ANTHOCYANIN

Anthocyanin Extraction

Ketaren (1986) explains that the extraction is a way to get the substance of the material suspected to contain the substance. Extraction can be done in various ways. Using solvent extraction based on the solubility of the component to other components in the mixture (Suyitno, 1989). Shriner et al. (1980) stated that polar solvents will dissolve polar solutes and non-polar solvents will dissolve the non-polar solute or so-called "like dissolve like".

In the fruit or vegetable, anthocyanin pigments are generally located in the cells near the surface (Markakis, 1982). Extraction of anthocyanin pigments from plant materials commonly used solvent extractors HCl in ethanol (Gao and Mazza, 1996). HCl in ethanol plants will denaturation of the cell membrane and then dissolving the pigment anthocyanin out of the cell. Anthocyanin pigments soluble in ethanol because both polar (Broillard, 1982).
In the study Saati (2002) for the extraction of anthocyanins from flowers girlfriend water, the best solvent used is ethanol 95%. So also with the research Wijaya (2001) on the extraction of the pigment from the skin of the fruit rambutan. This is due to the level of anthocyanin polarity similar to the 95% ethanol that can dissolve well in ethanol 95%. In addition to the solvent, according Pifferi and Vaccari (1998), factors that can affect the outcome of anthocyanin extraction is the extraction time, pH and temperature extraction.

The basic structure of anthocyanin is anthocyanidins. Anthocyanidins or aglycone consists of aromatic rings (A) that binds to the heterocyclic ring (C) that contains oxygen and bound by carbon-carbon bonds in the third aromatic ring (B). When anthocyanidins found in the form of glycosides, it is called anthocyanin. Anthocyanin is very unstable and susceptible to damage. Stability is affected by several factors such as pH, temperature, chemical structure, light, solvents, enzymes, flavonoids, protein and metal ions (Castañeda-Ovando et al. 2009).

Anthocyanins are synthesized in the shikimic biosynthetic pathway and use phenylalanine as a precursor. Enzymes that work is PAL

(phenylalanineammonialyase), CHS (Chalcone synthase), CHI (Chalcone isomerase), F3H (flavonone 3-hydroxylase), F3'H (flavonoid 30-hydroxylase), DFR (dihydroflavonol reductase), LDOX (anthocyanidin synthase), GST (glutathione-S-transferase) (Guo et al.2001).

Anthocyanins in plants serves as a veil to ultraviolet B light and protect chloroplasts against high light intensity. Anthocyanins may also act as a means of transport for the monosaccharides and as an osmotic regulator during periods of drought and low temperature. In general, anthocyanin antioxidants are believed to increase the response of plants to survival in biotic or abiotic stress. In addition, anthocyanins also plays an important role in the reproduction of plants that attract pollinators that can help in the pollination of flowers (Mori et al. 2007).

Anthocyanins are considered as an important component in human nutrition as a higher antioxidant than vitamins C and E. These compounds can capture free radicals by donating a hydrogen atom phenolic. Anthocyanins can be transported in the human body and show the antitumor activity, anticancer, antiviral, anti-inflammatory, inhibiting platelet aggregation, lowering blood capillary wall permeability and boost immunity (Stintzing and Carle 2004).

Materials for Extraction Process

Ethanol

Ethanol is a clear solution, colorless, volatile, and with a distinctive odor. In high concentrations, will cause a burning sensation when in contact with skin. Ethanol is an alcohol group, where the molecule contains a hydroxyl group (-OH) bonded to carbon atoms. Ethanol made since ancient times by the fermentation of sugar. This process is widely used in the industry with raw materials such as sugar. Ethanol is soluble in water and many organic solvents (Anonymous, 2004b)).

Ethanol is toxic, but the body will set immediately. More than 90% ethanol will be processed by the liver. In the liver, the enzyme alcohol dehydrogenase converts ethanol into acetaldehyde which is still toxic.

Ethanol Acetaldehyde

But acetaldehyde will be destroyed by the enzyme aldehyde dehydrogenase that megkonversinya into acetate ions.

Ethanol acetate ions

Meanwhile, according to the FDA, the residue levels of ethanol as a solvent in the extraction

is 50 ppm.

Hydrochloric acid (HCl)

Hydrochloric acid is a solution of hydrogen chloride (HCl) in water. The color varies from colorless to light yellow. The difference in color depending on its purity. At concentrations above 10%, hydrochloric acid produces a very pungent odor. Hydrochloric acid is highly corrosive and can damage metals such as iron and steel (Wikipedia, 2002).

Steam highly concentrated acid solutions can cause irritation to the eyes, while the direct contact can cause injury to the eyes and can cause blindness. If contact with the skin will cause burns. HCl is hygroscopic, these substances are generally present in the form of aerosols in the atmosphere (NRC, 2000). According to Revilla (1998), HCl can cause partial hydrolysis of the anthocyanins of red wine.

According to Maga and Tu (1994) allowed a marinade HCl food by FAO in 1974. HCl is also used to process that requires hydrolysis on materials such as proteins and starch. HCl can also be used for the production of "corn syrup" (Maga and Tu, 1994).

Citric Acid

Citric acid is an organic acid found in many fruits and vegetables. The highest concentration found in lemon and lime which is about 8% of the dry weight of the fruit. The acidity of citric acid by its three carboxyl groups COOH which can release protons into the solution. If this happens, the resulting ion called citrate ion (Wikipedia, 2004).
At room temperature, citric acid is a white crystalline powder form. Citric acid can be found in the form of "substance" (water-free) or monohydrate that contains one molecule of water per molecule of citric acid. Citric acid is used in food safe even in large amounts. It is based on national and international food regulations. Citric acid can be metabolized and excreted from the body (Wikipedia, 2004).

Food and beverage industry are using citric acid. Selection of type of acid is due to provide the typical incorporation of desirable properties and the market are available in large quantities. Citric acid is a food additive that has varied functions. Industrial food and beverage consumed mostly to reinforce the flavor and color. Another function is to control acidity. Control of the proper pH will prevent the growth of microorganisms and act as a preservative and helps prevent browning reaction (Hui, 1992).

FLAVONOID

What are Flavonoids?

 Flavonoids (or bioflavonoids), also collectively known as Vitamin P and citrin, are a class of plant secondary metabolites. According to the IUPAC nomenclature, they can be classified into:

• ''flavonoids'', derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure (examples: quercetin, rutin).
• ''isoflavonoids'', derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure
• ''neoflavonoids'', derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure.

The three flavonoid classes above are all ketone-containing compounds, and as such, are flavonoids and flavonols. This class was the first to be termed "bioflavonoids." The terms flavonoid and bioflavonoid have also been more loosely used to describe non-ketone polyhydroxy polyphenol compounds which are more specifically termed flavanoids, flavan-3-ols, or catechins (although catechins are actually a subgroup of flavanoids).

Flavonoids are widely distributed in plants fulfilling many functions.

Flavonoids are the most important plant pigments for flower coloration producing yellow or red/blue pigmentation in petals designed to attract pollinator animals.

Flavonoids secreted by the root of their host plant help ''Rhizobia'' in the infection stage of their symbiotic relationship with legumes like peas, beans, clover, and soy. Rhizobia living in soil are able to sense the flavonoids and this triggers the secretion of Nod factors, which in turn are recognized by the host plant and can lead to root hair deformation and several cellular responses such as ion fluxes and the formation of a root nodule.

They also protect plants from attacks by microbes, fungi and insects.

Flavonoids (specifically flavanoids such as the catechins) are "the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants". Flavonols, the original bioflavonoids such as quercetin, are also found ubiquitously, but in lesser quantities. Both sets of compounds have evidence of health-modulating effects in animals which eat them.

The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Resulting from experimental evidence that they may modify allergens, viruses, and carcinogens, flavonoids have potential to be biological "response modifiers", such as anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities shown from in vitro studies.

Antioxidant activity in vitro

Flavonoids (both flavonols and flavanols) are most commonly known for their antioxidant activity in vitro.

Consumers and food manufacturers have become interested in flavonoids for their possible medicinal properties, especially their putative role in prevention of cancers and cardiovascular diseases. Although physiological evidence is not yet established, the beneficial effects of fruits, vegetables, and tea or even red wine have sometimes been attributed to flavonoid compounds rather than to known micronutrients, such as vitamins and dietary minerals.

Alternatively, research conducted at the Linus Pauling Institute and evaluated by the European Food Safety Authority indicates that, following dietary intake, flavonoids themselves are of little or no direct antioxidant value. As body conditions are unlike controlled test tube conditions, flavonoids and other polyphenols are poorly absorbed (less than 5%), with most of what is absorbed being quickly metabolized and excreted.

The increase in antioxidant capacity of blood seen after the consumption of flavonoid-rich foods is not caused directly by flavonoids themselves, but most likely is due to increased uric acid levels that result from metabolism of flavonoids. According to Frei, "we can now follow the activity of flavonoids in the body, and one thing that is clear is that the body sees them as foreign compounds and is trying to get rid of them."

Other potential health benefits

Cancer

Physiological processing of unwanted flavonoid compounds induces so-called Phase II enzymes that also help to eliminate mutagens and carcinogens, and therefore may be of value in cancer prevention. Flavonoids could also induce mechanisms that may kill cancer cells and inhibit tumor invasion.

Research also indicated that only small amounts of flavonoids may be needed for possible benefits. Taking large dietary supplements likely provides no extra benefit and may pose risks. However, certainty of neither a benefit nor a risk has been proven yet in large-scale human intervention trials.

Capillary stabilizing agents

Bioflavonoids like rutin, monoxerutin, diosmin, troxerutin and hidrosmin have potential vasoprotective proprieties still under experimental evaluation.

 

Antioxidant Activities of Flavonoids

Summary: Flavonoids are compounds found in fruits, vegetables, and certain beverages that have diverse beneficial biochemical and antioxidant effects. Their dietary intake is quite high compared to other dietary antioxidants like vitamins C and E. The antioxidant activity of flavonoids depends on their molecular structure, and structural characteristics of certain flavonoids found in hops and beer confer surprisingly potent antioxidant activity exceeding that of red wine, tea, or soy.

Flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Over 4,000 flavonoids have been identified, many of which occur in fruits, vegetables and beverages (tea, coffee, beer, wine and fruit drinks). The flavonoids have aroused considerable interest recently because of their potential beneficial effects on human health-they have been reported to have antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor and antioxidant activities. 

Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. An imbalance between antioxidants and reactive oxygen species results in oxidative stress, leading to cellular damage. Oxidative stress has been linked to cancer, aging, atherosclerosis, ischemic injury, inflammation and neurodegenerative diseases (Parkinson's and Alzheimer's). Flavonoids may help provide protection against these diseases by contributing, along with antioxidant vitamins and enzymes, to the total antioxidant defense system of the human body. Epidemiological studies have shown that flavonoid intake is inversely related to mortality from coronary heart disease and to the incidence of heart attacks. 

The recognized dietary antioxidants are vitamin C, vitamin E, selenium, and carotenoids. However, recent studies have demonstrated that flavonoids found in fruits and vegetables may also act as antioxidants. Like alpha-tocopherol (vitamin E), flavonoids contain chemical structural elements that may be responsible for their antioxidant activities. A recent study by Dr. van Acker and his colleagues in the Netherlands suggests that flavonoids can replace vitamin E as chain-breaking anti- oxidants in liver microsomal membranes. The contribution of flavonoids to the antioxidant defense system may be substantial considering that the total daily intake of flavonoids can range from 50 to 800 mg. This intake is high compared to the average daily intake of other dietary antioxidants like vitamin C (70 mg), vitamin E (7-10 mg) or carotenoids (2-3 mg). Flavonoid intake depends upon the consumption of fruits, vegetables, and certain beverages, such as red wine, tea, and beer. The high consumption of tea and wine may be most influential on total flavonoid intake in certain groups of people. 

The oxidation of low-density lipoprotein (LDL) has been recognized to play an important role in atherosclerosis. Immune system cells called macrophages recognize and engulf oxidized LDL, a process that leads to the formation of atherosclerotic plaques in the arterial wall. LDL oxidation can be induced by macrophages and can also be catalyzed by metal ions like copper. Several studies have shown that certain flavonoids can protect LDL from being oxidized by these two mechanisms. 

Antioxidant flavonoids (listed in order of decreasing potency) Quercetin (a flavonol in vegetables, fruit skins, onions)  Xanthohumol (a prenylated chalcone in hops and beer) Isoxanthohumol (a prenylated flavanone in hops and beer)  Genistein (an isoflavone in soy) Pro-oxidant flavonoids Chalconaringenin (a non-prenylated chalcone in citrus fruits)  Naringenin (a non-prenylated flavanone in citrus fruits)

The capacity of flavonoids to act as antioxidants depends upon their molecular structure. The position of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant and free radical scavenging activities. Quercetin, the most abundant dietary flavonol, is a potent antioxidant because it has all the right structural features for free radical scavenging activity. 

Recently, chalcone and flavanone flavonoids with prenyl or geranyl side chains have been identified in hops and beer by Dr. Fred Stevens and Dr. Max Deinzer at Oregon State University. Hops are used in beer for flavor. Xanthohumol (a chalcone) and isoxanthohumol and 6-prenylnaringenin (flavanones) are the major prenyl-flavonoids found in beer. Although the antioxidant activities of these compounds have not been studied, these flavonoids may be responsible for the antioxidant activity of lager beer, which is higher than that of green tea, red wine, or grape juice as reported earlier by Dr. Joe A. Vinson from the University of Scranton in Pennsylvania. Xanthohumol is found only in beer but in small concentrations. 

To assess the antioxidant activity of the prenylated flavonoids, we-in collaboration with LPI researchers-evaluated the capacity of these flavonoids to inhibit the oxidation of LDL by copper. The antioxidant properties of the prenylflavonoids were compared to those of quercetin (a flavonol), genistein (the major isoflavone in soy), chalconaringenin (a non-prenylated chalcone), naringenin (a non-prenylated flavanone), and vitamin E. The possible interaction of xanthohumol, the major prenylchalcone in beer, with vitamin E to inhibit LDL oxidation induced by copper was also examined. 

Our results showed that the prenylchalcones and prenylflavones are effective in preventing LDL oxidation  initiated by copper and that the prenylchalcones generally have greater antioxidant activity than the prenylflavanones. Xanthohumol, the major prenylchalcone in hops and beer, is a more powerful antioxidant than vitamin E or genistein. However, xanthohumol was less potent than quercetin. The potency of xanthohumol as an antioxidant is markedly increased when combined with an equivalent amount of vitamin E. 

As reported in the Journal of Agricultural and Food Chemistry, we also found that the prenyl group plays an important role in the antioxidant activity of certain flavonoids. A flavonoid chalcone (chalconaringenin) and a flavanone (naringenin) with no prenyl groups act as pro-oxidants, i.e. they promote rather than limit the oxidation of LDL by copper. However, adding a prenyl group to these flavonoid molecules counteracted their pro-oxidant activities. 

Our work reveals that there are unique flavonoids in hops and beer that may be potentially useful in the preventionof human disease attributed to free radical damage. The observation that prenyl groups are important in conferring antioxidant activity to certain flavonoids may lead to the discovery or synthesis of novel prenylated flavonoids as preventive or therapeutic agents against human diseases associated with free radicals. Our encouraging results with xanthohumol suggest that this prenylchalcone should be further studied for its antioxidant action and protective effects against free radical damage in animals and humans. Preliminary studies have shown that xanthohumol is absorbed from the digestive tract in rats, and more studies are needed to evaluate the bioavailability of these interesting flavonoids in people. 

Further studies are also needed to establish the safety of xanthohumol or other flavonoids for use as dietary supplements since high doses of these compounds may produce adverse effects in humans, according to recent findings by Dr. Martyn Smith, professor of toxicology, University of California at Berkeley.