Wednesday, April 30, 2008

Structurally related antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxyg

Flavonoids exist extensively in plants and Chinese herbs, and several biological effects of flavonoids have been demonstrated. The antitumor effects in colorectal carcinoma cells (HT29, COLO205, and COLO320HSR) of eight flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, 7-OH flavanone, naringenin, nargin, and taxifolin were investigated. Results of the MTT assay indicate that 2'-OH flavanone showed the most potent cytotoxic effect on these three cells, and cell death induced by 2'-OH flavanone was via the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, all characteristics of apoptosis. Induction of caspase 3 protein processing and enzyme activity associated with cleavage of poly(ADP-ribose) polymerase (PARP) was identified in 2'-OH flavanone-treated cells, and a peptidyl inhibitor (Ac-DEVD-FMK) of caspase 3 attenuated the cytotoxicity of 2'-OH flavanone in COLO205 and HT-29 cells. Elevation of p21 (but not p53) and a decrease in Mcl-1 protein were found in 2'-OH flavanone-treated COLO205 and HT-29 cells. Elevation of intracellular reactive oxygen species (ROS) was detected in 2'-OH flavanone-treated cells by the 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) assay, and ROS scavengers including 4,5-dihydro-1,3-benzene disulfonic acid (tiron), catalase, superoxide dismutase (SOD), and pyrrolidine dithiocarbamate (PDTC) suppressed the 2'-OH flavanone-induced cytotoxic effect. Subcutaneous injection of COLO205 induced tumor formation in nude mice, and 2'-OH flavanone showed a significant inhibitory effect on tumor formation. The appearance of apoptotic cells with H&E staining, and an increase in p21, but not p53, protein by immunohistochemistry were observed in tumor tissues under 2'-OH flavanone treatment. Primary tumor cells (COLO205-X) derived from a tumor specimen elicited by COLO205 were established, and 2'-OH flavanone showed an significant apoptotic effect in COLO205-X cells in accordance with the appearance of DNA ladders, caspase 3 protein processing, PARP protein cleavage, and increasing p21 protein. These results revealed in vitro, ex vivo, and in vivo antitumor activities of 2'-OH flavanone via apoptosis induction, and indicates that 2'-OH flavanone is an active compound worthy of development for cancer chemotherapy.

Structural requirements for mutagenicity of flavonoids upon nitrosation. A structure—activity study

A wealth of promutagens can damage DNA provided metabolic/chemical reactions take place before an ultimate mutagen is formed. Nitrosation reactions are amongst those chemical reactions which may take place to render some chemical reactions which may take place to render some chemical classes of promutagens as ultimate mutagens. Flavonoids are amongst chemicals which can be rendered mutagenic upon nitrosation. In this study, 22 flavonoids were tested in the Ames assay for their mutagenicity upon nitrosation and the respective structural requirements for nitrosation-dependent mutagenicity were established. Nitrosatable chemicals present in the diet may play a role in the aetiology of gastric cancer and flavonoids are amongest the common molecules present in a variety of food items. Flavonoids such as quercetin and catechin were predicted to be non-mutagenic upon nitrosation by the CASE methodology and were shown in this study to be strong nitrosatable mutagens.

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.

Rutin-induced beta-glucosidase activity in Streptococcus faecium VGH-1 and Streptococcus sp. strain FRP-17 isolated from human feces: formation of the

A fecal isolate, Streptococcus sp. strain FRP-17, and strain VGH-1 of Streptococcus faecium were shown to contain beta-glucosidases which converted rutin (quercetin-3-O-beta-D-glucose-alpha-L-rhamnose) to quercetin and were active against o-nitrophenyl-beta-D-glucose. The activity against rutin could be measured by increased mutagenicity in the Ames assay or visualized on thin-layer chromatography plates. In both organisms, the beta-glucosidase activities were inducible by the addition of rutin to the growth media. Several closely related strains of Streptococcus spp. lacked any beta-glucosidase activity. In cell preparations of the active organisms, activities with rutin and o-nitrophenyl-beta-D-glucose were optimal at pH 6.8 and could be enhanced by increasing the ionic strength of the assay system. At low ionic strengths, both quercetin and a new product (intermediate between the polarities of rutin and quercetin) were formed by the incubation of rutin with cell preparations of either active organism. This product disappeared with increased ionic strength, suggesting that it may be a reaction intermediate, quercetin-3-O-beta-D-glucose. These results suggest that the beta-glucosidase active against rutin and that active against o-nitrophenyl-beta-D-glucose are the same.

Potential mutagenic activity of some vitamin preparations in the human gut

Rutin is a nonmutagenic flavonol glycoside, whereas its aglycone quercetin is mutagenic. Cell-free preparations from fecal cultures (fecal preparations) contain a beta-glucosidase that, when incubated with rutin, hydrolyzes it to quercetin. This activity can be further induced when rutin is added to the fecal culture from which the cell-free preparation is made. When vitamin pills that contain rutin are added to the cultures, this induction is equally effective. The vitamin extracts by themselves, like rutin, were nonmutagenic; however, when the vitamin extracts were incubated with fecal preparations containing induced beta-glucosidase, a great increase in mutagenicity was observed.

Fecalase: a model for activation of dietary glycosides to mutagens by intestinal flora

Many substances in the plant kingdom and in man's diet occur as glycosides. Recent studies have indicated that many glycosides that are not mutagenic in tests such as the Salmonella test become mutagenic upon hydrolysis of the glycosidic linkages. The Salmonella test utilizes a liver homogenate to approximate mammalian metabolism but does not provide a source of the enzymes present in intestinal bacterial flora that hydrolyze the wide variety of glycosides present in nature. We describe a stable cell-free extract of human feces, fecalase, which is shown to contain various glycosidases that allow the in vitro activation of many natural glycosides to mutagens in the Salmonella/liver homogenate test. Many beverages, such as red wine (but apparently not white wine) and tea, contain glycosides of the mutagne quercetin. Red wine, red grape juice, and tea were mutagenic in the test when fecalase was added, and red wine contained considerable direct mutagenic activity in the absence of fecalase. The implications of quercetin mutagenicity and carcinogenicity are discussed.

Mutagenecity of Plant Flavanoids

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.

Flavonoids





Flavonoid



Molecular structure of the flavone backbone (2-phenyl-1,4-benzopyrone)
The term flavonoid refers to a class of plant secondary metabolites. According to the IUPAC nomenclature,[1] they can be classified into:
flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure
isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure
neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure.
Flavonoids are most commonly known for their antioxidant activity. However, it is now known that the health benefits they provide against cancer and heart disease are the result of other mechanisms. Flavonoids are also commonly referred to as bioflavonoids in the media – the terms are largely equivalent and interchangeable, for most flavonoids are biological in origin.


Biosynthesis
Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA. This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones, which contain two phenyl rings (see polyphenols). Conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield flavanonesdihydroflavonolsanthocyanins. Along this pathway, many products can be formed, including the flavonols, flavan-3-ols, proanthocyanidins (tannins) and a host of other polyphenolics.



Biological effects
Flavonoids are widely distributed in plants fulfilling many functions including producing yellow or red/blue pigmentation in flowers and protection from attack by microbes and insects. 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. Flavonoids have been referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory , anti-microbial and anti-cancer activity.
Consumers and food manufacturers have become interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiovascular disease. The beneficial effects of fruit, vegetables, and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins.

Health benefits aside from antioxidant values
In 2007, research conducted at the Linus Pauling Institute and published in Free Radical Biology and Medicine indicates that inside the human body, flavonoids themselves are of little or no direct antioxidant value.Unlike in the controlled conditions of a test tube, flavonoids are poorly absorbed by the human body (less than 5%), and most of what is absorbed is quickly metabolized and excreted from the body.
The huge increase in antioxidant capacity of blood seen after the consumption of flavonoid-rich foods is not caused directly by the flavonoids themselves, but most likely is due to increased uric acid levels that result from expelling flavonoids from the body. 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. But this process of gearing up to get rid of unwanted compounds is inducing so-called Phase II enzymes that also help eliminate mutagens and carcinogens, and therefore may be of value in cancer prevention... Flavonoids could also induce mechanisms that help kill cancer cells and inhibit tumor invasion."
Their research also indicated that only small amounts of flavonoids are necessary to see these medical benefits. Taking large dietary supplements provides no extra benefit and may pose some risks.
Diarrhea
A study done at Children's Hospital & Research Center Oakland, in collaboration with scientists at Heinrich Heine University in Germany, has shown that epicatechin, quercetin and luteolin can inhibit the development of fluids that result in diarrhea by targeting the intestinal cystic fibrosis transmembrane conductance regulator Cl– transport inhibiting cAMP-stimulated Cl– secretion in the intestine.[6]

Important flavonoids
Quercetin




Quercetin
Quercetin is a flavonoid and, to be more specific, a flavonol (see below), that constitutes the aglycone of the glycosides rutin and quercitrin. In studies, quercetin is found to be the most active of the flavonoids, and many medicinal plants owe much of their activity to their high quercetin content. Quercetin has demonstrated significant anti-inflammatory activity because of direct inhibition of several initial processes of inflammation. For example, quercetin inhibits both the production and release of histamine and other allergic/inflammatory mediators. In addition, it exerts potent antioxidant activity and vitamin C-sparing action. It has been found to be anti-cancer. Quercetin can be found in the herbal products based on Hawthorn, which are used for acute symptoms of Congestive Heart Failure.

Epicatechin




Epicatechin (EC)
Epicatechin improves blood flow and thus seems good for cardiac health. Cocoa, the major ingredient of dark chocolate, contains relatively high amounts of epicatechin and has been found to have nearly twice the antioxidant content of red wine and up to three times that of green tea in in-vitro tests.[7] [8] But in the test outlined above it now appears the beneficial antioxidant effects are minimal as the antioxidants are rapidly excreted from the body.

Oligomeric proanthocyanidins
Proanthocyanidins extracts demonstrate a wide range of pharmacological activity. Their effects include increasing intracellular vitamin C levels, decreasing capillary permeability and fragility, scavenging oxidants and free radicals, and inhibiting destruction of collagen, the most abundant protein in the body.

Important dietary sources

Good sources of flavonoids include all citrus fruits, berries, ginkgo biloba, onions[citation needed], parsley[citation needed], legumes[citation needed], tea (especially white and green tea), red wine, seabuckthorn, and dark chocolate (with a cocoa content of seventy percent or greater).
Citrus


Grapefruit, a type of Citrus
The citrus bioflavonoids include hesperidin (a glycoside of hesperetin), quercitrin, rutin (two glycosides of quercetin), and tangeritin. In addition to possessing antioxidant activity and an ability to increase intracellular levels of vitamin C, rutin and hesperidin exert beneficial effects on capillary permeability and blood flow. They also exhibit some of the anti-allergy and anti-inflammatory benefits of quercetin. Quercetin can also inhibit reverse transcriptase, part of the replication process of retroviruses.[9] The therapeutical relevance of this inhibition has not been established. Hydroxyethylrutosides (HER) have been used in the treatment of capillary permeability, easy bruising, hemorrhoids, and varicose veins.

Ginkgo
Leaf extract from the Ginkgo tree is widely marketed as an herbal supplement. The active ingredients are flavoglycosides.

Tea

Bai Hao Yinzhen from Fuding in Fujian Province, widely considered the best grade of white tea
Green tea flavonoids are potent antioxidant compounds, thought to reduce incidence of cancer and heart disease. The major flavonoids in green tea are the kaempferol and catechins (catechin, epicatechin, epicatechin gallate, and epigallocatechin gallate (EGCG)).
In producing teas such as oolong tea and black tea, the leaves are allowed to oxidize, during which enzymes present in the tea convert some or all of the catechins to larger molecules. White tea is the least processed of teas and is shown to present the highest amount of catechins known to occur in camellia sinensis. However, green tea is produced by steaming the fresh-cut leaf, which inactivates these enzymes, and oxidation does not significantly occur.

Wine
Grape skins contain significant amounts of flavonoids as well as other polyphenols[10]. Both red and white wine contain flavonoids; however, since red wine is produced by fermentation in the presence of the grape skins, red wine has been observed to contain higher levels of flavonoids, and other polyphenolics such as resveratrol.

Dark chocolate
Flavanoids exist naturally in cacao, but because they can be bitter, they are often removed from chocolate, even the dark variety[11].

Subgroups
Over 5000 naturally occurring flavonoids have been characterized from various plants. They have been classified according to their chemical structure, and are usually subdivided into the following subgroups (for further reading see [3]):
Flavones
Flavones are divided into four groups:[12]
Flavones
Flavones use the 2-phenylchromen-4-one skeleton.
Examples: Luteolin, Apigenin, Tangeritin
Flavonols
Flavonols or 3-hydroxyflavones use the 3-hydroxy-2-phenylchromen-4-one skeleton.
Examples: Quercetin, Kaempferol, Myricetin, Fisetin, Isorhamnetin, Pachypodol, Rhamnazin
Flavanones
Flavanones use the 2,3-dihydro-2-phenylchromen-4-one skeleton.
Examples: Hesperetin, Naringenin, Eriodictyol, Homoeriodictyol.
3-Hydroxyflavanones or 2,3-dihydroflavonols
3-Hydroxyflavanones use the 3-hydroxy-2,3-dihydro-2-phenylchromen-4-one skeleton.
Examples: Dihydroquercetin, Dihydrokaempferol

Isoflavones
Isoflavones
Isoflavones use the 3-phenylchromen-4-one skeleton.
Examples: Genistein, Daidzein, Glycitein

Flavan-3-ols and Anthocyanidins
Flavan-3-ols
Flavan-3-ols use the 2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton.
Examples: Catechins (Catechin (C), Gallocatechin (GC), Catechin 3-gallate (Cg), Gallocatechin 3-gallate (GCg)), Epicatechins (Epicatechin (EC), Epigallocatechin (EGC), Epicatechin 3-gallate (ECg), Epigallocatechin 3-gallate (EGCg))
Anthocyanidins
Anthocyanidins are the aglycones of anthocyanins. Anthocyanidins use the flavylium (2-phenylchromenylium) ion skeleton
Examples: Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin

Availability through microorganisms
A number of recent research articles have demonstrated the efficient production of flavonoid molecules from genetically-engineered microorganisms[13][14].

References
1. ^ Flavonoids (isoflavonoids and neoflavonoids)., IUPAC Compendium of Chemical Terminology
2. ^ a b c d "Studies force new view on biology of flavonoids", by David Stauth, EurekAlert!. Adapted from a news release issued by Oregon State University. URL accessed .
3. ^ a b c Ververidis Filippos; Trantas Emmanouil, Douglas Carl, Vollmer Guenter, Kretzschmar Georg, Panopoulos Nickolas (October 2007). "Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: Chemical diversity, impacts on plant biology and human health". Biotechnology Journal 2 (10).
4. ^ Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer. Yamamoto and Gaynor 107 (2): 135 -- Journal of Clinical Investigation.
5. ^ Lotito SB, Frei B (2006). "Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon?". Free Radic. Biol. Med. 41 (12): 1727–46. PMID 17157175.
6. ^ Schuier, Maximilian; Helmut Sies, Beate Illek, and Horst Fischer (October 2005). "Cocoa-Related Flavonoids Inhibit CFTR-Mediated Chloride Transport across T84 Human Colon Epithelia" (PDF). Journal of Nutrition 135 (10).
7. ^ J. Agric.Food Chem. (2003) 51: Lee et al.
8. ^ Cocoa nutrient for 'lethal ills'. BBC News.
9. ^ Spedding, G., Ratty, A., Middleton, E. Jr. (1989) Inhibition of reverse transcriptases by flavonoids. Antiviral Res 12 (2), 99-110. PMID
10. ^ James A. Kennedy, Mark A. Matthews, and Andrew L. Waterhouse, Effect of Maturity and Vine Water Status on Grape Skin and Wine Flavonoids Am. J. Enol. Vitic. 53:4:) (abstract)
11. ^ Editorial. 'The devil in the dark chocolate.' The Lancet. 2007; 370:2070 [1]
12. ^ Phenolics:figure 4
13. ^ Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S. Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Appl Environ Microbiol. 2003 May;69(5): PMID
14. ^ Ververidis Filippos; Trantas Emmanouil, Douglas Carl, Vollmer Guenter, Kretzschmar Georg, Panopoulos Nickolas (October 2007). "Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part II: Reconstruction of multienzyme pathways in plants and microbes". Biotechnology Journal 2 (10).

Antioxidant Activities of Flavanoids




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.

Antiglycemic & Antilipemic Effect of Aqueous Extract of Cissus sicyoides







Abstract

Cissus sicyoides (Vitaceae) is a medicinal plant popularly known in Brazil as "cipó-pucá, anil-trepador, cortina, and insulina". The plant is used in several diseases, including rheumatism, epilepsy, stroke and also in the treatment of diabetes. In the present work, we studied the hypoglycemic and anti-lipemic effects of the aqueous extract prepared from fresh leaves of the plant (AECS), in the model of alloxan-induced diabetes in rats. In addition, hepatic enzyme levels were also determined.
Results
Results showed that the daily treatment of diabetic rats with AECS for 7 days (100 and 200 mg/kg, p.o.) significantly decreased blood glucose levels in 25 and 22% respectively, as compared to the same groups before AECS treatment. No significant changes were seen in control diabetic rats before (48 h after alloxan administration) and after distilled water treatment. While no changes were seen in total cholesterol levels, a significant decrease was observed in plasma triglyceride levels, in the alloxan-induced diabetic rats after AECS treatment with both doses, as compared to the same groups before treatment. Significant decreases in blood glucose (25%) and triglyceride levels (48%) were also observed in the alloxan-induced diabetic rats after 4 days treatment with AECS (200 mg/kg, p.o.). Aspartate (AST) and alanine (ALT) aminotransferases levels, in diabetic controls and AECS-treated rats, were in the range of reference values presented by normal rats.

Conclusions
The results justify the popular use of C. sicyoides, pointing out to the potential benefit of the plant aqueous extract (AECS) in alternative medicine, in the treatment of type 2 diabetes mellitus.

There are several species of medicinal plants popularly used in the treatment of diabetes mellitus, a disease responsible for serious complications, affecting a large number of people worldwide. Cissus sicyoides (L.) belongs to the Vitaceae family, and is a medicinal plant popularly used in Brazil in several diseases, such as epilepsy, stroke, as well as in abscesses and in the treatment of diabetes. Anti-inflammatory and anti-rheumatic activities are also attributed to the plant. Other species (C. succicaulis) presented anti-ulcer activity in the model of ethanol-induced gastric ulcer in rats. A survey carried out in Nigeria identified C. populnea, among other species, as being largely used for the treatment of trypanosomiasis . The aqueous extract from C. rubiginosa was shown to possess prominent antibacterial activity, which supports the ethno-medical use of this plant as an anti-diarrhea agent.
Earlier work showed that the aqueous extract from C. sicyoides contracts isolated guinea pig aortic rings, in a dose dependent manner. These authors concluded that the extract acts at the membrane level, increasing the calcium entry through the membrane as well as acting in the internal calcium deposits, such as the sarcoplasmic reticulum. In a recent work , the aqueous extract from C. sicyoides was shown to present an anti-inflammatory effect, as determined by the carrageenan-induced rat paw edema (a model for general inflammation), and in the mouse ear edema (a model of topical inflammation). These authors also observed a decrease in the level of myeloperoxidase in tissue samples from the inflammation area. While Saenz et al. demonstrated a cytostatic activity in C. sicyoides against Hep-cells, others did not detect any anti-viral activity against the influenza type A virus, in this species. According to these authors, the main chemical components of the aerial part of C. sicyoides were tannins, steroid-triterpenes, aminoacids, lipids and flavonoids.
Another phytochemical analysis of the plant showed the presence of alkaloids, triterpenes and/or steroids, flavonoids, tannins and saponins. Other compounds, namely onocer-7-ene 3 alpha 21 beta-diol, delta-amyrin, delta-amyrone, and 3,3', 4,4'-tetrahydroxybiphenyl were also isolated from C. quadrangularis. These compounds are used for plant extract standardization purposes. An earlier study detected the presence of steroids, terpenes, quinones and phenolic compounds in C. sicyoides, while others showed no alkaloid in the plant, and cyanidins only in the fruit . Recently, a new coumarin glycoside, 5,6,7,8 tetrahydroxycoumarin-5-beta-xylo-pyranoside was isolated from the aerial parts of C. sicyoides, together with a known coumarin, sabadin, two flavonoids, kaempferol 3-rhamnoside and quercetin 3-rhamnoside, and two steroids, sitosterol and 3-beta-O-beta-glupyranosylsitosterol.
We showed that the aqueous extract from C. sicyoides (AECS) presents an anti-nociceptive activity, as demonstrated by the tests of acetic acid-induced abdominal contractions, formalin, and hot plate in mice. Besides, AECS also has anticonvulsant properties as evidenced in the models of pentylenetetrazol and strychnine-induced convulsions in mice. Although the plant is popularly used, in Brazil, for the treatment of diabetes mellitus, there are only two reports in the literature on this subject. Furthermore, several plant constituents, including flavonoids (also detected in AECS), are known to decrease triglycerides and transaminases, which are usually increased in the serum of diabetic patients . Thus, in order to validate the popular use of the plant as an anti-diabetic, the objectives of the present work were to study the possible effects of the aqueous extract from leaves of C. sicyoides on glycemia, lipid profile, as well as on levels of ALT and AST hepatic enzymes, in the model of alloxan-induced diabetes, in rats.
Results
Table 1 shows the results of blood glucose values in alloxan-induced diabetic rats, after the daily treatment with the aqueous extract of C. sicyoides (AECS 100 and 200 mg/kg, p.o.), for 7 days. Significant decreases in the blood glucose levels, of 25 and 22% respectively, were observed in the groups treated with both doses of AECS, as compared to the same group before treatment. No significant changes were seen in diabetic (48 h after administration with alloxan) controls, before and after distilled water treatment.


Table 1
Effects of the aqueous extract from leaves of Cissus sicyoides (AECS) on blood glucose levels in alloxan-induced diabetic rats
Group
Glucose levels (mg/dL)
(Before treatment)
(After treatment)
Control (11)
313.1 ± 15.04
281.0 ± 15.07
AECS
100 mg/kg, p.o. (20)
300.1 ± 10.47
224.7 ± 13.13*
200 mg/kg, p.o. (13)
292.7 ± 8.05
227.8 ± 12.90*
Values are means ± SEM of the number of animals (in parenthesis). The blood was collected from the orbital sinus, 48 h after alloxan administration, 60 mg/kg, i.v., before and 1 h after the last administration of distilled water (control group) or of AECS, administered daily for 7 days. *p < href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0gBac1b_WrNRIZKZPWlzPQNzDw57MLDxSj49s1ArO&reftype=extlink-entrez-taxonomy&artid=443509&iid=10729&jid=56&FROM=Article%7CBody&TO=Entrez%7CTerm%7CTaxonomy&article-id=443509&journal-id=56&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=289665" target="pmc_ext">Cissus sicyoides (AECS) on blood total cholesterol and triglyceride levels in alloxan-induced diabetic rats
Group
Cholesterol (mg/dL) Before
Cholesterol (mg/dL) After
Triglycerides (mg/dL) Before
Triglycerides (mg/dL) After
Control (7)
87.0 ± 5.15
74.0 ± 2.93
244.0 ± 29.65
286.8 ± 58.23
AECS
100 mg/kg, p.o. (20)
76. 8 ± 3.94
78.1 ± 5.18
222.1 ± 26.40
111.5 ± 9.51*
200 mg/kg, p.o. (13)
86.4 ± 2.96
73.2 ± 3.27
294.0 ± 32.89
171.1 ± 19.13*
Values are means ± SEM of the number of animals (in parenthesis). The blood was collected from the orbital sinus, 48 h after alloxan administration, 60 mg/kg, i.v., before and 1 h after the last administration of distilled water (control group) or AECS, administered daily for 7 days. *p < href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0TtPPttqr6D9zZFvKqYV_GEwDXZH5g1BPW3hCjkD9&reftype=extlink-entrez-taxonomy&artid=443509&iid=10729&jid=56&FROM=Article%7CBody&TO=Entrez%7CTerm%7CTaxonomy&article-id=443509&journal-id=56&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=289665" target="pmc_ext">Cissus sicyoides (AECS) on biochemical parameters in the blood from alloxan-induced diabetic rats
Parameter
Group
Control
AECS 100 mg/kg
AECS 200 mg/kg
Glucose (mg/dL)
Before
264.8 ± 12.66
252.2 ± 8.44
250.8 ± 10.74
After
248.1 ± 11.65
218.1 ± 16. 21
188.0 ± 16.22*
Cholesterol (mg/dL)
Before
58.4 ± 3.92
56.6 ± 3.19
63.0 ± 2.55
After
55.4 ± 2.91
50.1 ± 5.89
54.6 ± 2.29
Triglycerides (mg/dL)
Before
169.8 ± 8.61
256.4 ± 24.60
165.4 ± 12.87
After
124.1 ± 8.02 *
132.5 ± 9.02 *
86.4 ± 7.56 *
ALT (IU/l)
Before
20.0 ± 1.33
28.6 ± 2.15
20.8 ± 1.62
After
34.7 ± 2.89 *
41.2 ± 2.71 *
29.8 ± 2.22
AST (IU/l)
Before
60.5 ± 3.90
62.2 ± 5.52
75.9 ± 4.74
After
85.5 ± 4.35 *
89.3 ± 7.03 *
88.3 ± 3.73
Values are means ± SEM of 8 to 18 animals per group. The blood was collected from the orbital sinus, 48 h after the alloxan administration, 60 mg/kg, i.v., before and 1 h after the last administration of distilled water (controls) or AECS, daily for 4 days. *p < href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0Yh4uc8yC3l8uTfuva50bXoOno9rwh3aZmIi37QSe&reftype=extlink-entrez-taxonomy&artid=443509&iid=10729&jid=56&FROM=Article%7CBody&TO=Entrez%7CTerm%7CTaxonomy&article-id=443509&journal-id=56&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=289665" target="pmc_ext">Cissus sicyoides (AECS)
Group
Body weight (g)
% Death
Before
After
Control
218.3 ± 4.30 (15)
169.3 ± 5.41 (10)*
33
AECS
100 mg/kg, p.o.
288.8 ± 6.63 (15)
264.3 ± 14.45 (8)
47
200 mg/kg, p.o.
243.7 ± 7.63 (15)
208.7 ± 9.61 (7)
53
Values are means ± SEM. Animals were administered daily with distilled water (controls) or AECS, for 7 days (see Methods, for details). *p < href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0KEqvhBlNS4-R_RbXNptudujSpVYKNLfz_JZV2kVT&reftype=extlink-entrez-taxonomy&artid=443509&iid=10729&jid=56&FROM=Article%7CBody&TO=Entrez%7CTerm%7CTaxonomy&article-id=443509&journal-id=56&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=289665" target="pmc_ext">Cissus sicyoides (AECS)
Parameter
Result
Reference Values
Glucose (mg/dL)
88.5 ± 1.55
63.3 – 86.4
Total cholesterol (mg/dL)
54.3 ± 2.74
51.1 – 71.5
Triglycerides (mg/dL)
54.2 ± 0.87
42.9 – 64.4
AST (IU/L)
78.0 ± 4.46
72.6 – 105.8
ALT (IU/L)
29.4 ± 1.20
19.7 – 31.7
Values are means ± SEM of 8 normal rats treated daily with 100 mg/kg, p.o., of AECS, for 7 days. In Reference Values, the intervals are means ± SD of 22 animals (male Wistar rats), from the Animal House of the Faculty of Medicine of Juazeiro do Norte – FMJ.

Table 6
Phytochemical profile of the fresh leaves from Cissus sicyoides
Chemical Group
Cissus sicyoides
Hydrolyzable tannin
+
Coumarin
-
Flavonoid
+
Saponin
-
Anthraquinone
-
Alkaloid
-
Steroid
-
For the phytochemical profile, specific reactions for chemical groups or thin layer chromatography (TLC) were performed. + = presence; - = absence.

The phytochemical analysis of the aqueous extract prepared from the fresh leaves of C. sicyoides revealed the presence of flavonoids and hydrolyzable tannins, and the absence of coumarin, anthraquinones, alkaloids, saponins and steroids (Table 6).
Discussion
In the present work, we investigated the hypoglycemic and anti-lipemic effects of the aqueous extract from C. sicyoides in the model of alloxan-induced diabetes in rats. Alloxan causes diabetes through its ability to destroy the insulin-producing beta cells of the pancreas. In vitro studies have shown that alloxan is selectively toxic to pancreatic beta cells, leading to the induction of cell necrosis. The cytotoxic action of alloxan is mediated by reactive oxygen species, with a simultaneous massive increase in cytosolic calcium concentration, leading to a rapid destruction of beta cells.
We showed that the aqueous extract from C. sicyoides (AECS, 100 and 200 mg/kg), administered orally for 7 days, produced significant decreases in plasma glucose in the model of alloxan-induced diabetes in rats, by comparing the results before and after the AECS treatment. A similar effect was observed after a shorter (4 days) treatment with the higher dose of AECS (200 mg/kg). However, AECS had no effect on glycemia in normal rats. Besides, no significant decrease was detected in diabetic animals administered with distilled water, for the same period of time (controls). Although the alloxan group (controls) presented a dramatic body weight reduction, weight losses were lower in the alloxan plus AECS-treated group, indicating another potential benefit of AECS.
Our findings that the extract of C. sicyoides reduces the glycemia of alloxan-induced diabetic rats, but had no effect on that of normal rats, are in agreement with a very recent work. These authors also showed that the leaf decoction from C. sicyoides significantly reduced the intake of both food and fluid, and the volume of urine excreted, as well as the levels of blood glucose, urinary glucose and urinary urea, as compared to controls. Others showed that, after oral administration, the leaf extract from C. sicyoides presented a potential hypoglycemic activity in hereditary diabetic mice, normal rats and rats with streptozotocin-induced diabetes. The authors showed that the extract, administered for 4 weeks, significantly lowered the mean plasma glucose level of mice, under feeding conditions. Besides, a single oral administration significantly lowered the plasma glucose level, 1 h after sucrose loading, in normal rats and rats with streptozotocin-induced diabetes.
However, Beltrame et al., 2001 failed to show any anti-diabetic activity in the hydroalcoholic extract obtained from the leaves of C. sicyoides, which instead intensified the decreased glucose tolerance promoted by dexamethasone, in rats. It is worthwhile to point out that the plant is popularly used as a decoction, which is similar to the aqueous extract used in the present work. Active principles responsible for the hypoglycemic activity presented by C. sicyoides are possibly better extracted by aqueous and more polar solvents.
We found that the aqueous extract is rich in carbohydrate type compounds, which are easily precipitated by ethanol (results not shown). Besides, recently a new coumarin glycoside was obtained from the aerial parts of C. sicyoides. Glycosides are sugar derivatives, and an overwhelming number of glycosides occur in nature, mainly in plants, and such compounds have received much attention for possessing a variety of biological activities. Thus, flour prepared from C. rotundifolia was shown to contain significant amounts of non-starch polysaccharides, the major fraction of which was water-soluble. These authors verified that, in humans, the species elicited significant reductions in plasma glucose levels, at post-prandial time points and for the area-under-the-curve (AUC) values. Significant reductions in plasma insulin levels, at various post-prandial time points and for AUC values, were also seen after C. rotundifolia administration. Water-soluble non-starch polysaccharides are certainly one of the components responsible for the glucose and insulin lowering effects.
Although, in the present work, we showed no changes in total cholesterol levels, a significant decrease was observed in plasma triglyceride levels in the alloxan-induced diabetic rats, after 4 and 7-day treatments with AECS administered orally. On the other hand, another work did not find any alteration in lipid metabolism, nor in levels of hepatic glycogen in streptozotocin-diabetic rats, after C. sicyoides treatment. According to them, these results indicate that the mechanism responsible for the improvement in carbohydrate metabolism, observed in animals treated with C. sicyoides decoction, does not involve inhibition of glycogenolysis and/or stimulation of glycogenesis.
We also measured plasma levels of AST and ALT, hepatic enzyme markers, and showed that these enzyme levels were significantly increased after AECS treatment. However, these effects were also observed in controls and, under both conditions, the values are in the range of those shown by our normal control rats. Elevated activities of serum aminotransferases are a common sign of liver disease, and are more frequently observed among people with diabetes, than in the general population. Furthermore, diabetic complications such as limited joint mobility, retinopathy and neuropathy are associated with liver enzyme activities, independently of alcohol consumption, body mass index, and metabolic control of diabetes. Besides, it has been shown that the alloxan injection causes a significant increase in the activity of several enzymes, such as beta-glucuronidase, N-acetyl-beta-glucosaminidase, lysosomal acid phosphatase, leucine aminopeptidase, and cathepsin D. Moreover, the activities of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes, among others, have been used as indicators of tissue toxicity in experimental diabetes. For instance, Mori et al., 2003] reported that levels of AST, ALT and alkaline phosphatase (ALP) were higher in streptozotocin-induced diabetic rats, over a 53-day period.
The duration of the alloxan-induced diabetic state is still a matter of concern. An earlier work showed that alloxan-induced diabetes of 4-day duration produced metabolite changes in the brain, compatible with severe reduction of cerebral metabolism and reduced phosphofructokinase activity. Alloxan-treated animals were also severely dehydrated. A more recent work showed disease-related abnormalities, such as ATPase activities in sciatic nerves, from rats with alloxan-induced diabetes of various durations (2 weeks up to 6 months). Others observed high glucose levels even after 2 weeks of alloxan injection. Under the conditions of the present investigation, diabetes was well maintained up to the 7th-day treatment with AECS, which began 48 h after a single alloxan injection.
Levels of glucose, insulin, triglycerides and total cholesterol were also shown to increase in experimental models of chemically-induced diabetes, including that with alloxan. A recent work reported the reversibility of the diabetic state, 12 days after the alloxan injection, as demonstrated by the reduction of glucose and triglyceride concentrations, and a positive reaction of the anti-insulin antibodies in the pancreatic tissue. In the present investigation, we followed the hepatic enzymes and lipid profile, for 4 and 7 days, when the diabetic state was still well maintained.
AECS administration to normal animals caused no changes in any of the measured parameters, similarly to results observed by others [16]. Their data and ours suggest that the mode of action of AECS in diabetic animals does not resemble those of sulfonyl ureas or insulin. It may, however, act in a similar way to biguanides, via the inhibition of gluconeogenesis.
Conclusions
In conclusion, we showed anti-diabetic and anti-lipemic effects of the aqueous extract prepared from the fresh leaves of C. sicyoides. The plant leaves are rich in polysaccharide type compounds, and some of the effects observed with the AECS were also demonstrated with the carbohydrate fraction. Our results suggest that, at least in part, AECS effects are due to this plant constituents.
Methods

Plant material and preparation of the aqueous extract
The plant was collected at the city of Barbalha, state of Ceará, Brazil, and identified by Prof. A. Fernandes from the Biology Department, of the Federal University of Ceará (UFC). The voucher (No. 32.240-EAC) is deposited at the Prisco Bezerra Herbarium, of the UFC. Three hundred grams of fresh leaves from C. sicyoides were blended with approximately 2 L of distilled water, in order to prepare an aqueous extract. The extract was heated for about 2 h at 60°C, filtered in a double layer of gauze, and reduced at 60°C to half of its original volume. A one milliliter sample was completely evaporated in the oven, and the solid residue was weighted, giving a final concentration of 20 to 30 mg per mL. The extract was kept at -20°C until use.

Animals
Male Wistar rats (250–300 g) from the Animal House of the Faculty of Medicine of Juazeiro do Norte (FMJ) were used. Animals were maintained in plastic cages and 12 h light/dark cycle, with free access to food and water. Experiments were performed according to the Guide for the care and use of laboratory animals, from the US Department of Health and Human Services.

Experimental protocol
Diabetes was induced by the intravenous administration of alloxan (60 mg/kg), after anesthesia with ethyl ether. Forty-eight hours later, the blood (1 mL) was collected from the orbital sinus into tubes containing separator gel (from Vacuette, Brazil). The serum was separated by centrifugation at 3,500 rpm for 10 min, and immediately used for biochemical assays. Only animals presenting glycemic levels equal to or above 200 mg/dL were submitted to treatments, which consisted of a daily administration of the AECS (100 and 200 mg/kg, p.o.) or an equivalent volume of distilled water (controls), for 4 or 7 days. The oral treatments (by gavage) of all groups were carried out at the same time (in the morning) and under the same conditions. One hour after the last administration, the blood was collected again for biochemical measurements. In another set of experiments, non-diabetic normal rats were administered daily with AECS (100 mg/kg, p.o.) for 7 days and, 1 h after the last administration, the blood was collected for biochemical measurements as described above.

Biochemical measurements
Glucose was determined according to a previously described method. Determinations of ALT and AST were carried out by methods described by Bergmeyer et al., 1978, and triglycerides and cholesterol were measured by standard enzymatic methods with a spectrophotometer Selectra II model, from Winner. The phytochemical profile was performed as described by Costa, 1977, through identification reactions based on the chemical group to be determined or by thin layer chromatography.

Statistical analysis
Results were expressed as means ± SEM. Data were analyzed with One-way ANOVA for the comparison between groups, followed by Tukey as a post hoc test. The significance level was set at p < href="http://www.pubmedcentral.nih.gov/redirect3.cgi?&&auth=0M4V-v41sUqPpfA94a1i1QMuih2eO2iu4cKRqY5Pw&reftype=pubmed&artid=443509&iid=10729&jid=56&FROM=Article%7CCitationRef&TO=Entrez%7CPubMed%7CRecord&article-id=443509&journal-id=56&rendering-type=normal&&http://www.ncbi.nlm.nih.gov/pubmed/11801393">PubMed]
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aKo si mArky!

MARK ANTHONY F. SUAREZ
258 P. Monroe St., Mariveles, Bataan/
123 Valino St., Magsaysay Norte, Cabanatuan City, Nueva Ecija
09163889761/ 09187492996
marky0072007@yahoo.com
Summary Profile

Name: Mark Anthony F. Suarez
Gender: Male
Date of Birth: January 30,1987
Place of Birth: Mariveles, Bataan
Address 1 : 258 P. Monroe St., Mariveles, Bataan
Address 2 : 123 Valino St., Magsaysay Norte, Cabanatuan City, Nueva Ecija
Religion: Roman Catholic
Language Spoken: Filipino ( Tagalog) and English
Parents:
Name of Father: Rodel M. Suarez, Sr. Occupation: N/A
Name of Mother: Ma. Melodie F. Suarez Occupation: Government Employee

EDUCATIONAL BACKGROUND

Tertiary: B.S. in Chemistry
Nueva Ecija University of Science and Technology
Gen. Tinio St., Cabanatuan City, Nueva Ecija
2004-present

Vocational : Certificate on Artificial Flower and Candle-Making
TESDA Manpower Training Center
Mariveles, Bataan
Summer 2003

Secondary: Mariveles National High School- Poblacion
Mariveles, Bataan
2000-2004

Elementary: Antonio G. Llamas Elementary School
Mariveles, Bataan
1994-2000

SPECIAL SKILLS
Computer Literate
Public Speaking
Research/ Experimentations

MEMBERSHIPS & LEADERSHIP BACKGROUND

Academic/ Non-Political Organizations

NEUST Chemical Society
2004-2008 Vice President

SIFE- NEUST ( Students in Free Enterprise)
2007-2008 Treasurer for Board of Ambassadors
2007-2008 President for Arts & Sciences

Junior Scientists Society
2006-2007 President

Junior Scientists & Technologists Society
2007-2008 Jr. Adviser

NEUST Camera Club
2007-2008 President

College Red Cross Youth, Member
NEUST Campus Dancers Club
2006-2008 Junior Adviser

University Political Student Organizations

NEUST College of Arts & Sciences Student Council
2006-2007 Chairman
2007-2008 Junior Adviser

SINAG ( Self-Reliant & Independent NEUSTians Aiming for Good-Governance)
2007-2008 President

SENTRO NEUST ( Samahan ng mga Estudyante na Naninindigan para sa Tunay na Reporma at Ordinansa), Present President


REFERENCES

Engr. Darwin U. Ong
Laboratory Technician, NEUST Physics Department

Prof. Mercedes Q, Cabling
Director, NEUST Research Office
President, Kapisanang Kimika ng Pilipinas

Mario Abesamis, M.S. Chem
Adviser, NEUST Chemical Society
Prof. Lolita F. Joson
Dean, CAS-NEUST