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=__**The Basics of Anthocyanins**__= Marisa Egan

__**Introduction**__

Many people believe there are medicinal qualities in everything, especially plants. Some researchers spend large amounts of their time and energy conducting experiments to find these qualities. Other researchers spend their time and energy trying to find alternative, less toxic methods to color the world based on those qualities. Anthocyanins are pigmented molecules that possess both medicinal qualities and non-toxic pigment. only are they ample in red flowers and fruits, they are ample in antioxidant characteristics and activity, as well as colorful affect. [20] Antioxidants are classified as naturally occurring molecular species that prevent the degradation and oxidation of stored food products. Oxidation is the movement of electrons away from molecules and can cause damaging free radicals, molecules often with odd numbers of electrons, within the human body that can rip through tissues and cause an array of undesired results including damage to DNA and mitochondria. One free radical can create an entire chain of mass destruction by creating more free radicals. It is thought that free radicals can do significant damage to tissues within the human or animal body by leading to peroxidation, the tendency of hydroxyl groups to become dangerous peroxides. [1] Antioxidants come in many forms. One specific group of antioxidants is the flavonoids, a common group of polyphenols with a flavone backbone in the molecule. They are members of the polyphenol group of molecules, like many antioxidants, and have been found to exist in over 4000 varieties. They are classified by their molecular shape: a three-carbon chain with benzene rings at both ends. Figure 1 shows the basic Flavone backbone of a polyphenol. Various oxygen containing groups attached cyclically to the chain and rings account for the overabundance of variety of flavonoids and referred to as benzo-pyrones. [13]

Figure 1. A basic flavone molecule. [6]

Flavonoids are found abundantly in plants and are pigmented molecules. The flavonoid pigments are useful for attracting pollinating animals, deterring certain herbivores, and for their antioxidant qualities. A further subset of flavonoids is the anthocyanins. As flavonoids, the anthocyanins are found in plants and fruits and vegetables, are pigmented, and possess antioxidant qualities. Anthocyanins possess non-toxic and non-mutagenic attributes, based on studies, and they also have positive therapeutic qualities. [29] Anthocyanins have been drawing more and more attention for these reasons and may even be linked to controlling diseases from memory loss to cancer. One fruit that has drawn a lot of research is the blueberry for its plentiful anthocyanins and vibrant color.

__**Antioxidants and Free Radicals**__

Humans need oxygen to survive. This is a basic fact of life. The deprivation of oxygen to the human body quickly and irrevocably leads to death. It is obvious then that people cannot live without oxygen but, as with all things, it can have detrimental effects to the body. One of these side effects is the typically unwanted oxidation of various chemicals within various essential systems and pathways in the body. This side reaction has been linked to several diseases, the most prominent of which is cancer. Despite this risk, the intake of oxygen is unavoidable and, needless to say, necessary. This poses as sort of a dilemma as oxygen must be ever present in the human body, yet it is its presence that is a risk. The reason oxygen is harmful to the body is because of its high electronegativity. Essentially, oxygen is a prime mover of electrons in transport chains because of its high affinity for negative charges. This affinity can sometimes lead to oxygen having an unpaired electron. This situation causes what is called free radicals or a Reactive Oxygen Species (ROS). One unpaired electron is incredibly reactive and it is a form that is not necessarily preferred in the chemical or biological world. This singularity causes the instability of free radical oxygen compounds and these free radicals will immediately react with available molecules or metals immediately around it to escape having a singular, and often harmful, electron conformation. Since oxygen is both the cause of these dangerous free radicals and necessary in the basic functions of human existence, the removal of oxygen is impossible. The only recourse is to prevent or hinder this unwanted oxidation and formation of free radicals within the human body. This prevention can be accomplished with antioxidants. Antioxidants are found in various health foods or so-called ‘super foods’ like blueberries. “Henry Mattill tendered the role of antioxidants in nutritional systems in 1927 when he used the term ‘antioxidizer’ to describe the action of dietary factors that prevented oxidation. [24] Antioxidants come in many forms and there is no distinct molecular structure to encompass antioxidants generally, although many of them fall into the polyphenol category. They are organic molecules and found in many fruits and vegetables. Vitamins A, C, and E are often commonly known as antioxidants, but there are countless others. For instance, tomatoes possess carotenoids, lycopene, and phenolics, as well as Vitamins C and E. It’s no secret that fruits and vegetables are essential to the human diet. Antioxidants are a large contributor to that fact. [28] In uncomplicated terms, antioxidants inhibit or prevent oxidation of another molecule susceptible to those changes. Of course it is far more complicated than that. Their main discipline is to remove or divert free radicals from destructive paths within the bodies they inhabit. They challenge free radicals by taking away the electron’s need to bind and continue to create further radical chains by intercepting the electrons and the deactivating molecules that are willing to be reduced by radicals. [3] As the human issue becomes more and more centered on being healthy, vibrant, and vain, more and more studies have and are being done on antioxidant qualities in foods. There is not yet a set amount of antioxidants that a person should take in on a daily basis to prevent heart attacks, strokes, and wrinkles, but investigation into those values are becoming ever more prominent in science. Studies have been conducted on all types of fruits, vegetables, grains, lipids and oils, beans, greens, alcoholic beverages, and teas. Foods are continually being tested and found to have antioxidant qualities, some of them more than others. Even wine has been tested and found to have an exceptional amount of antioxidant qualities, particularly that of anthocyanins. [21]

__**Flavonoids**__

Flavonoids are the antioxidants that possess the poly phenol structure. Found in plants, fruits and vegetables, they are the most common group of polyphenolic compounds in the human diet. [26] They occur most often as primary glycosides and polymers in food and break down within the digestive tract. They inhibit lipid peroxidation and have the abilities to chelate hard metls and offset the processes of oxygen reactive species, meaning they have antioxidant powers. [12] Flavonoids are most abundant in citrus fruits and have been studied at length for their cancer fighting qualities. Flavonoids go one step further than antioxidants in that they can act more aggressively on a cellular level. They interact with genes as well as enzymes and contribute to different types of metabolic processes that may prohibit certain activities in cells infected with cancer. For this reason, much research has been focused on flavonoids in the search for cancer therapies and cures. [5] Other studies are indicative of a flavonoids ability to fend off cardiovascular disease, as well. Flavonoids are capable of acting in three ways to disrupt heart disease. They can act as anti-coagulants, preventing the blood from thickening and clotting too readily. They can improve circulation in this same way and by dilating arterial pathways to reduce blood pressure. Of course, their antioxidant qualities allow them to prevent oxidation of Low-Density Lipids (LDL’s), the “bad” kind of cholesterol that is characteristic of clinging to arterial walls. [2] These are just two of the many benefits of flavonoids. They also possess anti-inflammatory properties. [9] Flavonoids are incredibly versatile. This accounts for the exceptional amount of research and diverse nature of that research. As diseases become more and more resistant to modern medicine, there is an ever-growing necessity to investigate and create more natural drugs with less side effects, complications, and toxicity. Flavonoids fit all these categories. They are naturally occurring. They are abundant in fruits and vegetables. Flavonoids have shown to have little to no effect on normal cells and cellular activity, only abnormal cells and molecules. This makes them virtually harmless and non-toxic. [19] The best part about ingesting flavonoids is where they come from. It is easy to add flavonoids into a daily diet simply by ingesting vegetables, fruits (especially citrus fruits), teas, wine, and even beer.

__**Anthocyanins**__

The word flavonoid encompasses over 4000, [13] it is necessary to break the group apart based on further molecular structure in order to study the effects of each type of flavonoid structure. Anthocyanin is one of those groups of flavonoids. It has the same basic benzo-pyrone structure as a flavonoid, two aromatic rings bound together with a 3-carbon chain and oxygen, but the substituent groups attached to the rings consist of oxygen containing groups. In fact, the differences between anthocyanins and their simpler, more basic form anthocyanidin, is the substitution on the rings. The substituent groups consist of different oxygen-containing groups, often hydroxyl groups, and glucose groups, as seen in Figure 2.

Figure 2. The general structure of an. ‘R’ groups most often contain oxygen. [14]

Also, the molecule itself is positively charged and unstable, as shown below in Figure 3. This is because anthocyanin exists as the cation of a flavilium ion salt in protic environments and its stability is based on several factors, including pH, concentration, solvent, etc. The molecule here in its ionic state does not look much different from its flavone backbone, polyphenol parent. Anthocyanidins are considered the basic form of anthocyanins because they are less substituted and do not contain glucose groups. [4]

Figure 3. The charged flavilium ion. [7]



Figure 4. Ir spectrum and molecular structures of an anthocyanin in its pigments form at pH 1.00 and its colorless form at pH 4.5. [30]

Anthocyanins are pigmented based on the pH of their surroundings. They can range in color from blue and purple to reds and browns, even orange and yellow. The word “Flavonoid” comes from the Latin word “Flavus” meaning “yellow.” Brown is more common upon degradation of the anthocyanin. The color changes are reversible as pH changes. This means that the colors may degrade over time. This is an interesting concept in commercial food coloring and another area of anthocyanin research. Scientists are working toward stabilizing anthocyanins in different solutions to add them to food to maintain their intended colors. Figure 4 shows the molecular structure of a particular anthocyanin that ranges from orange to purple depending on the acidity of its surrounding solution, in this case pH 1.00. The adjacent molecule is the same anthocyanin in a solution of pH 4.5 where it appears colorless. The IR spectrum for both is also included. This is an example of the morphology of the molecule in solvents with different pH properties. [30] The flavilium ion is no longer an ion at pH 4.5. Because the reaction of anthocyanins is based on the form of anthocyanin substituent groups, chemical mechanisms must be tailored to the anthocyanin. However, anthocyanins are capable of scavenging free radicals through movement of hydrogen atoms around substituent groups and solution, metal chelation and binding of proteins. [17] Research has been done to prove the antioxidant qualities of anthocyanins. Wang, et al. used varied antioxidant assays that work as indicators and showed the antioxidant activity of different forms of anthocyanins compared to their big brother counterparts the flavonoids. Although the flavonoids had higher reactivity in antioxidant behavior, the anthocyanin groups showed a direct correlation between concentration and free radical scavenging activity. Mostly, anthocyanins with more than one hydroxyl group reacted more with radicals by donating hydrogen atoms more readily. However, the data showed there may be a correlation between the type of glycosylation on the carbon rings. The theory behind this may be that different sugars allow anthocyanin molecules to take more stable forms based on the attached sugars to their base structures. An anthocyanin’s antioxidant ability to scavenge radicals is only as good as the structure it becomes to support the electron it takes. [29] Blueberries and Anthocyanins The deep purple and blue colors of blueberries have drawn the attention of adults and children alike to the sweetness of the fruit. People have been adding blueberries to their diets simply because they taste good. What they may not realize is the amazing health effects they are ingesting. In depth studies have been done specifically on the anthocyanins in blueberry skin and flesh. Certain studies have even shown that the anthocyanins in blueberries, when extracted, can aid in weight loss and prevent obesity. [22] Other studies are in progress to find better ways of extracting anthocyanins so that they can be used as commercial food additives. These studies are based around the methods of extraction of the anthocyanins to preserve their pigments so that their vibrant colors will last longer once they are added to foods. Different extraction techniques are in question to prolong color and defend against degradation of anthocyanin pigments in environments that may not be suited to the acidity levels necessary to maintain anthocyanins. As more and more studies reveal the extraordinary benefits of anthocyanins, there is a greater need developing to extract them from fruits faster and better. [25] The increase in production of blueberries as people learn the amazing attributes of this tiny, blue fruit is another aspect of the blueberry that is under review. Increased demand means increased farming and faster growing of blueberries. The genetic modification of the blueberry is a case to alter the fruit to encompass perhaps more of its finest qualities. [23] This may be due to the most astounding tests done on blueberry anthocyanins: the tests that are finding the cancer fighting qualities of the fruit. The cures and treatments being used for different types of cancer are in themselves lethal chemicals in near toxic doses. The plans of attack for cancer cells are often based around stimulating apoptosis of cancer cells, preventing proliferation, shrinking tumors, and harming the cancer DNA so that it cannot keep growing at its tremendous rate. Cutting off the blood line, the food supply seemed to be the best course of action. [10] But other plans are in the works. A study of the blueberry anthocyanins paired up against breast cancer showed significant results. The blueberry anthocyanins showed that, combined with an anthocyanin-pyruvic acid mix, it had anticancer properties. The anthocyanins inhibited cell proliferation of cancer cells. They also acted as antiinvasive factors for cells, preventing cancer from invading cells, and also as chemoinhibitors in two separate lines of breast cancer. Chemotaxis is a cell’s movement toward a nutrient source or other influence. The anthocyanins did not show any evidence of this action so it could not be influenced by the cancer cells. [8] Testing often targets certain blueberry types. There are many different types of blueberries. They are cultivated on almost every continent. In Europe, they are called Bilberries. One study conducted Chinese blueberries and their effects on colon and colorectal cancer. These studies also showed repression of cell proliferation and the possibility of certain blueberry anthocyanins to be feasible cancer suppressors. [31] Another study done on Highbush blueberries showed significant increases in apoptosis of cancer cells using two different methods: DNA fragmentation and caspase-3 activity. Some anthocyanins most abundant in blueberries are delphinidin, cyanidin, and malvidin, and they are often isolated for testing to see the effects of each on certain aspects being studied. [27] It’s possible that some day there will be a recommended daily intake of blueberries or anthocyanins.

__**Conclusion**__

Anthocyanins are stable flavilium ions when in acidic solutions. They need highly protic solutions to be stable that way, but they can survive in solutions that are not acidic by changing their structures and losing hydrogen. They are oxygen-containing aromatic species with two benzylic rings and glucose substituents. Their molecular structures give them pigments that transforms when hydrogen bonding, or the lack thereof, causes them to morph into non-ionic forms. The anthocyanins are known for their pigments and are under research to make safer colorants for commercial uses. Anthocyanins are the glucose-containing version of anthocyanidins and a subset of flavonoids. Flavonoids are a subset of antioxidants and anthocyanins possess the qualities of both flavonoids and antioxidants. They are interesting for the antioxidant qualities they possess by scavenging and removing free radicals. The antioxidant activities of anthocyanins are directly and proportionally based on two things: the number and type of hydroxyl substituent groups and concentration. There is also possibility that the types of sugars attached are in proportion to the amount of proton donating an anthocyanin can do, as the sugars attribute to the stability of the molecule.

Anthocyanins have amazing capabilities of fighting illness. They have been involved in cancer studies and blueberries show magnificent capabilities of playing a role in cancer treatments and possibly a path to a cure of the disease. They have taken part in studies and shown to repel obesity in lab mice. There are numerous types of blueberries, each possessing antioxidant characteristics. Not only are they delicious and sweet, tiny, blue pearls, the benefits they bestow on their consumers make them an integral part of the human diet, as well as the human condition.