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Antioxidant Activity

IntroductionThe 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 scavengersOxygen 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 DamageFree 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, -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

PrincipleThe 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 g/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 MethodsDPPH 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 materials2,2-diphenyl-1-picryldrazyl (DPPH) Beaker (100 & 200 ml)tert-butyl-1-hydroxytoluene (TBHT)

Test tube

Ascorbic acid Light-proof boxDistilled water Pipette (5 ml)Methanol Micropipette (50-200 l)UV-spectrophotometer Amber reagent bottleBeaker (100 & 200 ml) Weighing balanceTest 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 2.00n-Hexane soluble partitionate of ESAE HXSP 2.00Carbon tetrachloride soluble partitionate of ESAE CTSP 2.00

Dichloromethane soluble partitionate of ESAE DMSP 2.00Aqueous soluble partitionate of ESAE AQSP 2.00

A. chinensis

Methanol soluble bark extract (crude) MSBE 2.00n-Hexane soluble partitionate of MSBE HXSP 2.00Carbon tetrachloride soluble partitionate of MSBE

CTSP 2.00

Chloroform soluble partitionate of MSBE CFSP 2.00Aqueous soluble partitionate of MSBE AQSP 2.00

S. sesban

Methanol soluble leaves extract MSLE 2.00Pet. ether soluble partitionate of MSLE PESP 2.00Carbon tetrachloride soluble partitionate of MSBE

CTSP 2.00

Chloroform soluble partitionate of MSBE CFSP 2.00Aqueous soluble partitionate of MSBE AQSP 2.00

M. oleifera

Methanol soluble bark extract (crude) MSLE 2.00n-Hexane soluble partitionate of MSLE HXSP 2.00Carbon tetrachloride soluble partitionate of MSLE CTSP 2.00

Dichloromethane soluble partitionate of MSLE DMSP 2.00Aqueous 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 preparation20 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 preparationCalculated 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

g/ml) were mixed with 3.0 ml of a DPPH methanol solution (20 g/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) 100Where 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.

DPPH in methanol – 3.0 ml(conc.– 20 g/ml)

Purple color

Extract in methanol – 2.0 ml(conc.– 500 to 0.977 g/ml

Reaction allowed for 30 min in absence of light at room

temperature

Decolonization of purple color of DPPH

Absorbance measured at 517 nm using methanol as blank

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 paniculataDifferent 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 0.946ESAE 23.79 ± 1.17 y = 11.135Ln(x) + 14.706 0.9727HXSP 52.26 ± 2.1 y = 8.796Ln(x) + 15.194 0.9341CTSP 21.25 ± 0.59 y = 7.1105Ln(x) + 28.262 0.9773DMSP 19.33 ± 1.08 y = 10.469Ln(x) + 18.988 0.976AQSP 36.6 ± 1.63 y = 9.9965Ln(x) + 14.005 0.9658

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

IC50 values of different extractives of A. paniculata

15.08

23.79

52.26

21.25 19.33

36.6

0

10

20

30

40

50

60

TBHT ESAE HXSP CTSP DMSP AQ SP

Extractives

IC50

val

ues

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(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)

Free Radical Scavenging Activity of TBHT

y = 14.666Ln(x) + 10.202R2 = 0.946

0

20

40

60

80

100

120

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

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 93.671125 0.061 85.99362.5 0.09 79.24131.25 0.145 66.46815.625 0.228 47.3867.8125 0.306 29.4523.90625 0.343 21.0131.953125 0.344 20.7970.9765625 0.354 18.548

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

Abs ofBlank

Conc(g/ml)

Abs ofExtrac

%Inhibition

IC50

(g/ml)

Free Radical Scavenging Activity of ESAE

y = 11.173Ln(x) + 14.67R2 = 0.9716

0

10

20

30

40

50

60

70

80

90

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

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 79.193125 0.120 72.24362.5 0.166 61.75631.25 0.213 51.10215.625 0.272 37.24487.8125 0.279 35.74693.90625 0.311 28.3571.953125 0.338 22.19380.9765625 0.345 20.6632

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

Abs of Blank

Conc(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)

0.435 500 0.108 75.1152 52.26250 0.137 68.4285125 0.184 57.669262.5 0.225 48.124331.25 0.271 37.557615.625 0.292 32.71887.8125 0.301 30.6451

Free Radical Scavenging Activity of HXSP

y = 8.796Ln(x) + 15.194R 2 = 0.9341

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600

Concentration (microgram/ ml)

Figure 4.5: Chart for IC50 value of HXSP of A. paniculata

3.90625 0.305 29.72351.953125

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(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)

Free radical scavenging activi ty of CTSP

y = 7.1105Ln(x) + 28.262R2 = 0.9773

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.6: Chart for IC50 value of CTSP of A. paniculata

0.435

500 0,093 72.6206

21.25

250 0.105 68.8619125 0.123 61.427162.5 0.168 58.379131.25 0.177 53.272715.625 0.198 44.12437.8125 0.224 43.50573.90625 0.249 40.75861.953125

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(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)

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

Free radical scavenging activity of DMSP(AP)

y = 10.469Ln(x) + 18.988R2 = 0.976

0102030405060708090

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

Figure 4.7: Chart for IC50 value of DMSP of A. paniculata

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(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)

Fre e radical scavenging activi ty of AQSP

y = 9.9965Ln(x) + 14.005R2 = 0.9658

0102030405060708090

0 100 200 300 400 500 600Concentration (microgram/mi)

% I

nhib

ition

Figure 4.8: Chart for IC50 value of AQSP of A. paniculata

0.435

500 0.097 77.701149

36.6

250 0.122 71.954023125 0.135 64.76943362.5 0.203 53.33333331.25 0.243 43.13393415.625 0.275 36.7816097.8125 0.292 32.8735633.90625 0.307 29.4252871.953125

0.323 25.747126

0.9765625

0.331 23.904701

4.3.2 Anthocephalus chinensisFree 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 g/ml. Methanol soluble extract and aqueous soluble materials exhibit significant antioxidant capacity having IC50 value of 22.68 g/ml and 24.54 g/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)

IC50 Values of different Extractives of A. Chinensis

15.08 22.68

157.15

53.37

27.21 24.54

0

20

40

60

80

100

120

140

160

180

TBHT MSBE HXSP CTSP CFSP AQ SPExtractives

IC50

val

ues (

mic

rogr

am/m

l)

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(g/ml)

Abs ofExtract

%Inhibition

IC50

(g/ml)

Free radical Scavenging Activity of MSBE

y = 14.405Ln(x) + 5.0287R2 = 0.9426

0102030405060708090

100

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

Figure 4.10: Chart for IC50 value of MSBE of A. chinensis

0.435

500 0.054 87.586207

22.68

250 0.060 86.2068125 0.064 85.287362.5 0.135 68.965531.25 0.216 50.344815.625 0.246 43.44827.8125 0.322 25.9773.90625 0.345 20.68961.953125

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.(g/ml)

Abs. ofExtract

%Inhibition

IC50

(g/ml)

Free Radical Scavenging Activity of HXSP

y = 10.108Ln(x) - 1.1272R2 = 0.853

-10

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600

Concebtration (microgram/ml)

% In

hibi

tion

Figure 4.11: Chart for IC50 value of HXSP of A. chinensis

0.435

500 0.130 70.09152

157.15

250 0.151 65.39032125 0.255 41.3081762.5 0.289 33.56331.25 0.287 34.02215.625 0.377 13.3337.8125 0.390 10.3443.90625 0.378 13.1031.953125

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.(g/ml)

Abs.ofExtract

%Inhibition

IC50

(g/ml)

Free radical Scavenging Activity of CTSP

y = 10.535Ln(x) + 8.0922R2 = 0.9457

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.12: Chart for IC50 value of CTSP of A. chinensis

0.435

500 0.126 71.0164

53.37

250 0.136 68.7356125 0.167 61.609162.5 0.184 57.625231.25 0.289 33.563215.625 0.289 33.56327.8125 0.295 32.18393.90625 0.308 29.19541.953125

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.ofBlank

Conc.(g/ml)

Abs.ofExtract

%Inhibition

IC50

(g/ml)

0.435 500 0.085 80.2873 27.21250 0.121 72.1724125 0.138 68.275462.5 0.154 64.597731.25 0.202 53.5517

Free Radical Scavenging Activity of CFSP

y = 11.3Ln(x) + 12.661R2 = 0.9738

0

10

20

30

40

50

60

70

80

90

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.13: Chart for IC50 value of CFSP of A. chinensis

15.625 0.251 42.29887.8125 0.289 33.54043.90625 0.301 30.72311.953125

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. chinensisAbs.of Blank

Conc.(g/ml)

Abs.ofExtract

%Inhibition

IC50

(g/ml)Free Radical Scavenging Activity of AQ SP (AC)

y = 12.022Ln(x) + 11.518R2 = 0.9629

0

1020

3040

50

6070

8090

100

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

Figure 6.14: Chart for IC50 value of AQSP of A. chinensis

0.435

500 0.086 80.1438

24.54

250 0.114 73.57208125 0.096 77.7350662.5 0.159 63.5410631.25 0.172 60.459715.625 0.239 45.045137.8125 0.300 31.03443.90625 0.322 25.9771.953125 0.358 17.629010.9765625 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)

IC50 values of different extractives of S. sesban

14.18

48.5

25.73

69.49

17.8121.72

0

10

20

30

40

50

60

70

80

TBHT MSLE PESP CTSP CFSP AQ SPExtractives

IC50

(mic

rogr

am/m

l)

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.(g/ml)

Abs.of Extract

%Inhibition

IC50

(g/ml)

Free Radical Scavenging Activity of MSLE

y = 7.8153Ln(x) + 24.816R2 = 0.9876

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

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 69.214876125 0.173 64.25619862.5 0.206 57.43801731.25 0.251 48.14049615.625 0.274 43.388437.8125 0.288 40.4958683.90625 0.310 35.9504131.953125

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(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)Free Radical Scavenging Activity of PESP

y = 6.2183Ln(x) + 29.801R2 = 0.9874

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

Figure 4.17: Chart for IC50 value PESP of S. sesban

0.484

500 0.146 69.834711

25.73

250 0.180 62.809917125 0.193 60.12396762.5 0.210 56.6115731.25 0.232 52.06611615.625 0.272 43.8016537.8125 0.283 41.5289263.90625 0.301 37.8099171.953125 0.312 35.537190.9765625 0.337 30.371901

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

Abs.of Blank

Conc(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)Free Radical Scavenging Activity of CTSP

y = 6.0195Ln(x) + 24.466R2 = 0.9834

0

10

20

30

40

50

60

70

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.18: Chart for IC50 value of CTSP of S. sesban

0.484

500 0.190 60.743802

69.49

250 0.207 57.231405125 0.214 55.78512462.5 0.246 49.17355431.25 0.277 42.76859515.625 0.281 41.9421497.8125 0.291 39.8760333.90625 0.327 32.4380171.953125 0.351 27.4793390.9765625 0.370 23.553719

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

Conc(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)Free Radical Scavenging Activity of CFSP

y = 8.8342Ln(x) + 24.555R2 = 0.9829

0

1020

3040

5060

7080

90

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.19: Chart for IC50 value of CFSP of S. sesban

0.484

500 0.083 82.85124

17.81

250 0.126 73.966942125 0.158 67.35537262.5 0.189 60.95041331.25 0.236 51.23966915.625 0.264 45.4545457.8125 0.284 41.3223143.90625 0.310 35.9504131.953125 0.328 32.2314050.9765625 0.350 27.68595

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

Abs.of Blank

Conc(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)Free radical scavenging activity of aqueous soluble

fraction

y = 6.0164Ln(x) + 31.478R 2 = 0.8474

0102030405060708090

0 100 200 300 400 500 600

Conc. (microgram/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 61.570248125 0.200 58.67768662.5 0.246 49.17355431.25 0.253 47.72727315.625 0.268 44.6280997.8125 0.269 44.4214883.90625 0.277 42.7685951.953125 0.294 37.2561980.9765625 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)

IC50 values of different extractives of M. oleifera

14.18

44.348.47

35.7827.49

77.77

0102030405060708090

TBHT MSBE HXSP CTSP DMSP AQSPExtractives

IC50

val

ues

(mic

rogr

am/m

l)

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(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)Free Radical Scavenging Activity of MSBE

y = 11.156Ln(x) + 7.7007R2 = 0.9071

010

203040

506070

8090

0 100 200 300 400 500 600

Conc (microgram/ml)

% In

hibi

tion

Figure 4.22:Chart for IC50 value of MSBE of M. oleifera

0.395

500 0.101 78.961

44.3

250 0.118 75.5844125 0.143 70.389662.5 0.243 49.610331.25 0.306 36.623315.625 0.336 30.38967.8125 0.373 22.85713.90625 0.39 19.22071.953125 0.382 21.03890.9765625 0.398 17.6623

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

Abs.of Blank

Conc(g/ml)

Abs of Extract

%Inhibition

IC50

(g/ml)

0.395

500 0.164 66.1157

48.47

250 0.183 62.1901125 0.187 61.363662.5 0.217 55.165331.25 0.255 47.314115.625 0.266 45.04137.8125 0.336 30.57853.90625 0.348 28.09921.953125 0.394 18.5950

Free Radical Scavenging Activity of HXSP

y = 8.5434Ln(x) + 16.839R2 = 0.9684

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.23: Chart for IC50 value of HXSP of M. oleifera

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(g/ml)

Abs of Extract

% Inhibition

IC50

(g/ml) Free Radical Scavenging Activity of CTSP

y = 8.6283Ln(x) + 19.128R2 = 0.9723

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.24: Chart for IC50 value of CTSP of M. oleifera

0.395

500 0.099 74.285714

35.78

250 0.108 70.456378125 0.118 59.50413262.5 0.143 53.78313831.25 0.196 44.91356215.625 0.225 38.4591237.8125 0.269 36.9572153.90625 0.310 35.9504131.953125 0.321 23.4196830.9765625 0.305 20.638123

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

Abs.of Blank

Conc(g/ml)

Abs of Extract

% Inhibition

IC50

(g/ml)

0.385500 0.152 68.595041 27.49250 0.170 64.876033125 0.201 58.47107462.5 0.213 55.99173631.25 0.239 50.61983515.625 0.229 52.685957.8125 0.264 45.454545

Free Radical Scavenging Activity of DMSP

y = 6.9879Ln(x) + 26.84R2 = 0.9556

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600Concentration (microgram/ml)

% In

hibi

tion

Figure 4.25: Chart for IC50 value of DMSP of M. oleifera

3.90625 0.311 35.7438021.953125 0.351 27.4793390.9765625 0.364 24.793388

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

Abs.of Blank

Conc(g/ml)

Abs of Extract

% Inhibition

IC50

(g/ml) Free Radical Scavenging Activity of AQ SP (MO )

y = 7.4341Ln(x) + 17.628R2 = 0.9596

0

10

20

30

40

50

60

70

0 100 200 300 400 500 600

Concentration (microgram/ml)

% In

hibi

tion

Figure 4.26: Chart for IC50 value of AQSP of M. oleifera

0.385

500 0.186 61.570248

77.77

250 0.211 56.404959125 0.231 52.27272762.5 0.242 50.031.25 0.247 48.96694215.625 0.275 43.1818187.8125 0.329 32.0247933.90625 0.365 24.5867771.953125 0.388 19.8347110.9765625 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)

Slno.

Abs.ofBlank

Conc(g/ml)

Abs ofExtract Inhibition %

Inhibition

IC50

(g/ml)

1 0.484 500 0.175 0.6384298 63.84298 58.202 250 0.201 0.5847107 58.471073 125 0.221 0.5433884 54.33884

4 62.5 0.232 0.5206612 52.066125 31.25 0.236 0.5123967 51.239676 15.625 0.265 0.4524793 45.247937 7.8125 0.318 0.3429752 34.297528 3.90625 0.355 0.2665289 26.652899 1.953125 0.377 0.2210744 22.1074410 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 shown to increase reactive oxygen species and end products of oxidative damage in isolated cell cultures, in animals with diabetes, and in humans with diabetes. Measurement of the end products of oxidative damage to body fat, proteins, and DNA are commonly used to assess the degree of oxidative damage to body cells and tissues. Most studies show that these measures are increased in people with diabetes (Internet IV-I).The activities of key antioxidant enzymes are found to be abnormal in people with diabetes. In some studies, these enzyme activities are seen to be lower than normal. Some studies indicate that oxidative damage is greater in people with Type 2 diabetes compared to those with Type1.There is evidence that antioxidant protection is decreased and oxidative stress increased in some people even before the onset of diabetes. For instance, increased levels of oxidative stress have been found in people who have impaired glucose tolerance or pre-diabetes.

Evidence for antioxidant protection in people with diabetes

Overall, the evidence indicates that hyperglycemia creates additional oxidative stress, and that measures of oxidative damage are generally increased in people with diabetes. Therefore, the question arises as to whether antioxidant treatment may delay or prevent diabetes, or delay the onset of diabetes complications that include cardiovascular, kidney, and eye diseases. Cell culture and animal studies support the hypothesis that antioxidants can protect diabetic cells from some damage. However, two types of human studies must be examined to answer the question: population studies and clinical trials.Population, or epidemiologic, studies have looked at the relationship between antioxidant intake and the development of diabetes. Examination of the diets of some 4,300 Finnish adults (40-69 years old) without diabetes showed that those with low dietary intakes of vitamin E had a significantly greater risk of developing Type 2 diabetes over the next two decades. There was no relationship between intake of vitamin C and risk of future diabetes development. In another study of 81 male and 101 female Finnish adults at high risk for Type 2 diabetes, dietary carotenoids were associated with improved measures of glucose metabolism in men but not women. In a third study, blood levels of five carotenoids were measured in 1,597 Australian adults that were healthy or had varying degrees of impaired glucose metabolism. Those with higher blood levels of the carotenoids had a healthier profile of glucose metabolism tests- fasting plasma glucose levels, insulin concentrations, and glucose tolerance levels. Another study with flavonoids (a class of antioxidants found in fruits and vegetables) of 38,018 healthy U. S. women over an average of nine years. The results showed no relationship between intake of flavonoids and risk of developing Type 2 diabetes. However, there was a modest benefit for consumption of apples and tea.

Kaneto H. et al, were conducted a long experiment with antioxidants on mice for observing diabetes status. According to an intraperitoneal glucose tolerance test, the treatment with N- a c e t y l -L-cysteine [NAC] retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Vitamins C and E were not effective when used alone but slightly effective when used in combination with NAC. No effect on insulin secretion was observed when the same set of antioxidants was given to nondiabetic control mice. Histologic analyses of the pancreases revealed that the β-cell mass was significantly larger in the diabetic mice treated with the antioxidants than in the untreated mice. As a possible cause, the antioxidant treatment suppressed apoptosis in β-cells without changing the rate of β-cell proliferation, supporting the hypothesis that in chronic hyperglycemia, apoptosis induced by oxidative stress causes reduction of β-cell mass. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. Furthermore, expression of pancreatic and duodenal homeobox factor-1 (PDX-1), a β- c e l l – specific transcription factor, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. In conclusion, our observations indicate that antioxidant treatment can exert beneficial effects in diabetes, with preservation of in vivo β-cell function. This finding suggests a potential usefulness of antioxidants for treating diabetes and provides further support for the implication of oxidative stress in β-cell dysfunction in diabetes (Kaneto H. et al, 1999, D i a b e t e s 4 8 :2 3 9 8–2406).

Diabetes mellitus worsens antioxidant status in patients with chronic pancreatitis, especially diabetes mellitus (Quilliot D. et al., 2005, Am J Clin Nutr, 81(5), 1117-25).In in vivo studies also, pretreatment of rats with oleanolic acid (an antioxidant) displayed significant (p<0.05) antihyperglycemic activity in starch tolerance test however, administration of starch fortified with oleanolic acid to the rats could not exhibited antihyperglycemic activity (Tiwari et al. 2010). Oleanolic acid glycosides exhibited their

hypoglycemic activity by suppressing the transfer of glucose from the stomach to the intestine and by inhibiting glucose transport at the brush border of the small intestine [Chem Pham Bull (Tokyo), 1998].

In summary, population studies and some clinical trials have shown mixed results as to possible benefits of antioxidants to people with diabetes. Some show a benefit, others show no.

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

Two compounds oleanolic acid and methoxy genistein (isolated from S. sesban) has reported to possess potential antidiabetic and antioxidant properties. Genistein acts as an antioxidant, similar to many other isoflavones, counteracting damaging effects of free radicals in tissues. (Han et al., 2009; Borras et al., 2009). Genistein and daidzein, the two major isoflavones, principally occur in nature as their glycosylated or methoxylated derivatives, which are cleaved in the large intestine to yield the free aglycones and further metabolites possesses antioxidant activity (Arti et al., 1998). Isoflavones have the property to neutralize free radicals. Among the isoflavones, genistein has the highest antioxidant activity (Internet-IV-III).

4.4 Conclusion All the extractives and some compounds of the investigated plants were subjected to free radical scavenging activity by the method of Brand-Williams et al., 1995. Here, tert-butyl-1-hydroxytoluene (TBH000T) and ascorbic acid was used as reference standard. In this study, the DMSP and CTSP of A. paniculata showed significant free radical scavenging activity with IC50 values of 19.33 µg/ml and 21.25 µg/ml, respectively. The MSBE and AQSP of A. chinensis exhibited promising antioxidant capacity having IC50 values of 22.68 g/ml and 24.54 g/ml. In this investigation, the CFSP and AQSP showed the highest free radical scavenging activity with IC50 values of 17.81 µg/ml and 21.72 µg/ml. At the same time, PESP exhibited moderate antioxidant potential having IC50 value of 25.73 µg/ml, and MSLE and SS-02 exhibited low antioxidant activity having IC50 values of 48.5 and 58.20 µg/ml, respectively. The DMSP and CTSP of M. oleifera showed free radical scavenging activity with IC50 values of 27.49 µg/ml and 35.78 µg/ml. In this study, the IC50 value for the TBHT was found to be around 15.0 µg/ml.

The studied data have denoted that some of the extractives of A. paniculata, A. chinensis, S. sesban and M. oleifera possess significant free radical scavenging activity whereas the compound isolated from S. sesban revealed moderate antioxidant activity.