ANTIOXIDANT SCREENING OF

Averrhoa bilimbi

(Kamias)

Cananga odorata

(Ylang-Ylang), and

Plumiera alba

(Calachuchi)USING 2,2-diphenyl 1-picrylhydrazyl DPPH ASSAY

Ruth T. Libag

, Maria Jacquilyn F. Ancheta

, Gail P. Igaya

and Maribel V. Tolentino

Department of Chemistry Angeles University Foundation,

College of PharmacyOur Lady of Fatima University,

College of Engineering Angeles University Foundation

Scientific evidence suggests that antioxidants reduce risk for chronic diseases including cancer and heart disease. Primary sources of naturally occurring antioxidants are whole grains, fruits and vegetablewhile the synthetic antioxidants currently used have been found to exhibit various health effects. It is for this reason that the researchers focused on natural antioxidants present in medicinal plants. The samples used inthe study were the ethanolic extracts of Kamias, Ilang-ilang and Calachuchi using

,2-diphenyl 1-picrylhydrazyl

(

DPPH) assay. All haveantioxidant activity against the free radical DPPH at varying amounts. Theleaves of Kamias have the highest antioxidant activity, followed by theflowers of ilang ilang and calachuchi. All of the samples were initially measured at 0.125 mg/ml crude ethanolic extract and read at 520 nmusing spectrophotometry. The free radical scavenging activities of the plant samples using the parameter EC

also coincide with the amount of antioxidants present expressed in percentage. Thus, Kamias has thehighest EC

, followed by ilang ilang and calachuchi, respectively. Theresults showed that the given plant samples are potential sources of antioxidants.

Antioxidant compounds in food play an important role as a health-protecting factor. Scientific evidence suggests that antioxidants reduce risk for chronic diseases including cancer and heart disease. Primary sources of naturally occurring antioxidants are whole grains, fruits and vegetables. Thesynthetic antioxidants currently used have been found to exhibit various healtheffects. That is why researchers focused on natural antioxidants present inmedicinal plants (Castro 2006).Previous epidemiological and experimental evidence suggest thatantioxidants positively influence the defence of the body against cancer. Sincecancer can be induced by damage to DNA, deoxyribonucleic acid, it is postulatedthat free radicals produced from normal chemical reactions may be responsiblefor the development of some human cancers. Because antioxidants canneutralize free radicals or unpaired electrons, they are now of considerableimportance as chemopreventive agents (Beecher 2000).The main characteristic of an antioxidant is its ability to trap free radicals.Highly reactive free radicals and oxygen species are present in biologicalsystems from a wide variety of sources. These free radicals may oxidize nucleic

acids, proteins, lipids or DNA and can initiate degenerative disease. Antioxidantcompounds like phenolic acids, polyphenols and flavonoids scavenge freeradicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit theoxidative mechanisms that lead to degenerative diseases (Prakash 2001).Free radicals and oxidants can trigger lipid peroxidation, as well as theoxidation of proteins and DNA, causing extensive damage to body cells.Oxidative stress resulted from an imbalance of oxidising species and naturalantioxidants in the body has been thought to have contributed to aging, cellapoptosis, and severe diseases such as cancer, Parkinson’s disease,Alzheimer’s disease, and even cardiovascular disorders. Epidemiological studiesand intervention trials on prevention of cancer and cardiovascular disease inpeople taking antioxidant supplements are suggestive that dietary intake of antioxidants can help free radicals and oxidants and protect the body againstdiseases. It is also clear that most of the dietary antioxidants have low or minimal toxicity, and that intake can be increased without adverse effects (Frei1994). There are different kinds of antioxidant test like ABTS (2,2’-azonobis-3[-ethylbenzthiazoline-6-sulphonic acid]), DPPH (1-1-diphenyl-2-picrylhydrazyl),ORAC(oxygen radical absorbance capacity), β-carotene–linoleate model system,this test is to determine the free-radical scavenging activity of sample or plantsample. One such method that is currently popular is based upon the use of thestable free radical diphenylpicrylhydrazyl (Castro et. al. 2006).DPPH free radical (1,1-diphenyl-2-picryl-hydrazyl) DPPH and reducedform the molecule of 1,1-diphenyl-2-picryl-hydrazyl(

α,α-diphenyl-β-picrylhydrazyl) DPPH is characterised as a stable free radical byvirtue of the delocalisation of the spare electron over the molecule asa whole, so that the molecules do not dimerise, as would be t

he casewith most other free radicals. The delocalisation also give rise to deep violetcolor, characterised by an absorption band in ethanol solution centered at520nm.When a solution of DPPH is mixed with that of a substance that candonate a hydrogen atom, then this gives rise to the reduced form with the violetcolor from the picryl group still present). Representing the DPPH radical by Zand the donor molecule by AH, the primary reaction is Z + AH = ZH+ A [1]where ZH is the reduced form and A is free radical produced in this first step.This latter radical will then undergo further reactions which control the overallstoichiometry,that is, the number of molecule of DPPH reduce by one moleculeof the reactant. The reaction [1] is therefore intended to provide the link withthe reactions taking place in an oxidaizing system,such as the autoxidation of alipid or other unsaturated substance; the DPPH molecule Z is thus intended torepresent the free radicals formed in the system whose activity is to besuppressed by the substance AH. (Molyneux 2004)

As oxidative stress might be an important part of many human diseases,the use of antioxidants in pharmacology is intensively studied, particularly astreatments for stroke and neurodegenerative diseases. Antioxidants are alsowidely used as ingredients in dietary supplements in the hope of maintaininghealth and preventing diseases such as cancer and coronary heart disease.Although some studies have suggested antioxidant supplements have healthbenefits, other large clinical trials did not detect any benefit for the formulationstested, and excess supplementation may be harmful.In addition, there has been growing interest in natural antioxidant becausethey have greater application in food industry for increasing the stability andshelf-life of food products (Suja 2004).It is in this basis that the researchers under take to determine theantioxidant properties on plants and flowers readily available, they were able toidentify three plant samples. Among these are the following,

Plumeria alba

(Calachuchi),

Averrhoa bilimbi

(Kamias), and

Cananga odorata

(Ilang Ilang).

METHODOLOGYCollection of Plant Samples

The leaves and flowers were brought to the National Museum and wereproperly identified and were given authentication by professional botanists.After the collection of plant samples, fresh water was used to clean andwipe off dirt and contaminants on the samples. Plant specimens were air driedthrough room temperature to make sure that moisture was properly evaporatedoff and confine the important constituents of the plant samples.

Extraction

To extract the organic constituents from the plant samples, the driedmaterials were cut into smaller pieces and were grounded. By the use of solvent extraction method, diluted 80% ethyl alcohol was used to completelysubmerge the material. After forty eight hours of submerging materials tosolvent, the extracts were collected through filtration. To ensure that allimportant constituents of the materials were collected, washing is also done bythe use of fresh portions of alcohol. Plant residues were discarded.

Rotary Evaporation

To concentrate the filtrate, rotary evaporation was used. After which, theexact volume of the concentrated extracts were measured.

Preparation of Vitamin C Calibration Curve for DPPH Assay