Antioxidant Activity

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Antioxidant Activity

Introduction

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

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

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

Antioxidants: The free radical scavengers

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

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

Oxidative Damage

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

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

Antioxidant Protection

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

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

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

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

Antioxidant activity: DPPH assay

Principle

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

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

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

Color variation of DPPH solution after samples treatment

Materials and Methods

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

Materials and preparation of materials

Table 4.1: Test samples of experimental plants

Control preparation for antioxidant activity measurement

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

DPPH solution preparation

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

Test sample preparation

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

Methods

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

Where A_blank 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.

Results and Discussion

Andrographis paniculata

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

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

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

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

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

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

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

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

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

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

Table 4.8: List of absorbance against concentrations and 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 IC₅₀ value for the standard (TBHT) was found to be 15.08 mg/ml. Methanol soluble extract and aqueous soluble materials exhibit significant antioxidant capacity having IC₅₀ value of 22.68 mg/ml and 24.54 mg/ml (Table 4.9, Figure 4.9).

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

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

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

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

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

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

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

Table 4.14: List of absorbance against concentrations and 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Table 4.26: List of absorbance against concentrations and 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 IC₅₀ value of SS-02 (3,7-dihydroxy oleanolic acid, 104)

Antioxidant in diabetes management

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

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

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

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

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