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
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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.
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)
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
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
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
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)
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.