The Many Shades of Flavonoids

Flavonoids do not lack for scientific attention. Research continues to reveal numerous potent health mechanisms of these phytochemicals most known for their antioxidant properties. Amid this growing foundation in defense against diseases and other maladies, flavonoids are drawing more interest from manufacturers and consumers. And with so many eyes and hands on flavonoid ingredients, various production and formulation issues have surfaced, confronting suppliers and manufacturers with challenges in bringing quality flavonoid products to market that best represent the science behind the benefits.

Within the complex chemical matrices of plants is a large class of polyphenols called flavonoids, which serve various functions related to the plants’ survival from stress, disease and ultraviolet light. This protection from harm extends from plants to humans, as flavonoids have been found effective against diseases marked by oxidative damage, such as heart disease, cancer, diabetes and neurological diseases, as well as other health issues including diarrhea and weight loss. Antioxidant properties may be the most known of the researched flavonoid benefits, but additional investigation has considered other mechanisms of action of various forms of flavonoid extracts.

Most flavonoids in plants are bound to sugar molecules, forming glycosides; flavonoids not bound to sugar are called aglycones. Among the flavonoid subgroups are anthocyanins, flavanols, flavonols, flavanones, flavones and isoflavones.

Anthocyanin-rich blueberries have demonstrated protection against age-related neurodegenerative processes in the brain,¹ including damage to neurons.² Similar protection is provided by strawberry flavonoids,³ which also protect the overall central nervous system, in addition to chemoprotection of the liver and esophagus from cancer development.⁴ Another esophageal defender is the blackberry, which can inhibit tumor formation in the oral cavity,⁵ while also helping to curb colon cancer development.⁶ Anthocyanins in bilberries have shown the ability to protect the eyes from development of age-related macular degeneration (AMD). And, cranberries have been found to counter numerous health problems, including liver cancer, kidney stones, gum disease and urinary tract infection.^7,8,9,10

Combining several berries—wild blueberry, strawberry, cranberry, wild bilberry, elderberry and raspberry—is OptiBerry® (from InterHealth USA), a multi-berry extract containing standardized levels of anthocyanins, including malvidin, cyanidin, delphinidin and petunidin, in biologically active ratios. In early 2006, researchers reported OptiBerry provided total body antioxidant protection from oxidative stress in an animal study, with particular benefits to the liver and lungs.¹¹ Earlier research on this anthocyanin-rich extract revealed chemopreventive and hypocholesterolemic effects,^12,13 in addition to antiangiogenic properties.¹⁴ As might be expected, this combination of berries demonstrated a greater effect than did the individual berries on Heliobacter pylori infection,¹⁵ which has been increasingly linked to development of gastrointestinal ulcers.

Red wine and its grape predecessors certainly contain an array of flavonoids, including anthocyanins and proanthocyanidins, and have shown promise in limiting heart disease, cancer and inflammation; but, most of these health benefits are primarily attributed to the phytochemical resveratrol, which is not in the class of flavonoids, but is categorized with the stilbenes.However, grape seed extract (GSE) has a long list of benefits not necessarily credited to resveratrol. In various studies, GSE has discouraged atherosclerosis;¹⁶ enhanced the antioxidant status and decreased the incidence of free radical-induced lipid peroxidation in the central nervous system of aged rats;¹⁷ and demonstrated the potential to protect against future age-related dementia by influencing specific proteins in healthy brains.¹⁸

In fact, a wide spectrum grape extract comprising grape skins and seeds (as BioVin®, from Cyvex Nutrition) was found to deliver as much proanthocyanin, anthocyanin and trans-resveratrol content as one glass of red wine.¹⁹ Also, a IH636 grape seed proanthocyanidin extract (as Activin®, from San Joaquin Valley Concentrates) has demonstrated inhibition of cell adhesion molecules in inflammation; researchers concluded this GSE may help significantly in the treatment of skin rashes, dermatitis, psoriasis, acne, arthritis, cardiovascular disease and other inflammatory health concerns.²⁰ In other research, Activin GSE reduced systolic blood pressure (BP) in normal and hypertensive rats.²¹ Additionally, three proprietary studies funded by Polyphenolics Inc. (a division of Constellation Wines) in 2005 showed anthocyanin- and proanthocyanidins-rich GSE (as MegaNatural® Gold) reduced arterial cholesterol accumulation caused by a high-fat diet, and inhibited coconut oil saturated fatty acids from causing aortic endothelial dysfunction.

As evidenced, the flavonoid content of grapes, cranberries, bilberries and other fruits and plants are complex, as they not only contain anthocyanins, but also contain proanthocyanidins, which belong to another flavonoid group, the flavanols. Flavanols (also called flavan-3-ols) are the main source of aglycones, including catechins and proanthocyanins, and are commonly found in tea (primarily green and white), chocolate, berries and apples. Within the flavanols there are two types: monomers include catechin, epicatechin, epigallocatechin (EGC) and epigallocatechin gallate; while dimers and polymers include theaflavins, thearubigins and proanthocyanidins.

According to recent research, proanthocyanidin-rich fractions of cranberry extract inhibit growth of lung tumors, colon and leukemia cells in vitro.²² But flavanol supremacy has been given mostly to tea catechins and theaflavins, and cocoa epicatechin.

Epigallocatechin gallate (EGCG) and its shorter form EGC, both found in green and black tea, have been shown to reduce the risk of certain cancers, including prostate cancer,^23,24 owing to their abilities to scavenge free radicals before cancer-causing damage can occur,²⁵ reduce the size of existing tumors,²⁶ and inhibit tumor growth.²⁷ Tea catechins also help manage heart disease, reducing the fatality of heart attacks and lowering blood pressure in hypertension.^28,29 More recently, supplementation with EGCG (as Teavigo™, from DSM Nutritional Products Inc.) curbed body fat accumulation in obese mice, apparently by promoting fat oxidation, but possibly by reducing diet digestibility.³⁰

As have other flavonoids, tea catechins have addressed neurological health, proving useful in both Parkinson’s and Alzheimer’s diseases, and cerebral injuries.³¹ Other injurious and debilitating maladies impacted by these flavanols include kidney and liver ailments,^32,33 diabetes³⁴ and prostatitis.³⁵

Apparently, catechins also rule cocoa flavonoids, at least in the area of vascular health. In early 2006, scientists from the University of California, Davis, reported epicatechin and its metabolite epicatechin- 7-O-glucuronide are the keys to cocoa’s nitric oxide-related vasodilation properties.³⁶ This may explain, in part, cocoa’s beneficial effects on low-density lipoprotein (LDL) oxidation,³⁷ thrombosis formation,³⁸ high blood pressure³⁹ and platelet activation.⁴⁰

Cocoa may have benefits beyond vascular health, as recent research has discovered cocoa flavanols may inhibit diarrhea by inhibiting intestinal secretion of the electrolyte chloride.⁴¹ The researchers reported the addition of cocoa extract or cocoa flavanols to the mucosal side of colonic tissues caused partial inhibition of chloride ion transport; the flavonols quercetin and luteolin were similarly efficacious.

Chocolate and tea are comfort foods rich in flavanols, but supplemental sources also include herbs, such as French Maritime pine bark extract.⁴² This herbal extract contains proanthocyanidins, catechins and other flavonoids, and has been shown beneficial in numerous areas of health. As Pycnogenol® from Natural Health Science, this extract has achieved typical flavonoid results against heart disease and cancer risk;^43,44 but, it has further proven helpful against blood sugar factors of diabetes,⁴⁵ development of Alzheimer’s disease,⁴⁶ autoimmunity associated with lupus,⁴⁷ retinal deterioration in neuropathy,⁴⁸ venous thrombosis,⁴⁹ menstrual pain,⁵⁰ memory failure,⁵¹ male infertility,⁵² childhood asthma⁵³ and attention deficit disorder (ADD).⁵⁴

Flavonoids are often called bioflavonoids in the media and in product marketing, especially in the case of citrus fruits. Flavanones common to citrus fruits and juices including lemons, oranges, grapefruits and tangerines are among the widely researched citrus bioflavonoids, which include the flavonones hesperetin, naringenin, eriodictyol, tangeretin, as well as the flavones tangeretin, apigenin, luteolin, which are also abundant in foods such as parsley, thyme, celery and hot peppers.

Heart health is certainly a target of citrus bioflavonoids. Individual flavonoids have been shown to decrease aortic fat,⁵⁵ lower blood pressure and heart rate,⁵⁶ decrease plasma LDL cholesterol levels,⁵⁷ inhibit macrophage cell formation⁵⁸ and help treat hypertriglyceridemia.⁵⁹ As a group, citrus bioflavonoids lower total and LDL cholesterol and tricylglycerols.⁶⁰

In other areas of health, a recent trial showed hesperidin, an antioxidant flavonoid found in sweet orange and lemon, protected cells against damage induced by toxins paraquat and hydrogen peroxide.⁶¹ And, naringenin was found to help prevent generation of amyloid betainduced reactive oxygen species (ROS) in neurons and prevent neurodegeneration in mice in response to scopolamine injection.⁶² Additional recent studies have reported bioflavonoids from citrus oil limonene inhibited ozone, an oxidant that can cause pulmonary inflammation when inhaled, especially in asthmatics;⁶³ limonene also prevented bronchial obstruction via anti-inflammatory effects indicated by peribronchiolar and perivascular inflammatory infiltrates.⁶⁴

Polymethoxylated flavones (PMFs) are found in the peels of citrus fruits and are highly methoxylated, which can result in increased bioactivity, according to SourceOne Global, supplier of Sytrinol™, a combination of PMFs and tocotrienol developed by KGK Synergize. According to SourceOne, Sytrinol, which contains tangeretin and nobiletin, has been proven useful in heart disease via three mechanisms. It reduces apo-B levels, and inhibits liver enzymes that produce triglycerides and cholesterol. A recently completed, threephase clinical trial showed Sytrinol could significantly improve total cholesterol, LDL cholesterol and triglycerides by as much as 30 percent, 27 percent and 33 percent, respectively, compared to in hypercholesterolemic patients.

PMFs may also act on inflammation parameters of immune function. Citri-Z™ a polymethoxylated flavone (PMF) extract from Next Pharmaceuticals, was shown to modulate TNF-alpha and natural killer cell activity.⁶⁵

Citrus bioflavonoids also include the flavonol aglycone quercetin and its glycoside rutin.

Quercetin is the most researched of flavonols, most recently demonstrating an ability to inhibit oxidative stress and tissue damage associated with diabetes,⁶⁶ and showing promise in helping prevent bone loss.⁶⁷ In research from Columbia University, New York, lyophilized grape powder (LGP), which contained quercetin, as well as myricetin, kaempferol, flavans, anthocyanins and resveratrol, favorably altered lipoprotein metabolism (LDL, troglycerides and apoB/E), oxidative stress, and inflammatory markers in both pre- and post-menopausal women.⁶⁸ Similarly, vitamin E in conjunction with flavonoid supplementation, including quercetin, inhibited COX-2 activity, posttranscriptionally.⁶⁹ Lasoperin™ from Unigen Pharmaceuticals, a novel composition of free- B-ring flavonoids and flavans derived from Scutellaria and Acacia plants, recently exhibited an ability to suppress inflammation in the brain—a cause of cognitive dysfunction associated with aging.⁷⁰

Often considered separate from the main class of flavonoids, isoflavones found in soy and red clover, including daidzein, genistein and glycitein, are noted for their phytoestrogenic activity. The robust research profile on isoflavones includes documented benefits to cholesterol levels and endothelial function in heart disease;^71,72,73 risk of hormone-related cancers including prostate cancer;74,75,76 inhibition of bone loss and osteoporosis;^77,78 attenuation of various pre- and post-menopausal symptoms;^79,80,81 and various parameters of gut health.^82,83

An emerging popular supplement of late is pomegranate, which contains an abundance of phytonutrients possibly linked to its many scientifically established health benefits, including reduced cholesterol,⁸⁴ improved blood flow,⁸⁵ enhanced immunity⁸⁶ and prostate cancer inhibition.⁸⁷ Research has pointed to its flavonoids, ellagic acid, tannins and punicalagin, with researchers in one study suggesting a synergistic effect among the fruit’s multiple phenolic compounds.⁸⁸ This highlights a particular issue in flavonoid ingredients, especially extracts: to what phenol is it best to standardize?

As far as pomegranate goes, some suppliers are standardizing to ellagic acid while others are focusing on specific compounds. Cyvex Nutrition’s PomActiv™ is standardized to 70 percent ellagic acid; Geni Herbs standardizes its PomElla™ to punicalagin; P.L. Thomas’ newly launched POM40p is standardized to 40 percent punicosides; and Amoretti’s newest line of pomegranate ingredients feature different extracts standardized to ellagic acid, punicalagin and general polyphenolic content.

The antioxidant mechanisms of action are certainly the most well known, but some scientists have suggested other mechanisms of action, including cell signaling pathways, protein-dependent communications that regulate the growth, proliferation and apoptosis (death) of cells. As the theory goes, flavonoid metabolites selectively inhibit certain protein kinases that could disrupt normal cell signaling and cause disease. Flavonoids also may modify growth factor signaling, as in vitro studies have indicated the health benefits of flavonoids may be related to their modulation of these cell signal pathways. This may explain low plasma level of flavonoids, as the intracellular concentrations needed to impact cell signaling are much lower than those required for major antioxidant activity.

Cell signal pathway regulation by flavonoids has been linked in research to some of the health benefits regularly associated with flavonoid supplementation. By this alternate mechanism, flavonoids address heart health by decreasing vascular cell adhesion molecule expression,⁸⁹ increasing endothelial nitric oxide synthase (eNOS) activity,⁹⁰ decreasing platelet aggregation⁹¹ and reducing inflammation.⁹² And flavonoids can inhibit cancer development by stimulating phase II detoxification enzyme activity,⁹³ reducing tumor invasion and angiogenesis,⁹⁴ preserving normal cell regulation,⁹⁵ inhibiting proliferation,⁹⁶ inducing apoptosis⁹⁷ and decreasing inflammation.⁹⁸

Food sources may offer admirable flavonoid content, but absorption of these phytonutrients from food consumption is not so clear. Flavonoids are often absorbed quickly and eliminated just as fast, and blood levels of certain flavonoids—anthocyanins, flavanols and flavonols—following consumption have been scientifically determined to be low. Glycosides and aglycones are first absorbed in the small intestine, where they are quickly metabolized into various metabolites; flavonoids that reach the colon are metabolized by bacterial enzymes for absorption. Moreover, metabolites generated by absorbed flavonoids are not always as active as the parent compound, according to scientists at the Linus Pauling Institute at Oregon State University, Corvallis. A 2005 study discovered rapid degradation of flavonoids by gut microflora may result in lower overall bioavailability than slowly degraded flavonoids, because quickly degraded flavonoids are less likely to be absorbed intact.⁹⁹

As potent antioxidants, flavonoids are highly reactive with oxygen, which can degrade the polyphenolic content. Further degradation of flavonoids can result from exposure to high temperatures and humidity, as well as from processing methods, including wounding, freezing, freeze-drying and canning. Exposure to extreme moisture or water can also diminish flavonoid potency, but most flavonoids in dry form can be stored in a moisture-free environment for up to a year, according to InterHealth, which noted increased flavonoid content in glycone form, as is predominant in OptiBerry, can increase the lifespan, bioavailability and efficacy of the extract.

Antioxidants have been used for years as natural preservatives in packaged food products, which are highly susceptible to oxygen.

San Joaquin Valley Concentrates said its Activin GSE is a good candidate for use in food products. It has been affirmed GRAS (generally recognized as safe), which is required for inclusion of flavonoid ingredients in food products. Also, it is highly soluble, which helps avoid a haze in beverages, and is heat resistant, which is ideal for cereal or nutraceutical bars that undergo higher cooking temperatures.

Citrus bioflavonoids are also water soluble, making them more easily absorbed in the body, according to Matt Phillips, director of sales and marketing for BI Nutraceuticals, which produces rutin, quercetin and various bioflavonoids from lemons, oranges and grapefruits.The company’s bioflavonoid ingredients primarily target the supplement market. “Citrus bioflavonoids are often used in antioxidant formulas to improve the absorption of vitamin C,” Phillips said.

Flavonoids can enhance end products in even more visible ways. As the rich pigments in vibrantly colored fruits and vegetables, anthocyanins make good functional food colorings. The color palate achievable by these flavonoids ranges from deep purples to crisp reds and cool blues. However, anthocyanin-rich berry and grape ingredients can have a bitter, astringent taste at certain volumes, presenting a challenge to make the end products taste good. For GSEs, it depends on how much is used, explained Phil Castro, a trained chemist and extract manager for San Joaquin Valley Concentrates. “If you use 100 mg/100mL water, BSE can add a straw appearance—a golden amber look—and impart a tea flavor.”

Further, fiber content in some whole berry products or powders is quite high, which can be an issue for formulators, who also face the possibility these extracts might not dissolve as desired. Thus, formulators need to investigate the solubility of each berry extract considered for a product formula.

Another formulation consideration is the manner by which a flavonoid ingredient was extracted. Those extracted by common alcohol and water processes can undergo a significant loss of antioxidant power from the original fruit. Castro suggested a 100-percent water extraction for GSEs. “Many companies will use a solvent extraction to increase yields,” he said. “They can get up to 55-percent yields, but the solvents—such as hexane and benzene— remain and, at certain levels, can be carcinogenic.” Thus, it becomes crucial for formulators to seek out suppliers that use extraction processes designed to maximize safety as well as flavonoid content retention and antioxidant capacity, which is primarily reported as an ORAC (oxygen radical absorbing capacity) value.

Determining the content of an individual flavonoid extract is difficult, as considerations include quality control methods, extract standardization and tests on each specific extract’s flavonoid content and antioxidant capacity.

“There used to be different testing methods for GSE content but I think most are settling on the Singleton Folin-Ciocalteu method of analyzing for phenolics,” Castro said. The Folin- Ciocalteau method, which measures phenolic concentration, has been used in the wine industry for many years, but does not differentiate between phenol types. Other tests used include HPLC (High Pressure Liquid Chromatography), which measures the percentage available of monomers, oligomers and polymers; and GPC (Gel Permeation Chromatography), which measures total concentration of phenols.

But before ingredient suppliers extract and test their flavonoid rich plants, fruits and vegetables, aspects of sourcing and production can impact the flavonoid content. Because these compounds are produced by plants in defense of stress and other threats, there is no standard amount found in each fruit, vegetable, flower, root or piece of bark. The U.S. Department of Agriculture (USDA) has created a list of the flavonoid content of foods, but cautioned flavonoid content can vary widely due to multiple factors including cultivar, growing location, agricultural practices, processing and storage conditions, and preparation methods. Phillips noted two important questions to ask when sourcing fruit for flavonoid extracts are: How are they growing the fruit; are they using pesticides? And, what is the polyphenolic activity of the fruit? “Addressing flavonoid sourcing from this basis helps generate standardization of bioflavonoids in the extract,” he said.

To even better counter this variability, or at least control it first-hand, many companies source their raw materials from corporate sites or contracted farmers to help ensure consistent polyphenolic content. This is especially true for GSE companies, which are often rooted in wine-making origins with their own grape fields. “We know what we’re getting and when we’re getting it,” said Castro, whose company is owned by A.J. Gallo Wineries, which recognized a synergy between the byproducts of wine production and the raw materials needed for GSE production—seeds and skins. “When using outside sources, it is common for companies to get seeds that have been drying for two years, which decreases flavonoid content. Those ORAC values are minimized, because the OPC content is then larger than the monomeric content, which is what gives you the higher ORAC values.”

The science involved in flavonoids is complex, relative to the nature of their plant origins and creation. This complexity extends to the growing body of research on the health benefits of flavonoids, as positive results on flavonoid-rich fruit or juice does not necessarily translate to the corresponding dietary supplement form. Also, research done on a specific extract cannot be simply applied to similar ingredients, because each unique formulation and manufacturing processes can cause significant variations in potency and activity.

While the specific flavonoid powerhouses in food sources such green and black tea are relatively well defined, the compounds responsible for the benefits of other sources, such as pomegranates, are no fully defined. This makes it a challenge to supply or utilize an extract that will deliver the antioxidant capacity and health benefits observed in the flood of studies on these foods and extracts.

Scientists are still finding flavonoids, although more than 4,000 such phytochemicals have been discovered. In fact, recent healthy findings on common favorite foods and beverages, such as juices, teas, chocolate and even beer, are often linked to flavonoid content. The science of flavonoids’ mechanisms of action in human health and the related processing and formulation issues is the key to the future of this burgeoning ingredient market.