Executive oxidative damage to lipids, proteins, enzymes and

ExecutiveSummaryFree radical species produced during metabolism result inoxidative stress, which can cause damage of DNA, proteins, and lipids; andtheir occurrence has been associated with some diseases including cancer,diabetes, and several cardiovascular and neurodegenerative diseases.

At a stateof high free radical formation, internal antioxidants become insufficient forthe counteraction of free radicals; therefore, external antioxidants are anecessity in the prevention of the oxidative damages. In the present study, themain objective is to evaluate the total content of flavonoids, the antioxidantcapacity and the scavenging activity of methanolic plant extracts of Brassica oleracea var. sabauda, Helianthusagrestis, and Murraya Koenigi species. A preliminary phytochemicalscreening of the extracts will reveal the bioactive constituents of the plantsstudied. The total content offlavonoids for each plant species will be estimated by using the aluminumchloride colorimetric method.

The free radical scavenging capacity of theextracts will be determined by the 1,1,-diphenyl-2-picryl hydrazyl (DPPH)method. Finally, the phosphomolybdenum method will be used to evaluate thetotal antioxidant capacity of each plant species. Since flavonoids areprominent constituents of plants that possess an immense spectrum ofbiochemical activities necessary to maintain a stable human health, the presentstudy will lead to the future isolation and characterization of the activecompounds responsible for the pharmacological activity related to these plants.This further investigation will allow for a greater understanding of themechanism of action of the bioactive compounds from the extracts againstoxidative-stress-related diseases in order to develop it as a promising drugfor pharmaceutical and therapeutic distribution.

 Statement of NeedReactive oxygen species, such assinglet oxygen, hydroxyl ion, superoxide ion and hydrogen peroxide, are highlyreactive, toxic molecules, which are generated normally in cells duringmetabolism. These free radicals cause severe oxidative damage to lipids,proteins, enzymes and DNA by covalent binding and lipid peroxidation, withsubsequent tissue injury. (Baba, S. A., & Malik, S.

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A, 2015) In recentyears, natural antioxidant agents have attracted much interest because of theirability to tremendously diminish the damage due to oxidants by neutralizing thefree reactive species before they begin their onslaught on the cells, andeffectively obstruct damage to lipids, proteins, enzymes and DNA. (Saeed N., KhanM.R., & Shabbir M., 2012). Free radicals have been implicated in thedevelopment of a number of disorders, including neurodegeneration,inflammation and cancer, giving rise to studies of antioxidants for theprevention and treatment of diseases. (Peschel W.

, Sanchez-Rabaneda F., Dieckmann W., Plescher A.

, & Gartzia I., 2006). Flavonoids are phenolic substances that act in plants asantioxidants, antimicrobials, photoreceptors, visual attractors, feedingrepellants, and for light screening. Many studies have suggested thatflavonoids exhibit biological activities, including antiallergenic, antiviral, anti-inflammatory,and vasodilating actions. However, most interest has been devoted to theantioxidant activity of flavonoids, which is due to their ability to reducefree radical formation and to scavenge free radicals (Pietta P., 2000). Indeed,many recent investigations have reported a significant correlation between thetotal content of flavonoids and the antioxidant activity of plant species.

The present study hypothesizes that the species with thehighest content of flavonoids will also show the highest scavenging andantioxidant activity. The main objective of this investigation is to evaluatethe total content of flavonoids, antioxidant capacity and the scavengingactivity of methanolic plant extracts of Brassicaoleracea var. sabauda, Helianthus agrestis, and Murraya Koenigi species. Thesespecies are commonly used by the world population on a daily basis and can beeasily acquired in the local markets; they are accessible and inexpensive.Moreover, very little research on the antioxidant potential of these plant specieshave been done before, which makes them an excellent source for new discoveriesand innovation. This research has great significance because if the plants studiedare found to be rich in natural antioxidants, they can be further investigatedfor their effects in the maintenance of the human health (i.e.

as strong anticancerousand in the protection against heart disease), and in the food industry asconsumers move towards functional foods with specific health effects.  Background/LiteratureReviewSinceancient human civilizations, plant-based sources have been invaluable remedy inthe health care system to ease and cure diseases. For thousands of years, thewidespread use of plants for medicinal means have been embedded in our cultureand even showed high potential to be used in modern medicine and in otherfuture applications (Bhaigyabati, Bag, & Grihanjali Devi, 2014). According to Kamboj(2000), herbal medicine is still the mainstay of about 75–80% of the world population,mainly in the developing countries, for primary health care because of bettercultural acceptability, better compatibility with the human body and lesserside effects. They also offer therapeutics for age-related disorders likememory loss, osteoporosis, immune disorders, etc. for which no modern medicineis available (Kamboj, 2000). Most plantspecies are abundant source of potent free radical scavenging molecules(antioxidants), which are bioactive compounds such as carotenoids, flavonoids,dietary glutathione, and vitamins (Kalita , Barman Tapan , Pal Tapas , & Kalita , 2013).

Antioxidants are powerful preventive agents thatcan inhibit or delay reactive oxygen species and other forms of free radicalsin the human body. This characteristic featureof an antioxidant is due to its redox hydrogen donators and singlet oxygenquencher (Senguttuvan, J., Paulsamy, S.,& Karthika, K, 2014).Incontemporary research, antioxidants have gained popularity. Free radicalspecies produced during metabolism result in oxidative stress, which can causedamage of DNA, proteins, lipids, and so on.

Some diseases correlated to theoccurrence of free radicals include several cardiovascular andneurodegenerative diseases, cancer and diabetes (Chun, Frei, Gardner, Lee Alekel, & Killen Jr., 2013). At a state of highfree radical formation, internal antioxidants become insufficient to counteractthe effects of free radicals, so external antioxidants are needed in order toprevent the oxidative damages (Kalita et al., 2013).

In the present literature review, differentscientific methods to identify the bioactive phytochemical constituents ofplants, evaluate their total content of flavonoids, antioxidant capacity and scavengingactivity are investigated.  It ishypothesized that the species with the highest reported content of flavonoidswill also show the highest percent of scavenging and antioxidant activities.Furthermore, this literature review examines suitable methods for the mentionedtests, and attempts to demonstrate and support the stated hypothesis. In an effort to identify the bioactiveconstituents present in a variety of Argentine medicinal plants, Coussio, J., Rondina, R., Mendiondo, M., &Bandoni, A.

(1976) conducted a preliminary qualitative phytochemicalanalysis of all species studied. Tocomplete this analysis, Coussio et al., (1976)performed successive extractions with solvents of increasing polarity,from non-polar (hexane) to polar solvent (methanol) that drew high range ofplant constituents. Therefore, the methanolic extracts of each species wereused to estimate the total flavonoids content and antioxidant activity. Thetest revealed the presence of trepenoids, tannis, phenols, amino acids, terpenoids, anthraquiones, alkaloids, cardiacglycosides, reducing sugars, saponins, and flavonoids as the plants’ bioactiveconstituents. These secondary metabolites have many biological and therapeuticproperties; hence, the species tested were expected to have a great variety ofmedicinal uses (Senguttuvan et al., 2014). Flavonoids are defined as polyphenolic compounds comprisingfifteen carbons with two aromatic rings connected by a three-carbon bridge.

These compounds are the most numerous of the phenolics and are found throughoutthe plant kingdom (Cartea, M. E.,Francisco, M., Soengas, P.

, & Velasco, P., 2010). Flavonols are the mostwidespread of the flavonoids. Quercetin, is the most abundant dietary flavonol,and it is a potent antioxidant because it has all the right structural featuresfor free radical scavenging activity (Cartea et. al.

, 2010). Based on thisfact, quercetin was used as the standard to construct the calibration curve fortotal flavonoid content test in Chang, C., Yang,M., Wen, H., & Chern, J. (2002) experiment. Moreover, Chang et al.

, (2002)developed the aluminum chloride colorimetric method to identify thetotal content of flavonoids present in propolis, the material used by bees toprotect their hives. In this method, diluted standard solutions of quercetinwere separately mixed with 95% ethanol, 10% aluminum chloride, 1M potassiumacetate and distilled water; incubated at room temperature for 30 min; andtheir absorbance measured at 415nm (maximum wavelength of quercetin) with aShimadzu UV-160A spectrophotometer. As specified by Bhaigyabati et al., (2014),the principle involved in aluminum chloride (AlCl3) colorimetric method is thatAlCl3 forms acid stable complexes with the C-4 keto groups and either the C-3or C-5 hydroxyl group of flavones and flavonols. In addition, Bhaigyabati etal., (2014) explains that AlCl3 also forms acid labile complexes with theorthodihydroxyl groups in the A- or B-ring of flavonoids. Likewise, aluminumchloride colorimetric method was used to determine the total content offlavonoids in methanolic extract of Biophytum Sensitivum Linn in Kalita et al.

,(2013) research. In this experiment, the final concentration values of plantextracts were obtained from the quercetin standard curve by interpolating tothe x- axis. The total content of flavonoids was calculated in this experimentby using the following formula, where “R” represents the results obtained fromthe standard quercetin curve, “V” is the volume of stock solution, “D.F” thedilution factor, and “W” the weight of dry sample plant used in experiment.(Kalita et al., 2013)The second method investigated in this paper, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) freeradical method, aims to determine the scavenging activity of plant species. The DPPH is a stable radical with a maximum absorptionat 517 nm that can readily undergo scavenging by antioxidant (Lu and YeapFoo, 2001).

It has been widely used to test the ability of compounds as free-radicalscavengers or hydrogen donors, and to evaluate the antioxidative activity ofplant extracts (Porto et al., 2000). In the DPPH assayconducted in Sowndhararajan, K.

, & Kang, S. C. (2013) research, the absorbance at 517 nm decreased asthe concentration of the extracts increased, thus increasing the concentrationof the antioxidants. This reduction of absorbance is due to the odd electron ofDPPH that generally yields a strong absorbance at 517 nm while being quenchedby the introduced extract.

Lastly, the percent scavenging radical activity wascalculated for each extract sample using the formula in Zheleva-Dimitrova, D., Nedialkov, P., & Kitanov, G. (2010)publication:            Acorrelation between the increase in DPPHscavenging activity and the increase in flavonoid content is evident inall Zheleva-Dimitrova et al., (2010),Sowndhararajan, et al., (2013), and Bhaigyabati et al., (2014) experiments, which support the initial hypothesis.         The last method discussed in this paper involves thephosphomolybdenum complex, and aims todetermine the total antioxidant activity of plant extracts.

In Waheed,I., Ahmad, M., Syed, N., & Ashraf, R.

(2014) research article, the totalantioxidant potential of methanol extracts and fractions of Ballota limbata(Lamiaceae) was measured by employing phosphomolybdenum complex formationmethod. In this assay, the conversion of Mo (V) from Mo (VI) by reductionoccurred by various fractions of plants which were detected at an absorbance of695 nm by the spectrophotometer. This method was also used by Miladi S, DamakM.

, (2008) research team, who described the method to be simple and independentafter using it to successfully quantify vitamin E in seeds. More proof for theefficiency of the method is obtained in Esmaeili A.K., Taha R. M., Mohajer S., & Banisalam B.

, (2015) experiment, where the resultsreported show a correlation between phytochemical contents and the valuesobtained for both radical scavenging activity and antioxidant ability of theextract of red clover and its various soluble fractions. In this experiment,flavonoid contents exhibited excellent association with DPPH, superoxide, ABTSradical scavenging activities, and reducing power. In the same way, resultsreported by Senguttuvan et al., (2014) showed astrong correlation between the contents of total flavonoids and radicalscavenging activity, which indicates that flavonoids are major contributors tothe antioxidant potential of plant species. (Senguttuvan et al., 2014)Taken together, the present literature reviewconfirms that a preliminary phytochemical study provides invaluable informationof the different phytochemicals present in herbal extracts consumed on a dailybasis by the population.

This preliminary analysis is a necessary step thatpredicts whether or not the plant species studied is expected to contain highcontent of flavonoids and other bioactive constituents. Additionally, aluminumchloride colorimetric method, DPPH radical scavenging assay and phosphomolybdenummethod were found to be suitable techniques to assess the total flavonoidcontent, free radical scavenging capacity and overall total antioxidantactivity of methanolic extracts of plantspecies respectively. All findings in the present study support the viewthat many medicinal plants are promising sources of potential antioxidant andmay be efficient as preventive medication sources in many diseases.

Mostimportantly, the research articles analyzed support the initial hypothesis by explainingin multiple instances the direct relationship between the total content offlavonoids in plant extracts and their antioxidant and scavengingactivity.    ProjectDescriptionCollection of SampleFresh samples of B. oleraceavar. sabauda, H. agrestis, and M. koenigi were already collected from afarmer’s market at Homestead, Florida. The handpicked species were washed wellusing tap water and once again using distilled water.

They were cut into smallpieces and dried in shade for a period of ten days at room temperature of 22oC.The dried samples were grinded using a mortar and pestle. They were furtherstored in sterile flasks and refrigerated.  Preparation of extracts and Phytochemical ScreeningThe phytochemicals of B.oleracea var. sabauda, H. agrestis,and M.

koenigi will be determined bysuccessive extractions with solvents of different polarities (i.e hexane,methanol, chloroform, and water) according to the procedure described byRondina and J. D. Coussio, (1976).

Phytochemical screening of all species’extracts will be carried out to detect the presence of alkaloids, saponins,tannis and phenols, amino acids, terpenoids, anthraquinones, alkaloids, cardiacglycosides, reducing sugar and flavonoids. The extracts with the strongestintensity of flavonoids reported will be chosen, and evaporated in a vacuumrotary evaporator until dryness. The chosen extracts will be used to study thetotal content of flavonoids, antioxidant activity and scavenging capacity ofthe selected plants.

   Evaluation of Total Flavonoid Content The total content of flavonoids for each plant extract willbe estimated by using the aluminum chloride (AlCl3) colorimetricmethod. Quercetinwill be used as a standard to construct a calibration curve.  Stock solutions of plant extracts will beprepared by dissolving the extract of each species in methanol. Test solutionswill be prepared by adding 10% aluminum chloride, 1 M sodium acetate anddistilled water to the methanolic extracts. The blank sample will be preparedin a similar way with the exception that it would not contain 10% aluminumchloride. The absorbance of all test solutions will be measured against theblank at 415 nm. (Bag G.

C., Grihanjali Devi P., Bhaigabati. Th., 2014).Determination of Free Radical Scavenging Activity The free radical scavenging capacity of each species’extract will be determined by the 1,1,-diphenyl-2-picryl hydrazyl (DPPH)method. (Kalita , Barman Tapan , Pal Tapas , & Kalita , 2013) Ascorbic acid will be used as areference to construct a standard calibration curve.

Various concentrations ofthe methanolic extract of B. oleraceavar. sabauda, H. agrestis, and M. koenigi will be prepared withTris-HCl ph7 buffer and 0.1 mM DPPH solution. The negative control will be preparedwith methanol, tris-HCl ph7 buffer and DPPH solution. All extracts will beincubated for 30 minutes at room temperature and their absorbance measured at517 nm.

  Total Antioxidant Capacity The phosphomolybdenum method will be used to evaluate thetotal antioxidant activity of the extracts of B. oleracea var. sabauda,H. agrestis, and M. koenigi. (Bhaigyabati, Bag, & Grihanjali Devi, 2014) Ascorbic acidwill be used as a standard to plot a calibration curve. Test solutions will beprepared by adding ammonium molybdate reagent solution to each species’ methanolicextract.  All solutions will be incubatedat 95oC for one hour and then cooled down to room temperature.

Theirabsorbance will be measured at 695 nm using the ammonium molybdate reagentsolution as the control sample.  BudgetAnalysisThe project will be funded by the ARCOS UndergraduateResearch and Project Opportunity (URPO) Program at Miami Dade College. Allresources will be purchased by Miami Dade College and will be available to usein January 2018.  Table 1. Project Budget Item Description Supplier Catalog # Price ($) Quantity Total ($) Mercuric chloride (II)  Sigma Aldrich 215465-5G 24.

10 1 24.10 Potassium Iodide Sigma Aldrich 793582-100G 51.70 1 51.70 Methanol anhydrous, 99.8% Sigma Aldrich 322415-1L 60.30 2 120.

60 Chloroform Sigma Aldrich C2432-1L 73.50 1 73.50 Quercetin hydrate Sigma Aldrich 337951-25G 42.00 1 42.00 L-Ascorbic acid Sigma Aldrich A7506-100G 40.30 1 40.

30 2,2-Diphenyl-1picrylhydrazyl Sigma Aldrich D9132-1G 97.30 1 97.30 Aluminum chloride Sigma Aldrich 294713-5G 46.00 1 46.00 Sodium Phosphate Sigma Aldrich 342483-25G 29.20 1 29.20 Ammonium molybdate Sigma Aldrich 277908-5G 78.

10 1 78.10           $602.80   ConclusionThis preliminary study will provide invaluable informationof the different phytochemicals contained in herbal extracts available in thefarmer’s market and consumed regularly by the population. The results areexpected to show an equivalent relation between the total flavonoid content,the scavenging activity and the antioxidant capacity.

Since flavonoids and other of the metabolites studied inthis research are prominent constituents of plants that have a large range ofbiochemical activity necessary to maintain a healthy human being, furtherinvestigation will allow for the isolation and characterization of the activecompounds responsible for pharmacological activity related to these plants. Thisfurther examination will lead to a greater understanding of the mechanism ofaction of the bioactive compounds from the extracts against oxidative-stress-relateddiseases in order to develop it as a promising drug for pharmaceutical andtherapeutic distribution.