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View with images and charts 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 oxygencontaining 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 selfmedication 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, β-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 Âľg/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:

N

N + RH Antioxidant

N* NO2

NO2

+R* NH NO2

NO2

*DPPH (oxidized form)

•

NO2

NO2

DPPH (reduced form)

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 Methanol UV-spectrophotometer Beaker (100 & 200 ml) Test tube

Pipette (5 ml) Micropipette (50-200 µl) Amber reagent bottle Weighing balance Exts. of related plant

Table 4.1: Test samples of experimental plants Plant/ compounds

A. paniculata

A. chinensis

S. sesban

M. oleifera

From sesban

S.

Test samples

Code

Ethanol soluble aerial part extract (crude) n-Hexane soluble partitionate of ESAE Carbon tetrachloride soluble partitionate of ESAE Dichloromethane soluble partitionate of ESAE Aqueous soluble partitionate of ESAE Methanol soluble bark extract (crude) n-Hexane soluble partitionate of MSBE Carbon tetrachloride soluble partitionate of MSBE Chloroform soluble partitionate of MSBE Aqueous soluble partitionate of MSBE Methanol soluble leaves extract Pet. ether soluble partitionate of MSLE Carbon tetrachloride soluble partitionate of MSBE Chloroform soluble partitionate of MSBE Aqueous soluble partitionate of MSBE Methanol soluble bark extract (crude) n-Hexane soluble partitionate of MSLE Carbon tetrachloride soluble partitionate of MSLE Dichloromethane soluble partitionate of MSLE Aqueous soluble partitionate of MSLE

ESAE HXSP

Amount (mg) 2.00 2.00

CTSP

2.00

DMSP AQSP MSBE HXSP CTSP

2.00 2.00 2.00 2.00

CFSP AQSP MSLE PESP CTSP

2.00 2.00 2.00 2.00

CFSP AQSP MSLE HXSP

2.00 2.00 2.00 2.00

CTSP

2.00

DMSP AQSP SS-02

2.00 2.00

3,7-Dihydroxy oleanolic acid (104)

2.00

2.00

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 µg/ml) were mixed with 3.0 ml of a DPPH methanol solution (20 µg/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. DPPH in methanol – 3.0 ml (conc.– 20 µg/ml) Purple color

Extract in methanol – 2.0 ml (conc.– 500 to 0.977 µg/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 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 IC 50 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 Equation of Regression IC50 (µg/ml)# samples line TBHT 15.08 ± 0.52 y = 14.666Ln(x) + 10.202 ESAE 23.79 ± 1.17 y = 11.135Ln(x) + 14.706 HXSP 52.26 ± 2.1 y = 8.796Ln(x) + 15.194 CTSP 21.25 ± 0.59 y = 7.1105Ln(x) + 28.262 DMSP 19.33 ± 1.08 y = 10.469Ln(x) + 18.988 AQSP 36.6 ± 1.63 y = 9.9965Ln(x) + 14.005 # The values of IC50 are expressed as mean±SD (n=3)

R2 0.946 0.9727 0.9341 0.9773 0.976 0.9658

IC50 val ue s of di ffere nt extracti ve s of A. paniculata 60

52.26

IC50 values

50 36.6

40 30 20

23.79

21.25

19.33

C TSP

DMSP

15.08

10 0 TBHT

ESAE

HXSP

AQ SP

Extractive s

Figure 4.2: Chart for IC50 values of standard and different extractives of A. paniculata Table 4.3: List of absorbance against concentrations and IC 50 value of tert-butyl-1hydroxytoluene (TBHT) Abs Conc of (µg/ml) Blank 0.435 500

Abs of Extrac t 0.029

% IC50 Inhibition (µg/ml) 93.418

15.08


0.028 0.061 0.09 0.145 0.228 0.306 0.343 0.344 0.354

93.671 85.993 79.241 66.468 47.386 29.452 21.013 20.797 18.548

Fre e Radical Scavenging Activity of TBHT 120 100 % Inhibition

250 125 62.5 31.25 15.625 7.8125 3.90625 1.953125 0.976562 5

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

80 60 40

20 Table 4.4: List of absorbance against concentrations and IC 50 value of ESAE (crude) of A. paniculata 0 0

0.435

Conc (µg/ml)

Abs of Extrac

500

0.072

% Inhibitio n 83.23

250

0.091

79.193

125

0.120

72.243

62.5

0.166

61.756

31.25

0.213

51.102

15.625 7.8125 3.90625 .953125 0.97656 25

0.272 0.279 0.311 0.338

37.2448 35.7469 28.3571 22.1938

0.345

20.6632

100

200

300

400

500

600

Conce ntration (microgram/ml)

IC50 (µg/ ml)

Figure 4..3: Chart for IC50 value of tert-butyl-1Free Radical Scavenging Activity of ESAE hydroxytoluene (TBHT) 90 80 70 % Inhibition

Abs of Blank

23.79

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

60 50 40 30 20 10 0 0

100

200 300 400 500 Conce ntration (microgram/ml)

600

Figure 4.4: Chart for IC50 value of ethanol extract of A. paniculata

Table 4.5: List of absorbance against concentrations and IC 50 value of HXSP of A. paniculata Abs of Blank 0.435

Conc (µg/ml) 500 250 125 62.5 31.25 15.625

Abs of Extrac t 0.108 0.137 0.184 0.225 0.271 0.292

% Inhibitio n 75.1152 68.4285 57.6692 48.1243 37.5576 32.7188

IC50 (µg/ml ) 52.26


% Inhibition

Table 4.7: List of 0.301 absorbance against concentrations and IC 50 value of DMSP of A. 7.8125 30.6451 paniculata 3.90625 0.305 29.7235 1.95312 0.321 26.0368 Abs Conc Abs of % IC50 5 Free radical scavenging activity of DMSP(AP) of Extract Inhibition (µg/ml) (µg/ml 0.97656 0.355 18.2027 90 Blank 25 80 ) 70 500 0.065 85.057471 y = 10.469Ln(x) + 18.988 60 3 R2 = 0.976 50 Free Radical Scavenging Activity of HXSP 250 0.095 78.160919 40 80 5 30 70 125 0.123 71.724137 20 10 9 60 y = 8 . 7 9 6 Ln ( x ) + 15 . 19 4 0 62.5 0.134 62.7586 50 R = 0 . 9400 341 0 100 200 300 500 600 31.25 0.197 51.528710 Concentration (microgram/ml) 40 2 30 Figure 4.7: Chart for IC50 value of DMSP of 0.435 15.625 0.252 42.068965 19.33 A. paniculata 20 5 10 7.8125 0.266 38.850574 7 0 0 10 0 200 300 400 500 600 3.90625 0.285 34.482758 Co n c e n t ra t i o n ( m i c r o g r a m / m l ) 6 1.95312 0.302 30.574712 5 6 0.97656 0.311 28.46731 25 2

Table 4.8: List of absorbance against concentrations and IC 50 value of AQSP of A. paniculata

0.435

Conc (µg/ml) 500 250 125 62.5 31.25 15.625 7.8125 3.90625 1.95312 5 0.97656 25

Abs of % Extract Inhibition 0.097 0.122 0.135 0.203 0.243 0.275 0.292 0.307 0.323

77.701149 71.954023 64.769433 53.333333 43.133934 36.781609 32.873563 29.425287 25.747126

0.331

23.904701

IC50 (µg/ml )

Free radical scavenging activity of AQ SP 90 80 70 % Inhibition

Abs of Blank

36.6

60 50

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

40 30 20 10 0 0

100

200

300

400

500

600

Concentration (microgram/mi)

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

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 IC 50 value for the standard (TBHT) was found to be 15.08 µg/ml. Methanol soluble extract and aqueous soluble


materials exhibit significant antioxidant capacity having IC50 value of 22.68 µg/ml and 24.54 µg/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

Equation Regression line y = 14.666Ln(x) TBHT 15.08 ± 0.52 10.202 y = 14.405Ln(x) MSBE 22.68 ± 1.12 5.0287 y = 10.108Ln(x) HXSP 157.15 ± 2.08 1.1272 y = 10.535Ln(x) CTSP 53.37 ± 0.68 8.0922 y = 11.3Ln(x) CFSP 27.21 ± 2.3 12.661 y = 12.022Ln(x) AQSP 24.54 ± 1.47 11.518 # The values of IC50 are expressed as mean±SD (n=3) IC50 (µg/ml)#

of + + – + + +

R2 0.946 0.9426 0.853 0.9457 0.9738 0.9629

IC50 Values of different Extractives of A. Chinensis 180

157.15

IC50 values (microgram/ml)

160 140 120 100 80

53.37

60 40 20

15.08

22.68

27.21

24.54

C FSP

AQ SP

0 TBHT

MSBE

HXSP CTSP Extractive s

Figure 4.9: Chart for IC50 values of the standard and extractives of A. chinensis Table 4.10: List of absorbance against concentrations and IC 50 value of MSBE (crude) of A. chinensis Abs of Blank 0.435

Conc (µg/ml) 500

Abs of Extrac t 0.054

250 125 62.5

0.060 0.064 0.135

% Inhibitio n 87.5862 07 86.2068 85.2873 68.9655

IC50 (µg/m l) 22.68


0.216 0.246 0.322 0.345 0.335 0.354

50.3448 43.4482 25.977 20.6896 22.9885

Fre e radical Scavenging Activity of MSBE 100 90 80 70 % Inhibition

31.25 15.625 7.8125 3.90625 1.95312 5 0.97656 25

18.4739

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

60 50 40 30 20 10 0 0

100 200 300 400 500 C once ntration (microgram/ml)

600

Figure 4.10: Chart for IC50 value of MSBE of A. chinensis Table 4.11: List of absorbance against concentrations and IC 50 value of HXSP chinensis Conc. (µg/ml)

500 250 125 62.5 31.25 15.625 0.435 7.8125 3.90625 1.95312 5 0.97656 25

Abs. of Extrac t 0.130 0.151 0.255 0.289 0.287 0.377 0.390 0.378 0.391

% Inhibition

0.390

10.344

IC50 (µg/ml )

Free Radical Scavenging Activity of HXSP 80 70 60

70.09152 65.39032 41.30817 33.563 34.022 13.333 10.344 13.103 10.114

50 % Inhibition

Abs. of Blan k

of A.

40

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

30 20 10 0 -10 0

157.15

100

200

300

400

500

600

Concebtration (microgram/ml)

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

Table 4.12: List of absorbance against concentrations and IC 50 value of CTSP of A. chinensis Abs.o f Extrac t 0.126 0.136 0.167 0.184 0.289 0.289 0.295 0.308

% Inhibition

IC50 (µg/ml )

Free radical Scave nging Activity of CTSP 80 70 60

71.0164 68.7356 61.6091 57.6252 33.5632 33.5632 32.1839 29.1954

53.37

% Inhibition

Abs. Conc. of (µg/ml) Blan k 0.435 500 250 125 62.5 31.25 15.625 7.8125 3.90625

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

50 40 30 20 10 0 0

100

200

300

400

Concentration (microgram/ml)

500

600


1.95312 5 0.97656 25

0.387

11.0344

0.398

8.5057

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

Table 4.13: List of absorbance against concentrations and IC 50 value of CFSP of A. chinensis

500 250 125 62.5 31.25 15.625 0.435 7.8125 3.90625 1.95312 5 0.97656 25

Abs.o f Extrac t 0.085 0.121 0.138 0.154 0.202 0.251 0.289 0.301 0.324

% Inhibition

0.411

5.51772

80.2873 72.1724 68.2754 64.5977 53.5517 42.2988 33.5404 30.7231 25.4252

IC50 (µg/ml )

Fre e Radical Scave nging Activity of CFSP

90 80 70

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

60 50 40 30 20 10 0

27.21

Table 4.14: List of absorbance against chinensis Abs.of Conc. Abs.of % Blank (µg/ml) Extrac Inhibition t 500 0.086 80.1438 250 0.114 73.57208 125 0.096 77.73506 62.5 0.159 63.54106 31.25 0.172 60.4597 0.435 15.625 0.239 45.04513 7.8125 0.300 31.0344 3.90625 0.322 25.977 1.953125 0.358 17.62901 0.976562 0.382 12.1839 5

0

100

200

300

400

500

600

Conce ntration (microgram/ml)

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

concentrations and IC 50 value of AQSP of A. IC50 (µg/ml)

100

Fre e Radical Scave nging Activity of AQ SP (AC)

90 80 70 % Inhibition

Conc. (µg/ml)

% Inhibition

Abs. of Blan k

24.54

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

60 50 40 30 20 10 0 0

100

200 300 400 500 C oncentration (microgram/ml)

600

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

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 y = 14.776Ln(x) + TBHT 14.18 ± 1.01 10.812 MSLE 48.5 ± 0.78 y = 8.6915Ln(x) + 16.257 PESP 25.73 ± 2.3 y = 6.2183Ln(x) + 29.801 CTSP 69.49 ± 1.71 y = 6.0195Ln(x) + 24.466 CFSP 17.81 ± 0.86 y = 8.8342Ln(x) + 24.555 AQSP 21.72 ± 1.45 y = 6.0164Ln(x) + 31.478 # The values of IC50 are expressed as mean ± SD (n=3)

R2 0.9351 0.9877 0.9874 0.9834 0.9829 0.8474

IC50 val ue s of diffe re nt e xtracti ve s of S. se sban 80

69.49

IC50 (microgram/ml)

70 60

48.5

50 40

25.73

30 20

17.81

14.18

21.72

10 0 TBHT

MSLE

PESP CTSP Extractive s

C FSP

AQ SP

Figure 4.15: Chart for IC50 values of the standard and extractives of S. sesban Table 4.16: List of absorbance against concentrations and IC 50 value of MSLE (crude) of S. sesban Abs. Conc. of (µg/ml) Blan k 0.484 500 250 125 62.5 31.25

Abs.o f Extrac t 0.126 0.149 0.173 0.206 0.251

% Inhibition

IC50 (µg/ ml)

73.966942 69.214876 64.256198 57.438017 48.140496

48.5


0.274 0.288 0.310 0.335 0.356

43.38843 40.495868 35.950413 30.785124

Fre e Radical Scavenging Acti vity of MSLE 80 70 60 % Inhibition

15.625 7.8125 3.90625 1.95312 5 0.97656 25

26.446281

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

50 40 30 20 10 0 0

100 200 300 400 Conce ntrati on (microgram/ml)

500

600

Figure 4.16 Chart for IC50 value of MSLE (crude) of S. sesban Table 4.17: List of absorbance against concentrations and IC50 value PESP of S. sesban

500 250 125

0.484

Abs of Extrac t 0.146 0.180 0.193

62.5 31.25

0.210 0.232

15.625

0.272

7.8125

0.283

3.90625

0.301

1.953125 0.976562 5

0.312 0.337

% Inhibition

IC50 (µg/ml)

Fre e Radical Scavenging Activity of PESP 80 70

69.83471 1 62.80991 7 60.12396 7 56.61157 52.06611 6 43.80165 3 41.52892 6 37.80991 7 35.53719 30.37190 1

60 % Inhibition

Abs.of Conc Blank (µg/ml)

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

50 40 30 20 10 0 0

100

200

300

400

500

25.73

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

Table 4.18: List of absorbance against concentrations and IC50 value CTSP of S. sesban Abs.of Conc Blank (µg/ml) 0.484

500

Abs of Extrac t 0.190

250

0.207

600

Conce ntration (microgram/ml)

% Inhibition

IC50 (µg/ml)

60.74380 2 57.23140 5

69.49


0.214

62.5

0.246

31.25

0.277

15.625

0.281

7.8125

0.291

3.90625

0.327

1.953125

0.351

0.976562 5

0.370

55.78512 4 49.17355 4 42.76859 5 41.94214 9 39.87603 3 32.43801 7 27.47933 9 23.55371 9

Free Radical Scavenging Activity of CTSP 70 60 y = 6.0195Ln(x) + 24.466 R2 = 0.9834

50 % Inhibition

125

40 30 20 10 0 0

100

200

300

400

500

600

Concentration (microgram/ml)

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

% Inhibition

Table 4.19: List of absorbance against concentrations and IC50 value CFSP of S. sesban Free Radical Scavenging Activity of CFSP Abs Conc Abs of % IC50 of Extrac Inhibition (µg/ml) 90 (µg/ml) 80 Blank t 70 500 0.083 82.85124 y = 8.8342Ln(x) + 24.555 60 R = 0.9829 250 0.126 73.96694 50 2 40 30 125 0.158 67.35537 20 2 10 62.5 0.189 60.95041 0 3 0 100 200 300 400 500 600 31.25 0.236 51.23966 Conce ntration (microgram/ml) 9 Figure 4.19: Chart for IC50 value of CFSP of 0.484 15.625 0.264 45.45454 17.81 S. sesban 5 7.8125 0.284 41.32231 4 3.90625 0.310 35.95041 3 1.953125 0.328 32.23140 5 0.976562 0.350 27.68595 5 2

Table 4.20: List of absorbance against concentrations and IC50 value AQSP of S. sesban Abs.of Conc Blank (µg/ml) 0.484

500

Abs of Extrac t 0.093

% Inhibition

IC50 (µg/ml)

80.78512 4

21.72


250

0.186

125

0.200

62.5

0.246

31.25

0.253

15.625

0.268

7.8125

0.269

3.90625

0.277

1.953125 0.976562 5

0.294 0.296

61.57024 8 58.67768 6 49.17355 4 47.72727 3 44.62809 9 44.42148 8 42.76859 5 37.25619 8 34.01102 3

Fre e radic al s c a ve nging a c tivity o f aque o us s o luble frac tio n 90 80 70 60

y = 6 .0 164Ln(x) + 31.478 R 2 = 0.8 474

50 40 30 20 10 0

Moringa oleifera

0

10 0

200

3 00

4 00

50 0

Co nc . (mic ro gram/ ml)

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 IC 50 values of standard and test samples of M. oleifera Test samples

IC50 (µg/ml)#

Equation of regression line y = 14.776Ln(x) + TBHT 14.18 ± 1.01 10.812 y = 11.156Ln(x) + MSBE 44.3 ± 0.98 7.7007 y = 8.5434Ln(x) + HXSP 48.47 ± 2.41 16.839 y = 8.6283Ln(x) + CTSP 35.78 ± 1.83 19.128 y = 6.9879Ln(x) + DMSP 27.49 ± 0.87 26.84 y = 7.4341Ln(x) + AQSP 77.77 ± 2.62 17.628 # The values of IC50 are expressed as mean±SD (n=3)

R2 0.9351 0.9071 0.9684 0.9723 0.9556 0.9596

6 00


IC50 values (microgram/ml)

IC50 values of different extractives of M. oleifera 90 80 70 60 50 40 30 20 10 0

77.77

44.3

48.47 35.78 27.49

14.18

TBHT

MS BE

HXS P CTS P Extractives

DMS P

AQS P

Figure 4.21: Chart for IC50 value of the standard and extractives of M. oleifera Table 4.22: List of absorbance against concentrations and IC 50 value of methanol extract of M. oleifera

0.395

500 250 125 62.5 31.25 15.625 7.8125 3.90625 1.953125 0.976562 5

Abs of Extrac t 0.101 0.118 0.143 0.243 0.306 0.336 0.373 0.39 0.382 0.398

% IC50 Inhibition (µg/ml) 78.961 75.5844 70.3896 49.6103 36.6233 30.3896 22.8571 19.2207 21.0389 17.6623

Fre e Radical Scave nging Activity of MSBE 90 80 70 % Inhibition

Abs.of Conc Blank (µg/ml)

60

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

50 40 30 20

44.3

10 0 0

100

200

300

400

500

C onc (microgram/ml)

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

Table 4.23: List of absorbance against concentrations and IC 50 value of HXSP of M. oleifera Abs.of Conc Blank (µg/ml) 0.395

500 250 125 62.5 31.25 15.625 7.8125 3.90625

Abs of Extrac t 0.164 0.183 0.187 0.217 0.255 0.266 0.336 0.348

% IC50 Inhibition (µg/ml) 66.1157 62.1901 61.3636 55.1653 47.3141 45.0413 30.5785 28.0992

600

48.47


1.953125 0.976562 5

0.394 0.395

18.5950 18.3884

Free Radical Scave nging Activity of HXSP 80 70

% Inhibition

60

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

50 40 30 20 10 0 0

100

200

300

400

500

600

Concentration (microgram/ml)

Figure 4.23: Chart for IC50 value of HXSP of M. oleifera Table 4.24: List of absorbance against concentrations and IC 50 value of CTSP of M. oleifera

0.395

500

Abs of Extrac t 0.099

250

0.108

125

0.118

62.5

0.143

31.25

0.196

15.625

0.225

7.8125

0.269

3.90625

0.310

1.953125

0.321

0.976562 5

0.305

% Inhibition

IC50 (µg/ml)

Free Radical Scavenging Activity of CTSP 80 70

74.28571 4 70.45637 8 59.50413 2 53.78313 8 44.91356 2 38.45912 3 36.95721 5 35.95041 3 23.41968 3 20.63812 3

60 % Inhibition

Abs Conc of (µg/ml) Blank

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

50 40 30 20 10 0 0

100

200

300

400

500

600

Concentration (microgram/ml)

35.78

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

Table 4.25: List of absorbance against concentrations and IC 50 value of DMSP of M. oleifera


0.385

500

Abs of Extrac t 0.152

250

0.170

125

0.201

62.5

0.213

31.25

0.239

15.625 7.8125

0.229 0.264

3.90625

0.311

1.953125

0.351

0.976562 5

0.364

% Inhibition 68.59504 1 64.87603 3 58.47107 4 55.99173 6 50.61983 5 52.68595 45.45454 5 35.74380 2 27.47933 9 24.79338 8

IC50 (µg/ml)

Fre e Radical Scave ngin g Activity of DMSP 80 70 y = 6.9879Ln(x) + 26.84 R2 = 0.9556

60 % Inhibition

Abs.of Conc Blank (µg/ml)

50 40 30 20 10 0 0

27.49

100 200 300 400 500 Concentration (microgram/ml)

600

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

Table 4.26: List of absorbance against concentrations and IC 50 value of AQSP of M. oleifera

0.385

500

Abs of Extrac t 0.186

250

0.211

125

0.231

62.5 31.25

0.242 0.247

15.625

0.275

7.8125

0.329

3.90625

0.365

1.953125

0.388

0.976562 5

0.399

% Inhibition 61.57024 8 56.40495 9 52.27272 7 50.0 48.96694 2 43.18181 8 32.02479 3 24.58677 7 19.83471 1 17.56198 3

IC50 (µg/ml)

Fre e Radical Scave nging Activity of AQ SP (MO ) 70 60 50 % Inhibition

Abs.of Conc Blank (µg/ml)

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

40 30 20 10 0 0

100

200

300

400

500

600

C once ntration (microgram/ml)

77.77

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


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 IC 50 value of SS-02 (3,7dihydroxy oleanolic acid, 104) SS-02 (3, 7-Dihydroxyoleanolic acid, 103) Abs of Sl Abs.of Conc Extrac Inhibition no. Blank (µg/ml) t 1 500 0.175 0.638429 8 2 250 0.201 0.584710 7 3 125 0.221 0.543388 4 4 62.5 0.232 0.520661 2 5 31.25 0.236 0.512396 7 0.484 6 15.625 0.265 0.452479 3 7 7.8125 0.318 0.342975 2 8 3.90625 0.355 0.266528 9 9 1.953125 0.377 0.221074 4 10 0.976562 0.386 0.202479 5 3

% Inhibition

IC50 (µg/ml)

63.84298 58.47107 54.33884 52.06612 51.23967 45.24793

58.20

34.29752 26.65289 22.10744 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 Na 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 (PDX1), 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 exhibit ed 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-1hydroxytoluene (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 µg/ml and 24.54 µg/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.

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