [ 33 ].  Some 
5  Anthocyanins Absorption and Metabolism

65
anthocyanins metabolites have greater microbial and chemical stability, indicating 
their potential role in various biological effects and enhancement of antioxidant 
activity [ 33 ]. It is pertinent to mention that degradation of methylated anthocynains 
by the gut microbiota may yield de-methylated products [ 27 ]. 
 Since there are no endogenous esterases in humans to release phenolic acids, 
therefore the esterase activity of colonic microﬂ ora is required for the metabolism of 
acylated ﬂ avonoids [ 34 ]. Aura et al. [ 35 ] revealed that human fecal ﬂ ora immediately 
deconjugates quercitin glucosides, -rutinoside and glucuronide (glc). Simple pheno-
lics like 3,4-dihydroxyphenyl acetic acid and its derivatives are generated during ring 
ﬁ ssion of deglycosylated quercetin [ 36 ]. Aura et al. suggested that gut microﬂ ora can 
convert anthocyanins as revealed by fermentation of cyanidin-3- rutinoside and cyan-
idin-3-glucoside in the presence of human fecal slurry. Cyanidin- 3- glucoside was 
nearly hydrolyzed entirely after incubation of 2 h, and smaller than one third of the 
cyanidin-3-rutinoside present. Protocatechuic acid (PC), a ring- ﬁ ssion product of 
cyanidin-aglycone, was the main metabolite [ 35 ]. More than 90 % of the cyanidin-3, 
5-glucoside after incubation with human fecal suspension was degenerated after 2 h 
and fractional hydrolysis yielded cyanidin mono- glucoside as an intermediate of 
deterioration, which also degraded in the meantime. Resultant production and piling-
up of protocatechuic acid was observed again. Assessment of di-acylated anthocya-
nins from red radish indicated that the acyl group can be broken down by fecal 
microﬂ ora and the released acids were comparatively stable [ 37 ]. Deacylated antho-
cyanins can follow the same pathway of degradation as discussed above.  
5.6      Metabolism in Intestinal Mucosa and Tissues 
 The polyphenolic and cationic attributes of anthocyanins and their metabolites acti-
vate a variety of cellular responses. Some metabolites are responsible for bioactivity 
of ﬂ avonoids. For example, methylated cyanidin-3-glucoside is transformed to 
peonidin-3-glucoside [ 38 ]. Similarly, certain metabolic products of anthocyanins 
may have superior activity as compared to parent molecules.  
5.7      Tissue  Distribution 
 The knowledge of tissue distribution of anthocyanins is necessary to evaluate the 
health-promoting effects of anthocyanins. This information will also help to screen 
the suitable anthocyanins from various sources to assist development of supple-
ments and functional foods. The protective potential of anthocyanins associated 
with various tissue-diseases is proved in vitro by using various types of cell lines. 
However in vivo studies of same are limited. Distribution of anthocyanin in tissues 
has recently been evaluated in pig and rat models. Male Wistar rats were fed black-
berry extract (370 nmol anthocyanin/day) for 15 days and killed at 3 h after the 
5.7  Tissue Distribution

66
beginning of the last meal. Total anthocyanins averaged 605 nmol g 
−1
  in jejunum, 
68.6 nmol g 
−1
  in stomach, 3.27 nmol g 
−1
  in kidney, 0.38 nmol g 
−1
  in liver, and 
0.25 nmol g 
−1
  in brain [ 28 ]. In pigs fed diets supplemented with 0, 1, 2, or 4 % w/w 
blueberries for 4 weeks and fasted for 18–21 h before euthanasia, 1.30 pmol g of 
anthocyanins were identiﬁ ed in the liver, 1.58 pmol g 
−1
  in eyes, 0.878 pmol g 
−1
   in 
cortex, and 0.664 pmol g 
−1
  in cerebellum [ 39 ]. The results suggested that anthocya-
nins after crossing the blood-retinal and the blood-brain barrier can protect brain 
and eye tissues respectively. This hypothesis is also favored by another research in 
which aged blueberry-fed rats were used [ 40 ].  
5.8      Excretion 
 Urine is the major excretory pathway of intact anthocyanins and aglycones [ 41 ]. 
Although most conjugated ﬂ avonoid metabolites follow the same route [ 38 ],  how-
ever a small portion of them may re-enter the jejunum via bile, and are absorbed by 
the colon entering the entero-hepatic circulation again [ 42 ], or are defecated with 
feces. The lung is a chief excretion place for many bioactive constituents including 
quercetin [ 43 ]. However, there is no evidence indicating excretion of anthocyanin 
by respiration. 
 Recently biodegradation of anthocyanins into phenolic acids by enteric bacteria 
has gained much attention (Table  5.1 ). Various studies have documented the pres-
ence of phenolic acids, like protocatechuic acid, gallic acid, vanillic acid, phloroglu-
cinol acid, syringic acid and phloroglucinol aldehyde anthocyanin metabolites 
[ 45 – 47 ]. These phenolic acids are produced during anthocyanin metabolism either 
by enteric bacteria, or by chemical conversion, and have also been detected in 
humans [ 48 ].
   Anthocyanidin aglycones are degraded much faster as compared to anthocyanins 
monoglycosides [ 33 ]. Similarly anthocyanins degradation by intestinal microﬂ ora 
is also faster [ 44 ]. Unabsorbed and non-degraded anthocyanins are excreted in their 
intact forms. Unchanged anthocyanins are detected in fecal samples of humans 24 h 
after ingestion of blood orange juice [ 49 ], and in fecal samples of rats fed by grapes, 
bilberries and chokeberries [ 21 ].  
  Table  5.1     Degradation 
products of anthocyanidin 
aglycons [ 
44 ]  
 Anthocyanidin 
aglycon 
 Corresponding 
phenolic acid 
 Cyanidin   →  Protocatechuic acid 
 Delphinidin   →  Gallic acid 
 Pelargonidin   →  4-Hydroxybenzoic acid 
 Malvidin   →  Syringic acid 
 Peonidin   →  Vanilic acid 
 Petunidin   →  3-O-methylgallic acid 
5  Anthocyanins Absorption and Metabolism

5.9 Conclusions

The beneﬁ cial effects of anthocyanins have made the study of their bioavailability,

absorption and metabolism an area of considerable interest since humans can’t syn-

thesize these vital molecules de novo and obtain them from plant kingdom.

Understanding of anthocyanin chemistry, metabolism and their mechanism of

action has increased substantially in previous 10 years. Many older questions and

controversies have been resolved. Studies have been conducted in last decade to

develop a more thorough understanding of factors that inﬂ uence their uptake,

metabolism, and utilization in humans. This information however is only tip of the

iceberg. The exact molecular mechanism behind their metabolism remains to be

fully established. Although not all animals are good models for metabolic studies of

anthocyanins, rats are commonly used. There is need for validation of new animal

models especially induced mouse mutants in studying anthocyanin metabolism.

The effects of variety, growing practices, season, stage of maturity, storage and

processing condition are important for researchers to understand anthocyanin

metabolism in epidemiological studies.

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M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_6

Chapter 6

Biosynthesis and Stability of Anthocyanins

6.1 Introduction

Anthocyanins biosynthesis is one of the most studied secondary metabolite pathway

in plants. It is well-known now that anthocyanins biosynthesis is regulated by sev-

eral metabolic factors, majority of which are similar across various plant species.

Their biosynthetic pathway has been characterized at enzymatic as well as genetic

level with gene sequences available for all major biosynthetic steps of anthocyanins.

Further various mechanisms that regulate expression of these genes in plant cells

have also been studied.

Figure 6.1 summarizes the main steps of anthocyanins biosynthetic pathway. The

biosynthesis (phenylpropanoid metabolic pathway) starts from phenylalanine, an

amino acid, which yields 4-coumaroyl-CoA. This compound is then pooled with

malonyl-CoA to produce chalcones comprising two phenyl rings. The conjugate

ring-closure forms the chalcones, the well-known structure of ﬂ avonoids, repre-

sented by the three-ringed structure (ﬂ avanones). Flavanones are subjected to a

series of enzymatic modiﬁ cations that produces dihydroﬂ avonols, anthocyanins but

also ﬂ avonols, ﬂ avan-3-ols, proanthocyanidins (tannins) and other polyphenolics.

The dihydro-ﬂ avonols are converted to ﬂ avonols (myricetin) which further are gly-

cosylated by glycosyl-transferase to the corresponding anthocyanins. Based on the

known biosynthetic pathways of anthocyanins, optimal intervention on the involved

enzymes of the secondary metabolic pathways may lead to an increase of anthocy-

anin content of the plant, as shown by research on strawberries [ 3 ].

Anthocyanins are produced from precursors by two biosynthetic pathways:

shikimate, producing phenylalanine; and that generating malonyl-CoA. These two

precursors are interconnected by chalcone synthase by a polyketide folding mecha-

nism, to yield an intermediate chalcone, a substrate for chalcone isomerase

producing prototype pigment naringenin which is oxidized by a series of enzymes

(ﬂ avanone 3-hydroxylase, ﬂ avonoid 3′-hydroxylase and ﬂ avonoid 3′, 5′-hydroxylase)

to leucoanthocyanidins, and ﬁ nally converted to anthocyanidins by

leucoanthocyanidin- dioxygenase. These unstable anthocyanidins are joined to

various sugars by UDP-glucose/ﬂ avonoid 3- O -glucosyltransferase and O -methyl-

transferase to yield anthocyanins [ 4 ]. They accumulate in vacuoles of cells and

tissues of both reproductive and vegetative organs of plants. Out of 17 isolated

anthocyanins, majority are found in reproductive organs while 6 aglycones have

been identiﬁ ed in vegetative parts of plants. Enzymes involved in biosynthetic

Fig. 6.1 Anthocyanins biosynthetic pathway [ 1 , 2 ]

6 Biosynthesis and Stability of Anthocyanins

pathway of anthocyanins are localized on the cytosolic side of smooth endoplasmic

reticulum (SER). From SER, they are transported to cell vacuole by multidrug and

toxic compound extrusion (MATE) and ATP-binding cassette (ABC) membrane

transported and vesicles.

6.2 Stability of Anthocyanins

Stability of anthocyanins is a major topic of recent research on anthocyanins due to

their potential applications, beneﬁ cial effects and their use as alternative to artiﬁ cial

colors. More detailed discussion is available about the stability of anthocyanins in

their concerned chapter however in product formulation this point may be consid-

ered that the stability of anthocyanins increases with increasing the viscosity of their

solution and molecular associations within the solution also increase the stability by

preventing water based degradations in anthocyanins. Anthocyanins are extremely

unstable and very prone to degradation. Their stability depends on oxygen, light,

temperature, pH, chemical structure, concentration, solvents, and presence of metal

ions, enzymes, proteins and ﬂ avonoids [ 5 – 8 ]. Table 6.1 includes composition of

various anthocyanins.

Cyanidin and delphinidin are more stable than malvidin, peonidin and petuni-

din due to the blocking reactive OH group by methylation. Similarly anthocya-

nins with a 4-substituition are more stable than others. Glycosylation of 3

positions leads to stability while glycosylation of 5 position decreases stability.

Acylation of anthocyanins increases their stability by self-association reactions

and intra-molecular and intermolecular copigmentation. Therefore acylated

anthocyanins can offer the necessary stability for food applications. Acylated

anthocyanins can be obtained from black carrots, red cabbage, radish, purple

sweet potatoes and red potatoes [ 5 ].

Table 6.1 Edible sources of acylated anthocyanins [ 5 ]

Source Type of pigments

Radish ( Raphanus sativus ) Pelargonidin derivative acylated with one cinnamic acid and an

aliphatic acid

Potato ( Solanum tuberosum ) Pelargonidin derivatives acylated with one cinnamic acid

Black carrot ( Daucus carota

L.)

Cyanidin-3-Rutinoside-Glucoside-Galic acid acylated with one

cinnamic acid

Red cabbage ( Brassica

oleracea )

Cyanidin-3-Diglucoside-5-Glucoside acylated with one or two

cinnamic acids

Grape ( Vitis labrusca ) A mixture of ﬁ ve different aglycones, acylated and non-acylated

with p-coumaric acid

6.2 Stability of Anthocyanins

74
6.3      Relationships  Between  Structure  and  Stability 
 Anthocyanins are often partially degraded under the joint action of cellular and 
environmental factors (e.g. light, temperature, pH, metal ions, oxygen, coexisting 
sugar, etc.). Their molecular structure (e.g., the number and placement of the 
hydroxyl and methoxyl groups) affects their chemical behavior. The presence of 
hydroxyl groups on the rings makes the anthocyanins carry a positive charge in 
acidic solution. Although, the increased hydroxylation could stabilize the anthocy-
anidin, but excessive hydroxyl groups could also decrease the stability of the pig-
ment molecule, which makes it less stable than those with more methoxyl groups. 
In the glycosides of anthocyanidin, the types of glycosyl units and acyl groups 
attached to the aglycone have a signiﬁ cant effect on the structural stability together 
with their bonding site and number. Acylated anthocyanins are stable due to piling 
of acyl groups with the pyrylium ring of the ﬂ avylium cation, thus protecting the 
chromophores from nucleophile attack of water leading to synthesis of chalcone or 
pseudo-base [ 5 ]. This idea was also supported by Amiot and his coworkers [ 9 ],  this 
phenomenon is termed as intramolecular copigmentation. The color stability is 
attributed in unacylated anthocyanins to intermolecular copigmentation mechanism 
(including π–π overlap, dipole–dipole and hydrogen bonding interactions) [ 7 ,   10 ]. 
Table  6.2  presents a comparative stability of various anthocyanins.
   Table  6.2     Comparative stability of anthocyanins [ 10 ]   
 More  Stable   Stable   Reference 
 Pyranoanthocyanins   Anthocyanins > Anthocyanidins   [ 
5 ,   11 ] 
 Disaccharide  anthocyanins   Monosaccharidic  anthocyanins   [ 
5 ,   11 ] 
 Acylatedanthocyanins   unacylated  derivatives 
anthocyanins 
 [ 
5 ,   11 ] 
 Polyacylatedanthocyanins   Monoacylated  anthocyanins   [ 
5 ,   11 ] 
 Anthocyaninsacylated with aromatic acids 
(e.g. p-coumaric, caffeic, ferulic acids) 
 Acylated with aliphatic acids (e.g. 
acetic, malonic or oxalic acids) 
 [ 
5 ,   11 ] 
 Acylatedanthocyanins with caffeic acid   Acylated anthocyanins with 
p-coumaric acid 
 [ 
5 ,   11 ] 
 aromatic acids substitution in ring B of 
ﬂ avyliumcation 
 aromatic acids substitution in ring 
A of ﬂ avylium cation 
 [ 
12 ] 
 Color of anthocyanins with petunidin or 
malvidinaglycones 
 Color of anthocyanins with 
pelargonidin, cyanidin, or 
delphinidinaglycones 
 [ 
13 ] 
 Anthocyanins containing galactose   Anthocyanins containing arabinose   [ 
13 ] 
 Anthocyanins at low pH in acidic media   Anthocyaninswith high pH in 
alkaline solution 
 [ 
6 ] 
6  Biosynthesis and Stability of Anthocyanins

75
6.4         Factor  Affecting  Stability  of  Anthocyanins 
6.4.1      pH 
 Anthocyanins are susceptible toward change in pH [ 14 ] in media thus different 
chemical forms and colors [ 15 ]. In acidic aqueous solutions, anthocyanins occur in 
4 equilibrium species: the quinonoidal base (QB), the ﬂ avylium cation (FC), the 
carbinol or pseudo-base (PB) and the chalcone (CH) [ 16 ,   17 ]. Table  6.3   indicates 
the effect of pH on anthocyanins.
   Anthocyanidins are less stable at neutral pH while Pg is the most stable anthocy-
anidin among all, in anthocyanins, monoglycosides and diglycosides derivatives are 
more stable at pH 7 [ 18 ]. It is concluded that color variation with pH is more impor-
tant in the alkaline region due to their instability [ 7 ]. 
 Ozela et al. (2007) evaluated the stability of anthocyanin in the extract of spinach 
vine fruit ( Basella rubra  L.) in relation to degradative factors such as light, tempera-
ture and pH acting alone or in combination. Spinach vine extract is more stable at 
pH = 5.0–6.0 than at pH 4.0, in the absence as well as presence of light. This feature 
is different from other anthocyanins. This property indicates its potential use as a 
natural food color [ 19 ].  
6.4.2      Co-pigmentation  Effect 
 The anthocyanins color and intensity effects are the result of forming molecular or 
complex associations is a co-pigmentation phenomenon for anthocyanins [ 20 ]. 
Some investigators propose it as stabilization mechanism for anthocyanins color in 
plants [ 21 ]. The electron deﬁ cient ﬂ avylium ion associate with p-electrons rich co- 
pigment thus gives stability against nucleophilic attack of water on 2 position of 
ﬂ avylium [ 22 ] and for other species such as SO 
2
 and peroxides on 4 position [ 23 ]. 
Figure  6.2  indicates anthocyanin interactions.
   The co-pigmentation effect is pH dependent, because at low pH, anthocyanins 
occur in ﬂ avylium form, and at high pH values, they exist in colorless carbinol pseu-
dobase form. The example of charge transfer complex or p–p complex is the antho-
cyanins and tannins complexation in wines, producing pigmented tannins [ 
24 ]. 
Similar reactions occur between ﬂ avan-3-ols and tannins [ 
7 ,   25 ]. 
 Metals and ﬂ avylium salts chelates were considered for variety of colors in 
ﬂ owers [ 26 ]. Anthocyanin-metal complexations constitutes possible substitute for 
color stabilization especially if the metals have no toxic effects. The complex for-
mation occurs with o-di-hydroxyl groups in the B ring (Cy, Dp, Pt) of anthocyanins 
and anthocyanidins [ 20 ]. Some authors suggest that the blue color in plants is due 
to complexation between anthocyanins and certain metals like Al, Fe, Cu and Sn 
[ 27 ] or Mg and Mo [ 28 ]. Anthocyanin-molybdenum complexation causes the 
6.4   Factor Affecting Stability of Anthocyanins

Table 6.3 Effect of pH on anthocyanins [ 7 ]

pH of the medium Predomanint chemical form Structure

1 Flavylium cation (red color)

gly

Between 2 and 4 Quinoidal blue species

gly

Between 5 and 6 Carbinolpseudobase and a chalcone (colorless)

gly

Chalcone

Psedobase carbinol

>7 Corresponding degraded products

Pyrocatechol

6 Biosynthesis and Stability of Anthocyanins

stabilization of blue color in Hindu cabbage tissue [ 28 ]. Yoshida et al. (2006) also

conﬁ rmed the role of metal complex in plants by complexation between anthocya-

nins and proved that Mg (II) or Fe (III) at pH 5 are important for the development

of blue color in plants [ 7 , 29 ].

6.4.3 Solvent Effects

Studies on synthetic ﬂ avylium salts in different solvents (acetonitrile, dioxane, etha-

nol, propylene-glycol, water and 2-butanone) have shown that variations in color

depend on the ﬂ avylium salts concentration and solvent used [ 30 ]. In protic sol-

vents, the ﬂ avylium salts display red color due to monomer, while in aprotic sol-

vents the solutions are yellow due to dimer while increasing concentration of

ﬂ avylium salts, the red color is preferred. Another study suggests that increasing

O concentration in mixture of water:acetonitrile, the monomer is changed to a

green color dimer (monomer with charge-transfer property). Water therefore plays

an important role in dimerization of ﬂ avylium salts [ 7 ].

ANs-

copigment-

interactions

Charge-transfer

complex

formation e.g.

another phenolic

compound

Self association

or an

intramolecular

co-pigmentation

e.g. other

anthocyanin

More complex

co-pigmintation

e.g. aglycon,

sugar, co-

pigment and

protons

Intermolecular

co-pigmentation

e.g. free

electrons

Compelexation

e.g., metals

Fig. 6.2 Anthocyanins (ANs) interaction [ 7 ]

6.4 Factor Affecting Stability of Anthocyanins

78
6.4.4      Temperature 
 The increase in temperature cause the decrease in stability of anthocyanins and 
other plant pigments during processing and storage [ 8 ]. Rise in temperature cause 
thermal degradation which yields brown products in the presence of oxygen [ 31 ]. 
Various researchers have conﬁ rmed that heating has detrimental effect on anthocya-
nins stability [ 32 – 34 ].  
6.4.5      Concentration  Effects 
 Anthocyanin concentration has direct effect on color stability [ 5 ] such as strawberry 
syrup color was greatly enhanced by mounting the anthocyanins contents. Total 
anthocyanins are more color stabilizing than individual anthocyanins [ 35 ].  
6.4.6      Oxygen 
 The presence of oxygen ampliﬁ ed the degradation process, and the condition is 
worsening if heating is there. Oxygen and increased temperature were the most 
harmful combinations ever found in color degradation of various berry juices and 
isolated anthocyanins [ 36 ] demonstrated that oxygen-free environment remained 
best condition for the antioxidant activity and anthocyanins concentrations of straw-
berries (fresh-cut) during cold storage. Some investigators have reported that if the 
atmosphere is enriched with 60–100 % oxygen results increase in phenolic and 
anthocyanin content during the onset (0–7 days) of cold storage [ 37 ].  Yellow  col-
ored 3-deoxyanthocyanins (due to dehydroxylation of the carbon at C-3) are more 
stable than the corresponding red colored 3-hydroxy anthocyanins.  
6.4.7      Light 
 Light is crucial for the biosynthesis of anthocyanins, but it also speeds up their deg-
radation. Dark has safeguarding effect on anthocyanins in comparison to light [ 38 ]. 
Amr and Al-Tamimi [ 
39 ] in 2007 studied the effect of light on anthocyanins stabil-
ity; their work veriﬁ ed that light had a very detrimental effect on anthocyanins stabil-
ity during storage particularly in the presence of sugar. Light exposure cause 
degradation of anthocyanins in incoherent manner which is source (plant) and antho-
cyanins dependent degradation [ 40 ,   41 ]. Another study under neon light showed that 
the phenolic and anthocyanin content was decreased during storage [ 42 ]. 
6  Biosynthesis and Stability of Anthocyanins

79
 Ozela et al. (2007) [ 19 ] evaluated that various factors like pH, temperature and 
light affect the anthocyanins stability of the spinach vine fruit ( Basella rubra   L.) 
extract. Results showed that, independent of pH, the extract exposed to light have 
anthocyanin degradation kinetics with the average half-life is smaller compared to 
an average half-life in dark storage. They found that in the presence of light, 
increased temperature leads to increased anthocyanin degradation.  
6.4.8      Enzymes 
 Glycosidases are the most common anthocyanins degrading enzymes that break 
the glucosidic linkage and results unstable anthocyanidin [ 43 ]. Other anthocya-
nins degrading enzymes like peroxidases (phenol oxidases) and phenolases (poly-
phenol oxidases), are present in berries and fruits [ 44 ]. Inactivation of enzymes 
increases anthocyanin stability [ 45 ]. Fang et al. (2007) [ 46 ] reported that bayberry 
phenolase alone cannot react with directly with cyaniding-3-glucoside but 
Mathew and Parpia [ 47 ] and Kader et al. (1999) [ 44 ], demonstrated that polyphe-
nol oxidase can react directly with anthocyanins although they acted as fairly poor 
substrates.  
6.4.9      Ascorbic  Acid 
 Anthocyanins are degraded in the presence ascorbic acid by direct condensation 
mechanism [ 48 ] and by free radical mechanism the pyrilium ring undergo oxidative 
cleavage resulting in loss of color of anthocyanins. Ascorbic acid and its degrada-
tion products increase the degradation arte of anthocyanins [ 49 ].  
6.4.10      Sugars 
 The results of adding sugar to anthocyanin stability depends mainly on the the num-
ber and nature of sugars attached to the ﬂ avylium cation and number and nature of 
acids linked to the glycosylic moiety [ 50 ]. Sugars and their degradation products 
decrease anthocyanin stability. The addition of sucrose to mulberry extract increases 
the stability of anthocyanins by increase in polymeric and co-pigmented anthocya-
nins during heating before heating the effect was hyperchromic [ 51 ].  Similarly 
anthocyanins content of refrigerated berries extracts were more stabilized by 20 % 
sugar addition however by increasing the concentration the protective action was 
decreased [ 52 ].  
6.4   Factor Affecting Stability of Anthocyanins

80
6.4.11      Sulﬁ tes 
 Sulphates and sulﬁ tes in foods items form colorless sulfur derivatives with antho-
cyanins which may be reversed by heating or acidiﬁ cation if the concentration of 
sulﬁ tes are not too high (>10 g/kg) [ 53 ].   
6.5      Stability  of  Anthocyanins  in  Food  Products 
 Due to plentiful applications, valuable effects and their use as alternative to artiﬁ cial 
colorants in foods; anthocyanins are the main focus of recent investigations. Food 
processing involves extreme temperature up to 150 °C that may affect the stability 
of anthocyanins during shelf life. Apart from temperature, other factors like pH, 
storage temperature, chemical structure, concentration of anthocyanins present, 
light, oxygen, the presence of enzymes, proteins and metallic ions also affect the 
shelf lives of anthocyanins enriched products [ 54 ]. Generally high temperature may 
affect concentration of anthocyanins in corresponding food items and products, 
elevated temperature results in formation of polyphenolic degradation products that 
may have low antioxidant potentials. Studies are required to conﬁ rm whether these 
thermal induced degradation products have decreased or increased antioxidant 
capacity. Since the degradation mechanism of anthocyanins is rather complex and 
perplexing, it is possible that thermal processing could induce some un/expected 
and un/desired chemical reactions which in directly inﬂ uence food quality so fur-
ther investigations are required in this area to predict optimum processing condi-
tions and to opt advance processing rather conventional system for respective 
anthocyanins products to ensure maximum anthocyanins concentration, and longer 
shelf lives products to the consumers [ 54 ].  
6.6      Anthocyanins  Degradation  in  Plants 
 Anthocyanins degradation is controlled and induced when beneﬁ cial to the plant. 
Anthocyanins degradation is dependent on environmental conditions such as light 
quality, light intensity and growth temperature. Several enzymes have also been 
isolated that degrade anthocyanins in postharvest fruit that may be candidates for 
in vivo degradation. Peroxidase, α-glucosidase and polyphenol oxidase are the main 
enzyme groups that regulate anthocyanins degradation in fruit juices and extracts 
[ 55 ]. All research studies whether carried out in model systems or in extracts show 
that anthocyanin degradation follows ﬁ rst order kinetics. 
 Anthocyanins usually accumulate in young leaves and degrade as the leaves 
become mature [ 56 ]. Anthocyanins protect the leaves especially their photosynthetic 
6  Biosynthesis and Stability of Anthocyanins

81
apparatus from harmful UV light as well as from photo-inhibitory high intensities of 
visible light [ 57 ]. They also act as antioxidants thus protecting the cells from oxida-
tive damage [ 58 ]. The decrease in anthocyanins content may be due to increase 
chlorophyll accumulation as leaf expand and grow, and no longer is photoprotection 
required [ 55 ]. 
 Anthocyanins degradation is also observed in developing fruit e.g.  Capsicum  
spp. and Sicilian sweet orange varieties. Anthocyanins accumulation in fruit pro-
tects the photosynthetic apparatus in the developing fruit [ 55 ]. 
 Flowers often change color during development, acting as a signal for pollina-
tors. In most cases, the change in color is due to induction of anthocyanins synthe-
sis, but in others, such as  Brunfelsia calycina , anthocyanin is degraded, resulting in 
a change of ﬂ ower color from dark purple to white after synthesis [ 59 ].  
6.7      Drawback  and  Derivatives  of  Anthocyanins 
 As previously discussed, anthocyanins are unstable and their colors varies depend-
ing upon light, temperature, pH and presence of metals in media [ 60 ]. There are 
some limitations like decreased stability to processing, storage and formulation 
conditions which can impart un-wanted ﬂ avor or odor characteristics that have 
limited their use as natural colorants in food systems. The area required attention 
to developed processes and mechanisms to reduce the incompatibility of anthocya-
nins with pH, temperature and other environmental changes. For example there 
may be few coloring principles in red cabbage and radish extracts so techniques 
and procedures must be adopted to isolated these few necessary components as 
coloring agents [ 5 ].  
6.8      Anthocyanins  Stabilization  Mechanisms 
 As mentioned before, naturally anthocyanins are unstable, however they can be 
stabilized i.e. by applying various mechanisms like acetylations, association (co- 
pigmentation) and microencapsulation when a “co-pigment” is added. The stabili-
zation of grape skin anthocyanins by co-pigmentation with enzymatically modiﬁ ed 
isoquercitrin (EMIQ) was increased signiﬁ cantly [ 61 ]. 
 Encapsulation by spray-drying is a cost-effective way for protection of anthocya-
nins by coating the ingredients [ 62 ]. Thus bioavalability and the shelf lives of antho-
cyanins may be improved by providing protection against oxygen, water and light 
[ 53 ]. The encapsulation from diverse sources is given in Table  6.4  and the details of 
steps during process and their advantages are also given in Table  6.5 .
6.8   Anthocyanins Stabilization Mechanisms

Table 6.4 Anthocyanins encapsulation from various sources [

53 ]

Anthocyanin

source

Encapsulation

technique Coating material Stabilization improvement

Concord grape Spray drying Maltodextrin Not measured

Cranberry

pomace

Spray drying Maltodextrin Not measured

Roselle calyces Spray drying Maltodextrin Not measured

Roselle calyces Inclusion

complexation

β-cyclodextrin The addition of β-cyclodextrin

does not ↑ anthocyanin

stabilization

Apple pomace Spray drying Maltodextrin Anthocyanins have↑ stability

The ↑ in the shelf life is due to

↓ water activity

Roselle calyces Liophilization Pullulan Encapsulated anthocyanins have

slightly ↑ stability as compared

to the free anthocyanin extract

Black currant Thermal gelation Glucan gel Encapsulation of anthocyanins

followed by appropriate

processing ↑ the stability of

anthocyanins

Black carrot Spray drying Maltodextrin Not measured

‘Isabel’ grape

bagasse

Spray drying Maltodextrin and

Arabic gum

Not measured

Blackberry Thermal gelation Sodium alginate

and pectin

Not measured

Blackberry Ionic gelation Curdlan Not measured

Jabuticaba fruit

skin

Ionic gelation Sodium alginate Not measured

Table 6.5 Some common microencapsulation processes for anthocyanins characteristics [

63 ]

Technology

Particle

size

(μm) Advantages Disadvantages

Spray-drying 10–400 Low process cost, wide

choice of wall material,

good encapsulation

efﬁ ciency, good stability of

the ﬁ nished product and

potential of industrial

production continuously

Can degrade at highly

temperature-sensitive

compounds, control of the

particle size is difﬁ cult and

yields for small batches are

moderate

Freeze drying 20–

5000

Best for thermo-sensitive

substances in aqueous

solutions

More processing time, costly

process, costly transport and

storage of the capsules

Fluid bed coating 20–200 Low cost process and

allows speciﬁ c capsule size

distribution and low

porosities into the product

Degradation of temperature-

sensitive molecules

(continued)

6 Biosynthesis and Stability of Anthocyanins

6.9 Conclusions

A lot of data is available on molecular biology of anthocyanin biosynthesis. The

genes regulating the various steps and the inﬂ uence of phytohormones on this

genetic regulation are further clariﬁ ed now. Their intra-cellular transportation and

accumulation are also better understood. Although substantial progress has been

made in our understanding of anthocyanin biosynthesis in plants, many key issues

are yet to be addressed in detail. For example the interaction between anthocyanin

biosynthetic pathway and other pathways and the effects of such interaction on

plant growth and development are not fully understood. It is hoped that next decade

will clarify remaining ambiguities in this regard.

References

1. De Pascual-Teresa, S., & Sanchez-Ballesta, M. T. (2008). Anthocyanins: From plant to health.

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Technology

Particle

size

(μm) Advantages Disadvantages

Emulsiﬁ cation 0.2–

5000

Polar, non-polar (apolar),

and amphiphilic can be

incorporated

Low number of emulsiﬁ ers that

can be utilized and hard to

control formation of capsules

Melt extrusion 300–

5000

The material is completely

shielded by the wall

material, remaining core is

washed externally, low

temperature entrapping

assay

The capsule should be

essentially separated from liquid

bath and dried

Hard to obtain capsules in highly

viscous carrier materials melts

Rapid expansion

of supercritical

ﬂ uid (RESS)

10–400 Easy removal of solvent

and non-toxic method,

works at ↓temperature and

in in-active atmosphere

thereby avoiding product

degradation

Both the core and the wall

material must be very soluble in

supercritical ﬂ uids, low or no

solubility of high molecular

weight, polar compounds CO

poor control over the

precipitated crystal morphology

and size distribution

Ionic Gelation Organic solvents and

extreme conditions of

temperature and pH are

avoided

Mostly used on lab-scale

The capsules are highly porous

which leads to intensive burst

Thermal Gelation The same of ionic gelation Same as mentioned above

Table 6.5 (continued)

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6 Biosynthesis and Stability of Anthocyanins

M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_7

Chapter 7

The Role of Anthocyanins in Health

as Antioxidant, in Bone Health and as Heart

Protecting Agents

7.1 Introduction

Anthocyanins are one of the most well-known sub-groups of pigmented ﬂ avonoid

compounds in the plant kingdom. They can be found in almost all land plants, but

found in relatively high levels in 27 plant families. The dietary consumption of

anthocyanins is high due to their occurrence in fruits and vegetables. They function

as phytoprotective substances, have a role in plant, animal interactions and as such

are important in eco-physiology or plant defense mechanisms. Recently, interest in

anthocyanins as well as in their biological and therapeutic properties has strongly

increased. Nemours in vitro studies, animal models and human clinical trials sup-

port their medicinal beneﬁ ts to human. These studies suggest that anthocyaninshave

anti-carcinogenic and anti-inﬂ ammatory effects, provide cardiovascular disease

prevention, promote obesity and diabetes control beneﬁ ts, and also improve visual

and brain functions. To understand the role of anthocyanins in prevention of dis-

eases, it is essential to have a working-knowledge of anti-oxidants and their regula-

tory role on the human immune system.

Reactive species is a collective term used to indicate oxygen radicals, nitrogen

radicals and their associated nonradicals species that act as oxidizing agents. These

species are known as reactive oxygen species (ROS) and reactive nitrogen species

(RNS) respectively or commonly as oxidants. These radicals can be produced by a

variety of mechanism in human body and their uncontrolled production can be

harmful for biomolecules, cells, tissues and body organs known as oxidative dam-

age. The in vivo generation of ROS and RNS elicits the radical scavenging or

antioxidant mechanism collectively known as antioxidant defense which minimizes

and repairs this oxidative damage. This oxidant-antioxidant balance is required for

proper functioning of cells and during disease; this balance is sloped in favor of

oxidants thus generating oxidative stress and increasing oxidative damage. The

ROS and RNS are involved in pathology of more than 100 various diseases and

metabolic disorders. This damage may be countered by oral administration of

antioxidants from antioxidant rich sources of fruits, vegetables, nuts, grains and

cereals. It has led to new antioxidant based preventive/curative nutrition based ther-

apies. These antioxidants modulate innate as well as adaptive immunity and reverse

various age-related immune deﬁ ciencies by stimulating and boosting the human

immune system. Although more signiﬁ cant effects have been noted in elderly, the

antioxidant rich diets have same effects in youngers. It is therefore recommended to

have a sufﬁ cient intake of antioxidant rich foods regularly to prevent and delay the

onset of various age-related degenerative disorders. Some of these food-related

antioxidants are ascorbic acid, tocopherols, carotenoids and anthocyanins.

It is well-established now that anthocyanin stimulates immunity against tumor

growth. Anthocyanin intervention can be helpful strategy in modulating the immune

response. Whether the immune modulatory effects of anthocyanins translate into

health beneﬁ ts should be studied with various biological and animal models. It must

be kept in mind that these studies have their drawbacks as well. For example, studies

using one pure anthocyanin, the aspect of interaction of various dietary bioactive

constituents and complete array of various anthocyanin are missing. Similarly for

studies involving plant material or extract, which contain collection of other

constituents and so results can’t be attributed to anthocyanin only.

Different signaling pathways like nuclear factor κB, Wnt/β-catenin, AMP-

activated and mitogen-activated protein kinase as well as certain central cellular

processes, such as cell cycle, apoptosis, autophagy and biochemical metabolism are

involved in these valuable effects of anthocyanins and can provide potential thera-

peutic strategies for treatment of broad spectrum of diseases in future [ 1 ]. Figure 7.1

gives a general overview of pharmacological effects of anthocyanins on various

organs and tissues of human body.

7.2 Presumed Health-Promoting Effects of Anthocyanins

Due to their health-promoting and immunity-boosting properties, anthocyanins are

appropriate nutraceuticals and supplementary treatments for various aspects of

chronic diseases. Dieto-therapeutic applications of anthocyanin-rich foods optimize

health and performance constituting an important largest market of nutraceutical

products throughout the world. Therefore, it is strongly recommended to identify

these health-promoting components in anthocyanin extracts that led to new opening

of opportunities of using these extracts in a variety of food applications.

A lot of research has been performed during last decade to identify non-essential

nutrients including anthocyanin responsible for observed health-beneﬁ ts and to

verify various claimed physiological effects on human body. Their beneﬁ cial effects

include anti-inﬂ ammatory and anti-cancer activities and protective effects against

various metabolic, degenerative and cardiovascular diseases and vision improve-

ments. There are numerous studies in animal/human cell lines, animal models and

7 The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

human clinical trials that support above mentioned effects of anthocyanins. Their

peculiar chemical diversity, suitable molecular weight, speciﬁ c three dimensional

conformation and resulting physical and biochemical effects allow them to interact

with various targets in cells producing desirable effects. Data retrieved from various

studies support the health-promoting and immunity boosting effects of anthocya-

nins. Their and their metabolites role in maintaining optimal functions of various

physiological and biochemical processes is supported by epidemiological studies,

in vitro studies, cell-culture studies and recently in vivo studies. These efforts have

led to better understanding of the role of anthocyanin and their derivatives in the

Fig. 7.1 General Overview of pharmacological effects of anthocyanins

7.2 Presumed Health-Promoting Effects of Anthocyanins

90
process of various chronic diseases, with special focus to their metabolism and 
 biological actions, dose-effects and organ speciﬁ c effects. Table  7.1   indicates 
healthy effects of uses of anthocyanins on various tissues, organs and organ systems 
of human body.
   Interest in anthocyanins has increased after the recognition of their health bene-
ﬁ ts [ 16 ]. Due to their anti-inﬂ ammatory and anti-oxidant potential, epidemiologic 
studies suggest that ingestion of anthocyanins reduces the risk of diabetes, arthritis, 
cancer and cardiovascular disease [ 17 ]. Their effects are due to their effects at 
    Table  7.1     Healthy effects of uses of anthocyanins   
 Anthocyanins   Health  use   References 
 Anthocyanins  extracts   Sight  acuteness   [ 
2 ] 
 Enhancement of antioxidant capacity   [ 
3 ,   4 ] 
 Cured several blood circulation ailments 
due to capillary fragility 
 [ 
5 ] 
 Vaso-protective and anti-inﬂ ammatory 
effects 
 [ 
6 ] 
 Platelet aggregation inhibition effects   [ 
7 ] 
 Preservation of normal vascular 
permeability 
 [ 
5 ] 
 Anti-diabetic, anti-neoplastic and chemo-
protective effects 
 [ 
8 ,   9 ] 
 Radiation-protective  agents   [ 
10 ] 
 Antioxidant properties and notable effects 
against Cardiovascular hypertension, 
chronic inﬂ ammation, Cancer and metabolic 
syndrome 
 [ 
11 ,   12 ] 
 Red orange extract ( Citrus sinensis  
varieties: Moro, Tarocco, 
Sanguinello) (Anthocyanins, 
ﬂ avanones, hydroxycinnamic 
acids, and ascorbic acid) 
 Anti-inﬂ ammatory activity was assessed in 
human keratinocytes (lineage NCTC 2544) 
exposed to IFN-γ and histamine 
 [ 
13 ,   14 ] 
 Red orange extract at various 
concentrations 
 Halted expression of ICAM-1 and secretion 
of MCP-1 and IL-8 
 [ 
13 ,   14 ] 
 Grape seed proanthocyanidins   Human keratinocytes irradiated with UVB 
and treated with GSP’s inhibited formation 
of UVB-induced H 
2
 O 
2
 , protein oxidation, 
lipid peroxidation, DNA damage and 
exhaustion of antioxidant components like 
catalase, superoxide dismutase, glutathione 
peroxidase and glutathione 
 [ 
14 ,   15 ] 
 Grape seed proanthocyanidins   Prevented phosphorylation of JNK, p38, 
ERK1/2and proteins of MAPK family and 
UVB-induced activation of NF-κB/p65. It 
suggests that GSP canreduce UV-induced 
oxidative stress in human skin 
 [ 
14 ,   15 ] 
7  The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

91
 various molecular levels by directly scavenging ROS [ 18 ] or inducing phase II 
detoxifying enzymes or to induce phase II detoxifying enzymes [ 19 ].  
7.3      Toxicity  of  Anthocyanins 
 No adverse health effects have been associated with anthocyanins consumption 
[ 20 ,   21 ]. Even if consumption in large quantities in countries of Europe and USA, 
no toxicity has been observed, probably due to their low overall absorption. 
However the over-increasing usage as supplements may prove problematic. In tox-
icity studies in animal models, using bilberry extracts (36 % anthocyanindins), 
LD 
50
  values were over 2000 mg/kg without any toxic indications [ 22 ]. Similarly in 
dogs, no mortality or toxic effects were observed at 3000 mg/kg. No adverse or 
toxic effects were observed in rats (@ 125–500 mg/kg) and dogs (@80–320 mg/kg) 
daily for 6 months. The extract did not exhibit any teratogenic or mutagenic effects 
thus conﬁ rming clinical safety studies [ 22 ]. People consuming 160 mg/kg twice 
daily for 1–2 month tolerated the extract very-well. Only some (4 %) complained 
side effects, mostly gastrointestinal (GIT) and nervous system and cutaneous 
annexes and skin [ 23 ]. 
 Anthocyanins have been consumed since ancient times but no toxic effects 
regarding health were reported [ 24 ]. That is the reason that Europe, Japan, USA 
and many other countries allowed the use of anthocyanins as food color in 
foods and beverages [ 25 ]. The Joint FAO/WHO Expert Committee on Food 
Additives (JECFA) determined on early toxicological studies that anthocyanin-
containing extracts had a very low toxicity [ 26 ]. The no-observed-effect-level 
(NOEL) for young rats was determined to be approximately 225 mg kg 
−1
   body 
weight in a two-generation reproduction study. Based on the above result, the 
estimated acceptable daily intake (ADI) for human was estimated to be 
2.5 mg kg 
−1
  body weight per day in 1982, using the equation of ADI = NOEL/100 
[ 27 ]. The studies presented in Tables  7.1  and  7.2  have confirmed the safety of 
anthocyanins.
   Table  7.2     Various mutagenicity studies are described by JECFA [ 28 ]   
 Anthocyanins   Model  tested   Genotoxicity   References 
 Cyanidin  and 
delphinidin 
 Ames assay system for ﬁ ve different 
strains of  Salmonella typhimurium  
 Not  mutagenic   [ 
29 ] 
 Cyanidin   Ames assay using  Salmonella 
typhimurium strain  TA98 
 Not  mutagenic   [ 
30 ] 
 Anthocyanins 
(compounds not 
speciﬁ ed) 
 Ames test using  Salmonella 
typhimurium TA1538 
 Not  mutagenic   [ 
31 ] 
 Anthocyanins 
(compounds not 
speciﬁ ed) 
  Escherichia coli  WP2 for induction of 
DNA damage 
 Not  mutagenic   [ 
31 ] 
7.3   Toxicity of Anthocyanins

92
7.4         Reproductive  and  Developmental  Toxicity 
 Grape-skin extract containing 3 % anthocyanins were evaluated for 2-generation 
reproduction study in Sprague–Dawley rats conﬁ rmed its safety in reproduction and 
growth; there were no corresponding compound-related histopathological effects 
[ 32 ]. Anthocyanins (an extract from currants, blueberries and elderberries) were 
reported not to be a developmental toxicant in rats, mice or rabbits when given over 
three successive generations [ 28 ]. Table  7.3  shows effects of processing conditions 
on anthocyanin contents (mg/g DW) of fruits.
7.5         Anthocyanins  Biological  and  Pharmacological  Activities 
 Anthocyanins are not only beneﬁ cial to its producers, i.e. plants but also to humans and 
other animals as nutraceutical and coloraceutical. In present era numerous  scientists 
have focused anthocyanins, most of them have found anthocyanins as useful and 
 beneﬁ cial reporting wide range of biological activities [ 11 ]. These activities include 
anti-inﬂ ammatory [ 34 ] antimicrobial [ 35 ] anti-carcinogenic [ 11 ,  36 ] vision- improvement 
[ 37 ], stimulation of apoptosis [ 36 ]; neuroprotective effects [ 38 ,   39 ] effects on blood 
 vessels  [ 40 ,   41 ] platelets [ 42 ] and decrease the risk of coronary heart disease [ 43 ]. 
   Table  7.3     Effects of processing conditions on anthocyanin contents (mg/g DW) of fruits [ 33 ]   
 Fruits 
grower 
 Fresh 
 Heating (98 °C, 
10 min)   Freezing (20 °C)   Freeze-drying 
 Cyd-
3- glu 
 Cyd-
3- rut 
 Cyd-
3- glu 
 Cyd-
3- rut 
 Cyd-
3- glu 
 Cyd-
3- rut 
 Cyd-
3- glu 
 Cyd-
3- rut 
  Cherries  
 Cromwell   207   276   419   475   570   737   158   211 
 Roxburgh   546   698   470   615   548   706   522   628 
 Dunedin   46   49   126   172   62   89   22   28 
  Nectarines  
 Cromwell   24   25   27   18   33   29   58   7 
 Roxburgh   16   16   12   12   19   18   71   9 
 Dunedin   10   15   5   4   9   7   69   7 
  Peaches  
 Cromwell   7   7   12   10   17   18   10   12 
 Roxburgh   16   17   32   27   33   29   6   8 
 Dunedin   14   15   10   9   14   8   11   15 
  Plums  
 Cromwell   51   52   38   40   58   63   14   18 
 Roxburgh   12   13   37   46   46   59   7   8 
 Dunedin   18   23   48   60   66   85   19   24 
   Cyd-3-glu  cyaniding-3-glucoside,  Cyd-3-rut   cyaniding-3-rutinoside  
7  The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

93
 The purposed mechanism of actions include antioxidant effects, modulation of 
hormonal and enzyme system, stimulation of the immune system and their inter- 
related mechanisms. As mentioned in Chap.   5    , anthocyanins show less  bioavailability, 
due to their instability under physiological and pH conditions at absorption site in 
human body, it was difﬁ cult to attest their tagged health-promoting effects in animal 
models in vivo. Although mechanism of action and various health- promoting effects 
need further investigation, anthocyanins certainly have a key role in a healthy and 
well-being oriented diet.  
7.6      Antioxidant  Activity 
 Anthocyanins exhibit excellent in vitro and in vivo antioxidant activity protecting 
human body against oxidative stress by scavenging free radicals and suppressing 
lipid peroxidation. Despite various limitations, much progress has been done during 
last decade especially in assessing the dynamics of the interplay of ROS and tran-
scription. However the full extent to which anthocyanin is functionally signiﬁ cant 
in maintaining the cellular redox homeostasis still remains to be understood. The 
core strategy for improving immune suppression and slowing-down the progression 
of associated diseases is decreasing the extra production of ROS. 
 The structure activity relationships of anthocyanins may be considered through 
various angles like the position and numbers of hydroxyl and methyl groups in 
anthocyanins nucleus. For example the increase antioxidant effect and higher pro-
apoptotic activity in human leukemia cells is considered to be associated with 
greater number of hydroxyl group. Similarly the antiproliferative activity is thought 
to be more potent with the hydroxyl or methyl groups substitution or placement on 
ring B. The phenolic structure of anthocynains imparts antioxidant property to 
them. Their antioxidant activity depends on many factors mainly; the number of OH 
group; the catechol moiety in B-ring and oxonium ion in C-ring, the methylation 
and hydroxylation pattern as well as glycosylation and acylation [ 44 ]. Glycosylation 
decreases antioxidant activity as it decreases the capacity of anthocyanin to delocal-
ize electrons [ 45 ]. The contribution of B-ring substituition in antioxidant activity is 
OH > OCH3  ≫ H  [ 46 ]. The positively charged O-atom in anthocyanin makes it a 
potential H-donating antioxidant [ 11 ]. 
 Anthocyanins can express their antioxidant effects directly or indirectly. Directly 
they exhibit antioxidant activity by direct free radical scavenging potential due to 
electron/hydrogen donating capacity of anthocyanin structure [ 47 ,   48 ] which can 
bind reactive free radical species. Indirectly, anthocyanins can (i) restore or increase 
the activities of glutathione peroxidase or superoxide dismutase thus increasing 
 glutathione content [ 49 ], (ii) activate genes that code for these enzymes [ 50 ]  and 
(iii) decrease the generation of oxidative abducts in DNA decreasing the generation 
of ROS by inhibiting xanthine oxidase and NADH oxidase or by modiﬁ cation of 
arachidonic metabolism and mitochondrial respiration [ 1 ,   51 ]. 
7.6  Antioxidant Activity

94
 The anthocyanidins and anthocyanins have shown a higher antioxidant activity 
than vitamins C, butylated hydroxyl anisole (BHA), butylated hydroxyl toulene 
(BHT), α-tocopherol and Vitamin-E [ 48 ,   52 ] by capturing free radicals through the 
donation of hydrogen atoms [ 53 ,   54 ]. 
 Anthocyanidins have greater antioxidant capacity than anthocyanins i.e. the radi-
cal scavenging activity decreases with glycosylation [ 48 ]. However, Kähkönen and 
Heinonen [ 55 ] concluded that in vitro effect of glycosylation on antioxidant activity 
is dependent on the environment e.g. anthocyanidin and oxidation models in which 
oxidations is occurring [ 56 ]. 
 The antioxidant ability of anthocyanins depends on the chemical structure of a 
anthocyanins. It is not necessary that all of them possess similar activities for scaveng-
ing different ROS and reactive nitrogen species (RNS) [ 56 ,   57 ] for example the 
 reactivity against superoxide anion follows the order delphinidine > cyaniding > pelar-
gonidin while pelargonidin is the most effective against the hydroxyl radical [ 58 ,   59 ]. 
 Number and position of sugar residues in the anthocyanidin also affects the anti-
oxidant activity of anthocyanins [ 55 ,   59 ]. The smaller the number of sugar units at 
C3, higher is the antioxidant potential however the effect is method dependent [ 55 , 
 60 ]. Pyranoanthocyanins of malvidin, petunidin, pelargonidin and cyanidin exhib-
ited higher superoxide anion radicals (O 
2−
 ) quenching activity but did not quench 
hydroxyl radicals [ 61 ], however integration of pyruvic acid into malvidin-3- 
monoglucoside and delphinidin-3-monoglucoside decreased signiﬁ cantly the anti-
oxidant activity in aqueous-phase systems as suggested by some reports [ 57 ,   60 ]. 
 Greater the number of free OH around the pyrone ring, greater is antioxidant 
activity. Anthocyanins with 3′, 4′-dihydroxy groups are good chelating agent for 
metal ions [ 62 ]. The chelating capacity also increases at pH 2–4, anthocyanins 
exist as ﬂ avylium cations and due to charge distribution, these are vulnerable to 
nucleophilic attack on 2 and 4 positions [ 63 ]. Not only the hydroxyl group but the 
position and degree of methoxyl groups on pyrone ring inﬂ uenced antioxidant 
potential of anthocyanins [ 60 ]. For example cyanidin-3-rutinoside and delphini-
din-3-glucoside have greater antioxidant capacity than malvidin-3-glucoside and 
petunidin-3- glucoside  [ 55 ,   57 ]. 
 The antioxidant potential of berries like red raspberries, black raspberries, straw-
berries and black berries, is directly proportional to the anthocyanins content and 
has strong scavenging capacity against chemically generated reactive oxygen spe-
cies [ 64 – 66 ]. Various drinks like whiskeys, sake, Cavas, Chilean Cabernet Sauvignon 
red wine, port wine or by-products such as grape pomace and fruit and vegetable 
juices have been reported for antioxidant activity due to presence of anthocyanins 
[ 67 – 71 ] and a reason behind the French paradox. Red wine fractions having highest 
anthocyanins substantially decreased reactive oxygen  species (ROS) in human red 
blood cells treated with H 
2
 O 
2
  as in vitro oxidative model [ 72 ]. Recent days the cus-
tomers expect natural pigment in wine, food and pharmaceutical industry with anti-
oxidant potentials as well [ 73 ]. 
 Wang and Mazza reported that cyaniding, pelargonidin, peonidin, malvidin, 
 delphinidin, malvidin-3,5-diglucoside and malviding-3-glucoside possess strong inhibi-
tory effects >50 % comparable quercetin on NO production in LPS/IFN-γ- activated 
7  The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

95
RAW 264.7 macrophage without showing any cytotoxicity at the range of 16–500 μM 
[ 34 ]. Anthocyanin-rich berry extracts inhibited NO-production and inhibitory effects are 
directly related to the content of total anthocyanins. The antioxidant capacity of serum 
increases with the consumption of red wine, strawberries and blueberries the report was 
conﬁ rmed by measuring antioxidant capacity as ORAC, TEAC and TRAP [ 74 ,   75 ]. 
 Anthocyanins are powerful antioxidants in vitro. Using the oxygen radical absor-
bance capacity (ORAC) assay, antioxidant potential of 14 anthocyanins including 
delphinidin, cyanidin, pelargonidin, malvidin, peonidin, and their glycosylated 
derivatives was measured in aqueous phase at neutral pH [ 5 ].  Cyanidin-3-glucoside 
was found to the highest antioxidant (ORAC value, 3.5 times as potent as Trolox) 
and pg exhibited the lowest ORAC value. 
 In linoleic acid autoxidation, liposome rabbit erythrocyte membrane, and rat 
liver microsomal systems, cyanidin-3-glucoside and its aglycone cyanidin were 
shown to have similar antioxidant potency as vitamin E (α-tocopherol) [ 76 ]. 
 Cyanidin-3-glucoside diminished powerfully the changes of biomarkers in hepatic 
injury in vivo (rat model) hepatic ischemia-reperfusion as an oxidative stress models 
thus revealing protective effect of anthocyanins on oxidative stress–induced damage 
[ 77 ]. Ramirez-Tortosa et al. found that anthocyanins enhanced plasma antioxidant 
potential and reduced the level of 8-oxo-deoxyguanosine and hydroperoxides in 
study of 12 week interval on rats, feeding them vitamin E–deﬁ cient diets for followed 
by supplementation with puriﬁ ed anthocyanin-rich extracts thus indicating signiﬁ cant 
reductions of the vitamin E deﬁ ciency–induced lipid peroxidation and DNA damage 
[ 78 ]. The plasma antioxidant status of Dahl–SS rats, measured by a total antioxidant 
status (TAS) assay, was restored by a diet supplemented with 1 % freeze-dried whole 
tart cherry powder for 90 days [ 79 ]. Various antioxidant assays used for assessment 
of antioxidant potential of anthocyanins are shown in Table  7.4 .
   Tyrosine nitration activity of anthocyanins declined as: cyanidin-3- rutinoside > mal-
vidin-3-monoglucoside ≈ delphinidin-3- monoglucoside > petunidin- 3-
monoglucoside [ 60 ]. Antioxidant activity is dependent on pH and anthocyanins 
isomeric forms [ 77 ]. The peroxynitrite (ONOO 
−
 ) quenching activity of anthocyanins 
at pH 7.4 declinedas: cyanidin-3-rutinoside  > malvidin-3- monoglucoside  ≈ delphinidin-  
3-mono-glucoside > petunidin-3-monoglucoside  [ 60 ].  
7.7      Protection  Against  Cardiovascular  Diseases 
 The development of cardiovascular disease is mainly due to hypertension, platelet 
aggregation, dysfunctioning of vascular endothelium and high plasma LDL choles-
terol. Anthocyanin exerted cardio-protection under ischemia-perfusion condition 
besides lowering the oxidative stress in vascular endothelium [ 97 ]. Youdim et al. 
(2000) have proved that anthocyanins are incorporated into the cytosol and 
 membrane of vascular endothelial cells thus protecting against oxidative stress and 
 protecting endothelial function thereby halting the vascular diseases [ 98 ]. In a study 
reporting direct effects of bilberry anthocyanins on whole rat hearts under 
7.7   Protection Against Cardiovascular Diseases

96
ischemia- reperfusion situation, the results indicated that perfusion with small 
amounts of bilberry anthocyanins (0.01–1 mg/L) substantially reduced ischemia-
perfusion injury by increasing post-ischemic coronary ﬂ ow, decreasing the rate of 
lactate dehydrogenase as well as chances and length of reperfusion arrhythmias 
[ 99 ]. Anaerobic metabolism can decrease intracellular pH under reperfusion condi-
tions thus affecting radical-quenching activities of anthocyanins [ 100 ]. All these 
results suggest that anthocyanin despite their low bioavailability act as cardio- 
protectants. In in vivo hamster animal model of ischemia-reperfusion, bilberry 
anthocyanin decreased micro-vascualr injuries by preserving endothelium and 
enhanced capillary perfusion [ 101 ]. In 8 week dietary ingestion of plant-derived 
anthocyanin in rats, made myocardium less vulnerable to ischemia-reperfusion 
impairments in vivo as well as ex vivo [ 49 ]. Figure  7.2  indicates the relationship 
between  consumption of anthocyanins and their effects on cardiovascular health.
   As mentioned above, over production of free radicals and reactive oxygen spe-
cies (ROS) are the key elements that led to activation of  pro- inﬂ ammatory media-
tors that ultimately led to cardiovascular disorders like ischemic heart problem, 
cardiomyopathy and coronary heart problems. Anthocyanins are most consumed 
subgroup of ﬂ avonoids that control/reduce this heart associated chronic abnormali-
ties via antioxidant, anti-inﬂ ammatory and free radical  scavenging mechanisms. 
   Table  7.4     Various antioxidant assays reported for anthocyanins   
 Name of antioxidant assay   Reference 
 Oxygen radical absorbance capacity (ORAC)   [ 
3 ,   5 ,   65 ,   74 ,   75 , 
 
80 ,   81 ] 
 Hydrogen transfer-based assay   [ 
3 ,   5 ,   65 ,   80 ,   82 ] 
 Ferric reducing antioxidant potential (FRAP)   [ 
83 – 88 ] 
 Trolox equivalent antioxidant capacity (TEAC)   [ 
74 ,   75 ,   81 , 
 
83 – 88 ] 
 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity   [ 
83 – 88 ] 
 Electron transfer-based assays   [ 
83 – 88 ] 
 Superoxide scavenging assay   [ 
89 ] 
 Peroxynitrite (ONOO−) scavenging activity   [ 
90 ] 
 Inhibition of human low-density lipoprotein   [ 
64 ] 
 Inhibition of lipid peroxidation   [ 
90 ] 
 Induction of antioxidant enzymes assay e.g. gluthatione- S -transferase 
(GST), gluthationereductase (GR), gluthationeperoxidise (GPx) and 
superoxide dismutase 
 [ 
91 ,   92 ] 
 Heavy metal binding assay e.g. iron, zinc and copper   [ 
93 ] 
 NO  assay   [ 
94 ] 
 TRAP   [ 
74 ,   75 ,   81 ] 
 Human red blood cells treated with H 
2
 O 
2
  as in vitro oxidative model   [ 95 ] 
 Hepatic ischemia-reperfusion as an oxidative stress model   [ 
77 ] 
 Vitamin E deﬁ ciency–induced lipid peroxidation   [ 
78 ] 
 Serum antioxidant capacity (SAOC)   [ 
96 ] 
 Total antioxidant status (TAS) assay   [ 
79 ] 
7  The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

97
Despite the high fat intake there is low incidence of cardiovascular disease amongst 
the French people (French paradox) which was supposed to be due to the daily con-
sumption of red wine, rich in polyphenols like anthocyanins [ 43 ,   103 ].  Fresh, 
freeze-dried and puriﬁ ed extracts and juice of chokeberries, cranberries, blueber-
ries, and strawberries anthocyanins have veriﬁ ed signiﬁ cant step up in low density 
lipoprotein (LDL) oxidation, lipid peroxidation, dyslipidemia, glucose metabolism 
and total plasma antioxidant status in human blood [ 104 ,   105 ]. 
 Mink et al. (2007) reported in a speciﬁ c epidemiological study that strawberry 
and blueberry intake caused reduction of mortality due cardiovascular diseases in 
postmenopausal woman [ 106 ]. The effect was considered to be due to presence of 
anthocyanins, a question was raised that other ﬂ avonoids may also contribute but 
later in another epidemiological study, Cassidy et al. reported that high consump-
tion of anthocyanins can decrease MI risk in predominantly young women in a 
comparison of those with lower intake. They conﬁ rmed that other ﬂ avonoids are not 
associated with such effects in their study [ 107 ] however further details trials are 
required to note the impact of anthocyanins enriched food. 
 According to several epidemiological studies, coronary heart disease mortality 
may be reduced by reasonable intake of red wine [ 108 ]. The reduction in mortality 
due to coronary heart disease may be due to reduction in platelet coagulation [ 43 ], 
  Fig.  7.2     Anthocyanins and cardiovascular health (actions and mechanisms) [ 102 ]        
 
7.7   Protection Against Cardiovascular Diseases

98
increase in circulatory high-density lipoprotein (HDL) [ 108 ], free radical scaveng-
ing, modulation of eicosanoid metabolism and inhibition of lipoprotein oxidation 
[ 109 ]. The last three mechanisms are also considered to be responsible for reduction 
of atherosclerosis [ 75 ]. Anthocyanins are responsible for reduction in coronary 
heart diseases and other health beneﬁ cial effects so complete metabolomic proﬁ le is 
required in properly designed long term intervention studies. This area has much 
space to be investigated [ 75 ]. 
 Daily intake of concentrated red grape juice (@125 mL) increase serum anti-
oxidant potential thus reducing susceptibility of LDL to oxidation. Thus red grape 
juice was proposed to possess beneﬁ cial effects equal to red wine [ 110 ].  Similarly 
other anthocyanins rich foods e.g. black currant consumption cause a rapid growth 
in plasma antioxidant capacity until 2 h [ 111 ]. Abuja et al. investigated that spray- 
dried elderberry juice comprising greater anthocyanins content has shielding 
effect on human LDL in vitro [ 112 ]. Tsuda et al. (1996) demonstrate that puriﬁ ed 
 anthocyanins like pelagonidin-3-glucoside, cyanidin-3-glucoside, delphenidin- 3- 
glucoside and their aglycones in a UV-induced lipid peroxidation model, showed 
strong inhibition of lipid peroxidation [ 58 ]. Even anthocyanin colonic metabolite, 
i.e. protocatechuic acid improved atherosclerosis progression by acting as 
 anti- inﬂ ammatory agent and also exhibited antiplatelet activity [ 113 ,   114 ].  
7.8      Anthocyanins  and  Bone  Health 
 Bone health is one of the prime concerns in human health specially aged people; 
one of the most common occurring bone diseases is osteoporosis. Osteoporosis is 
characterized by decrease in bone mineral content including decrease in calcium 
content; age related factors like hormonal imbalance, chronic inﬂ ammation and 
increase in oxidative stress are the main causes [ 115 ]. All these factors are linked to 
reactive oxygen species and to prevent bone loss, direct or indirect control of reac-
tive oxygen species is required. Anthocyanins containing foods are thought to have 
important role in the prevention of osteoporosis. Different studies reported the con-
ﬁ rmed the role of anthocyanins in bone diseases prevention (Fig.  7.3 ).
   Kaume et al. (2015) carried out a study in 9 months old female Sprague–Dawley 
rats, it was conﬁ rmed that feeding blackberries to rats prevent bone desorption 
especially in ovariectomized rat models, the author observed the effect in dose 
dependent manner, cyanidin 3- O - β - D -glucoside may be responsible for the bone 
loss preventive effect [ 116 ]. Women who consumed fruit in greater quantity, in 
childhood have high bone mineral density compared to normal, similar is the case 
with all women and men who are consumer of fruit, vegetables and grains [ 115 , 
 117 ]. Welch et al. (2013) carried out an observational study in women that were 
habitual to ﬂ avonoids and anthocyanins intake, they observed increased bone min-
eral density [ 118 ]. Similarly the decrease risk of fracture was observed by 
Langestmo et al. (2011) in individuals that were frequents consumers of fruit, 
 vegetables and grains [ 119 ]. 
7  The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

Tanabe et al. (2011) applied cranberry extract on human bone marrow cells

(pre- osteoclastic) at a dose of 10, 25, 50, 100 μg/mL for 4 days duration, the rate of

bone degradation by inhibition of RANKL dependent osteoclasts decreased [ 120 ].

500 ng/mL blueberry extract for 72 h duration increased proliferation of human

bone marrow cells (CD34+ or CD133) while decreased tartrate resistant acid phos-

phatase (TRAP) staining and RANKL dependent osteoclast numbers [ 121 ]. Six

months old SHAM and OVX (ovariectomized) female rats, were used in this study,

Fig. 7.3 Anthocyanins osteoporosis control mechanism

7.8 Anthocyanins and Bone Health

100
they were treated in three groups as (i) SHAM Control (ii) OVX-Control and (iii) 
OVX + 5 % blueberry for 100 day duration, it was found that OVX + 5 % blueberry 
group increased whole body bone mineral density (BMD) and serum alkaline phos-
phatase (ALP) [ 122 ]. 
 Chen et al. (2010) and Zhang et al. (2011) studied the affect of blueberry enriched 
diet in Sprague–Dawley rats and it was found that bone mass and bone mineral 
density increases, Early blueberry supplementation prevented osteoblast senescence 
and adult bone loss [ 123 ,   124 ]. 
 Berries are rich source of anthocyanins so the bone loss preventive effect are 
considered to associated with the antioxidant, and anti-inﬂ ammatory effect of 
anthocyanins however as berries also contain vitamin C so it’s could not be excluded. 
 Dou et al. (2014) studied the effect of cyanidin on bone for the ﬁ rst time, they 
used RAW264.7 cells and primary bone monocytes in their study design, they found 
that cyanidin has dual effect on RANKL induced osteoclastogenesis, dose less than 
1 μg has promoting while greater than 10 μg has inhibitory effect. Fusogenic and 
osteoclast related genes are regulated by cyanidin by the same dual pattern thus 
osteoclast related bone disorders may be controlled with the use of cyanidin [ 125 ]. 
Related group members of anthocyanins delphinidins have been shown to support 
bone mineralization by suppressing the differentiation and function of osteoclasts, 
the cells resorbing bone, this statement is supported by study in ovariectomized 
mice model through oral administration of delphinidin [ 126 ,   127 ]. These investiga-
tions suggest a potential beneﬁ t from regular consumption of anthocyanins rich 
sources for preventing bone demineralization at higher age.  
7.9      Conclusions 
 Despite all above mentioned effects, it is occasionally difﬁ cult to discriminate the 
effects of anthocyanins from other constituents or even isolation of effect of a 
single anthocyanin molecule from total pool. Further the doses mentioned in lit-
erature for animal models are sometimes far from those compatible with realistic 
values for human body due to bioavailability differences in both. There are entic-
ing glimpses of health-promoting effects however many questions remain unan-
swered since most of these studies are in vitro. There is need of balanced, 
data-driven answers for questions raised by nutritionists and health professionals 
regarding the totality of evidence rather than ﬁ ndings of any single study. There is 
still unceratininity about some mechanisms. There is plenty of information about 
role of anthocyanins in prevention of major diseases however many tropical and 
emerging diseases remain un- attentioned. Considering complex biological 
 functions and their efﬁ cacy in prevention of various chronic human diseases, 
combination therapies with other natural antioxidants can be a useful approach for 
various diseases.   
7  The Role of Anthocyanins in Health as Antioxidant, in Bone Health and as Heart…

101

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M. Riaz et al., Anthocyanins and Human Health, SpringerBriefs in Food,

Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_8

Chapter 8

The Role of Anthocyanins in Obesity

and Diabetes

8.1 Introduction

Food and phytomedicines are now becoming a trusted remedy and therapy for vari-

ous types of chronic diseases. The observed interfaces among substances present in

a phytochemical mixture strongly support the dogma of ‘eating the whole food’

rather than puriﬁ ed compounds, extracts, nutraceuticals, or formulation. In case of

formulation, phytochemicals can loose interactions during the product development

phase leading to decreased potency of extract. As noted in previous chapters, antho-

cyanins possess a wide spectrum of biological and pharmacological effects, such as

antioxidants, anti-cancer, anti-inﬂ ammatory, liver support by induction of detoxiﬁ -

cation enzymes, and induction of apoptosis. As known for other phytochemicals,

mixtures of interacting anthocyanins offer boosted synergistic curative and thera-

peutic potential by multi-pronged pathways of intercession concurrently. Numerous

mechanisms of action of anthocyanins at cellular and biochemical levels can be

complementary and overlapping, and a blend of these pathways leads to the

observed health beneﬁ ts of ingested anthocyanins. A lot of available studies indicate

potentiating interactions between various anthocyanins, and between anthocyanins

and other bioactives.

As discussed in Chaps. 1 and 7 , many studies suggest anthocyanins as promising

molecules for treatment of various diseases such as cancer, diabetes, ulcer, cardio-

vascular disorders and various metabolic disorders. While observational and lab

experimental studies appear to link dietary intake of anthocyanins to improvement

in health outcomes, data from randomized controlled trials is limited. In this regard,

two factors should be kept in mind, ﬁ rst only small portion of ingested anthocyanins

are absorbed to exert any biological effect and second stability of anthocyanins is

regulated by various factors. Since most studies describing beneﬁ cial effects of

anthocyanins are performed in vitro, more studies should be conducted to clarify the

mechanism of action as well as dose required to get that effect.

110
8.2      Anthocyanins  and  Obesity 
 Obesity is deﬁ ned as an excessive accumulation of adipose tissue due to disparity 
of energy intake and its disbursement. This condition is linked with several meta-
bolic disorders representing a strong risk factor for hypertension, heart disease, 
hyperlipidemia and type 2 diabetes. Lack of physical activity and imbalanced 
diet is a major road to obesity. Studies suggest that consumption of anthocyanins 
improve the job of adipocytes and inhibit the obesity. They increase the levels of 
adiponectin, an important adipocytokines, that is found at decreased levels in 
obesity [ 1 ]. 
 Animal experiments on mice conﬁ rmed the obesity preventive effect of corns 
containing anthocyanins [ 2 ]. Similar anti-obesity effects were observed in rats fed 
with anthocyanins from black soybean and blueberry. Results obtained by these 
experiments indicate an increase in serum high-density lipoprotein (HDL)-and a 
decrease in cholesterol triglyceride and cholesterol levels [ 3 ]. 
 Tsuda et al. (2005) studied the effect of anthocyanins on gene expression of 
cultured adipocyte, it has been reported that whole blueberry results increase in 
obesity while puriﬁ ed blueberry anthocyanins has signiﬁ cant anti-obesity action 
[ 4 ]. The total RNA isolated from the adipocytes was analyzed using GeneChip 
microarray. A total of 633 or 427 genes were up-regulated, after the treatment of 
adipocytes with 100 μM of cyanidin-3-glucoside or cyanidin, respectively. Based 
on the gene expression proﬁ le, the up-regulation of hormone-sensitive lipase and 
enhancement of the lipolytic activity were suggested to be the result of anthocy-
anin treatment on adipocytes [ 4 ]. An excessive adipose tissue (AT) accumulation 
has metabolic consequences, as adipocyte dysfunction, strongly associated with 
the development of obesity and diabetes, involving insulin resistance. However, 
few studies suggest that AT is an important site of  Vaccinium  species actions to 
ameliorate obesity complications [ 5 ]. Anthocyanins were shown to regulate obe-
sity and insulin sensitivity associated with adipocytokine secretion and PPARc 
activation in adipocytes [ 6 ]. High fat diet along with blueberry attenuates insulin 
resistance and hyperglycemia in mice coincident with reductions in adipocyte 
death [ 7 ]. In another animal study, Prior et al. (2010) concluded that consumption 
of puriﬁ ed anthocyanins (0.2 mg/ml) in the drinking water (0.49 mg/mouse/day) 
improved β-cell function and, the rate of fat deposition was decreased, however, 
blueberry juice was not effective in preventing obesity. Interestingly enough, 
lower serum leptin concentrations were found in anthocyanins treatments, which 
retarded the development of obesity [ 
3 ]. 
 According to Titta et al. (2009) [ 
8 ], orange juice has anti-obesity effect on fat 
accumulation but only anthocyanins are not responsible, multiple components pres-
ent in the orange juice might act synergistically to inhibit fat accumulation. Animal 
studies using  Vaccinium asheii  cultivars, results in reduction in food intake and, 
consequently a decrease in body weight gain [ 9 ].  
8  The Role of Anthocyanins in Obesity and Diabetes

111
8.3      Anthocyanins  and  Diabetes 
 Diabetes is a metabolic disease that occurs by a combination of several genetic and 
life-style patterns, which leads to hyperglycemia and associated symptoms includ-
ing polydipsia, polyuria and polyphagia (Fig.  8.1 ). Ultimately, hyperglycemia seri-
ously damages nerves and blood vessels and can lead to irritability and blurred 
vision. The WHO estimates that number of diabetic patients will be doubled within 
coming two decades. This exponential rise is mainly due to lack of healthy balanced 
diet, sedentary life style and increasing prevalence of obesity [ 10 – 14 ].
   Diabetes may arise due to persistent stress on pancreas, degeneration of β-cells, 
decreased insulin secretion, and increased insulin resistance [ 15 – 19 ]. Various scien-
tists have demonstrated anti-diabetic action of anthocyanins, which can be ascribed 
to several and concurrent effects of anthocyanins, including decreasing glucosuria, 
Hb A1c and blood glucose, increasing insulin secretion, improving insulin resis-
tance and preventing excessive generation of free radicals [ 20 ,   21 ]  however  plasma 
insulin or blood glucose concentrations remain the same in healthy humans [ 22 ].  In 
addition, anthocyanins may contribute to the prevention of type-2 diabetes through 
its antioxidant activity which may protect β-cells from glucose induced oxidative 
stress [ 23 ]. Sugimoto et al. (2003) reported that boysenberry anthocyanins rein-
stated or favored to reinstate the biomarkers of oxidative stress to the level of the 
control group in STZ-induced diabetic rats [ 24 ]. 
  Fig.  8.1     Pathogenesis  of  diabetes  and  glucose  regulation  by  pancreas       
 
8.3   Anthocyanins and Diabetes

112
 Stull et al. (2010) suggested that consumption of blueberries increase insulin 
action or sensitivity effectiveness in increasing insulin action in vivo, in obese and 
insulin resistant (non-diabetic) human subjects, without changes in adiposity or 
inﬂ ammatory biomarkers [ 25 ]. However, more cellular mechanistic studies are 
needed to clarify the speciﬁ c cellular pathway involved in improving sensitivity to 
insulin when blueberries were consumed. Furthermore, hypoglycemic activity has 
been reported by various scientists as anthocyanins and anthocyanidins induce insu-
lin secretion and the order of affect observed was delphinidin-3-glucoside > cyanidin- 
3- glucoside > and  pelargonidin-3-galactoside  [ 20 ]. Grace et al. (2009) reported 
anthocyanins enriched fraction from low bush black berries for hypoglycemic activ-
ity in acute mouse model of type 2 diabetes however the fruit extract has no hypo-
glycemic effect [ 20 ]. Among these studies, Tsuda et al. (2003) evaluated that 
supplementation with cyaniding-3-O-α- D -glucoside rich purple corn color improves 
hyperglycemia induced by high fat diet in mice [ 26 ]. Another study by Vuong et al. 
(2010) showed that biotransformed blueberry juice decreases hyperglycemia in a 
diabetic animal model, by partly reversing adiponectin levels thus protects young 
pre-diabetic mice from developing diabetes and obesity [ 27 ]. 
 Tsuda et al. (2003) demonstrated that dietary anthocyanins signiﬁ cantly normal-
ized hypertrophy of the adipocytes in the epididymal white adipose tissue with 
increase in adiponectin and leptin secretion, thus diminishing hyperglycemia 
inﬂ icted by the high fat diet in mice (C57BL/6J) [ 26 ]. Later in 2005 Tsuda et al. 
reported that if we treat adipocytes with cyaniding-3-glucoside or cyanidin, the hor-
mone sensitive lipase gene expression elevation occur that results in lipolytic activ-
ity and insulin sensitivity [ 4 ,   26 ]. 
 Suzuki et al. (2011) determined that bilberry extracts are capable of inhibiting 
adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner and to dimin-
ish lipid accumulation with concomitant down-regulation of peroxisome proliferator- 
activated receptor gamma (PPARc). Additionally, all tested single anthocyanins 
(pelargonidin, cyanidin, delphinidin, peonidin, and malvinidin) also inhibit lipid 
accumulation in 3T3-L1 cells but delphinidin was most effective in down-regulating 
PPARc and sterol regulatory element-binding protein 1c (SREBP1c) mRNA levels 
[ 5 ]. Bilberry extracts inhibit adipocyte differentiation via insulin pathway and these 
effects are mainly due to the presence of anthocyanins. Clinical trials involving 
blueberry consumption or blueberry extract are recommended to get beneﬁ ts of 
obesity prevention and to understand it mechanism [ 9 ]. A simpliﬁ ed way of antho-
cyanins to control diabetes is shown in Fig.  8.2 .
8.3.1        Oxidative  Stress 
 There is a vital linkage among oxidative stress, inﬂ ammatory response and insulin 
activity. Excessive generation of free radicals occurs in hyperglycemic and hyperlipid-
emic state. In order to reduce free radicals and ultimately stress, antioxidants like 
anthocyanins are required to be consumed [ 24 ,   28 ].  Ischemia-reperfusion-induced 
8  The Role of Anthocyanins in Obesity and Diabetes

113

oxidative stress leads to high concentrations of thiobarbituric acid reactive substances

(TBARS) and diminished levels of reduced glutathione [ 26 ], the details of anti-diabetic

studies in animals which involves reduction of oxidative stress are given in Table 8.1 .

Anthocyanins enriched freeze-dried blueberry powder increased plasma antioxi-

dant levels after ingestion of fat diet in healthy men [ 33 ]. Similarly juice prepared

from freeze-dried strawberry powder lowers lipid peroxidation, leading to decreased

plasma levels of oxidized LDL, MDA and 4-hydroxy-noneal in women with meta-

bolic syndrome after consuming two glasses for 4 weeks [ 22 ].

8.3.2 β Cells

When diabetes type 2 develops pancreatic β cells stop working due to high oxidative

stress. But anthocyanins can decrease oxidative stress by their antioxidant action

and β cells may be protected [ 31 ]. Blueberry ( Vaccinium angustifolium ) fruit extract

augmented the proliferation of pancreatic β TC-tet cells exerting a substantial anti-

diabetic effect [ 34 ] that may be due to anthocyanins [ 35 ]. Jayaprakasam et al. (2006)

Mechanism to control diabetes

β−cells

Oxidative stress

Insulin resistance

α-Glucosidase inhibitory

action

Pancreatic β TC-tet cells

Anthocyanins

pancreatic islets protection

modulation of pro-apoptotic

proteins

insulin secretion

reduced blood glucose,

TNF-α and MCP-1

inhibition of maltase

inhibition of sucrase

competitive inhibition

α-Glucosidase

change in the molecular

configuration

Increased GLUT4

mRNA expression

reduction in plasma

levels of MCP-1

Fig. 8.2 Mechanism to control diabetes via anthocyanins

8.3 Anthocyanins and Diabetes

114
reported that cornelian cherry anthocyanins protected the pancreatic islets of 
C57BL/6 CB mice and insulin secretion was increased [ 36 ]. Treatment with antho-
cyanins from black soybean seed coats prevented the apoptosis of β cells through 
modulation of pro-apoptotic proteins caspase 3 and Bax which ampliﬁ ed Bcl2 
expression which indicates anti-apoptotic effects [ 32 ].  Puriﬁ ed anthocyanins were 
reported to increase β cell function in C57BL/6J mice [ 3 ]. Anthocyanins from 
Chinese bayberry extract protected pancreatic β INS-1 cells and primary islets from 
damage induced by H 
2
 O 
2
   [ 37 ].  
8.3.3      Insulin  Secretion 
 In type 2 diabetes, insulin secretion may be impaired due to the dysfunction of β cells 
[ 38 ]. Synthetic agents like sulfonyl urea increase insulin secretion but anthocyanins, 
an important natural bioactives may act as insulin secretagogue without producing 
such side effect [ 31 ,   32 ]. Delphinidin-3- glucoside in INS-1 832/13 cells led to 
   Table  8.1     Anthocyanins that results reduction in oxidative stress   
 Anthocyanins   Model   Mechanism   References 
 Diet containing 0.2 % 
cyanidin-3-glucoside 
 Ischemia  reperfusion 
rat model 
 ↓ Serum thiobarbituric acid 
reactive substances (TBARS) 
 [ 
26 ] 
 ↑ Hepatic reduced glutathione 
in rats 
 Diet containing 0.1 % 
mulberry anthocyanins 
 Streptozotocin(STZ)-
induced diabetic 
Wistar rats 
 ↓ Liver oxidized glutathione 
and tended ↓ TBARS levels 
 [ 
24 ] 
 Diet with 0.5 % of black 
rice anthocyanins-rich 
extract 
 Sprague–Dawley  rats   ↓ Blood concentrations of 
TBARS ↓ glutathione 
 [ 
29 ] 
 Diet supplemented with 
0.2 % chokeberry fruit 
extract 
 Pre-diabetic  Wistar 
rats 
 Normalize  the 
concentrations of TBARS in 
the liver, kidneys and lungs 
and no effect on SOD 
 [ 
30 ] 
 One i.p. injection of 
pelargonidin at a dose of 
3 mg/kg body weight 
 STZ-induced  diabetic 
Wistar rats 
 Restoration of exhausted 
serum levels of SOD and 
catalase and normalization 
of the serum levels of MDA 
 [ 
31 ] 
 Anthocyanins from black 
soybean seed coats 
 STZ-diabetic rats   Reinstatement of the 
depleted serum levels of 
catalyze and SOD enzymes 
and Normalization of the 
serum levels of MDA 
 [ 
32 ] 
 Anthocyanins  rich 
ethanolic extract of 
 Vaccinium arctostaphylos  
fruit, Orally 
 Alloxan-induced 
diabetic Wistar rats 
 ↑ The concentration of 
glutathione peroxidase, 
catalase and superoxide 
dismutase in red blood cells 
 [ 
28 ] 
8  The Role of Anthocyanins in Obesity and Diabetes

115
decreased insulin secretion more than cyaniding- 3-glucoside which is more potent 
than pelargonidin-3-galactoside. Among various anthocyanins evaluated, only pelar-
gonidin showed secretagouge activity. Pelargonidin-3-glucoside and anthocyanins 
extracted from black soybean increased insulin secretion in STZ-induced-diabetic 
Sprague–Dawley and Wistar diabetic rats respectively [ 31 ,   32 ]. Extract of  V. arcto-
staphylos  increases insulin expression in cardiac and pancreatic cells of alloxon-
induced-diabetic Wistar rats [ 28 ].  
8.3.4      Insulin  Resistance 
 Anthocyanins have been reported for anti-inﬂ ammatory potential and can 
decrease the expression of monocyte chemotactic protein-1 (MCP-1), tumor 
necrosis factor α (TNF-α), interleukin-6 (IL-6), thus improving T2D and insulin 
resistance [ 7 ,   39 ]. The proposed hypoglycemic activity of anthocyanins can be 
ascribed partly by the activation of insulin receptors generated [ 32 ]. Anthocyanins 
can modify the expression of adipocytokines, intensify the expression of GLUT4, 
reduce the expression of RBP4, stimulate AMPK and decrease the oxidative 
stress to improve insulin sensitivity, thus positively modulating the glycemic 
control. Table  8.2  indicates improvement of insulin resistance in anthocyanins 
treated animal models.
8.3.5         α-Glucosidase Inhibitory Action 
 α-Glucosidases cause hydrolysis of carbohydrates in small intestine, thus inhibit-
ing these enzymes is one therapeutic approach to control diabetes specially after 
meal [ 42 ]. Matsui et al. (2002) reported that anthocyanins extract of purple sweet 
potato reduces the blood glucose through inhibition of maltase activity in healthy 
Sprague–Dawley rats after carbohydrate loading (sucrose, maltose and glucose) 
[ 43 ]. Another study by Jurgoński et al. (2008) reported that anthocyanins from 
chokeberry lowered the high glucose level by inhibiting maltase and sucrase in 
Wistar rat model of prediabetes obtained through a high-fructose diet and simulta-
neous single injection of streptozotocin (20 mg/kg) [ 30 ]. Anthocyanins cause inhi-
bition of α-glucosidases [ 43 ] but the exact mechanism of inhibition is unknown, 
however it is considered to be competitive, since normal substrates of the enzyme 
and glucosyl groups linked to anthocyanins are structurally similar [ 44 ].  Another 
possible mechanism may be change in the molecular conformation of the enzyme 
that polar groups of enzyme active site interact with hydroxyl groups on anthocya-
nins, leading to a modiﬁ cation in enzyme activity [ 45 ]. Another important aspect to 
be considered is that the use of synthetic inhibitors of α-glucosidases and anthocya-
nins might have synergistic effect and the α-glucosidases inhibition of anthocya-
nins is directly related to its stability [ 46 ].  
8.3   Anthocyanins and Diabetes

116
8.3.6      Obesity  and  Diabetes 
 Due to sedentary life-style and changed dietary patterns especially large intake of 
reﬁ ned food has led to a dramatic rise in obesity and diabetes. Obesity and diabetes 
are inter connected disorders and anthocyanins have been reported for health ben-
eﬁ cial effect in association with both for example Tsuda et al. (2003, 2008) reported 
high fat diet if supplemented with cyanidin-3-glucoside cause reduction in fat 
   Table  8.2     Insulin resistance improvement in various models   
 Anthocyanins   Model 
 Insulin resistance improvement 
mechanism  Reference 
 High-fat  and 
anthocyanins 
supplemented diet 
 C57BL/6J mice   Reduced blood glucose, TNF-α  [ 
26 ] 
 Anthocyanins 
supplemented diet 
 KK-Ay mice   Reduced blood glucose, TNF-α 
and MCP-1 
 [ 
40 ] 
 High-fat  diet 
supplemented with 
tart cherry powder 
 Zucker rats   Reduced glycemia and insulinemia 
as well as improved insulin 
resistance due to a decrease in 
plasma levels of IL-6 and TNF-α 
 [ 
39 ] 
 High-fat  diet 
supplemented with 
freeze-dried whole 
blueberry powder 
 C57BL/6 mice   Reduced levels of fasting glucose 
and TNF-α expression in adipose 
tissue and improved insulin 
tolerance test (ITT) 
 [ 
7 ] 
 Freeze-dried  powders 
from Different 
anthocyanins-rich 
whole berries in 
hypo- and hyper-
lipidemic diets 
 C57BL/6 J mice   No changes in plasma levels of 
TNF-α, IL-6 and PAI-1 
 [ 
41 ] 
 High-fat diet and 
drinking water 
supplemented with 
anthocyanins puriﬁ ed 
from blueberries 
 C57BL/6 J mice   ↓ Plasma levels of MCP-1   [ 
41 ] 
 Anthocyanins  extracted 
from black soybean 
 Sprague–Dawley 
rats 
 ↑Autophosphorylation of insulin 
receptors 
 [ 
32 ] 
 Anthocyanins  from 
different plant species 
 Animal  models   ↓ Glycemia and ↑expression of the 
glucose transporter type 4 (GLUT4) 
 [ 
21 ,   32 , 
 
40 ] 
 Extract of  Vaccinium 
arctostaphylos  
 Heartcells  of 
alloxan-induced 
diabetic Wistar 
rats 
 IncreasedGLUT4  mRNA 
expression 
 [ 
28 ] 
 Billberry extract in diet   DiabeticKK-Ay 
mice 
 ↓ Plasma glucose and ↑ insulin 
sensitivity in activation of AMPK 
in white adipose, skeletal muscle 
and liver tissues 
 [ 
21 ] 
8  The Role of Anthocyanins in Obesity and Diabetes

117
accumulation in C57BL/6J mice [ 2 ,   26 ] and suppression of elevated serum glucose 
levels induced by high-fat meals. Table  8.3  shows simultaneous effects of anthocy-
anin in diabetes and obesity.
8.4          Improvement  of  Eye  Vision 
 The mounting evidence supported by clinical trials indicates anthocyanins as a 
potential future drug to treat various opthalmological diseases. Bilberry anthocya-
nins increase night vision by interacting with rhodopsin [ 52 ] or phosphodiesterase 
(PDE) on photo-transduction [ 53 ,   54 ]. 
 Berry anthocyanins improve vision by multiple mechanisms by (i) reducing 
molecular degeneration and diabetic retinopathy (ii) increasing blood circulation in 
retina capillaries (iii) improving night-vision by increased production of retinal pig-
ments and by (iv) preventing cataracts, retinitis pigmentosa and glaucoma [ 55 ,   56 ]. 
   Table  8.3     Anthocyanins simultaneous effects on obesity and diabetes   
 Anthocyanins   Model   Effect   Reference 
 High-fat diet (45 % of energy) 
with anthocyanins extracted 
from blueberries 
 C57BL/6 
mice 
 Inhibit weight gain and body fat 
accumulation 
 [ 
47 ] 
 Whole blueberry powder   C57BL/6 
mice 
 Promote body fat accumulation   [ 
47 ] 
 Blueberry juice high fat diet 
(45 % of energy) 
 Mice   Reduction in body weight gain and 
percentage of white adipose tissue 
 [ 
3 ] 
 Supplementing a high-fat diet 
(60 % of energy) with WBP 
 C57BL/6 
mice 
 Did  not  ↓ body weight gain   [ 
7 ] 
 Supplementing a high-fat diet 
(45 % of energy) with 2 % 
WBP 
 Zucker 
fatty rats 
 Reduced abdominal fat mass and 
increased the activity of the adipose 
tissue and skeletal muscle 
peroxisome proliferator-activated 
receptor 
 [ 
48 ] 
 Supplementing a high-fat diet 
(45 % of energy) with 2 % 
WBP 
 Zucker 
lean rats 
 WBP-induced body weight gain   [ 
48 ] 
 Black raspberry anthocyanins 
(as a juice or in powder form) 
high-fat diet (60 % of energy) 
 Mice   Didnot  signiﬁ cantly reduce body fat 
accumulation or body weight gain 
 [ 
3 ,   41 , 
 
49 ] 
 Mulberry water extracts high 
concentration of anthocyanins 
 Decrease body weight gain   [ 
50 ] 
 Tart cherry powder   Zucker 
fatty rats 
 Decrease body weight gain   [ 
39 ] 
 Anthocyanins-rich  berry, 
chokeberry fructose rich diet 
 Mice   Suppress the increase in epididymal 
white adipose tissue and blood 
glucose level 
 [ 
51 ] 
8.4   Improvement of Eye Vision

118
 Anecdotal evidence in Japan describes Royal Air Force pilots during World War 
II who experienced enhanced vision in the dark after ingesting blueberry jam indi-
cating that anthocyanin ingestion can increase eye vision [ 57 ]. Decreasing the dark- 
adaptation threshold was noted in a placebo-controlled cross-over double-blind 
study involving healthy human subjects that were fed blackcurrant anthocyanin 
concentrate [ 58 ]. The effect with the highest dose (50 mg per subject) had a statisti-
cally signiﬁ cant effect (P = 0.011). Recently, very low (pmol g 
−1
 ) concentration of 
blueberry ANs in pig eyes were found after feeding for 4 weeks [ 59 ]. 
 Another placebo-controlled double-blind study indicated that oral administration 
of anthocyanin is vital for the production of visual purple, which helps in conversion 
of light into electrical signals for brain. Adapto-electro-retinograms of two sets of 
six subjects were performed before treatment and 1 and 3 h after administration; the 
subjects consuming bilberries adapted to the light in 6.5 min compared to 9 min for 
the control group [ 55 ,   56 ]. In another clinical trial of 4 months, 50 patients suffering 
from sensile cataract were given a mixture of bilberry extract, containing 25 % 
anthocyanins (@180 mg twice/day) and vitamin E (@100 mg twice/day). This com-
bination stopped the development of cataract in 96 % of the subjects as compared to 
76 % in control group [ 60 ]. 
 Sundalius (2008) during her PhD research work investigated the ability of blue-
berry anthocyanins to inhibit the uptake of  N -retinyl- N -retinylidene  ethanolamine 
(A 
2
 E) by retinal pigment epithelial (RPE) cells as quantiﬁ ed by pigment epithelial 
derived factor (PEDF) levels in RPE cells. The average PEDF results showed that 
blueberry anthocyanins stimulated and sustained PEDF levels as compared to con-
trol RPE cells and RPE cells treated with A 
2
 E thus it was evaluated that blueberry 
anthocyanins preserved RPE cells from oxidation as indicated by PEDF values [ 61 ]. 
 Bilberry anthocyanins have been shown to exhibit neuroprotective effects in ocu-
lar tissue and suppressive effects on diabetic retinopathy via a reduction of angio-
genesis [ 62 ,   63 ]. Furthermore, several studies have demonstrated that blackcurrant 
anthocyanins improve visual function; for example, BA inhibit transient myopia, 
reduce eye fatigue, improve dark adaptation, and increase retinal blood ﬂ ow in glau-
coma [ 58 ,   64 ]. The ﬁ rst three beneﬁ ts have been shown in humans, and effective 
daily BA intake was reported as 50 mg [ 58 ]. Two studies have indicated the possible 
mechanism underlying inhibition of myopia. The blackcurrant anthocyanins con-
centration was higher in ocular tissues (sclera, choroid, ciliary body, retina, iris, and 
cornea) than in blood in a rat fed a diet containing blackcurrant anthocyanins, and 
in particular, concentrations in the sclera and choroid were 100-fold higher than the 
blood blackcurrant anthocyanins concentration [ 65 ]. In addition, it was shown that 
an anthocyanins in the concentration range of 10 
−7
  to 10 
−8
  M relaxed endothelin- 1- 
induced contraction of bovine ciliary smooth muscle [ 66 ], which plays an important 
role in modulating refraction of the lens through contraction and relaxation, which 
in turn control accommodation. Thus, it is suggested that anthocyanins stimulate the 
endothelin-1 receptor to induce production of NO, thereby relaxing ciliary smooth 
muscle, which in turn ﬂ attens the lens and consequently inhibits myopia. 
  Vaccinium myrtillus  extracts containing anthocyanins were reported to improve 
the night vision, particular diabetic retinopathy, through the inﬂ uence on the perme-
8  The Role of Anthocyanins in Obesity and Diabetes

119

ability of retinal vessels which also decrease the permeability of the blood-brain bar-

rier to toxic substances [ 67 ]. The differences in results and conclusions are probably

due to variations in dose and source of anthocyanin, type of subjects and the tech-

niques used to evaluate vision.

8.5 Conclusions

Health is a key priority of people of all age and income group and a vital feature of

a prosperous life. Health-concerned consumers of modern society are now moving

from curative to preventive health measures. It has led to the generation of dietary

recommendation and food composition tables. It is well- known now that plant-

based foods and bioactive compounds found in them like anthocyanins help in

remaining healthy. Anthocyanins act as preventive and curative agents for various

diseases and disorders as presented in this chapter. Works that suggested protective

actions of anthocyanins are mainly based on in vitro experiments, a fact that com-

promises the robustness of these ﬁ ndings. More details are required to establish the

real implications of anthocyanins and the mechanism by which they exert their bio-

logical effects.

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Health, and Nutrition, DOI 10.1007/978-3-319-26456-1_9

Chapter 9

Anthocyanins Effects on Carcinogenesis,

Immune System and the Central Nervous

System

9.1 Introduction

As mentioned in Chap. 1 , anthocyanins are phytochemicals that are not required for

the immediate survival of the plant but which are synthesized to increase the ﬁ tness

of the plants to survive by allowing them to interact with their environment, includ-

ing pathogens and herbivorous and symbiotic insects. In many cases, the effects of

these secondary metabolites on the human immune system and central nervous sys-

tem might be linked either to their ecological roles in the life of the plants or to

molecular and biochemical similarities in the biology of plants and higher animals.

The health professionals now recognize that in addition to the macromolecules, i.e.

carbohydrates, lipids and proteins, there is dearth of phytochemicals like anthocya-

nins and carotenoids which have major heath promoting effects. Age-related disor-

ders like hypertension, diabetes, cardiovascular diseases, Alzheimer’s disease,

cataracts, neurodegenerative problems and macular degeneration, and improvement

of vision and brain functions may be prevented by taking ample amount of these

bioactive constituents.

9.2 Anti-Inﬂ ammatory Activity

Inﬂ ammation is a multifaceted biological response of vascular tissue to stimulants, irri-

tants or injuries and is linked with instigation and progression of various chronic dis-

eases like cardiovascular diseases, Alzheimer’s, disease, diabetes mellitus (type 2) and

various types of cancers [ 1 – 9 ]. Therefore, anti-inﬂ ammatory agents also can act as

anticancer agents. Anti-inﬂ ammatory properties of anthocyanins, anthocyadin (puri-

ﬁ ed) and their concerned sources were reported by various scientists. For example,

Cy-aglycone was found to exhibits higher anti-inﬂ ammatory effects than aspirin in

COX-assays [ 10 ]. Puriﬁ ed anthocyanin fractions from bilberries, blueberries,

126
blackberries, cranberries, sweet cherries, raspberries, elderberries, strawberries and tart 
cherries exhibit anti-inﬂ ammatory properties as assessed by COX-1 and COX-2 inhibi-
tory assays and it may be due to cyanidin glycosides [ 11 ]. Strawberry, blackberry, and 
raspberry showed the highest anti-inﬂ ammatory activity, comparable to that of ibupro-
fen and naproxen at 10 μM concentrations. In an in vivo study, the therapeutic efﬁ cacy 
of blackberry anthocyanins (Cyanidin-3-glucoside accounts for 80 %) was investigated 
in rats with carrageenan-induced lung inﬂ ammation [ 12 ]. Anthocyanins effectively 
reduced all parameters of inﬂ ammation dose-dependently. 
 Anthocyanidins possess structure dependent anti-inﬂ ammatory properties. The 
action mechanism of this effect is believed to be mediated through inhibiting 
COX-2 in lipo-poly-saccharide (LPS)-activated cells (RAW-264) or inhibiting 
inducible nitric acid synthase (iNOS) and mRNA expression in LPS-activated 
murine-J774 macrophages [ 13 ]. Cyclooxygenase-2 is involved in many inﬂ amma-
tory actions. Delphinidinis are the strongest inhibitor of COX-2 expression at 
mRNA and protein levels and exerts inhibitory effect on degradation of nuclear 
translocation of p65 and IκB-α [ 14 ]. 
 Anthocyanins or anthocyanin-containing extract inhibit pro-inﬂ ammatory cyto-
kines in vitro by suppressing NF-κB through down-regulation of mitogen-activated 
protein kinase (MAPK) pathways [ 15 ]. Some authors reported that these actions are 
due to antioxidant effect but some authors reported anthocyanins can exhibit sub-
stantial anti-inﬂ ammatory property through signaling pathways responsible for 
anti-inﬂ ammatory action without affecting the in vivo anti-oxidative status or its 
signaling pathways [ 16 ,   17 ]. Wang and his colleagues [ 17 ] found that cyanidin- 3- 
glucoside inhibit COX-2 expression and iNOS by induction of liver-X-receptor-α 
(LRX-α) activation in THP-1 macrophages. The signaling pathways of the nuclear 
receptors are activated by anthocyanins effectively, for example LRX α and PPAR γ 
(peroxisome proliferator-activated receptor γ) that antagonizes in vitro inﬂ amma-
tory gene expression [ 14 ]. In carrageenan-induce inﬂ ammation in rat lungs, blue-
berry anthocyanins reduced all inﬂ ammation parameters dose-dependently [ 12 ]. 
Anthocyanins obtained from red wine inhibited TNF-α-induced inﬂ ammation by 
modulating the endothelial monocyte chemo-attractant protein-1 [ 18 ].  
9.3      Anthocyanins  and  Cancer 
 Cancer is a principal reason of causality and mortality globally. Numerical ﬁ gures 
specify that cancer affects more than 1/3rd of world population and more than 20 % 
death occur due to this malady. In both benign and malignant cancers, cells divide 
abnormally by escaping the regulatory control of cell resulting in invasion of other 
body tissues. Till now, more than 100 various type of cancer have been discovered 
which have been classiﬁ ed depending upon affected tissue or organ like blood can-
cer, colon cancer and breast cancer etc. Cancer results from DNA abnormalities 
Inﬂ ammation, unhealthy diet and stress-induced oxidative damages are one of major 
reasons of cancer. Diet and phytochemicals have been used since dawn of civilization 
9  Anthocyanins Effects on Carcinogenesis, Immune System and the Central Nervous…

127
as therapeutic strategy to cure various diseases including cancer. Bioactive constitu-
ents present in food commodities like grains, fruits, vegetables, nuts, legumes and 
herbs encompass effective protection against various types of cancers. Therefore 
dietary chemo-prevention is now focus of attention globally [ 19 – 21 ]. 
 Anthocyanins, although discovered in 1918, their anticancer effects have been 
studied and reported only recently. Table  9.1  lists anticancer effects of various antho-
cyanins. Modulation of carcinogenesis occurs through variety of biological func-
tions of anthocyanins. Anthocyanins are believed to exert anti-cancer effects by 
multiple mechanisms like by (i) arresting cell cycle by arresting the G1/G0 and 
G2/M phase (ii) inducing apoptosis and anti-angiogenesis (iii) inhibiting oxidative 
DNA damage (iv) inducing phase II enzymes for detoxiﬁ cation and (v) by inhibiting 
COX-2 enzymes. The chemopreventive action of anthocyanins is due to their anti-
oxidant property.
   Anthocyadins are more effective in inhibition of cell proliferation than anthocya-
nins [ 40 ]. The latter are more potent anti-cancer agents than other ﬂ avonoids [ 23 ]. 
In another study by Kamei and his colleagues, anthocyanins from red wine showed 
higher anticancer activity than other ﬂ avonoids of red or white wine [ 24 ]. 
Anthocyanins fraction from 4 cultivars of muscadine grape exhibited higher antip-
roliferative activity than crude extract or phenolic acid fraction [ 28 ]. Antiproliferative 
effects of anthocyanins on the colon cancer cells are structure dependant [ 27 ].  The 
proposed mechanism of anticancer effects of anthocyanins is shown in Fig.  9.1 .
   The work of Jing et al. [ 27 ] advocated the additive effect of anthocyanins and 
other phenolics in combinational usage in antiproliferative studies. Various animal 
studies showed that anthocyanins have chemopreventive effects in gastrointestinal 
tract like oral cavity, the esophagus [ 41 ], and the colon [ 30 ]. This chemopreventive 
effect may be due to anthocyanins contact directly with the epithelial layer [ 42 ] 
while non-gastrointestinal organs required anthocyanins availability through blood 
delivery. It was conﬁ rmed by strawberry anthocyanins failure to inhibit 
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-and benzo[a]pyrene-induced lung 
cancer in a mice model [ 43 ]. 
 Anti-cancer activity of anthocyanins may be attributed to the additive effect of 
multiple mechanisms [ 30 ]. Possible mechanisms that have been suggested are given 
in Table  9.2 . Various studies showed that anthocyanins can inhibit the growth of 
different cancer cells and embryonic ﬁ broblasts, indicating their potential as chemo-
preventive agents in the form of cheap and safe anticancer dietary supplements [ 50 ]. 
Anthocyanins exert antiproliferative activity against human cancer cells derived 
from malignant tissues [ 40 ]. Anthocyanins were also potent and selective 
proliferation- inhibitor of human promyelocytic leukemia cells [ 51 ]. The inhibitory 
effect on cancer cells of berry extracts deepens upon the contents and not the com-
position of anthocyanins [ 52 ].
   Antiangiogenic effect, increased apoptosis and decreased proliferation were 
observed in patients suffering from colon cancer administered black raspberry pow-
der daily for several weeks [ 16 ]. In another clinical study, 25 colorectal cancer 
patients were given standardized anthocyanin bilberries extract (mirtocyan) 
 administered daily for 1 week, decrease in proliferation index, and increase in apop-
9.3   Anthocyanins and Cancer

128
   Table  9.1     Anticancer activities reported from different anthocyanins   
 Anthocyanins  source   Model   Effect   Reference 
 Anthocyanin-rich 
extract from 
chokeberry 
 Colon cancer HT-29 cells   Induce cell cycle 
block at G1/G0 and 
G2/M phases but not 
in NCW460 normal 
colonic cells 
 [ 
22 ] 
 Anthocyanins 
extracted from 
ﬂ ower petals 
 Intestinal carcinoma derived 
HCT-15 cell line 
 Cell  growth 
inhibition 
 [ 
23 ] 
 Anthocyanins  from 
red wine or white 
wine 
 Humangastric cancer HCT-15 
cell line and AGS cell line 
 Cell  growth 
inhibition 
 [ 
24 ] 
 Commercial  bilberry, 
chokeberry, and grape 
extracts 
 HT-29 cell line   Cell growth 
inhibition 
 [ 
25 ] 
 Four  anthocyanins 
isolated from 
strawberry 
 Human oral (CAL-27, KB), colon 
(HT29, HCT-116), and prostate 
(LNCaP, DU145) cancer cells 
 Reduction in cell 
viability 
 [ 
26 ] 
 Anthocyanins  rich 
extract of purple corn, 
purple carrot, and red 
radishes 
 HT-29 cell line   Cell growth 
inhibition 
 [ 
27 ] 
 Anthocyanins  fraction 
from muscadine 
grapes (4 cultivars) 
 Human colon cancer derived cell 
liens, HT-29 and Caco-2 
 Cell  growth 
inhibition 
 [ 
28 ] 
 Cranberry  extract 
versus its ﬂ avonol 
glycosides (gly), 
anthocyanins, 
proanthocyanidins, 
and organic acids 
fractions 
 Human oral (KB, CAL27), colon 
(HT-29, HCT116, SW480, 
SW620), and prostate (RWPE-1, 
RWPE-2, 22Rv1) cancer cell 
lines 
 Anthocyanins and the 
proanthocyanidin 
fraction exhibited 
substantial inhibitory 
effect except SW480 
cell lines, but 
combination of both 
exhibited inhibition 
of all comparatively 
 [ 
29 ] 
 Dietary anthocyanins   Colon carcinogen azoxymethane 
(AOM)-induced rat colon cancer 
model 
 Inhibitory effect on 
cell proliferartion 
 [ 
30 ] 
 Anthocyanins  rich 
extracts of grapes, 
bilberries and 
chokeberries 
 Human malignant HT-29 colon 
cancer cells 
 Cell  growth 
inhibition 
 [ 
25 ] 
 Anthocyanins  rich 
extracts of grapes, 
bilberries and 
chokeberries 
 Low and high tumorigenic colon 
cancer cell lines, LoVo/Adr and 
LoVo 
 Cell  growth 
inhibition 
 [ 
31 ] 
(continued)
9  Anthocyanins Effects on Carcinogenesis, Immune System and the Central Nervous…

129
totic index was observed [ 53 ]. No substantial association was observed between 
anthocyanidins intake and risk for pharyngeal or oral cancer [ 
54 ]. Similarly no pro-
tective effect has been reported against the development of prostate cancer [ 55 ].  To 
get optimum results the anthocyanins containing berry are consumed before, during 
and after chemotherapy of cancer may maximize chemopreventive effectiveness in 
humans. The difference in response against various tumors may be due to its bioaval-
ibility to the site [ 
56 ]. Studies are required to evaluate different anthocyanins in com-
bination with various chemotherapeutic agents to maximize rational treatment of 
cancer. Anthocyanins from highbush blueberry and mulberry fruits ( V. angustifolium ) 
stopped the proliferation activities of cells [ 57 ]. Delphinidin inhibited invasion of 
human ﬁ brosarcoma cells by down regulation of gene expression of MMP-2/9 [ 58 ]. 
Delphinin is more potent antioxidant due to highest number of OH groups on B ring.  
Table 9.1 (continued)
 Anthocyanins  source   Model   Effect   Reference 
 Anthocyanins  from 
tart cherries 
 Human colon cancer cells HT29 
and HCT-116 
 Reduced  proliferation   [ 
32 ] 
 Anthocyanins  extract 
from  Vaccinium 
uliginosum  
 Human colorectal cancer cells 
DLD-1 and COLO205 
 Growth  suppression 
in a dose-dependent 
manner 
 [ 
33 ] 
 Red grape pomace 
extract (oenocyanin) 
 Adenoma development in the 
ApcMin mouse 
 Suppress  adenoma 
cell proliferation and 
↓-regulation of 
expression of the PI3 
pathway component 
Akt, 
 [ 
34 ] 
  Vaccinium 
myrtillus , Vaccinium 
vitis - idaea  and  Rubus 
chamaemorus , rich in 
anthocyanins 
 Min/1  mice   Chemopreventive  as 
signiﬁ cant ↓ in the 
number of intestinal 
tumors 
 [ 
35 ] 
 Grape  juice 
comprising 15 
different anthocyanins 
 Rats mammary tumors   Incidence, 
multiplicity and ﬁ nal 
mass reduction 
 [ 
36 ] 
 Lyophilized  black 
raspberries 
 NMBA 
(Nitrosomethylbenzylamine)-
induced esophageal tumors 
 Prevention of tumor 
development 
 [ 
37 ] 
 Anthocyanins 
containing 
pomegranate extract 
 DMBA 
(7,12-dimethylbenzanthracene)-
induced skin tumors in CD-1 
mice. 
 Deferred the onset 
and reduced the 
incidence of 
 [ 
16 ] 
 Black  rice 
anthocyanins 
 Human colon cancer cells HT-29 
and HCT-116 
 Reduced  the 
expression of MMP-2 
and MMP-9 in cancer 
cells 
 [ 
38 ] 
 Georgian  Grown 
Blueberries 
 HT-29 colon cancer cells   Induce apoptosis 
decrease GST 
activities 
 [ 
39 ] 
9.3   Anthocyanins and Cancer

130

Fig. 9.1 Chemopreventive effect of anthocyanins with mechanism

Table 9.2 Mechanisms of anticancer activity of anthocyanins

Mechanism Reference

Antimutagenic activity [

44 ]

Inhibition of oxidative DNA damage [

45 ]

Induction of phase-II enzymes for detoxiﬁ cation and activation of carcinogen [

46 ]

Cellcycle arrest [

47 ]

Induction of apoptosis; inhibition of COX-2 enzymes [

28 ]

Antiangiogenesis e.g. inhibition of H

and tumor necrosis factor α (TNF-α)-

induced VEFG-expression, inhibition of VEGF and VEGF-receptor expression

[ 48 ]

Increased glutathione S transferase (GST) activity [

49 ]

9 Anthocyanins Effects on Carcinogenesis, Immune System and the Central Nervous…

131
9.4      Anthocyanins, Alzheimer Diseases and Brain Function 
 Anthocyanins prevent Alzheimer’s diseases and neuro-degeneration by increasing 
neuronal signaling in brain, mediating memory function and improving glucose 
utilization [ 59 ]. Anthocyanins have very beneﬁ cial effects on memory, cognition 
and motor function because of delaying the deterioration of neural function [ 59 , 
 60 ]. Goyarzu et al. (2004) found in Fisher rats, that blueberry intake suppressed an 
aging-associated increase in nuclear factor-kappa B levels (NF-κB) [ 61 ]  while 
upregulated TNF-α, IL-1β and NF-κB expression in the hippocampus [ 62 ]. Williams 
et al. [ 63 ] (2008) demonstrated that blueberry intake induced activation of cyclic 
AMP-response element-bonding protein and an increase in the level of brain- 
derived neurotrophic factor. Improvement of cerebrovascular blood ﬂ ow may also 
be implicated in the favorable effects of berries on brain function [ 64 ]. Ameloriative 
effects of anthocyanins in neurodegenerative processes of Alzheimer’s and 
Parkinson’s disease is chieﬂ y due to their ability to decrease inﬂ ammation and oxi-
dative stress in brain. It was observed that berries enhance release of dopamine in 
human brain leading to improved communication between brain cells. More con-
sumption of anthocyanin-rich diets like vegetables and fruits and can prevent or 
reverse age-related changes in brain and behaviour [ 65 ]. Berries not only stop but 
also reverse oxidative-stress-induced and age-related deterioration in neural func-
tion increasing dopamine release thus improving the ability of neurons to increase 
intracellular communication [ 66 ,   67 ]. Bilberry-fed animals exhibited improved 
memory and control of sensory inputs than control animals [ 66 ] while rats given 
lyophilized berries indicated improved working memory and increased short-term 
memory [ 68 ]. In a brain injury mice-model with middle cerebral artery occlusion 
showed neuroprotective effects when fed mulberry fruit extract containing cyanidin- 
3- glucoside  [ 69 ] (Table  9.3 ).
9.5         Miscellaneous  Activities 
 Anthocyanins can express their antimicrobial activity by causing morphological 
damages to bacterial cells or by destroying the structural integrity of the wall, 
membrane, intracellular matrix or by cell deformation [ 71 ,   72 ]. Four anthocya-
nins namely cyanidin chloride, delphinidin chloride, pelargonidin chloride and 
cyanidin- 3- glucoside possess strong anti-bacterial activity against  E. coli   (gram 
−ve) strain CM-871 (DNA repair-deﬁ cient strain) but did not inhibit normal 
 Escherichia coli  and beneﬁ cial probiotic bacteria (gram +ve). That shows the 
action association with DNA. In another antibacterial study, anthocyanin exhib-
ited more potential in decreasing the viability of  Salmonella enterica   var. 
 typhimurium  than other berries phenolic fractions [ 73 ]. The effect was due to 
anthocyanins induction of lipopolysaccharide molecules release from the outer 
membrane of the gram-negative bacteria. 
9.5  Miscellaneous Activities

132

Blueberry and billberry extracts showed growth-inhibitory effects on gram nega-

tive bacteria ( Escherchia coli , Enterococcus faecali , Pseudomonas aeuruginosa ,

Citrobacter freundi and Salmonella enterica ser. Typhimurium ) and gram positive

bacteria ( Staphylococusaureus , Listeria monocytogenes , Enterococcus faecalis and

Bacillus subtilis ). Of all bacteria, E. faecalis and C. fruendii were the most sensitive

and E. coli showed the highest resistance [ 74 ]. American cranberry and European

cranberry showed similar anti-bacterial effects [ 75 , 76 ]. Cranberries decreased

growth of E. coli below detectable limits when treated with anthocyanins at native

pH. Anthocyanins exhibited low antimicrobial activity at neutral pH, probably due

to their instability [ 71 ]. Cranberry extract showed no effect on yeast. Blackcurrent

concentrates arrested the growth of E. faecium and S. aureus strains while minor

effects were observed against E. coli [ 77 ]. In another comparative study, microbial

strains exhibited different susceptibility to berry extracts; the order of antimicrobial

Table 9.3 Mechanisms of action of anthocyanins [ 70 ]

Mechanism Anthocyanins

Oxidative stress Scavenges superoxide radicals

Scavenges hydroxyl radicals

Increases antioxidant enzyme activity

Lowers ROS

Decreases NO production

Cardiomyocytes and the endothelium Improves endothelial dysfunction

Mediates vasodilation

Anti-inﬂ ammatory effects Decreases P-selectin, MCP-1, TNF-α

and IL-10 expression

Decreases VEGF and ICAM-1 expression

on endothelial cells

Decreases VLA-4, CD40 and CD36

expression on monocytes

Metabolic effects Decreases LDL-cholesterol

Increases HDL-cholesterol

Inhibits LDL oxidation

Increases faecal expression of acidic

and neutral sterols

Alters sugar and lipid metabolism

Improves insulin resistance

Decreases NFκB levels

Decreases iNOS and COX

expression

Anti-ischemic and cardioprotective effects Decreases the magnitude of ischemia–

reperfusion injury

ROS reactive oxygen species, NO nitric oxide, MCP monocyte chemotactic protein, TNF tumour

necrosis factor, IL interleukin, VEGF vascular endothelial growth factor, ICAM intracellular adhe-

sion molecule, VLA very late antigen, CD cluster of differentiation, LDL low density lipoprotein,

HDL high density lipoprotein, NFκB nuclear factor, iNOS inducible nitric oxide synthase, COX

cyclooxygenase

9 Anthocyanins Effects on Carcinogenesis, Immune System and the Central Nervous…

133
effects observed was cloudberries > raspberries > strawberries [ 73 ]. In another inves-
tigation, berry extracts showed bactericidal effects, inhibiting growth of  Helicobacter 
pylori . All berries extracts showed >70 % inhibition at 1 % concentration with bill-
berry, blueberry and cranberry, extract showing >90 % inhibition [ 78 ]. 
 Anthocyanins of berry extracts (bilberry, blueberry, strawberry, cranberry, rasp-
berry) have shown protective effects against liver damage [ 79 ], ulcer [ 80 ]  and 
UV-radiation-induced damages especially cyaniding acts as skin-protective agent [ 81 ]. 
 Several enzymes in vitro may be inhibited by anthocyanins [ 82 ] like aldo- 
reductase in the lens of rats can be inhibited by both pelargonidin and delphinidin 
[ 83 ]. Anthocyanin-3-monoglycosides (namely delphinidin-, petunidin-and malvi-
din-) extracted from grapes inhibited hexokinase, malate dehydrogenase and glyc-
erol dehydrogenase while increased the activity of glutamic acid decarboxylase and 
 α -glucan-phosphorylase  [ 84 ]. 
 Tobacco bud worm larva growth was reported to be inhibited by cyaniding- 3- 
glucoside, which may be a useful biological controlling agent [ 85 ].  
9.6      Pharmaceutical  Products 
 Anthocyanin containing diets have been used since long as therapeutic remedy 
against various diseases in different parts of the world. For example crude extracts 
of  Vaccinium myrtillus  administered intramuscularly and intravenously or ingested 
orally can decrease the fragility and permeability of capillaries of blood system in 
humans. A health-promoting product, OptiBerry is made form the synergistic com-
bination of 6 selected extracts of wild blueberry and bilberry ( Vaccinium myrtillus  
and  Vaccinium corymbosum ), elderberries, strawberries, cranberries and raspber-
ries. This product exhibits very high anti-angiogenic, anticarcinogenic, antioxidant, 
antibacterial, and anti-atherosclerotic properties [ 86 ]. OptiBerry shows very low 
cytotoxicity and can be used as a dietary supplement and safe food. Anthocyanin 
powder of red cabbage, grape and tomatoes is used as natural food additives in soft 
drinks and confectionary items. Anthocyanins of red cabbage are stable over a 
broader pH range and therefore used as substitutes to synthetic blue colorings for 
foods with neutral pH [ 87 ]. Anthocyanins of  Hibiscus  are utilized in herbal formula-
tions as well as in soft drinks [ 88 ].  
9.7      Conclusions 
 With better understanding of cancer subtypes and their risk factors, human clinical 
trials should be designed to obtain maximum beneﬁ ts from dietary anthocyanins. 
Research in epigenetics and personalized medicines should be carried out to deci-
pher speciﬁ c anthocyanins compounds and determine dosage that may modulate 
risk of various age-related diseases in humans. Taking into account the efﬁ cacy and 
9.7  Conclusions

134

pharmacological effects of anthocyanins in humans, it is believed that combining

them with other antioxidants like ascorbic acid will be a useful approach for preven-

tion of various diseases. As they target different signaling pathways providing a

complementary and synergistic protective effects against chronic diseases.

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