Antioxidant From Various Sources Of Plants Biology Essay

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Antioxidants protect cell from damage caused by unstable molecules known as free radicals. The deleterious reactions are controlled by antioxidant that eliminate through oxygen and scavenge free radical. Carotenoid lutein may reduce the oxidative damage or minimize the damage due to oxidative stress by limiting the degree to which oxygen penetrates the membrane. Dietary lutein reduces inflammation and immunosuppression and protects human cells against oxidant induced damage. Naturally occurring antioxidants in leafy vegetables and seeds such as vitamin C, vitamin E, β-Carotene, Xanthophylls are preferred over synthetic antioxidants such as Butylated hydroxytoluene (BHT) and Butylated hydroxyl anisole (BHA) as they cannot be used in food due to their carcinogenic nature. Thus, there is a need for isolation of antioxidants from natural products. The aim of our study is to extract lutein from various sources, estimate its yield using different solvents and study the effect of standing time on concentration of lutein in different solvents. This study focuses on screening some potential plants to determine the most suitable source which offers the higher yield of antioxidant and its affordability.

Keywords: Antioxidants, carotenoids, Lutein, Extraction, free radical.

Introduction

Antioxidants are the substances which when present at lower concentrations than those of an oxidizable substrate can delay or prevent oxidation of that substance (Halliwell et al. 1995). Antioxidants such as phenolics, terpenoids, carotenoids, steroids and alkaloids are receiving increased attention due to their demonstrated health benefit as they reduce the oxidative damage associated with diseases like cancer, cardiovascular diseases, cataracts, artherosclerosis, diabetes and Age Related Macular Degeneration (ARMD). Natural antioxidants play a decisive role in plants and biological systems (Willcox et al. 2004). Carotenoids, possessing an antioxidant property, are a group of red, yellow and orange pigments (most commonly C40) that have extended conjugated double - bond systems. This effective structural diversity has evolved in relation to many functions of carotenoids, which include acting as structural component of membranes and photo systems, accessory light - harvesting pigments, components for photoprotection and substrates for hormone synthesis. They are derived from isoprenoid precursors and are basically divided into two groups: the Carotenes- acyclic or cyclic hydrocarbons and the Xanthophylls - oxygenated derivatives of carotenes (Zhen-Xing Tang 2010). The double bond of carotenoids reacts with ROS to scavenge radicals.

Lutein (3R, 3′R,6′R)-β, ɛ-carotene- 3,3′-diol ) is a naturally occurring oxygenated derivative of hydrocarbon carotenoids (Figure 1). There are many applications of lutein which include pigmentation of animal tissues and products, drugs and cosmetics, coloration of food, prevention of ARMD (Bone R. A. et al. 1985, Bieri J. G. et.al. 1985, Dufosse L. et al. 2005).

Figure 1: Lutein ; trans-lutein;4-[18-(4-hydoxy-2,6,6-tri methyl-1 cyclohexenyl)-3,7,12,16-tetramethyl octa deca-1,3,5,7,9,11,13,15,17 nonaenyl] -3,5,5-trimethyl-cyclohex-2-en-1-ol.

("The Merck index" 8th Ed .1968)

Lutein is mainly found in central part of retina along with zeaxanthin, where its function is to filter out damaging UV light. Its antioxidant activity is to protect outer retina, which is rich in polyunsaturated fats, from light induced free radicals. As animals cannot synthesize lutein, they must obtain it from diet.

A lot of information regarding the importance of lutein on human health has been gathered. Lutein can be obtained from various sources such as fruits and vegetables. To prevent ARMD a minimum of about 6mg of lutein is needed which we may not get from our diet, so we need an additional supplement of lutein. However, there is no much data available in the literature pertaining to lutein production such as its extraction, purification and phytochemical analysis.

Materials and methods

Chemicals: Acetone, Ethanol, Butanol, Methanol, Hexane, Silica, Sodium nitrite, Sulphuric acid was purchased from High Media chemicals. Standard lutein was obtained in the form of capsule from Elnova Pharma.

Sample preparation: Flowers such as Marigold, Gerbera, fruits such as orange, grapes, papaya, vegetables such as spinach, cabbage, corn, carrot, and medicinal plant neem were used in the study. They were purchased from the local market of Vellore and washed under tap water to remove all the dirt particles and were air dried at room temperature. The raw material chosen were based on their local availability and also on the basis of information gathered during literature studies.

Test for carotenoids: The colour of solution containing sample in solvent disappears after successive addition of 5% solution of sodium nitrite and 0.5M sulphuric acid.

Extraction: 10 grams of each sample were used. The samples were then grinded with a mortar and pestle and mixed in a solvent. 40 ml of three different solvents i.e. acetone, ethanol and butanol were used. The solution was filtered using muslin cloth. The filtrate was centrifuged at 10,000 rpm for 1 minute. The aqueous phase was collected and stored at 4ËšC.

Measurement of Absorbance: Absorbance was measured at 446 nm at zeroth hour and after 6 hrs of standing time using a spectrophotometer.

Concentration of Lutein was calculated by the following formula:

Conc of Lutein (µg/g of sample) = A X V (ml) X dilution factor

€ X W (g)

Where,

A= Absorbance at 446 nm

V= Volume of extract

€= Absorption coefficient (2589)

W= Dry weight of sample

(R. G. Alcides Oliveira et al, 2010)

Detection Method: To detect the lutein content in the extracted material Thin Layer Chromatography (TLC) was performed. The extracted samples were concentrated under vacuum using rotary evaporator. High temperature causes destruction of lutein. Thus the temperature is kept less than the boiling point of the solvents. Air dried samples were then used in TLC.

TLC plates were made using silica slurry, mobile phase used was hexane. The samples were dissolved in methanol solvent and spot was placed on the TLC plate along with standard lutein. The plates were then kept in the beaker containing the mobile phase and allowed to travel up to ¾ th of the length. The spots were detected under UV light and Rf value was calculated using the formula:

Rf = ­­­­­­­­­­­­­­­­­­­­­ Length of spot travelled

Length of mobile phase travelled

Result and Discussion: The screening experiment was carried out to look for the most suitable raw material for the extraction of lutein. The relative amount of lutein was measured using UV Spectrophotometer at 446 nm. Among all the samples selected marigold showed the highest yield and corn showed the lowest yield.

Absorbance of various extracts:

Source

Standing time (hrs)

Acetone

Ethanol

Butanol

OD

Con

OD

Con

OD

Con

Marigold

0

0.541 0.301

0.301 1.163

0.221 0.854

3

0.631 2.437

0.622 2.402

0.341 1.317

6

0.978 3.778

0.878 3.391

0.432 1.669

9

0.990 3.823

0.880 3.398

0.435 1.680

Neem

0

0.332 1.282

0.297 1.147

0.211 0.815

3

0.458 1.769

0.451 1.742

0.444 1.715

6

0.912 3.523

0.676 2.611

0.632 2.441

9

0.915 3.534

0.679 2.623

0.637 2.460

Spinach

0

0.345 1.333

0.279 1.078

0.201 0.776

3

0.464 1.792

0.315 1.217

0.394 1.522

6

0.818 3.159

0.610 2.356

0.493 1.904

9

0.820 3.167

0.615 2.375

0.499 1.927

Papaya

0

0.311 1.201

0.204 0.788

0.196 0.757

3

0.530 2.047

0.256 0.989

0.280 1.081

6

0.661 2.553

0.398 1.537

0.339 1.309

9

0.685 2.695

0.399 1.541

0.341 1.317

Carrot

0

0.215 0.830

0.197 0.761

0.132 0.509

3

0.381 1.472

0.202 0.780

0.139 0.537

6

0.431 1.664

0.212 0.819

0.145 0.444

9

0.431 1.665

0.213 0.823

0.148 0.571

Gerbera

0

0.198 0.764

0.111 0.428

0.091 0.351

3

0.261 1.008

0.134 0.517

0.101 0.390

6

0.362 1.398

0.145 0.560

0.156 0.603

9

0.380 1.468

0.150 0.579

0.158 0.610

Grape

0

0.106 0.409

0.095 0.367

0.064 0.247

3

0.211 0.815

0.100 0.386

0.099 0.382

6

0.311 1.201

0.137 0.529

0.111 0.428

9

0.316 1.221

0.139 0.537

0.111 0.429

Orange

0

0.098 0.378

0.065 0.251

0.033 0.127

3

0.111 0.429

0.081 0.313

0.045 0.174

6

0.376 1.452

0.103 0.398

0.096 0.371

9

0.377 1.456

0.109 0.421

0.099 0.382

Cabbage

0

0.067 0.258

0.059 0.228

0.026 0.100

3

0.111 0.429

0.075 0.286

0.031 0.119

6

0.298 1.151

0.096 0.371

0.075 0.289

9

0.300 1.158

0.099 0.382

0.076 0.294

Corn

0

0.066 0.172

0.021 0.081

0.012 0.046

3

0.011 0.042

0.032 0.124

0.028 0.108

6

0.113 0.436

0.054 0.209

0.043 0.166

9

0.141 0.544

0.054 0.209

0.044 0.169

Table 1: Absorbance at 446 nm

Graph1 shows the concentration of lutein immediately at zeroth hour of standing time. Graph 2, 3 and 4 shows the amount of lutein extracted after 3 h of standing time, 6h of standing and 9h of standing time, respectively. Within the range of standing time covered in this study, it was observed that the increment of standing time increased the amount of lutein extracted. This is due to the longer duration allowed for process to take place. But after the standing time was increased from 6 to 9 h rate of increase in lutein concentration decreased. Thus further increment in time was not considered. In case of marigold the concentration of lutein in acetone increased from 0.541 µg/g of sample to 3.778 µg/g of sample after 6 hours.

Graph 1

Graph 2

Where, 1-Mariegold; 2-Neem; 3-Spinach; 4-Papaya; 5-Carrot; 6-Gerbera

7-Grape; 8-Orange; 9-Cabbage; 10-Corn

Graph 4

Graph 3

Comparing the performance of various solvents used in study, acetone extracted highest amount of lutein followed by ethanol and then butanol (Graph 1, 2, 3 & 4). It is because acetone has a polarity which is lower than that of ethanol and butanol and also it has been found that a good solvent for a solute has a polarity to that of the solute.

Table2: shows Rf values for sample and standard lutein with different solvents.

TLC was done to identify lutein in the extract by comparing Rf values of the standard with the Rf value of sample. Results obtained from different sources showed Rf values for acetone to be around 0.42, ethanol to be around 0.30 and butanol to be around 0.55. These values matched with the results obtained from the standard source of lutein, proving that the extract contained lutein.

source

Acetone

Ethanol

Butanol

Marigold

Standard

Sample

0.40

0.42

0.29

0.30

0.58

0.55

Neem

Standard

Sample

0.45

0.44

0.27

0.26

0.57

0.56

Spinach

Standard

Sample

0.39

0.40

0.30

0.31

0.55

0.55

Papaya

Standard

Sample

0.42

0.43

0.25

0.25

0.58

0.56

Carrot

Standard

Sample

0.47

0.45

0.32

0.30

0.60

0.56

Gerbera

Standard

Sample

0.49

0.50

0.35

0.36

0.52

0.50

Grape

Standard

Sample

0.43

0.42

0.31

0.30

0.57

0.59

Orange

Standard

Sample

0.38

0.39

0.28

0.26

0.60

0.61

Cabbage

Standard

Sample

0.40

0.42

0.30

0.32

0.58

0.54

Corn

Standard

Sample

0.45

0.47

0.28

0.28

0.56

0.56

Sample

Standard Lutein (marigold in acetone )

2

1

Where, 1- distance travelled by lutein

2-distance travelled by mobile phase

TLC SLIDE

Conclusion:

Our study is carried out to determine the best source for extraction of lutein from locally available sources. Among all the sources used for the extraction of lutein, Marigold showed the highest content of lutein which was 3.82 µg/g of sample. Also the literature studies shows that other carotenoids in marigold are less compared to lutein which is about 80% of total carotenoid content. The rate of extraction and yield on the basis of solubility and polarity of all the three solvents when evaluated showed that Acetone yielded the highest concentration of lutein. As standing time was increased to 9 hours, there was increase in concentration of lutein as the solvent was in contact with the sample for a longer time period. However after a certain time interval, the effect of standing time was constant. Therefore optimum time for extraction was 6 hours. TLC result showed the presence of lutein in extract. Although, lutein is administered for macular degeneration, use of this carotenoid to reduce many systemic diseases and ease of purification needs further study.

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