Flavonoids as Nutraceuticals
Muhammad Kaleem; Asif Ahmad PMAS-Arid Agriculture University, Rawalpindi, Pakistan
Abstract
Excessive use of synthetic chemicals for the treatment of different diseases is associated with several adverse health effects. Because of this, scientists are searching for natural sources of therapeutic agents and other substances which protect the body from various ailments and disease. A diverse group of bioactive compounds from plants can be extracted and may be used for health purposes. Use of bioactive compounds is an appropriate alternative to synthetic medicines. Flavonoids are polyphenolic secondary metabolite phytochemicals which can be effectively used as nutraceutical agents. In order to shed light on the importance of flavonoids, this chapter will focus on the potential use of flavonoids against different diseases such as diabetes, cancer, hepatitis, osteoporosis, as well as other microbial, and viral infections. The possible mechanism of action of flavonoids will also be discussed. This chapter will also focus on the sources of these flavonoids, their extraction from these sources, and their usage in different food products as nutraceutical ingredients. The pharmacological importance of flavonoids which may be supplemented in staple food to develop different nutraceutical products will also be reviewed.
Keywords
Flavonoids; Cancer; Diabetes; Nutraceutical; Hepatoprotective; Osteoporosis
1 Introduction
Flavonoids are polyphenolic secondary metabolites, which are not required for the survival of a plant, but do impart color and aroma to flowers, leaves, and fruits. They may also help the plant withstand adverse conditions such as insect attacks or bacterial and viral infestation. Examining the protective role of flavonoids, many studies have been carried out to investigate their antibacterial, antiviral, antioxidant, and antifungal properties. Additionally, much research has also been conducted to evaluate their pharmacological properties against different maladies such as diabetes, cardiovascular diseases, cancer, neurodegenerative, osteoporosis (especially post-menopausal), as well as several bacterial, and viral diseases (Ahmad et al., 2015).
The basic skeletal unit of a flavonoid consists of two aromatic rings (A & B) which are linked to each other via heterocyclic ring C. based on the functional group attached, which is the type of derivatization and position of attachment of B ring to the C ring. Flavonoids are classified into various groups which include flavonols, flavan-3-ols, flavones, flavanones, isoflavones, and anthocyanins. Individual compounds present within the subclasses are further characterized by the pattern of hydroxylation and conjugation (Babu et al., 2013). One of the most prevalent groups of plant metabolites is flavonoids. A large number of flavonoid compounds have already been explored; research continues to expand this existing knowledge base. At present, about 9000 flavonoid compounds are known (Ahmad et al., 2015).
Flavonoids are present in almost all parts of the plant. Sources of flavonoids in the human diet include tea, wine, blackberries, blueberries, propolis, honey, red beans, and nuts. Black rice is an excellent source of anthocyanins, having 116.58 mg/g (Pedro et al., 2016; Tsuchiya, 2010; Ahmad et al., 2015). Vegetables, fruits, berries, and red wine are good sources of anthocyanidins, while parsley, celery, and herbs are excellent sources of flavones (Crozier et al., 2009). Genistein and daidzein are major isoflavones which are found in Genista tinctoria, a Chinese medicinal herb, and other leguminous plants (Veitch, 2013). Excluding fungi and algae, flavonols such as quercetin, kaempferol, isorhamnetin, and fisetin can be commonly found throughout the plant kingdom (Babu et al., 2013). Hesperidin and naringin are examples of flavanones mostly present in the citrus fruits. Naringin gives bitter taste to grapefruit (Jung et al., 2006). Sources of anthocyanidins and proanthocyanidins include cabbage, currants, barley, banana, berries (strawberries, raspberries, cranberries, blueberries, black berries), chocolate, tea (black and green), wine, beer, spices, onions, plums, peas, grapes, peaches, nuts (walnuts, peanuts, cashews, pistachio, almonds), mangos, and lentils (Kruger et al., 2014).
Flavonoids are the secondary metabolites of different fruits and vegetables having a critical role in performing numerous metabolic activities. Flavonoids have gained lot of attention because of their effects to beneficial health. The lowest levels of coronary heart diseases have been reported in France in spite of pervasive spoking and high fat intake. This is due to frequent consumption of red wine. Flavonoids have recently been identified as the major constituent preventing cancer in daily diet. Low risk of coronary heart disease and premature aging prevention is also attributed to daily consumption of flavonoid rich food. In addition, flavonoids reveal a lot of biological activities, including antihepatotoxic, anti-inflammatory, antiviral, antibacterial, antiosteoporotic, and antiulcer actions.
At present, health consciousness has increased, driving the attention of researchers and industrialists towards natural and nutraceutical foods. This has encouraged fortification of processed foods with flavonoids to boost the immune system. Following high population growth, the food processing industry is expanding at high rate, which makes safe and healthy products necessary. With the expansion of industries, industrial waste and byproducts of industries are increasing. These byproducts include such things as seeds and peels. These industrial byproducts happen to be a rich source of certain valuable constituents, including flavonoids, and minerals. Such valuable constituents of industrial byproducts can be extracted and incorporated into other products to increase their nutraceutical potential. Purification and concentration of these flavonoids is valuable to pharmaceutical industries as well. There is continued exploration of their potential and utility in concentrated or purified forms as therapeutic agents (Stalikas, 2007; Routray and Orsat, 2012).
2 Extraction of Flavonoids
Flavonoids are collected from dry, frozen, or lyophilized material due to the fact that enzymes act on and destroy flavonoids when the plant material is fresh or not dried. The plant material is generally dried at low temperatures to minimize the loss of flavonoids due to heat exposure. After drying, the plant material is usually ground to fine powder. Grinding increases the surface area of plant material and helps in the efficient extraction of flavonoids. After grinding, the next step is extraction with a solvent. This is a critical step because there is a huge diversity of flavonoid compounds that vary in structure and type of derivatization. Flavonoids can be broadly categorized into two groups for the choice of solvent for extraction. One group of flavonoids is less polar and includes flavanones, flavonols, methylated flavones, and isoflavones. The other groups of flavonoids are flavonoid glycosides and polar aglycones. Nonpolar or less polar flavonoids can be extracted with ethyl acetate, chloroform, diethyl ether, and dichloromethane, while polar flavonoids can be extracted with water alcohol mixture or alcohol alone. Factors affecting extraction of flavonoids from plant material include time of extraction, extraction temperature, pressure, and solvent (Andersen and Markham, 2005).
There are two types of extraction techniques: (1) Conventional extraction and (2) Modern extraction techniques.
2.1 Conventional Extraction Techniques
Conventional extraction techniques include maceration, hydrodistillation, and Soxhlet extraction. Maceration technique has been used for a long time in extraction of essential oils for medicinal purposes. The process of maceration involves grinding of material, addition of an appropriate amount of a solvent, followed by straining and pressing to remove occluded residue from the solution. The efficiency of this process was improved by shaking and by removing the concentrated solution. In the last step, filtration is carried out to remove impurities (Azmir et al., 2013). Hydrodistillation is another conventional technique which does not use organic solvents and uses water or steam for the extraction of flavonoids and other bioactive compounds (Silva et al., 2005). Soxhlet extraction was designed for the separation of lipids, but adapted for the extraction of other constituents from the food material such as flavonoids (Azmir et al., 2013).
2.2 Modern Extraction Techniques
There are several problems associated with conventional extraction techniques which include longer time of extraction, high cost of more pure solvent, decomposition of heat sensitive compounds, and less extraction selectivity. To overcome these problems, several new techniques have been developed which include microwave assisted extraction, ultrasound assisted extraction, pressurized liquid extraction, and supercritical fluid extraction.
In microwave assisted extraction, microwave energy is used for the extraction of important constituents of plant material into liquid solvent. The frequency of microwaves ranges between 300 MHz and 300GHz. In this type of extraction, electromagnetic energy is converted into heat energy. Microwave assisted extraction takes place in three steps: (1) solute separation from the sample matrix under high pressure and temperature, (2) solvent diffusion against sample matrix, and (3) solute is released to solvent from the matrix of the sample. Extraction has been shown to yield an increase of four times the polyphenol in microwave assisted extraction as compared to conventional techniques. Besides that, it reduces the time of extraction and reduces the volume of solvent used for extraction (Routray and Orsat, 2012). Microwave assisted extraction, using comparable concentration of solvent for extraction and temperature, requires much less extraction time as compared to solvent extraction and ultrasound assisted extraction (Li et al., 2011).
Comparison between different extraction techniques is described in Fig. 1., showing the time required for extraction, extraction temperature, and the concentration of solvent used for extraction. Factors affecting the microwave extraction include solvent system, time of microwave application, temperature of extraction, and surface contact area. The choice of solvent is among the most important factors, especially in the case of microwave extraction. One of the important factors for the solvent to be considered is the dielectric constant of solvent, which affects its rate of heating and its extraction capability. The properties of solvent can be improved by combining different solvents. For thermo-labile compound extraction, the combination of solvents to be made must have a lower dielectric constant; this ensures the maintenance of lower temperature, otherwise the desirable compounds may be lost (Casazza et al., 2010). Another factor to be considered in the choice of solvent is solubility of the desired compound in the solvent. Depending upon the polarity of flavonoid to be extracted, polar or nonpolar solvent may be chosen. Polar solvents are generally used for flavonoid glycosides, while for flavonoid aglycones, nonpolar solvents are used. It is mostly reported, with increase in time of extraction, extraction yield increases, but this only remains true for a certain period, after which extraction yield tends to decrease. In many studies in which extraction condition was optimized by response surface techniques, it was concluded the effect of extraction time on extraction yield is quadratic rather than linear. This is described by degradation of flavonoid compounds by prolonged heating (Xiao et al., 2008; Ghafoor et al., 2009). As temperature increases, the solubility of compounds in solvent increases, so high temperature extraction can be advantageous over low temperature extraction. High temperature also exerts pressure on the cellular walls, thus helping in the release of desirable compounds. In addition to this, the viscosity of solvent used also decreases, thus improving the solubility and mobility. However, it has been reported extraction efficiency increases with increase in temperature, but this rule only applies up to an optimum level, beyond which it starts to decrease. Effect of temperature is different for extraction of different compounds; it depends upon the nature of compound. If the compound is sensitive to heat, it may be extracted at low temperatures. Similarly, prolonged high temperature along with high power is not desirable because it may degrade the desirable compound (Khajeh et al., 2010). Extraction efficiency increases with the increase in surface area, so most of the sample preparation steps include milling, grinding, and homogenization. This is also the case in microwave extraction; greater surface area results in more efficient extraction (Kothari and Seshadri, 2010).
Ultrasound assisted extraction uses ultrasound waves having a frequency of 20 kHz–100 MHz for the extraction of different constituents from the matrix of the substance. UAE improves the extraction efficiency by producing cavitation. Heat is produced when ultrasound waves pass through the material because kinetic energy is converted into heat energy. Factors affecting the extraction efficiency of UAE include time, temperature, and frequency of ultrasound used for extraction (Khan et al., 2010). A study was conducted to compare the Soxhlet extraction with ultrasound assisted extraction for oil and polyphenols from powder of grape seed. It was found that ultrasound assisted extraction reduced the time of extraction from 6 h to 15 min, with consumption of less extraction solvent (Da Porto et al., 2013).
Supercritical fluid is the state in which gas or liquid is not distinguishable, due to having a density similar to liquid and viscosity as gas. Supercritical fluids have more diffusivity as compared to liquid in solid material, and possess good transport properties which help to reduce the extraction time. Several advantages associated with supercritical fluid extraction include improving the extraction efficiency in terms of low extraction time, and greater yield, and it can also be coupled with chromatographic techniques like supercritical fluid chromatography and gas chromatography (Herrero et al., 2006; McHugh and Krukonis, 2013).
3 Absorption, Metabolism and Bioavailability of Flavonoids
The absorption and metabolism of dietary flavonoids have continued to be a controversial issue for some time. In the past, the consensus regarding flavonoids was that they were fairly large and polar molecules which could not be absorbed after ingestion, and were hydrolyzed to corresponding aglycones by bacterial enzymes in the lower part of the intestine. Most flavonoids occur as glycosides in food (Drzikova et al., 2005). Different glycosidic units are present. Among these, glucose is the most common. Other glycosidic units include arabinose, rhamnose, and galactose (Cook and Samman, 1996). β Linkage present in these sugars resist hydrolysis by pancreatic enzymes, so it was thought that intestinal microflora hydrolyze this β linkage in sugars. However β endoglucosidases, which can hydrolyze flavonoid glycosides, have been characterized in the small intestine of humans. These β glucosidases include lactase phlorizin hydrolase and cytosolic enzyme, which, it is believed, deglycosylate flavonoids create a site for conjugation (Lazaridou and Biliaderis, 2007; Welch, 1995). Some flavonoid constituents, such as kaemferol-3-glucoside and luteolin-7-glucoside, are absorbed in the small intestine after hydrolysis, supporting the activity of β-glucosidase. Luteolin-3-glucoside is converted to aglycone during the passage from intestine (Day et al., 1998). Some studies showed anthocyanins are absorbed unchanged (Cao and Prior, 1999). Absorption kinetics of flavonoids varies from food to food, due to presence of different sugars and the variety of functional groups around the flavan nucleus. Absorption also depends upon the dose, vehicle of administration, gender and colon microbial population (Hollman et al., 1999; Hollman and Katan, 1999; Erlund et al., 2001). Flavonoids that are absorbed from the small intestine are metabolized in the liver as compared to compounds which are absorbed by colon.
Due to high molecular weight, flavonoids must to be degraded into smaller molecules to be absorbed across the intestinal epithelium. Procyanidin oligomers that consist of 7 units are not capable of translocating across the small intestinal epithelium, while procyanidin dimmers and trimmers can translocate across intestinal epithelium (Déprez et al., 1999).
In addition to flavonoid glycoside hydrolysis, cecal microbes degrade polymers and breakdown flavonoids into monophenolic acids. Quercetin metabolism by intestinal microbes produces 3,4-dihydroxyphenylacetic acid and phloroglucinol through the breakdown of C3-C4 bond of the heterocycle (Winter et al., 1991). Cultured colon flora convert tannins into aromatic compounds (Kim et al., 1998). Deprez and coworkers incubated C14 labeled proanthocyanidins with human colonic bacteria under anaerobic conditions that were similar to an enteric environment. All the substrate was degraded to phenylpropionic, phenylvaleric, and monohydroxylatedphenylacetic acids (Hollman et al., 1999). Phenolic acid degradation of catechin produces similar metabolites. Existing data suggest that flavonoids are altered structurally in vivo, but it is unclear whether it is dominated by phenolic acid or flavonoid isomers. After oral administration of quercetin in humans, it was recovered in plasma but minimum amount was detected in urine (Olthof et al., 2000).
Genistin and its octylglucosides are dominant isoflavones found in soy foods (Wiseman et al., 2002). Lactase phlorizin hydrolase mammalian enzyme hydrolyzes ingested isoflavones and released aglycones daidzein, glycitein, and genistein. Gut microflora act on these constituents and convert them into isoflavan equol, and convert genistein into p-ethyl phenol (Hur et al., 2000). Isoflavonoids which are unconjugated are quickly absorbed from the upper parts of the small intestine while conjugated glycoside are slowly absorbed. This is dependent on their hydrolysis in the distal site of the intestine (Sfakianos et al., 1997; King and Bursill, 1998). Once isoflavonoids are absorbed, they are converted to β-glucuronides. It is understood that these conjugates circulate in plasma and are excreted through urine because intact glucuronides isoflavones have been found in urine of human after consumption of soy foods (Clarke et al., 2002).
In vivo study shows that the cell wall of intestines are permeable to dimers and trimmers of proanthocyanidins (Holt et al., 2002). Proanthocyanidins with a higher degree of polymerization are broken into monomers and dimers in the form of epicatechin in the stomach. After being broken into monomers and dimers, they are absorbed in the cell wall of small intestine. Those proanthocyanidins which have a degree of polymerization greater than 10 and are not broken into simpler molecules, are not absorbed by the small intestine and pass through the small intestine unchanged. In the colon these polymeric proanthocyanidins are degraded by colon microflora which is present in the large intestine (Holt et al., 2002; Deprez et al., 2001; Kruger et al., 2014). Overall absorption and metabolism of flavonoids is shown in Fig. 2.
3.1 Bioavailability of Flavonoids
The study of bioavailability of dietary flavonoids is of great importance due to their health promoting effects. Absolute bioavailability, which is expressed in the percentage of non nutrient part of food, is estimated from the part of a molecule that gets absorbed from intestine and found in blood circulating in the system after ingestion, followed by their passage through the liver (Hollman et al., 1997). Bioavailability is not entirely indicated by the extent of absorption, but other factors as well, such as distribution and metabolism (bioconversion which takes place in the gut and biotransformation which takes place in the liver) and also effects bioavailability of non nutrient plant factor following ingestion. It can be concluded that bioavailability quantifies the exposure of the body (not including the liver or the gut) to the non-nutrient plant factor in question (Wiseman, 1999).
Bioavailability of flavonoids is low; consumed constituent in circulation is less than 10% at nanomolar or low micro molar range after few hours of consumption. Isoflavones are the most bioavailable subclass of flavonoids comparatively, while flavan-3-ols and anthocyanins are least absorbed (Manach et al., 2005; Barnes et al., 2011). Factors which affect the bioavailability of flavonoids include the presence of fiber, micro- and macronutrients, gastrointestinal transit time and gut microflora.
Flavonoid bioavailability is dependent on the proportion of taken quantity which is absorbed, varying from 0.2% to 0.9% of tea catechins, to 20% for quercetin and isoflavones (Field, 2001). A large proportion of flavonoids remain unabsorbed and a high concentration of these compounds interacts with gastrointestinal mucosa. Absorbed flavonoids are conjugated in the liver by the process of glucuronidation, methylation or sulfation, or are metabolized into smaller compounds (Avila et al., 1989).
Little information exists regarding the relation of bioavailability of flavonoids to interaction between food constituents and flavonoids. Some studies show fat improves bioavailability of flavonoid absorption due to micellarization of solubilized polyphenols (Ortega et al., 2009). Flavonoids that bind to dietary fiber are not accessed by hydrolytic enzymes in the small intestine, but can be degraded by colonic microbes later on (Pérez-Jiménez et al., 2009). Processing and homogenization of food may enhance the bioaccessibility of flavonoids, for example the flavanones naringenin from tomato products are absorbed better when compared to fresh fruits (Porrini and Riso, 2008).
4 Toxicity of Flavonoids
Diets containing flavonoids are generally thought to be safe because many foods that are naturally rich in flavonoids have been consumed for a long time with no ill effects. Studies that are based on observation demonstrate that there is no correlation between flavonoids (normally quercetin, up to 68 mg) as part of a routine diet and various cancers, including cancer of the breast, lung, respiratory tract, gastrointestinal tract (Harwood et al., 2007). Although these associations provide strong evidence for the safety of flavonoids, there has actually been little experimental research to track the possibility of adverse effects (Erdman et al., 2007). Hooper and Colleagues reviewed the effects of different flavonoids subclasses on cardiovascular diseases and other risk factors. They selected those flavonoids subclasses that are normally present in the human diet. Except from acute rise of blood pressure after black tea consumption that was observed in four studies, and they presume that this may be due to caffeine, and no other adverse effects were detected among the foods tested (Hooper et al., 2008; Erdman et al., 2007).
The proceedings of the International Life Sciences Institute North America Flavonoids Workshop describe that if flavonoids are consumed in low amounts, then they are not detrimental to health, however some evidence shows that flavonoids could cause adverse effects when consumed in high amounts or by some vulnerable groups or populations (Lambert et al., 2007). The potential risks due to flavonoids in the geriatric population include thyroid toxicity, antinutritional effects, carcinogenicity and genotoxicity (Egert and Rimbach, 2011).
The consumption of a test meal showed acute intake of flavonol rich beverages like coffee, tea, red wine and cocoa leads to impaired absorption of non heme iron in humans. Intake of beverages containing 20–25 mg flavonoids per serving reduces the absorption of iron by 50%–70%, while consuming beverages containing 100-400 mg total flavonoids per serving reduces the absorption of iron by 60%–90% (Corcoran et al., 2012). It is evident that high consumption of flavonoid increases the risk of iron deficiency in marginal iron status populations like the elderly. Because populations in western countries consume adequate amounts of iron, the chance of developing anemia due to intake of flavonoids is lower (Erdman et al., 2007).
In vitro studies and animal models show that flavonoids may possess antithyroid and goitrogenic activity. Genistein possess more pronounced antithyroid effects in animals where iodine status is low. However, no evidence has been provided to suggest soy foods or isoflavones affect thyroid function adversely in normal humans (Messina and Redmond, 2006). Additionally, Bitto and colleagues showed no effect of genistein on serum thyroid hormone after three years of treatment in postmenopausal and osteopenic women. Flavonoid toxicity depends upon the dose, duration of intake and type of flavonoids (Moon et al., 2006). Safety concerns of isoflavones due to their high intake are related to estrogenicity, though prior exposure duration may have an affect and lead to adverse outcomes. High concentrations of isoflavones that are not readily achieved through diet result in genotoxicity (Jerome-Morais et al., 2011). In vitro studies show that quercetin is mutagenic and genotoxic, but this is not confirmed with in vivo studies (Harwood et al., 2007).
Observance to dietary recommendations, recommending greater intake of plant foods to promote health, results in higher consumption of flavonoids. However, due to the putative health benefits of flavonoids, increased consumption has been encouraged, not only of naturally occurring flavonoid-rich foods, but also of foods that are fortified with flavonoids and dietary supplements like phytochemicals (Corcoran et al., 2012). For example, quercetin is marketed as a dietary supplement with dose recommendations exceeding 1000 mg/day, while daily intake of this flavonol from food is anticipated at 10–100 mg. There is no evidence suggesting quercetin toxicity resulting from consuming supplements (Egert and Rimbach, 2011). It is mostly older adults who use supplements and likely take prescribed medicines as well. Therefore the focus of research ensures safety of high intake of flavonoids specifically in older people (Lambert et al., 2007; Prasain et al., 2010).
5 Antioxidant Activity of Flavonoids
Flavonoids possess several properties, but one of the most important that is their described ability to scavenge free radicals and act as antioxidants. The difference in the antioxidant capacities of different flavonoids is different, depending on the type of functional group and its arrangement around the flavonoid skeleton. The number of hydroxyl groups, their configuration and substitution, affect the mechanism by which flavonoids act as antioxidants, like chelation of metal ion or scavenging free radicals (Pandey et al., 2012). One of the important factors which affect their ability to act as an antioxidant is the configuration of the hydroxyl group on ring B, because this ring has the ability to donate an electron and hydrogen to hydroxyl and scavenge reactive oxygen species (Cao et al., 1997).
Several mechanisms are described for the antioxidant activity of flavonoids. They include: scavenging reactive oxygen species, enzyme inhibition and trace element chelation that contributes in the generation of free radicals. In this way they suppress formation of reactive oxygen species, antioxidant defense protection and up regulation. The enzymes which are inhibited by flavonoids and help in the generation of reactive oxygen species include NADH oxidase, mitochondrial succinoxidase, glutathione S-transferase and microsomal monooxygenases (Kumar and Pandey, 2013).
Oxidative stress is associated with peroxidation of lipids. The protective effect of flavonoids against the peroxidation of lipids is described frequently (Kumar et al., 2013). Reactive oxygen species formation is enhanced by metal ions, the mechanism involved in this reaction is that hydrogen peroxide is reduced by these metal ions resulting in the generation of hydroxyl radical which is highly reactive (Mishra et al., 2013a). Among several flavonoids constituents, quercetin is the most commonly described flavonoid with antioxidant properties. The antioxidant properties of quercetin are due to chelation of iron and iron-stabilizing properties (Kumar and Pandey, 2013).
Because flavonoids mostly occur as flavonoid glycosides, in which a flavonoid molecule is attached to sugar molecule, its position and the number of attached sugar molecules also effect its antioxidant properties. By comparing the antioxidant activities of flavonoid aglycones and glycosides, it has been reported that aglycones have greater antioxidant potential. These findings are supported by the fact that the flavonol glycosides, which are found in tea, decrease with the increase in number of glycosidic moieties. Although aglycones are stronger antioxidant when compared to glycosides, their bioavailability is low (Hollman et al., 1999). As the degree of polymerization increases, the antioxidant activity or scavenging of reactive species in procyanidins is improved. As compared to monomeric procyanidin, dimers, and trimmers of procyanidin are more effective in scavenging superoxide anion. Similarly, the tetramers of procyanidin show greater activity against superoxide and peroxynitrite mediated oxidation. In the same way, hepta and hexamers are strong antioxidants (Kumar and Pandey, 2013).
Experiments carried out on animals have shown that flavonoids might be a valuable anti-inflammatory, owing in part to the fact that they inhibit blood-vessel damage (Harborne and Williams, 2000). Research recommend flavonoids for their potential to prevent chronic inflammation (Funakoshi-Tago et al., 2011) related to heart disease, arthritis, type 2 diabetes, Alzheimer’s disease, dementia, and many other diseases. Flavonoids may increase blood sugar metabolism and studies have shown that flavonoids might have a positive result on abnormal collagen making, a symptom linked to diabetes that subsidizes poor blood sugar control (Kumar and Pandey, 2013). A study demonstrated that flavonoids showed activities for inhibiting retinopathy, a diabetic disorder which can occur prior to blindness. (Kumar and Pandey, 2013).
6 Flavonoids and Cardiovascular Diseases
In the western world, the most common cause of death is cardiovascular diseases (CVD); worldwide about one third of all the deaths are caused by cardiovascular diseases. There are two types of factors involved in the development of cardiovascular diseases. The primary factors can be modified, like diet, life style, environment, smoking, and exercise. The second factors cannot be modified: genetic factors, gender, history, and age. The common phenomenon involved in the development of CVD is the atherosclerotic plaque formation which is initiated by endothelium damage. Inflammation and oxidative stress are the key factors contributing to the damage of endothelium.
Consumption of fruits and vegetables is inversely associated with incidence of CVD, due to the presence of bioactive compounds like flavonoids. Current research has focused on diet containing bioactive compounds, as an alternative to pharmaceutical medication, in the maintenance of cardiovascular health,. It can be concluded from the analysis of multiple studies that as the mean consumption of flavonoids increases, mortality due to cardiovascular diseases decreases. Another epidemiological study was conducted to determine the effect of blueberry and strawberry intake for 16 years follow up period on the mortality due to CVD in postmenopausal woman. It was concluded that the result demonstrated a significant connection between the two (Mink et al., 2007). Similarly, another study showed that the cardio-protective effects of Nigella sativa are due to the presence of phenolic compounds like thymoquinone, by reducing low density lipoprotein, total cholesterol and triglycerides (Shafiq et al., 2014).
Several mechanisms have been described for the mode of action of flavonoids as cardio protective agents. The ability of flavonoids to control oxidative stress and act as anti-inflammatory agents is responsible for their cardio-protective properties. The anthocyanins present in black rice grain and the proanthocyanidins found in red rice and grape seeds scavenge hydroxyl radicals and superoxide ion (Walter and Marchesan, 2011; Kruger et al., 2014). Production of NO is stimulated by proanthocyanidins which are present in red grapes. The availability of NO in acute oxidative stress like reperfusion/ischemia is protective to cardiomyocytes, because it inhibits the cardiomyocytes apoptosis (Jones and Bolli, 2006). Similarly, the supplementation of blueberry, which is rich in proanthocyanidins and anthocyanins, improved the endothelial dysfunction and decreased blood pressure in animals which were fed a high cholesterol/high fat diet (Rodriguez-Mateos et al., 2013). In a human study, cranberry juice consumption for 4 weeks did not affected the function of vascular endothelial as well as the level of NO generated. It was concluded that anthocyanins might be undetectable in plasma after 12 h due to rapid clearance. Another reason for no activity observed might be due to a lower concentration of anthocyanins in the juice (Rodriguez-Mateos et al., 2013). Similar results were also reported by Riso et al. (2013) who studied the effect of wild berry juice in humans.
Among CVDs, the effect of flavonoids on stroke is not clear. The association between intake of five flavonoid classes (flavan-3-ols, flavonols, anthocyanidins, flavanones, flavones) and risk of stroke and mortality caused by stroke was studied by Mursu et al. (2008) in eastern Finish men, age 42–60 years. During the follow up time, 153 deaths occurred due to CVD and 102 patients suffered from ischaemic stroke. The men consuming the highest amount of flavonol and flavan-3-ol had low risk for ischemic stroke, which is 0.55 and 0.59 as compared to the lowest quartile. So it was concluded that a greater intake of flavonoids decreased the chances of ischemic stroke as well as mortality caused by CVD. This reduction in the risk for ischemic stroke and CVD mortality was significant. Similarly, high intake of cocoa or chocolate reduced the systolic blood pressure by 5.9 mmHg, at the population level it has the capacity to reduce risk of stroke by 8%, mortality due to coronary artery disease by 5%, mortality caused by all factors at 4% (de Pascual-Teresa et al., 2010).
6.1 Anti-Inflammatory Activities of Flavonoids
The protective action of tissue in response to the invasion of pathogens, irritation, cell injury and for removal of necrotic and damaged cells is known as inflammation. For a short period, the process of inflammation helps to maintain the tissue integrity by minimizing the effect of injury or invasion. But if the inflammation takes place for a long period of time, it can mediate the development of several chronic diseases such as CVD, cancer, arthritis, neurodegenerative diseases, and pulmonary diseases (Rubio-Perez and Morillas-Ruiz, 2012). Several studies show the anti-inflammatory activity of flavonoids. Chronic inflammation is caused by the excessive production of chemokines and cytokines. Cytokines and chemokines act as regulatory proteins under normal physiological conditions, but their excessive production disrupts the gradient balance and more ROS are produced. It has been shown that the grape flavonoids control chronic inflammation by reducing ROS level and by modulating pathways of inflammation. As flavonoids are natural compounds, they can target multiple steps in the inflammation pathway as compared to mono-targeted synthetic anti-inflammatory drugs (Sung et al., 2012). In the same way, proanthocyanidins extracted from the seeds of grapes have been shown to modulate the immune system in inflammatory conditions and induce production of prostaglandin E2 and nitric oxide (Terra et al., 2007).
6.2 Atherosclerosis
Atherosclerosis is characterized by the plaque formation in large arteries, and it is one of the major factors contributing to incidence of stroke and myocardial infarction. Atherosclerosis is caused by high level of lipoprotein and cholesterol in plasma (Hackam and Anand, 2003). Factors which contribute to development of atherosclerosis include hypertension, diabetes, diet, obesity, smoking, and aging. Beside therapeutic treatment, the risk for cardiovascular diseases persist which necessitates the search for therapeutic agents that control the risk factors of atherosclerosis. Seeking the French Paradox, many studies have been carried out to evaluate the potential of flavonoids against atherosclerosis. In addition to the French Paradox, several epidemiological studies showed negative correlation between the incidence of atherosclerosis and intake of flavonoids. High intake of fruits and vegetables rich in flavonoids reduces several risk factors for development of atherosclerosis including: high tolerance to glucose, maintaining good body mass index, lowering blood pressure (Mulvihill and Huff, 2010).
In a mice model study, the effect of pomegranate juice, which is rich in proanthocyanidins and anthocyanidins, decreased the accumulation of macrophage CE and lipid peroxides without affecting the level of cholesterol in plasma. Supplementation of pomegranate for a period of three months reduced the risk of atherosclerosis up to 44%. In a similar animal model study, supplementation of resveratrol in the lab chew for four months reduced the total cholesterol level of plasma along with LDL-C and increased the HDL cholesterol. In addition to this resveratrol prevented the lipid peroxidation and increased cholesterol efflux. Another study showed that risk for atherosclerotic plaque development was significantly reduced by the consumption of resveratrol in mice (Aviram et al., 2008).
Naringenin plays an important role to overcome the metabolic problem that is connected to dyslipidemia and resistance to insulin. Consequently it was shown to prevent atherosclerosis development in mice fed a high fat diet. Naringenin treatment attenuated the adverse effects caused by hyperinsulinemia and hyperlipidemia which was induced by western style diet. In mice that were fed a western diet, hyperlipidemia led to development of atherosclerosis in the aortic sinus evidenced by the development of plaque is that increased 10 times as compared to chow fed animals. Naringenin treatment decreased the incidence of atherosclerosis by 70% (Mulvihill and Huff, 2010).
7 Antidiabetic Activity of Flavonoids
One of the most widely prevalent metabolic disorders is diabetes, which is characterized by hyperglycemia which may be the result of either no excretion of insulin, or production of non-functional insulin. High levels of sugar in the blood result in short term protein and lipid metabolism changes and irreversible long term changes in the vascular system (Brahmachari, 2011). The long term manifestation of insulin results in damage and dysfunction of various organs like nerves, kidneys, eyes, blood vessels, and heart. In the past few decades, it has been observed that there is a rapid increase in the incidence of coronary artery diseases (CAD) (2010). Several studies reporting anti-diabetic activities of flavonoids are described in Table 1.
Table 1
| Flavonoid | Source | Model of study | Diabetes caused by | Results | Reference |
|---|---|---|---|---|---|
| Quercetin, luteolin-7-o-glucoside, rutin, chlorogenic acid, isorhoifolin | Pilea microphylla | Mice | High fat diet and streptozotocin induced diabetes | dipeptidyl peptidase IV was inhibited with IC50 of 520.4 μg/mL | Bansal et al. (2012) |
| Total flavonoids | Litsea coreana | Male Sprague-Dawley rats (Type II diabetes) | Diabetes induced by Streptozotocin | Extract treatment increased sensitivity of insulin, HDL-C and increased the body weight | Lu et al. (2010) |
| Total flavonoids | Selaginella tamariscina | Rats | Diabetes induced by high fat diet and low concentration of STZ | Treatment with total flavonoids decreases the triglycerides, glycosylated hemoglobin A1C, free fatty acids, Total cholesterol, fasting blood glucose level, and LDL-C, with significant increase in SOD | Zheng et al. (2011) |
| Total flavonoids | Sanguis draxonis | Rats | Type II diabetes induced by high fat diet and streptozotocin | Showed hypoglycemic activity and increased the tissue steatosis and dyslipidemia. In addition islet protecting effects was also observed | Chen et al. (2013) |
| Fruit extract rich in flavonoids | Carissa carandas | Rats | Diabetes induced by alloxan | Reduced the high level of glucose in blood by 48%, by oral intake of extract at rate of 400 mg/kg | Itankar et al. (2011) |
| Quercetin | Rats | Diabetes induced by streptozotocin | Intraperitoneal injection at the rate of 50 mg/kg showed protective effects against diabetes induced by (STZ) and decreased the activity of antioxidant enzymes | Abdelmoaty et al. (2010) | |
| Extract containing flavonoids like chrysoeriol 7-o-β-D-galactopyranosyl and luteolin 7-O-[6″-O-α-L-rhamnopyranosyl]-β-D-galactopyranoside | Hyphaene thebaica | Rats | Alloxan | Lowered glycosylated hemoglobin level in blood and improved the tolerance of insulin and glucose. | Salib et al. (2013) |
Currently, diabetes is treated by several anti-diabetic agents, which include biguanides, sulfonylureas, glinides, and α-glucosidase inhibitor, along with insulin to control regulation of blood sugar level. These therapies are associated with several adverse health effects, so research has been focused to find new therapeutic agents which have minimum or no adverse health effects. Plants are a natural source of drugs; several drugs which are widely used have been obtained directly or indirectly from plant sources. Several studies have reported that either plant parts or extracts of plant parts possess antidiabetic properties when assessed through experimental trials. This antidiabetic activity of plants is due to the presence of phytochemicals which are termed as flavonoids (Brahmachari, 2011).
Meta-analysis and several epidemiological studies showed that there is inverse correlation between consumption of a diet rich in flavonoids and several disorders related to aging, such as osteoporosis, cardiovascular diseases, cancer, and neurodegenerative diseases (Graf et al., 2005; Arts and Hollman, 2005). Several studies have been conducted that showed consumption of flavonoid rich diet regulate digestion of carbohydrates, secretion of insulin and uptake of glucose in insulin sensitive tissue by regulating several intracellular pathways (Hanhineva et al., 2010).
The anti-diabetic effect of flavon-3-ols are reported by several studies. Epigallocatechin gallate (EGCG), a flavon-3-ol, at a concentration of 0.1-10 μM improved the viability of β-cells along with improving their insulin secretory function in rat cells in which glucose toxicity was induced. In addition, the function of mitochondria was also improved by maintaining its functional integrity in pancreatic b-cells exposed to glucose toxicity (Erdman et al., 2007). A high level of free fatty acids in the plasma plays an important role in impairing insulin resistance and development of type II diabetes. This insulin resistance induced by fatty acid can by minimized by the consumption of ECG and EGCG (Deng et al., 2012; Boden et al., 2001). Similar to EGCG and ECG, naringin and hesperidin minimized the oxidative stress and hyper glycemia in male albino rats in which diabetes is induced by streptozotocin, by oral administration at the dose of 50 mg/kg for a period of 1 month (Mahmoud et al., 2012). In the same way, a diet supplementation of either hesperidin or naringin at the rate of 200 mg/kg reduced the glucose level in the blood along with increasing leptin and plasma insulin concentration (Goldwasser et al., 2010; Jung et al., 2003).
Bilberry extract, a rich source of anthocynins, has been studied for anti-diabetic effect in mice with type II diabetes. Insulin sensitivity and hyperglycemia was improved by bilberry extract, along with down regulation of gluconeogenic enzyme expression like G6Pase and PEPCK (Takikawa et al., 2010). The seed coat of black soybeans is rich in delphinidin, cyanidin and petunidin. Soybean seed coat extract treatment by gavage ameliorated insulin resistance improved the insulin concentration in the serum, along with improving tissue glucose utilization in a rat model study (Nizamutdinova et al., 2009).
8 Hepato-Protective Effects of Flavonoids
One of the vital organs of the body is the liver. It regulates several physiological processes and plays an important role in the vital processes of body like secretion, storage, metabolism and detoxification of exogenous as well as endogenous toxins (Adewusi and Afolayan, 2010). In addition, the liver takes part in biochemical processes such as growth, nutrient provision, supply of energy and reproduction. It helps in the metabolism of fats and carbohydrates by the secretion of bile and storing vitamins (Adewusi and Afolayan, 2010). Due to these vital functions, hepatic disease is one the greatest threats to the world population. Structural or functional damage to the liver is termed hepatic disease. Liver damage is caused by autoimmune disorders like primary biliary cirrhosis, immune hepatitis as well as by other biological factors including viruses, bacteria or other parasites. Liver damage can also be caused by chemicals found in certain drugs such as antituberculosis drugs and CCl4. Despite enormous achievements in medicine, until now, there are no drugs available which stimulate complete liver function or help in the regeneration of liver cells. Beside, that there are several side affects as well as adverse health effects associated with these drugs. Several studies have investigated the efficacy of plant extract against hepatic diseases and positive results are found. Besides that, consumption of certain foods protects against liver damage. Hepatoprotective properties of plant based foods are mostly attributed to bioactive compounds like flavonoids. Following these successes, several studies have been conducted to check the hepatoprotective activities of plant extract rich in flavonoids or individual flavonoid compounds (Madrigal-Santillán et al., 2014).
Grapefruit is an excellent source of the flavonoid compound naringin which is metabolized as naringenin in the body. The hepatoprotective activity of naringin was reported in 2004 in rats in which hepatic damage was induced by dimethylnitrosamine (DMN). Oral administration of naringenin at the dose of 20 and 50 mg/kg per day for the period of 4 weeks protected the mice from damage induced by DMN. This was shown via liver weight as well levels of aspartate transaminase (ASAT), alanine transaminase (ALAT), and bilirubin. Other studies since then have shown that naringenin has the ability to reduce the serum level of ASAT, ALAT, gamma-glutamyltranspeptidase (GGT), lipid hydroperoxides, ALP, protein carbonyl content, conjugated dienes, superoxide dismutase (SOD), glutathione-s-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), and alcohol dehydrogenase (Lee et al., 2004). The hepatoprotective activities of flavonoids extracted from plants are described in Table 2.
Table 2
| Flavonoid | Source | Liver Damage was induced by | Model of Study | Liver Damaged Assessed by | Dose of Flavonoid Supplementation | Reference |
|---|---|---|---|---|---|---|
| Citromitin | Citrus depressa | D-galactosamine | rats | alanine aminotransferase and aspartate aminotransferase activities | 300 mg/kg body wt 4 h | Akachi et al. (2010) |
| Tangeretin | Citrus depressa | D-galactosamine | rats | alanine aminotransferase and aspartate aminotransferase activities | 300 mg/kg body wt 4 h | Akachi et al. (2010) |
| Nobiletin | Citrus depressa | D-galactosamine | rats | alanine aminotransferase and aspartate aminotransferase activities | 300 mg/kg body wt 4 h | Akachi et al. (2010) |
| Hirsutin, quercetin, avicularin | Lespedeza cuneata G. Don | tert-butyl hyperoxide | HepG2 cells | Cytotoxicity | 10 μg/mL | Kim et al. (2011) |
| Total flavonoids | Rosa laevigata Michx fruit | Paracetamol | Mice | alanine aminotransferase, aspartate aminotransferase, malondialdehyde, superoxide dismutase, GSH | Liu et al. (2011) | |
| Total flavonoid | Abelmoschus manihot (L.) Medic Flowers | CCl4 | Mice | ALT, AST, ALP, SOD, GPx, CAT and GST | 125, 250, 500 | Ai et al. (2013) |
| Total flavonoid content | Solanum melongena | tert-butyl hydroperoxide (t-BuOOH) | human hepatoma cell lines, HepG2 | Improved viability of cells | 14.49 ± 1.14% to 44.95 ± 2.72% | Akanitapichat et al. (2010) |
| Catechin glycoside, miricitrin-3-O-glucoside, astragalin, isoquercitrin, hyperin, quercetin-3-O-rhamnoside | Nelumbo nucifera Gaertn | CCl4-induced liver toxicity | rats | 300 and 500 mg/kg | Huang et al. (2010) | |
| Luteolin, isorhamnetin | Viola odorata | Paracetamol | Mice | Serum hepatic enzymes and bilirubin | 250 mg/kg and 500 mg/kg | Qadir et al. (2014) |
| Quercetin | Convolvulus arvensis | Paracetamol | Mice | Serum hepatic enzymes and bilirubin | 200 mg/kg and 500 mg/kg | Ali et al. (2013) |
As described above berries are good source of flavonoids and are consumed at large scale worldwide. Currently berry extract is used as a functional food ingredient and dietary supplement. The administration of cranberry at the rate of 7 mg/kg is effective in minimizing the adverse effects on the liver caused by acute and chronic administration of CCl4, as evidenced by normalizing the level of ASAT, ALAT, and bilirubin. In addition to this, it also prevented the lipid peroxidation products from accumulation in the liver of rats, with apparent mitochondrial ultrastructure preservation (Cheshchevik et al., 2012). The hepatoprotective effects of anthocyanins and proanthocyanidins was investigated by Shin et al. (2010) in rats. Hepatic damage was induced by DMN followed by the oral intake of anthocyanins and proanthocyanidins at a dose of 20 mg/kg for a period of 4 weeks. This minimized the adverse effects caused by DMN and reduced the serum level of ALP, ASAT, ALAT and bilirubin. In addition, it also minimized the accumulation of collagen which is induced by DMN. This was demonstrated by histological analysis of red stained tissue.
Grapes and grape seeds are rich source of flavonoids like resveratrol, proanthocyanidin, anthocyanidins, epicatechins, and catechins. The hepatoprotective effect and antioxidant activity of grape seeds was observed in rats in which hepatitis was induced by oxidative stress and assessed by marker enzymes like GGT, ASAT, LDH, ALAT, SOD, GSH, MDA, GPx, and GST (Dogan and Celik, 2012). The animals were divided into four groups: (I) control, (II) 20% ethanol, (III) 15% grape seed, and (IV) 20% ethanol + 15% GS. The ethanol treated group was observed to contain a significantly high amount of marker enzymes in the serum as compared to the control group. This level of marker enzymes was significantly decreased in group IV, which was fed ethanol along with grape seed. This showed that the adverse effects caused by the oxidative stress of ethanol were minimized by the consumption of grape seed. Several other studies also described the hepatoprotective effect of grape or seeds of grapes (Hassan, 2012; Dogan and Celik, 2012; Oliboni et al., 2011; Liu et al., 2012; Madrigal-Santillán et al., 2014; Madi Almajwal and Farouk Elsadek, 2015).
A flavonoid named Silymarin has three structural components: silydianine, silibinin, and silychristine. These are extracted from the seeds and fruit of milk thistle Silybum marianum (Compositae) and have been reported to stimulate enzymatic activity of DNA-dependent RNA polymerase 1 and subsequent biosynthesis of RNA and protein which results in biosynthesis of DNA and cell proliferation resulting in liver regeneration in damaged livers (He et al., 2004). The phytochemical properties of this flavonoid are also involved in regulation of permeability, integrity, ROS scavenging, and inhibition of leukotriene and collagen production (Saller et al., 2001; Kumar and Pandey, 2013).
Different flavonoids such as quercetin, rutin, catechin, naringenin, and venoruton have been reported for their hepatoprotective effect (Tapas et al., 2008). Several chronic maladies such as diabetes can cause clinical hepatic manifestations. Diabetic mice have decreased levels of ROS, glutathione, and ligase catalytic subunits expressions in their liver. As a result, anthocyanins are gaining attention due to the discovery that increased levels of these antioxidants have a preventive effect against many diseases. Zhu et al. (2012) explained that anthocyanin increases Gclc expressions by elevating cAMP levels to activate PKa (Protein kinase), which further increases Gclc transcription resulting in decrease in hepatic ROS levels. Moreover, C3G treatment reduces hepatic lipid peroxidation, inhibiting release of pro-inflammatory cytokines, and protecting against hepatic steatosis development (Zhu et al., 2012).
Additionally, flavonoids extracted from Laggera alata were also observed for their hepatoprotective effect against carbon-tetrachloride induced liver damage in rats. Flavonoids fed at 100 μg/ML concentration inhibited cellular leakage of AST, ALT, and also improved cell viability (Wu et al., 2006). Similarly, flavonoids administrated at 50, 100, and 200 mg/kg in an in-vivo experiment significantly reduced AST, ALT, albumin, total protein levels, and hydroxyproline levels in liver. Histopathological observations showed improvement in damaged liver cells. Many clinical experiments have also revealed the effect of flavonoids on hepatobiliary dysfunctions including feelings of bloating, less appetite, abdominal pain, and nausea. Hirustrin and avicularin flavonoids extracted from many sources were reported to have protection against induced hepatotoxicity. Additionally, luteolin and quercetin suppress phosphorylation of VEGF receptor 2 which is induced by VEGF (Carmeliet and Jain, 2000).
9 Anticancer Activity of Flavonoids
As flavonoids are a natural product, they are considered a safe and ideal candidate for chemoprevention or the treatment of cancer (Szliszka et al., 2011; Yoshimizu et al., 2004). The synthetic agents used for the treatment of cancer are highly toxic and destructive to healthy cells. An ideal anticancer agent is one which has a maximum capacity to inhibit tumor growth or to kill cancer cells, but causes minimum adverse health effects (Zhao et al., 2012). Because long term consumption of flavonoids is not toxic, and due to their inherent biological activity, flavonoids are ideal future candidates for cancer therapy. Many studies have shown that cytotoxic effects of flavonoids on cancer cells, with no or minimum adverse health effects. These findings have simulated the research to develop flavonoid based chemotherapies (Sak, 2014).
Due to the presence of polyphenol aromatic rings in flavonoids, it has been found that flavonoids possess pro and anti-oxidant properties (Leung et al., 2007). Most studies report the beneficial effect of flavonoids due to their reactive oxygen scavenging properties, but recent studies show anticancer properties of flavonoids may be due to their pro-oxidant properties (Li et al., 2008; Habtemariam and Dagne, 2010). Higher oxidative stress is observed in the cancerous cells as compared to normal cells, making them more susceptible to be killed by a substance which enhances reactive oxygen species level like flavonoids (Valdameri et al., 2011; Yuan et al., 2012). A flavonoid acting as pro-oxidant or antioxidant is dependent on the concentration, type of cell and culture condition in which it is grown (Pacifico et al., 2010).
One of the important factors that plays a significant role in the prevention of cancer is diet. It has been shown that consumption of vegetables and fruits which are rich in flavonoids prevent the development of cancer (Mishra et al., 2013b). The consumption of apple and onion, good sources of quercetin, are negatively associated with incidence of prostate, stomach, lung, and breast cancer. Similarly, it has been observed that wine drinkers are less susceptible to the development of colon, lung, and stomach cancer (Kumar and Pandey, 2013).
Flavonoids, along with their synthetic analogs, are currently being studied for their therapeutic potential against cervical, breast, ovarian, and prostate cancer. Some flavonoid compounds, like genistein, quercetin, and flavopiridol, are at the late phase of clinical trials for cancer treatment (Lazarevic et al., 2011). The anticancer activity of flavonoids is reportedly due to modulation of several factors like epidermal growth factor receptors (EGFRs), protein kinases, vascular endothelial growth factor receptors (VEGFRs), platelet derived growth factor receptors (PDGFRs) and cyclin dependent kinases (Ravishankar et al., 2013; Singh and Agarwal, 2006).
Cancer is one of the most fatal diseases that exists. The incidence of cancer is increasing continuously, not only in developing countries, but also in developed countries like United States of America, the United Kingdom, France, and Italy (Ahmad et al., 2015). Cancer incidence and deaths caused by cancer decreased with the use of radiotherapy, chemotherapy, and immunosuppressants, however deaths caused by cancer are still greater as compared to cardiovascular diseases in persons having age greater than 85 years (Ahmad et al., 2015).
Several studies have been carried out to check the antiproliferative activity of flavonoids in vitro and in vivo. The antiproliferative activity of flavonoid constituents isolated from Dracocephalim kotschyi, an ethnobotanical remedy, was studied in vitro against normal and malignant cell lines. The hydroxyflavone isolates, including apigenin, luteolin, and isokaempferol, showed antiproliferative effects comparable in both normal and malignant cells. Hydroxyl flavones, which are methylated like penduletin, calycopterin, cirsimaritin, and xanthomicrol, showed preferential antiproliferative activities against malignant cells (Moghaddam et al., 2012). This study showed that there might be a structure activity relationship. Flavonoid constituent may be modified in such a way to enhance their anti-cancer potential without affecting normal cells. Similarly, in another study six flavonoids were isolated from litchi leaf and studied for anticancer activity. Among six flavonoids which include kaempferol-3-o-β-gluscoside, rutin, procyanidin A2, epicatechin, kaempferol 3 o-a-rhamnoside, and luteolin, procyanidin A2 showed maximum anticancer activity against human cervical carcinoma HeLa cells and human hepatoma HePG2 cells (Wen et al., 2014).
Several mechanisms has been described for the anticancer activity of flavonoids which include protein tyrosine kinase inhibition, phosphatidylinositol-3-kinase inhibition, topoisomerase inhibition, antiangiogenic effects, as well as antioxidant and pro-oxidant activities. Byun et al. (2010) showed that flavonols quercetin, fisetin, and myrecetin, while the flavone luteolin is the potent inhibitor of protein tyrosine kinase. Among other flavonoids which have been reported to inhibit protein tyrosine kinase, include apigenin, luteolin, and quercetin (Ravishankar et al., 2013; Huang et al., 1999).
Phosphatidylinositol 3-kinase is also inhibited by quercetin, apigenin, luteolin, and myricetin. In addition to that, antiangiogenic effect is induced by flavonoids by vascular endothelial growth factor expression regulation and NFkB (Ravishankar et al., 2013).
10 Effect of Flavonoids on Osteoporosis
Osteoporosis and fragile fracture are common world problems, primarily afflicting aging people. Approximately one in two women and one in five men suffer from osteoporosis related fractures in their lives (van Staa et al., 2001) and these osteoporosis associated fractures are on the rise (Hardcastle et al., 2011). Diet is one changeable therapy likely play an important role in osteoporosis development and treatment. Recent research has shown that a healthy diet plan, rich in vegetables and fruits, is linked with low bone resorption whereas a poor diet plan consisting of processed foods results in less bone mineral density (Welch and Hardcastle, 2014).
The occurrence of osteoporosis is much more common in women as compared to men. In the female population, incidences of osteoporosis are much higher in postmenopausal women as compared to premenopausal women. This is due to hormonal imbalance, and in most cases, it is termed estrogen dependent osteoporosis. In the maintenance of bone health, estrogen plays a vital role by decreasing cytokines while simultaneously increasing the osteoclast apoptosis. Consequently, estrogen deficiency results in bone loss. In the same way, estrogen deficiency decreases the life span of osteoblast (Chiechi and Micheli, 2005). There are several flavonoid compounds which are capable of generating estrogen response. Chemicals capable of producing estrogenic response are referred to as phytoestrogen. Among flavonoids, lignans and isoflavones are important phytoestrogens. Genistein, which is an isoflavone obtained from soy. Following a regimen of genistein with a consumption rate of 56 mg/d, results showed a similar effect as by hormone replacement therapy which is currently the most widely used for treatment of postmenopausal osteoporosis. This treatment increases the bone mineral density insignificantly with the hormone replacement therapy (Morabito et al., 2002). Bone strength, as well as bone mineral density, increases with increasing consumption of isoflavones. It has also been shown that fructooligosaccharides consumption along with isoflavones, enhances the action of isoflavones. This effect of fructooligosaccharides is due to its prebiotic properties (Ahmad and Kaleem, 2016; Mathey et al., 2004).
10.1 Mechanism of Action
Bone is in a continuous state of modeling and remodeling to maintain a balance between osteoclast and osteoblast cells. This balance is responsible for maintaining physiological structure and mineral contents of bone. After the achievement of peak bone mass, there is gradual loss of bone. This loss of bone mass is higher in women, occurring more rapidly after menopause (Welch et al., 2004). However, men have more loss of bone mass as compared to women, but this loss is occurs at a slower rate with age. Mass loss predicts fractures in both men and women (Khaw et al., 2004). This loss with age is usually due to imbalance in the anabolic and resorptive activities of osteoblasts and osteoclasts respectively. The osteocyte cells are embedded within mineralized bone and give signal to osteoclasts to start resorption and are then activated to send signals through mechanical loading and microbial damage (Nishimura et al., 2012). Several signaling pathways maintain the activities of osteoclasts and osteoblasts which includes RANK ligand and BMP (Bone Morphogenetic Protein) pathways. These signals also maintain formation of osteoclasts and their survival in modeling and remodeling in normal bone. However, these osteoclasts are negatively regulated by osteoprotegerin (Nishimura et al., 2012). These RANKL pathways are also regulated by parathyroid hormone. Moreover, inflammatory cytokines may stimulate osteoclast formation and resorption of bone by inducing expressions of RANKL. Many different reviews have demonstrated the protective effect of flavonoid phytochemicals on bone protection by reducing RANKL and matrix metalloproteinases which digests bone collagen and reduces osteoblasts activities (Welch and Hardcastle, 2014).
10.2 Epidemological studies
Flavonoids are bioactive polyphenols found in fruit and vegetables. These flavonoids have anti-oxidant and anti- inflammatory properties which are strongly associated with bone health. Epidemiological linkages between bone health and flavonoid-intake have been observed in different studies. A positive link between flavonoid intake and spine, demerol and neck bone mineral density was observed while negative link was observed between flavonoids intake and markers of bone resorption in Scottish peri-menopausal women (Welch and Hardcastle, 2014). Moreover, catechins and flavanones were negatively associated in resorption of bone markers.
In another study, positive association of total flavonoid intake and BMD of spine in women in TwinsUK cohort was observed. In subclasses of flavonoids, anthocyanins were strongly linked with hip and spine BMD. Flavonols and polymers were linked with higher BMD of hip (Welch and Hardcastle, 2014). Additionally, tea which is rich in catechins, has also been reported for providing protection against hip fracture in different epidemiological studies (Welch and Hardcastle, 2014).
11 Antibacterial Effect of Flavonoids
The flavonoids extracted from plants are also known to have a strong effect against microbial infection. They have been found effective against microbes in many in vitro studies. Many flavonoids, such as flavone, flavonol glycosides, flavanones, apigenin, and chalcones, have been studied for their potent antimicrobial effect. However, these flavonoids have many different cellular targets as site of action except one. The most common molecular action of flavonoids is the formation of complex with proteins through different non-specific forces such as hydrophobic effect, covalent bonding and hydrogen bonding, so their antibacterial effect might be related with their capability to deactivate microbial adhesions, cell envelope proteins, enzymes and many others. Beyond that, lipophilic flavonoids have the capability to disrupt microbial membranes. Moreover, catechins (a class of flavonoid compounds) have been widely studied for their antibacterial effect. They have been studied for in vitro-antibacterial effect against streptococcus, Vibrio cholera, shigella and many others ailments (Gerdin and Svensjö, 1982). Catechins were found to have a lethal effect against cholera toxin Vibrio cholera and also inhibit glucosyltransferases in S mutants. Another study explained the inhibitory effect of apigenin, quercetin and pentahydroxyflavone against E. coli (Ohemeng et al., 1993). Moreover, other flavonoids, such as Naringenin and sophoraflavanone G, were also found effective against streptococci and Staphylococous aureus. This effect of flavonoids might be due to reduction in membrane fluidity of hydrophilic and hydrophobic regions and inner and outer layers of membranes. Furthermore, the relationship between the antibacterial concept and membrane interference supports the idea that flavonoids can lower the membrane fluidity of bacteria (Ahmad et al., 2015). The antimicrobial effect of two other flavonoids, licochalcones A and C extracted from roots of Glycyrrhiza inflata, were also studied against Staphylococcus aureus and Micrococcus luteus, showing that licochalcone A has a strong inhibitory effect against incorporation of radioactive precursors in proteins, DNA and RNA (Kumar and Pandey, 2013). The antibiotics have a similar effect for inhibition of respiratory chain, as energy is a primary component for biosynthesis of macromolecules and for absorbance of various metabolites. Further studies suggested that CoQ and cytochrome are inhibition sites for flavonoids in bacterial electron transport chain. Several studies support the antimicrobial effects of these phytochemicals extracted from different medicinal and edible plants (Kumar and Pandey, 2013).
12 Antiviral Activity
Natural compounds extracted from different plant sources are gaining importance for the development of different antiviral medicines due to their having fewer side effects. Flavonoids extracted from these compounds were recognized for antiviral effects since 1940 and different scientific studies have revealed their efficacy. There is a pressing need to develop effective drugs against HIV (human immunodeficiency virus). The effect of these antiviral compounds depend on inhibition of many enzymes linked with life cycle of these viruses (Gerdin and Svensjö, 1982). Flavan-3-ol was demonstrated be much more effective than flavonoids and flavones for their inhibition against HIV-1,2. The flavonoid Baicalin, which is extracted from Scutellaria baicalensis, was also found effective against inhibition of HIV-1 infection and replication. Similarly, catechins were also found to inhibit the DNA polymerases of HIV-1. It has also been demonstrated that flavonoids acacetin, chrysin, and apigenin, inhibit HIV-1 activation through a unique mode of action which usually involves prevention of viral transcription (Cushnie and Lamb, 2005). Moreover, the anti-dengue effect of hesperetin, quercetin and daidzein was also studied at different levels of dengue virus type-2 infection. Among these flavonoids, quercetin was observed most effective against dengue virus type-2 infection. Several other flavonoids, such as dihydrofistein, dihydroquercetin, pelargonidin and catechin, also revealed activities effective against many types of viruses, including HSV, polio virus and sindbis virus (Gerdin and Svensjö, 1982). Prevention of viral polymerase binding of viral capsid proteins was suggested as an antiviral mode of action.
13 Conclusion
The potential of flavonoids to prevent several ailments and diseases is well known. Good sources of flavonoids include cocoa, tea, berries and several other fruits and vegetables. As a result of mass production and processing of food products, a large quantity of byproducts such as seeds and peels are obtained which are a rich source of flavonoids. Due to increasing demand of nutraceutical products, and products free from synthetic ingredients, research has been focused towards the extraction and utilization of flavonoids. The previously mentioned facts highlight the potential of flavonoids as nutraceutical agents for food manufacturing industries. Besides having nutraceutical properties, flavonoids have great potential to be used as therapeutic agents against various diseases with no or minimum adverse/side effects.