Antioxidant Screening Of Averrhoa Bilimbi (kamias) Cananga Odorata (ylang-ylang), And

ANTIOXIDANT SCREENING OF Averrhoa bilimbi (Kamias) Cananga odorata (Ylang-Ylang), and Plumiera alba (Calachuchi) USING 2,2-diphenyl 1-picrylhydrazyl DPPH ASSAY Ruth T. Libag1, Maria Jacquilyn F. Ancheta2, Gail P. Igaya2 and Maribel V. Tolentino3 Department of Chemistry Angeles University Foundation, 2 College of Pharmacy Our Lady of Fatima University, 3College of Engineering Angeles University Foundation 1

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 vegetable while 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 in the study were the ethanolic extracts of Kamias, Ilang-ilang and Calachuchi using 2,2-diphenyl 1-picrylhydrazyl (DPPH) assay. All have antioxidant activity against the free radical DPPH at varying amounts. The leaves of Kamias have the highest antioxidant activity, followed by the flowers of ilang ilang and calachuchi. All of the samples were initially measured at 0.125 mg/ml crude ethanolic extract and read at 520 nm using spectrophotometry. The free radical scavenging activities of the plant samples using the parameter EC50 also coincide with the amount of antioxidants present expressed in percentage. Thus, Kamias has the highest EC50, followed by ilang ilang and calachuchi, respectively. The results showed that the given plant samples are potential sources of antioxidants.

Antioxidant compounds in food play an important role as a healthprotecting 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. The synthetic antioxidants currently used have been found to exhibit various health effects. That is why researchers focused on natural antioxidants present in medicinal plants (Castro 2006). Previous epidemiological and experimental evidence suggest that antioxidants positively influence the defence of the body against cancer. Since cancer can be induced by damage to DNA, deoxyribonucleic acid, it is postulated that free radicals produced from normal chemical reactions may be responsible for the development of some human cancers. Because antioxidants can neutralize free radicals or unpaired electrons, they are now of considerable importance 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 biological systems from a wide variety of sources. These free radicals may oxidize nucleic

acids, proteins, lipids or DNA and can initiate degenerative disease. Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases (Prakash 2001). Free radicals and oxidants can trigger lipid peroxidation, as well as the oxidation of proteins and DNA, causing extensive damage to body cells. Oxidative stress resulted from an imbalance of oxidising species and natural antioxidants in the body has been thought to have contributed to aging, cell apoptosis, and severe diseases such as cancer, Parkinson’s disease, Alzheimer’s disease, and even cardiovascular disorders. Epidemiological studies and intervention trials on prevention of cancer and cardiovascular disease in people taking antioxidant supplements are suggestive that dietary intake of antioxidants can help free radicals and oxidants and protect the body against diseases. It is also clear that most of the dietary antioxidants have low or minimal toxicity, and that intake can be increased without adverse effects (Frei 1994). 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 plant sample. One such method that is currently popular is based upon the use of the stable free radical diphenylpicrylhydrazyl (Castro et. al. 2006). DPPH free radical (1,1-diphenyl-2-picryl-hydrazyl) DPPH and reduced form the molecule of 1,1-diphenyl-2-picryl-hydrazyl(α,α-diphenyl-βpicrylhydrazyl) DPPH is characterised as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecules do not dimerise, as would be the case with most other free radicals. The delocalisation also give rise to deep violet color, characterised by an absorption band in ethanol solution centered at 520nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the violet color from the picryl group still present). Representing the DPPH radical by Z and 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 overall stoichiometry,that is, the number of molecule of DPPH reduce by one molecule of the reactant. The reaction [1] is therefore intended to provide the link with the reactions taking place in an oxidaizing system,such as the autoxidation of a lipid or other unsaturated substance; the DPPH molecule Z is thus intended to represent the free radicals formed in the system whose activity is to be suppressed 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 as treatments for stroke and neurodegenerative diseases. Antioxidants are also widely used as ingredients in dietary supplements in the hope of maintaining health and preventing diseases such as cancer and coronary heart disease. Although some studies have suggested antioxidant supplements have health benefits, other large clinical trials did not detect any benefit for the formulations tested, and excess supplementation may be harmful. In addition, there has been growing interest in natural antioxidant because they have greater application in food industry for increasing the stability and shelf-life of food products (Suja 2004). It is in this basis that the researchers under take to determine the antioxidant properties on plants and flowers readily available, they were able to identify three plant samples. Among these are the following, Plumeria alba (Calachuchi), Averrhoa bilimbi (Kamias), and Cananga odorata (Ilang Ilang).

METHODOLOGY Collection of Plant Samples The leaves and flowers were brought to the National Museum and were properly identified and were given authentication by professional botanists. After the collection of plant samples, fresh water was used to clean and wipe off dirt and contaminants on the samples. Plant specimens were air dried through room temperature to make sure that moisture was properly evaporated off and confine the important constituents of the plant samples. Extraction To extract the organic constituents from the plant samples, the dried materials were cut into smaller pieces and were grounded. By the use of solvent extraction method, diluted 80% ethyl alcohol was used to completely submerge the material. After forty eight hours of submerging materials to solvent, the extracts were collected through filtration. To ensure that all important constituents of the materials were collected, washing is also done by the use of fresh portions of alcohol. Plant residues were discarded. Rotary Evaporation To concentrate the filtrate, rotary evaporation was used. After which, the exact volume of the concentrated extracts were measured. Preparation of Vitamin C Calibration Curve for DPPH Assay

Standard Vitamin C solutions were prepares starting at an initial concentration of 0.25mg/mL. This standards were used to establish a calibration curve that was used as the basis of conclusion whether the plant sample has an antioxidant activity or none. The DPPH Assay was patterned on the procedure used by Molyneux. The DPPH reagent was prepared by dissolving 0.0100g of DPPH in 80% ethanol to make 250mL and a concentration 100µM. Preparation of Calibration Curve for Vitamin C. Volume in mL

Tub e1

Tub e2

Tub e3

Tub e4

Tub e5

Tub e6

Tub e7

DPPH Solution Vitamin C Standard Distilled Water

2

2

2

2

2

2

2

0.10

0.20

0.40

0.60

0.80

1.00

1.10

1.90

1.80

1.60

1.40

1.20

1.00

0.90

Total Volume

4.00

4.00

4.00

4.00

4.00

4.00

4.00

The absorbances of the Vitamin C standards were taken at 520 nm using a spectrophotometer. DPPH alone was used as blank. Diagram of Methodology

RESULTS AND DISCUSSION The following graph shown below shows the regression equation of the calibration curve for Vitamin C. Graph 1. Vitamin C Standard Calibration Curve 0 .3 5 y = -3 .0 2 7 4 x +0 .2 8 3 R2 = 0 .9 5 0 3

0.3 Absorbance (520 nm)

0 .2 5 0.2

0 .1 5

c

0.1

0 .0 5 0 -0 .05

0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0.1

0.1 2

Concentration of Vitamin C in mg/mL

Graph 1 above shows a good correlation between absorbance and Vitamin C concentration with an R2 = 0.9503 while the slope of the line gives a regression equation of y = -3.0374x + 0.283 (y=mx+b) and expressed as AEAC. Thus, the slope for the calibration curve is reliable for computing the antioxidant capacity of the crude ethanolic extracts of Calachuchi, Kamias, and Ilang-ilang. Table 2 shows the mean absorbances of the crude ethanolic extracts at 0.125 mg/mL concentration against 100 μM DPPH. It is noted from table 1 that the absorbance of the blank reads 0.3095 at 520nm. Table 2. Plant Sample Mean Absorbances & Antioxidant Activity in mg/mL Plant Sample

Mean

mg/mL

Absorbance

Antioxidant

Calachuchi

0.214

0.0228

Kamias

0.105

0.0588

Ilang-ilang

0.170

0.0373

Using the regression equation from graph 1 (y = - 3.0274 x + 0.283) with R2 = 0.9503. The Ascorbic Acid Equivalent Antioxidant Capacity (AEAC) were taken. The table above showed that Kamias has the highest antioxidant capacity against DPPH with an AEAC of 0.0588 mg/mL, followed by Ilang-ilang and Calachuchi at 0.0373 and 0.0228 mg/mL, respectively. The concentration of Antioxidant in the crude extracts were computed using the regression equation from the vitamin C calibration curve using the formula of the slope of the line, y = mx + b Where: y = the absorbance of samples m = slope of the line b = blank absorbance x = the antioxidant activity in mg/mL Graph 2. The Antioxidant Activities of Plant Extracts Against DPPH

Amount of Antioxidants in mg/mL

0.0 7 0 0 0 . 0 60 0 0 . 0 50 0 0 . 0 40 0 0.0 3 0 0 0.0 2 0 0 0.0 1 0 0 0.0 0 0 0

Calachuchi

Kam ias

Ilang-ilang

Pla nt Sa m ple s

A 100 μL sample was taken from the 2.5 μL/mL initial concentration of the crude ethanolic extracts and was added to 1.9 mL distilled water to make the

resulting concentration of extract to 0.125 mg/mL. From this concentration of extract, the percentage of antioxidant present was computed and presented in Graph 3.

Graph 3. Percentage of Antioxidant Present in 0.125 mg/mL Crude Samples

Percentage of antioxidant

50 . 0 0 % 45. 0 0 % 40 . 0 0 % 3 5. 0 0 % 3 0. 0 0% 2 5. 0 0 % 2 0. 0 0% 1 5. 0 0 % 1 0. 0 0% 5. 0 0 % 0.0 0%

Calachuchi

K am ias

Ilang-ilang

Pla nt Sa m ple s

Graph 3 above showed corresponding percentage of antioxidant present in 0.125mg/mL crude samples. The results showed that Kamias has the highest antioxidant content at 47.04%. This means that almost half of the crude extract of Kamias has antioxidant capacity. Ilang-ilang follows at 29.84% and Calachuchi at 18.24% at the given concentration of extracts. Percentage antioxidant present in each sample were taken using the following formula %Ao = (Ao Concentration ÷ 0.125 mg/mL) x 100 Another parameter which tests the antioxidant capacity of the samples is by computing its effective concentration, EC50 , also known as IC 50 , is defined as the concentration of substrate that causes 50% loss of the DPPH activity.

Graph 4. EC50 of the Crude Extracts 7 0 .0 0 60 . 0 0 EC 50 Value

50 . 0 0 40 . 0 0 3 0 .0 0 2 0 .0 0 1 0 .0 0 0 .0 0

Calachuchi

Kamias

Ilang-ilang

Plant Sample

Percent inhibition expressed as EC50 values at 0.125 mg/mL of crude extract showed that Kamias has the highest inhibitory concentration and was able to inhibit DPPH resulting to 66.07% loss. This is followed by Ilang-ilang at 45.07% and Calachuchi at 30.86% at the concentration of 0.125mg/mL crude ethanolic extracts of the samples. EC50 (Q) for each of the sample was computed using the following formula. Q = 100 ( Ao – Ac ) Ao Where: Ao = the absorbance of positive control containing DPPH only Ac = absorbance of solution containing extracts and DPPH. CONCLUSION All the plant samples namely Kamias, Ilang-ilang and Calachuchi have antioxidant activity against the free radical DPPH at varying amounts. With the

leaves of Kamias having the highest antioxidant activity, followed by the flowers of ilang ilang and calachuchi. Consequently, the given samples Kamias, Ilangilang and Calachuchi could be used as a natural source of antioxidant since all of them gave antioxidant capacities in varying degrees starting at 0.125mg/ml concentration of the crude ethanolic extracts. The free radical scavenging activities of the plant samples using the parameter EC50 also coincide with the amount of antioxidants present expressed in percentage. Thus, Kamias has the highest EC50, followed by ilang ilang and calachuchi, respectively. The results showed that the given plant samples are potential sources of antioxidants. REFERENCES Beecher, G. (2000). Measurement of “new” health related food components. Food Composition Laboratory, USDA/ARS/FCL Beltsvelle MD 20705. Castro, I A. et.al. (2006). 2,2 –Diphenyl-1-picrylhydrazil free radical scavenging activity of antioxidant mixtures evaluated by response surface methodology. International Journal of Food Science and Technology 2006, 41 (Supplemental 1), 59-67. Castro, I.A. et.al. (2006). Free Radical Scavenger and antioxidant capacity corrrelation of alpha-tocopherol and Trolox measured by three in vitro methodologies. Intenational Journal of Food Science and Nutrition, 57, 75-82. Molyneux, P. (2004). The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol., 2004, 26(2) : 211-219 Prakash, A. (2001) Antioxidant Activity. Progress 2001, Vol 19 No. 2.

Medallion Laboratories: Analytical

Frei, B. (1994). San Diego: Academic Press. Natural antioxidants in human health and disease. Suja, K.P., Jayalekshmy, A. & Arumughan, C. (2004). Free radical scavenging behavior of antioxidant compounds of Sesame (Sesamum indicum L.) in DPPH System. Journal of Agricultural and Food Chemistry,52, 912-915.