Antioxidant Activity

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Antioxidant Activity

Introduction

The largest parts of the diseases are mainly linked to oxidative stress due to free radicals (Gutteridgde, 1995). Antioxidants can interact with the oxidation process by reacting with free radicals, chelation, catalyzing metals, and also by acting as oxygen scavengers (Buyukokuroglu et al., 2001).

Literature reviews have shown that there was much effort to invent medicine to overcoming the death. But until recently the actual cause of aging was not known. There is considerable recent evidence that free radical induce oxidative damage to biomolecules. This damage causes aging, diabetes, cancer, malaria, neurodegenerative diseases and other pathological events in living organisms (Halliwell et al. 1992). Antioxidants which scavenge free radicals are known to posses an important role in preventing these free radical induced-diseases. There is an increasing interest in the antioxidant effects of compounds derived from plants, which could be relevant in relations to their nutritional incidence and their role in health and diseases (Steinmetz et al., 1996; Aruoma, 1998; Bandoniene et al., 2000; Pieroni et al., 2002; Couladis et al., 2003). A number of reports on the isolation and testing of plant derived antioxidants have been described during the past decade. Natural antioxidants constitute a broad range of substances including phenolic or nitrogen containing compounds and carotenoids (Shahidi et al., 1992; Velioglu et al., 1998; Pietta et al., 1998). The medicinal properties of plants have been investigated throughout the world, due to their potent antioxidant activities, minimum or no side effects and economic viability (Auudy et al., 2003).

Lipid peroxidation is one of the main reasons for deterioration of food products during processing and storage. Synthetic antioxidant such as tert-butyl-1-hydroxitoluene (TBHT), tert-butylhydroquinone (TBHQ), butylated hydroxianisole (BHA) and propyl gallate (PG) are widely used as food additives to increase shelf life, especially lipid and lipid containing products by retarding the process of lipid peroxidation. However, TBHT and BHA are known to have not only toxic and carcinogenic effects on humans (Ito et al. ,1986; Wichi, 1988), but also abnormal effects on enzyme systems (Inatani et al. 1983). Thus, the interest in natural antioxidant, especially of plant origin, has greatly increased in recent years (Jayaprakasha et al., 2000). Plant polyphenols have been studied largely because of the possibility that they might underlie the protective effects afforded by fruit and vegetable intake against cancer and others chronic diseases (Elena et al., 2006).

Antioxidants: The free radical scavengers

Oxygen is the highest necessary substance for human life. But it is a Jeckyl and Hyde (both pleasant and unpleasant) element. We need it for critical body functions, such as respiration and immune response, but the element’s dark side is a reactive chemical nature that can damage body cells. The perpetrators of this “oxidative damage” are various oxygen-containing molecules, most of which are different types of free radicals—unstable, highly energized molecules that contain an unpaired electron.

Since stable chemical bonds require electron pairs, free radicals generated in the body steal electrons from nearby molecules, damaging vital cell components and body tissues. Oxidative damage in the body is akin to the browning of freshly cut apples, fats going rancid, or rusting of metal. Certain substances known as antioxidants, however, can help prevent this kind of damage. The following section describes the special relationship between oxidative damage, antioxidant protection and diabetes (Internet IV-I).

Oxidative Damage

Free radicals and other ‘reactive oxygen species’ are formed by a variety of normal processes within the body (including respiration and immune and inflammatory responses) as well as by elements outside the body, such as air pollutants, sunlight, and radiation. Whatever their sources, reactive oxygen species can promote damage that is link to increased risk of a variety of diseases and even to the aging process itself.

Oxidative damage to LDL (low-density lipoprotein or “bad cholesterol”) particles in the blood is believed to be a key factor in the progression of heart disease. Oxidative damage to fatty nerve tissue is linked to increased risk of various nervous system disorders, including Parkinson’s disease. Free radical damage to DNA can alter genetic material in the cell nucleus and, as a result, increase cancer risk. Oxidative damage has also been linked to arthritis and inflammatory conditions, shock and trauma, kidney disease, multiple sclerosis, bowel diseases, and diabetes (Internet- IV-II).

Antioxidant Protection

As a defense against oxidative damage, the body normally maintains a variety of mechanisms to prevent such damage while allowing the use of oxygen for normal functions. Such “antioxidant protection” derives from sources both inside the body (endogenous) and outside the body (exogenous). Endogenous antioxidants include molecules and enzymes that neutralize free radicals and other reactive oxygen species, as well as metal-binding proteins that sequester iron and copper atoms (which can promote certain oxidative reactions, if free). The body also makes several key antioxidant enzymes that help “recycle,” or regenerate, other antioxidants (such as vitamin C and vitamin E) that have been altered by their protective activity.

Exogenous antioxidants obtained from the diet also play an important role in the body’s antioxidant defense. These include vitamin C, vitamin E, carotenoids such as beta-carotene and lycopene, and other plant nutrients, or substances found in fruits, vegetables, and other plant foods that provide health benefits. Vitamin C (ascorbic acid), which is water-soluble, and vitamin E (tocopherol), which is fat-soluble, are especially effective antioxidants because they quench a variety of reactive oxygen species and are quickly regenerated back to their active form after they neutralize free radicals.

Morever, recent years have witnessed a renewed interest in plants as pharmaceuticals. This interest has been focused particularly on the adoption of extracts of plants, for self-medication by the general people. Within this context, considerable interest has arisen in the possibility that the impact of several major diseases may be either ameliorated or prevented by improving the dietary intake of natural nutrients with antioxidant properties, such as vitamin E, vitamin C, b-carotene and plant phenolics like tannins and flavonoids. The use of plant extracts in traditional medicine by old Indian and Chinese people have been going on from ancient time. Herbalism and folk medicine, both ancient and modern, have been the source of much useful therapy (Rashid et al., 1997).

The purpose of this study was to evaluate extractives as well as isolated compounds as new potential sources of natural antioxidants and phenolic compounds.

Antioxidant activity: DPPH assay

Principle

The free radical scavenging activities (antioxidant capacity) of thecccccc plant extracts on the persistent radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) were estimated by the method of Brand-Williams et al., 1995.

Here 2.0 ml of a methanol solution of the extract at different concentration were mixed with 3.0 ml of a DPPH methanol solution (20 mg/ml). The antioxidant potential was assayed from the bleaching of purple colored methanol solution of DPPH radical by the plant extract as compared to that of tert-butyl-1-hydroxytoluene (TBHT) by a UV spectrophotometer. The reaction mechanism is shown below:

  • DPPH = 2,2-diphenyl-1-picrylhydrazyl

Color variation of DPPH solution after samples treatment

Materials and Methods

DPPH was used to evaluate the free radical scavenging activity (antioxidant potential) of various compounds and medicinal plants (Choi et al., 2000; Desmarchelier et al., 1997).

Materials and preparation of materials

2,2-diphenyl-1-picryldrazyl (DPPH) Beaker (100 & 200 ml)
tert-butyl-1-hydroxytoluene (TBHT) Test tube
Ascorbic acid Light-proof box
Distilled water Pipette (5 ml)
Methanol Micropipette (50-200 ml)
UV-spectrophotometer Amber reagent bottle
Beaker (100 & 200 ml) Weighing balance
Test tube Exts. of related plant

Table 4.1: Test samples of experimental plants

Plant/

compounds

Test samples Code Amount (mg)
A. paniculata Ethanol soluble aerial part extract (crude) ESAE 2.00
n-Hexane soluble partitionate of ESAE HXSP 2.00
Carbon tetrachloride soluble partitionate of ESAE CTSP 2.00
Dichloromethane soluble partitionate of ESAE DMSP 2.00
Aqueous soluble partitionate of ESAE AQSP 2.00
A. chinensis Methanol soluble bark extract (crude) MSBE 2.00
n-Hexane soluble partitionate of MSBE HXSP 2.00
Carbon tetrachloride soluble partitionate of MSBE CTSP 2.00
Chloroform soluble partitionate of MSBE CFSP 2.00
Aqueous soluble partitionate of MSBE AQSP 2.00
S. sesban Methanol soluble leaves extract MSLE 2.00
Pet. ether soluble partitionate of MSLE PESP 2.00
Carbon tetrachloride soluble partitionate of MSBE CTSP 2.00
Chloroform soluble partitionate of MSBE CFSP 2.00
Aqueous soluble partitionate of MSBE AQSP 2.00
M. oleifera Methanol soluble bark extract (crude) MSLE 2.00
n-Hexane soluble partitionate of MSLE HXSP 2.00
Carbon tetrachloride soluble partitionate of MSLE CTSP 2.00
Dichloromethane soluble partitionate of MSLE DMSP 2.00
Aqueous soluble partitionate of MSLE AQSP 2.00
From S. sesban 3,7-Dihydroxy oleanolic acid (104) SS-02 1.0

Control preparation for antioxidant activity measurement

Ascorbic acid and tert-butyl-1-hydroxytoluene (TBHT) were used as positive control. Calculated amount of ascorbic acid or TBHT was dissolved in methanol to get a mother solution having concentration of 1000 µg/ml. Serial dilution was made using the mother solution to get different concentrations ranging from 500.0 to 0.977 µg/ml.

DPPH solution preparation

20 mg DPPH powder was weighed and dissolved in methanol to get a DPPH solution having a concentration 20 µg/ml. The solution was prepared in the amber colored reagent bottle and kept in the light proof box.

Test sample preparation

Calculated amount of different extractives were measured and dissolved in methanol to get a mother solution (1000 µg/ml). Serial dilution of the mother solution provided different concentrations from 500.0 to 0.977 µg/ml which were kept in the dark flasks.

Methods

  • 2.0 ml of a methanol solution of the extract at different concentration (500 to 0.977 mg/ml) were mixed with 3.0 ml of a DPPH methanol solution (20 mg/ml).
  • After 30 min of reaction period at room temperature in dark place, the absorbance was measured at 517 nm against methanol as blank by using a suitable spectrophotometer.
  • Inhibition of free radical DPPH in percent (I%) was calculated as follows: (I%) = (1 – Asample/Ablank) ´ 100

Where Ablank is the absorbance of the control reaction (containing all reagents except the test material).

  • Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotted by inhibition percentage against extract/compound concentration (Figure 4.1).

The experiments were carried out in triplicate and the result was expressed as mean ± SD in every cases.

Calculation of IC50 value from the graph plotted inhibition percentage against extract concentration
Figure 4.1: Schematic representation of the method of assaying free radical scavenging activity

Results and Discussion

Andrographis paniculata

Different partitionates of ethanolic extract of the aerial part of A. paniculata were subjected to free radical scavenging activity assay by the method of Brand –Williams et al., 1995. Here, tert-butyl-1-hydroxytoluene (TBHT) was used as reference standard.

In this investigation, the dichloromethane soluble partitionate (DMSP) of crude ethanolic extract (ESAE) showed the highest free radical scavenging activity with IC50 value 19.33 µg/ml. At the same time the carbon tetrachloride soluble partitionate (CTSP) also exhibit moderate antioxidant potential having IC50 values 21.25 and 23.79 µg/ml, respectively. The IC50 value for the TBHT was found to be 15.08 µg/ml (Table 4.2, Figure 4.2).

Table 4.2: List of IC50 values and equation of regression lines of standard and the test samples of A. paniculata

Test samples IC50 (µg/ml)# Equation of Regression line R2
TBHT 15.08 ± 0.52 y = 14.666Ln(x) + 10.202 0.946
ESAE 23.79 ± 1.17 y = 11.135Ln(x) + 14.706 0.9727
HXSP 52.26 ± 2.1 y = 8.796Ln(x) + 15.194 0.9341
CTSP 21.25 ± 0.59 y = 7.1105Ln(x) + 28.262 0.9773
DMSP 19.33 ± 1.08 y = 10.469Ln(x) + 18.988 0.976
AQSP 36.6 ± 1.63 y = 9.9965Ln(x) + 14.005 0.9658

#The values of IC50 are expressed as mean±SD (n=3)

Figure 4.2: Chart for IC50 values of standard and different extractives of A. paniculata

Table 4.3: List of absorbance against concentrations and IC50 value of tert-butyl-1-hydroxytoluene (TBHT)

Abs of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4..3: Chart for IC50 value of tert-butyl-1-hydroxytoluene (TBHT)
0.435 500 0.029 93.418 15.08
250 0.028 93.671
125 0.061 85.993
62.5 0.09 79.241
31.25 0.145 66.468
15.625 0.228 47.386
7.8125 0.306 29.452
3.90625 0.343 21.013
1.953125 0.344 20.797
0.9765625 0.354 18.548

Table 4.4: List of absorbance against concentrations and IC50 value of ESAE (crude) of A. paniculata

Abs of

Blank

Conc

(mg/ml)

Abs of

Extrac

%

Inhibition

IC50 (mg/ml) Figure 4.4: Chart for IC50 value of ethanol extract of A. paniculata
0.435 500 0.072 83.23 23.79
250 0.091 79.193
125 0.120 72.243
62.5 0.166 61.756
31.25 0.213 51.102
15.625 0.272 37.2448
7.8125 0.279 35.7469
3.90625 0.311 28.3571
.953125 0.338 22.1938
0.9765625 0.345 20.6632

Table 4.5: List of absorbance against concentrations and IC50 value of HXSP of A. paniculata

Abs of Blank Conc

(mg/ml)

Abs of Extract %

Inhibition

IC50

(mg/ml)

Figure 4.5: Chart for IC50 value of HXSP of A. paniculata
0.435 500 0.108 75.1152 52.26
250 0.137 68.4285
125 0.184 57.6692
62.5 0.225 48.1243
31.25 0.271 37.5576
15.625 0.292 32.7188
7.8125 0.301 30.6451
3.90625 0.305 29.7235
1.953125 0.321 26.0368
0.9765625 0.355 18.2027

Table 4.6: List of absorbance against concentrations and IC50 value of CTSP of A. paniculata

Abs of Blank Conc

(mg/ml)

Abs of Extract %

Inhibition

IC50

(mg/ml)

Figure 4.6: Chart for IC50 value of CTSP of A. paniculata
0.435 500 0,093 72.6206 21.25
250 0.105 68.8619
125 0.123 61.4271
62.5 0.168 58.3791
31.25 0.177 53.2727
15.625 0.198 44.1243
7.8125 0.224 43.5057
3.90625 0.249 40.7586
1.953125 0.308 29.1954
0.9765625 0.302 30.5747

Table 4.7: List of absorbance against concentrations and IC50 value of DMSP of A. paniculata

Abs of Blank Conc

(mg/ml)

Abs of Extract %

Inhibition

IC50

(mg/ml)

Figure 4.7: Chart for IC50 value of DMSP of A. paniculata
0.435 500 0.065 85.0574713 19.33
250 0.095 78.1609195
125 0.123 71.7241379
62.5 0.134 62.7586
31.25 0.197 51.5287102
15.625 0.252 42.0689655
7.8125 0.266 38.8505747
3.90625 0.285 34.4827586
1.953125 0.302 30.5747126
0.9765625 0.311 28.46731

Table 4.8: List of absorbance against concentrations and IC50 value of AQSP of A. paniculata

Abs of Blank Conc

(mg/ml)

Abs of Extract %

Inhibition

IC50

(mg/ml)

Figure 4.8: Chart for IC50 value of AQSP of A. paniculata
0.435 500 0.097 77.701149 36.6
250 0.122 71.954023
125 0.135 64.769433
62.5 0.203 53.333333
31.25 0.243 43.133934
15.625 0.275 36.781609
7.8125 0.292 32.873563
3.90625 0.307 29.425287
1.953125 0.323 25.747126
0.9765625 0.331 23.904701

4.3.2 Anthocephalus chinensis

Free radical scavenging activities of different partitionates of A. chinensis have been examined. The obtained results have been listed in Table 4.9. The IC50 value for the standard (TBHT) was found to be 15.08 mg/ml. Methanol soluble extract and aqueous soluble materials exhibit significant antioxidant capacity having IC50 value of 22.68 mg/ml and 24.54 mg/ml (Table 4.9, Figure 4.9).

Table 4.9: List of IC50 values and equation of regression lines of standard and test samples of A. chinensis

Test samples IC50 (µg/ml)# Equation of Regression line R2
TBHT 15.08 ± 0.52 y = 14.666Ln(x) + 10.202 0.946
MSBE 22.68 ± 1.12 y = 14.405Ln(x) + 5.0287 0.9426
HXSP 157.15 ± 2.08 y = 10.108Ln(x) – 1.1272 0.853
CTSP 53.37 ± 0.68 y = 10.535Ln(x) + 8.0922 0.9457
CFSP 27.21 ± 2.3 y = 11.3Ln(x) + 12.661 0.9738
AQSP 24.54 ± 1.47 y = 12.022Ln(x) + 11.518 0.9629

#The values of IC50 are expressed as mean±SD (n=3)

Figure 4.9: Chart for IC50 values of the standard and extractives of A. chinensis

Table 4.10: List of absorbance against concentrations and IC50 value of MSBE (crude) of A. chinensis

Abs of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.10: Chart for IC50 value of MSBE of A. chinensis
0.435 500 0.054 87.586207 22.68
250 0.060 86.2068
125 0.064 85.2873
62.5 0.135 68.9655
31.25 0.216 50.3448
15.625 0.246 43.4482
7.8125 0.322 25.977
3.90625 0.345 20.6896
1.953125 0.335 22.9885
0.9765625 0.354 18.4739

Table 4.11: List of absorbance against concentrations and IC50 value of HXSP of A. chinensis

Abs.of

Blank

Conc.

(mg/ml)

Abs. of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.11: Chart for IC50 value of HXSP of A. chinensis
0.435 500 0.130 70.09152 157.15
250 0.151 65.39032
125 0.255 41.30817
62.5 0.289 33.563
31.25 0.287 34.022
15.625 0.377 13.333
7.8125 0.390 10.344
3.90625 0.378 13.103
1.953125 0.391 10.114
0.9765625 0.390 10.344

Table 4.12: List of absorbance against concentrations and IC50 value of CTSP of A. chinensis

Abs.of

Blank

Conc.

(mg/ml)

Abs.of

Extract

%

Inhibition

IC50 (mg/ml) Figure 4.12: Chart for IC50 value of CTSP of A. chinensis
0.435 500 0.126 71.0164 53.37
250 0.136 68.7356
125 0.167 61.6091
62.5 0.184 57.6252
31.25 0.289 33.5632
15.625 0.289 33.5632
7.8125 0.295 32.1839
3.90625 0.308 29.1954
1.953125 0.387 11.0344
0.9765625 0.398 8.5057

Table 4.13: List of absorbance against concentrations and IC50 value of CFSP of A. chinensis

Abs.of

Blank

Conc.

(mg/ml)

Abs.of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.13: Chart for IC50 value of CFSP of A. chinensis
0.435 500 0.085 80.2873 27.21
250 0.121 72.1724
125 0.138 68.2754
62.5 0.154 64.5977
31.25 0.202 53.5517
15.625 0.251 42.2988
7.8125 0.289 33.5404
3.90625 0.301 30.7231
1.953125 0.324 25.4252
0.9765625 0.411 5.51772

Table 4.14: List of absorbance against concentrations and IC50 value of AQSP of A. chinensis

Abs.of

Blank

Conc.

(mg/ml)

Abs.of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 6.14: Chart for IC50 value of AQSP of A. chinensis
0.435 500 0.086 80.1438 24.54
250 0.114 73.57208
125 0.096 77.73506
62.5 0.159 63.54106
31.25 0.172 60.4597
15.625 0.239 45.04513
7.8125 0.300 31.0344
3.90625 0.322 25.977
1.953125 0.358 17.62901
0.9765625 0.382 12.1839

Sesbania sesban

Five extractives and one isolated compound from S. sesban were subjected to assay for free radical scavenging activity. In this study, the CFSP and AQSP showed the highest free radical scavenging activity with IC50 value 17.81 µg/ml and 21.72 µg/ml. At the same time petroleum ether soluble materials exhibit moderate antioxidant potential having IC50 value 25.73 µg/ml. The crude methanolic extract and CTSP exhibit low antioxidant activity having IC50 values 48.5 and 69.49 µg/ml, respectively. IC50 value for TBHT was 14.18 µg/ml (Table 4.15, Figure 4.15).

Table 4.15: IC50 values and equation of regression lines of standard and test samples of S. sesban

Test sample IC50 (µg/ml)# Equation of regression line R2
TBHT 14.18 ± 1.01 y = 14.776Ln(x) + 10.812 0.9351
MSLE 48.5 ± 0.78 y = 8.6915Ln(x) + 16.257 0.9877
PESP 25.73 ± 2.3 y = 6.2183Ln(x) + 29.801 0.9874
CTSP 69.49 ± 1.71 y = 6.0195Ln(x) + 24.466 0.9834
CFSP 17.81 ± 0.86 y = 8.8342Ln(x) + 24.555 0.9829
AQSP 21.72 ± 1.45 y = 6.0164Ln(x) + 31.478 0.8474

#The values of IC50 are expressed as mean ± SD (n=3)

Figure 4.15: Chart for IC50 values of the standard and extractives of S. sesban

Table 4.16: List of absorbance against concentrations and IC50 value of MSLE (crude) of S. sesban

Abs.of

Blank

Conc.

(mg/ml)

Abs.of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.16 Chart for IC50 value of MSLE (crude) of

S. sesban

0.484 500 0.126 73.966942 48.5
250 0.149 69.214876
125 0.173 64.256198
62.5 0.206 57.438017
31.25 0.251 48.140496
15.625 0.274 43.38843
7.8125 0.288 40.495868
3.90625 0.310 35.950413
1.953125 0.335 30.785124
0.9765625 0.356 26.446281

Table 4.17: List of absorbance against concentrations and IC50 value PESP of S. sesban

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.17: Chart for IC50 value PESP of S. sesban
0.484 500 0.146 69.834711 25.73
250 0.180 62.809917
125 0.193 60.123967
62.5 0.210 56.61157
31.25 0.232 52.066116
15.625 0.272 43.801653
7.8125 0.283 41.528926
3.90625 0.301 37.809917
1.953125 0.312 35.53719
0.9765625 0.337 30.371901

Table 4.18: List of absorbance against concentrations and IC50 value CTSP of S. sesban

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.18: Chart for IC50 value of CTSP of S. sesban
0.484 500 0.190 60.743802 69.49
250 0.207 57.231405
125 0.214 55.785124
62.5 0.246 49.173554
31.25 0.277 42.768595
15.625 0.281 41.942149
7.8125 0.291 39.876033
3.90625 0.327 32.438017
1.953125 0.351 27.479339
0.9765625 0.370 23.553719

Table 4.19: List of absorbance against concentrations and IC50 value CFSP of S. sesban

Abs of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.19: Chart for IC50 value of CFSP of S. sesban
0.484 500 0.083 82.85124 17.81
250 0.126 73.966942
125 0.158 67.355372
62.5 0.189 60.950413
31.25 0.236 51.239669
15.625 0.264 45.454545
7.8125 0.284 41.322314
3.90625 0.310 35.950413
1.953125 0.328 32.231405
0.9765625 0.350 27.68595

Table 4.20: List of absorbance against concentrations and IC50 value AQSP of S. sesban

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.20: Chart for IC50 value of AQSP of S. sesban
0.484 500 0.093 80.785124 21.72
250 0.186 61.570248
125 0.200 58.677686
62.5 0.246 49.173554
31.25 0.253 47.727273
15.625 0.268 44.628099
7.8125 0.269 44.421488
3.90625 0.277 42.768595
1.953125 0.294 37.256198
0.9765625 0.296 34.011023

Moringa oleifera

Different extractives of bark of M. oleifera were subjected to evaluation for free radical scavenging activity by previously described method. Here, the dichloromethane (DMSP) and carbon tetrachloride soluble materials (CTSP) showed the highest free radical scavenging activity with IC50 value 27.49 µg/ml and 35.78 µg/ml. At the same time, methanol soluble extract (crude) and hexane soluble partitionates (HXSP) did not exhibit promising antioxidant activity (Table 4.21, Figure 4.21).

Table 4.21: List of absorbance against concentrations and IC50 values of standard and test samples of M. oleifera

Test samples IC50 (µg/ml)# Equation of regression line R2
TBHT 14.18 ± 1.01 y = 14.776Ln(x) + 10.812 0.9351
MSBE 44.3 ± 0.98 y = 11.156Ln(x) + 7.7007 0.9071
HXSP 48.47 ± 2.41 y = 8.5434Ln(x) + 16.839 0.9684
CTSP 35.78 ± 1.83 y = 8.6283Ln(x) + 19.128 0.9723
DMSP 27.49 ± 0.87 y = 6.9879Ln(x) + 26.84 0.9556
AQSP 77.77 ± 2.62 y = 7.4341Ln(x) + 17.628 0.9596

#The values of IC50 are expressed as mean±SD (n=3)

Figure 4.21: Chart for IC50 value of the standard and extractives of M. oleifera

Table 4.22: List of absorbance against concentrations and IC50 value of methanol extract of M. oleifera

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.22:Chart for IC50 value of MSBE of M. oleifera
0.395 500 0.101 78.961 44.3
250 0.118 75.5844
125 0.143 70.3896
62.5 0.243 49.6103
31.25 0.306 36.6233
15.625 0.336 30.3896
7.8125 0.373 22.8571
3.90625 0.39 19.2207
1.953125 0.382 21.0389
0.9765625 0.398 17.6623

Table 4.23: List of absorbance against concentrations and IC50 value of HXSP of M. oleifera

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.23: Chart for IC50 value of HXSP of M. oleifera
0.395 500 0.164 66.1157 48.47
250 0.183 62.1901
125 0.187 61.3636
62.5 0.217 55.1653
31.25 0.255 47.3141
15.625 0.266 45.0413
7.8125 0.336 30.5785
3.90625 0.348 28.0992
1.953125 0.394 18.5950
0.9765625 0.395 18.3884

Table 4.24: List of absorbance against concentrations and IC50 value of CTSP of M. oleifera

Abs of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.24: Chart for IC50 value of CTSP of M. oleifera
0.395 500 0.099 74.285714 35.78
250 0.108 70.456378
125 0.118 59.504132
62.5 0.143 53.783138
31.25 0.196 44.913562
15.625 0.225 38.459123
7.8125 0.269 36.957215
3.90625 0.310 35.950413
1.953125 0.321 23.419683
0.9765625 0.305 20.638123

Table 4.25: List of absorbance against concentrations and IC50 value of DMSP of M. oleifera

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.25: Chart for IC50 value of DMSP of M. oleifera
0.385 500 0.152 68.595041 27.49
250 0.170 64.876033
125 0.201 58.471074
62.5 0.213 55.991736
31.25 0.239 50.619835
15.625 0.229 52.68595
7.8125 0.264 45.454545
3.90625 0.311 35.743802
1.953125 0.351 27.479339
0.9765625 0.364 24.793388

Table 4.26: List of absorbance against concentrations and IC50 value of AQSP of M. oleifera

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

%

Inhibition

IC50

(mg/ml)

Figure 4.26: Chart for IC50 value of AQSP of M. oleifera
0.385 500 0.186 61.570248 77.77
250 0.211 56.404959
125 0.231 52.272727
62.5 0.242 50.0
31.25 0.247 48.966942
15.625 0.275 43.181818
7.8125 0.329 32.024793
3.90625 0.365 24.586777
1.953125 0.388 19.834711
0.9765625 0.399 17.561983

SS-02 (3, 7-Dihydroxyoleanolic acid, 104)

SS-02 (3, 7-dihydroxyoleanolic acid (104) isolated from leaves of S. sesban was subjected to evaluation for free radical scavenging activity by previously described method. It showed free radical scavenging activity with IC50 values of 58.20 µg/ml in the DPPH assay as compared to blank for the standard antioxidant agent TBHT.

Table 4.27: List of absorbance against concentrations and IC50 value of SS-02 (3,7-dihydroxy oleanolic acid, 104)

SS-02 (3, 7-Dihydroxyoleanolic acid, 103)
Sl

no.

Abs.of

Blank

Conc

(mg/ml)

Abs of

Extract

Inhibition %

Inhibition

IC50

(mg/ml)

1 0.484 500 0.175 0.6384298 63.84298 58.20
2 250 0.201 0.5847107 58.47107
3 125 0.221 0.5433884 54.33884
4 62.5 0.232 0.5206612 52.06612
5 31.25 0.236 0.5123967 51.23967
6 15.625 0.265 0.4524793 45.24793
7 7.8125 0.318 0.3429752 34.29752
8 3.90625 0.355 0.2665289 26.65289
9 1.953125 0.377 0.2210744 22.10744
10 0.9765625 0.386 0.2024793 20.24793

Antioxidant in diabetes management

There is recent evidence that free radical induce oxidative damage to biomolecules. This damage causes aging, diabetes, cancer, neurodegenerative diseases and other pathological events in living organisms (Halliwell et al. 1992). Antioxidants which scavenge free radicals are known to posses an important role in preventing these free radical induced-diseases (Jayaprakasha et al., 2000).

There have the close relationship between oxidative damage, antioxidant protection, diabetes and complications of diabetes. Oxidative damage has been link to arthritis, shock and trauma, kidney disease and diabetes.

There have two types of antioxidants, synthetic (chemically synthesized) and natural (plant derived). Some synthetic antioxidant such as tert-butyl-1-hydroxitoluene (TBHT), butylated hydroxianisole (BHA) are known to have not only toxic and carcinogenic effects on humans (Ito et al. ,1986; Wichi, 1988), but also abnormal effects on enzyme systems (Inatani et al. 1983). Thus, the interest in natural antioxidant, especially of plant origin, has greatly increased in recent years (Jayaprakasha et al., 2000).

Not only endogenous antioxidants, exogenous antioxidants obtained from the diet also play an important role in the body’s antioxidant defense. These include vitamin C, vitamin E, carotenoids such as beta-carotene and lycopene, and other phytonutrients, or substances found in fruits, vegetables, and other plant foods that provide health benefits.

There is substantial evidence that people with diabetes tend to have increased generation of reactive oxygen species, decreased antioxidant protection, and therefore increased oxidative damage. High blood glucose level (hyperglycemia) has been show