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RADICAL SCAVENGING AND ANTIOXIDANT ACTIVITIES OF SUCCESSIVE SOLVENT EXTRACTS OF INDIGOFERA ASTRAGALINA


Shiju V Mathew1, Senthil Kumar R2 And Manivannan R3*
1. Research Scholar, Department of Pharmacy, Pacific Academy of Higher Education & Research University, Udaipur, India.
2. Swamy Vivekanandha College of Pharmacy, Tiruchengode, Tamilnadu, India.
3. JKK Munirajahh Institute of Health Sciences, College of Pharmacy, Gobi, Tamilnadu, India.

ABSTRACT

In the present study, the antioxidant activity of successive solvent extracts of Indigofera astragalina was evaluated by different in vitro antioxidant assay models. The prepared solvent extracts were screened for the antioxidant and radical scavenging activity on ABTS radical cation, DPPH free radical, hydrogen peroxide, superoxide radical and hydroxyl radical. Among the prepared extracts, ethanol and ethyl acetate extracts showed prominent activity against the tested models. Aqueous extract showed moderate activity in all the models next to ethyl acetate. Hexane extracts fails to exhibit the antioxidant and radical scavenging activity in all the tested models. Chloroform extract did not produce significant activity. The antioxidant and free radical scavenging activities of the extracts were compared with standard antioxidants like ascorbic acid and rutin. Preliminary phytochemical screening revealed that ethanol and ethyl acetate extracts are rich in flavonoids, polyphenols, tannins and saponins. The radical scavenging and antioxidant activity of the extracts may be due to the presence of these kinds of phytochemicals in the extract. Therefore, the plant could be considered as a very good antioxidant source with therapeutic potential.

Key Words: Indigofera astragalina, antioxidant, radical scavenging, flavonoids, phenols, phytochemicals.

INTRODUCTION

Oxidation and reduction reactions are essential to many living organisms for the production of energy to biological purposes. However, oxygen free radicals and other reactive oxygen species (ROS) which are continuously produced in vivo, result in cell death and tissue damage. These species can react with biological substrates such as DNA and proteins, leading to several diseases including cancer, diabetes, cardiovascular diseases, aging, arthritis and atherogenesis1,2. Antioxidants are vital substances which provide protection to living organisms from damage caused by uncontrolled production of ROS and the concomitant lipid peroxidation, protein damage and DNA strand breaking3,4. Several anti-inflammatory, antinecrotic, neuroprotective, chemopreventive and hepatoprotective drugs have recently been shown to have antioxidant and radical scavenging mechanism as part of their activity5,6.

There is an increased interest in natural antioxidants present in medicinal and dietary plants, which might help to prevent oxidative damage7. Indigofera astragalina (Leguminosae), is a small trailing herb distributed throughout India. Various parts of this medicinal plant were used in Indian System of Medicine to treat various illnesses such as rheumatism, arthritis, inflammation, tumor and liver diseases. Several members of the species of Indigofera like Indigofera trita, Indigoera linnaei, Indigofera cassioides, Indigofera cordifolia, Indigofera aspalathoides etc. are used traditionally for a wide variety of ethnomedical properties such as antitumor, hepatoprotective, antioxidant, antimicrobial, anti-inflammatory and analgesic8-20.

Literature review revealed that the proximate and mineral composition of the leaves of Indigofera astragalina was determined and the plant contains essential nutrients like vitamin C and iron. The plant was also have high calorific value and considered as safe for consumption21.

Indigofera astragalina leaves are used to treat diarrhoea and the results highlighted the traditional uses of the plant22. Apart from these studies no phytochemical and pharmacological studies have been carried out in this plant. Based on the above facts, the present study is focused to evaluate the radical scavenging and antioxidant potential of successive solvent extracts of Indigofera astragalina using various in vitro assay models.

MATERIALS AND METHODS

Chemicals

2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) was obtained from Sigma Aldrich Co., St. Louis, USA. Rutin and p–nitroso dimethyl aniline (p–NDA) were obtained from Acros Organics, New Jersy, USA. Ascorbic acid and nitro blue tetrazolium (NBT) were obtained from S.D. Fine Chem, Ltd., Biosar, India. 2- Deoxy–D-ribose was from Hi–Media Laboratories Ltd., Mumbai. All other chemical used were of analytical grade.

Collection and Extraction

Entire plants of I. astragalina were collected from Tiruchengode, Tamilnadu. The plant was authenticated by Dr. G.V.S. Murthy, Joint Director, Botanical Survey of India, Coimbatore, Tamilnadu, India. A voucher specimen is preserved in our laboratory for future reference (Voucher No.: PCIAS 007). The plant material was shade dried, pulverized and successively extracted (500 g) with the solvents of increasing order of polarity viz. hexane, chloroform, ethyl acetate and ethanol using soxhlet extractor for 72 h. The residue obtained after extraction with ethanol was extracted with water by cold maceration process for 72 h. The prepared extracts were filtered and concentrated to dryness under reduced pressure and controlled temperature in a rotary evaporator and yield was calculated. The extracts were stored in a refrigerator until further use.

Preliminary phytochemical screening

Prepared plant extracts of I. astragalina were analyzed for the presence of various phytochemical constituents employing standard procedures23. Conventional protocol for identifying the presence of steroids, alkaloids, tannins, flavonoids, glycosides, etc., was used.

Preparation of test and standard solutions

The successive solvent extracts of I. astragalina and the standard antioxidants (ascorbic acid and quercetin) were dissolved in distilled dimethyl sulfoxide (DMSO separately and used for the in vitro antioxidant assays except the hydrogen peroxide method because it interferes with the method. For hydrogen peroxide method, the extracts and the standards were dissolved in distilled methanol and used. The stock solutions were serially diluted with the respective solvents to obtain the lower concentrations.

In vitro antioxidant activity

Successive solvent extracts of I. astragalina was tested for their in vitro antioxidant activity using the standard methods. In all these methods, a particular concentration of the extracts or standard solution was used which gave a final concentration of 1000-15.625 μg/ml after all the reagents were added. Absorbance was measured against a blank solution containing the extract or standards, but without the reagents. A control test was performed without the extract or standards. Percentage scavenging and IC50 values were calculated.  

ABTS radical scavenging activity

In a final volume of 1 ml, the reaction mixture comprised 950 μl of ABTS* solution and 50 μl of the plant extracts at various concentrations. The reaction mixture was homogenized and incubated for 20 min.  Absorbances of these solutions were measured spectrophotometrically at 734 nm24.

DPPH radical scavenging activity

The DPPH assay method depends on the reduction of purple DPPH to a yellow colored diphenyl picrylhydrazine and the remaining DPPH which showed maximum absorption at 517 nm was measured. About 2 ml of various concentrations of the plant extracts or standards were added to 2 ml of DPPH solution (0.1 mM, 2 ml). After 20 min of incubation at 37 °C in the dark, the absorbance was recorded at 517 nm17. c

Hydroxyl radical scavenging activity

By p-NDA method

Various concentration of the extracts or standards in 0.5 ml of distilled DMSO were added to a solution mixture containing 0.5 ml of ferric chloride (0.1 mM), 0.5 ml of EDTA (0.1 mM), 0.5 ml of ascorbic acid (0.1 mM), 0.5 ml of hydrogen peroxide (2 mM) and 0.5 ml of p-NDA (0.01 mM) in phosphate buffer (pH 7.4, 20 mM) to produce a final volume of 3 ml. Absorbance was measured spectrophotometrically at 440 nm20.

Deoxyribose method

To the reaction mixture containing deoxyribose (0.2 ml, 3 mM), ferric chloride (0.2 ml, 0.1 mM), EDTA (0.2 ml, 0.1 mM), ascorbic acid (0.2 ml, 0.1 mM) and hydrogen peroxide (0.2 ml, 2 mM) in phosphate buffer (pH, 7.4, 20 mM), added 0.2 ml of various concentrations of extracts or standard in freshly distilled DMSO to give a total volume of 1.2 ml. The solutions were then incubated for 30 min at 37 °C. After incubation, ice–cold trichloro acetic acid (0.2 ml, 15 %w/v) and thiobarbituric acid (0.2 ml, 1 %w/v), in 0.25 N HCl were added. The reaction mixture was kept in a boiling water bath for 30 min, cooled and the absorbance was measured at 532 nm20.

Superoxide radical scavenging activity by alkaline DMSO method

In this method, superoxide radical is generated by the addition of sodium hydroxide to air saturated dimethyl sulfoxide. The generated superoxide remains stable in solution, which reduces nitroblue tetrazolium (NBT) into formazan dye at room temperature and that can be measured at 560 nm. Briefly, to the reaction mixture containing 1 ml of alkaline DMSO (1 ml DMSO containing 5 mM NaOH in 0.1 ml water) and 0.3 ml of the extracts in freshly distilled DMSO at various concentrations, added 0.1 ml of NBT (1 mg/ml) to give a final volume of 1.4 ml. The absorbance was measured at 560 nm17.

Hydrogen peroxide radical scavenging method

In this method, when a scavenger is incubated with hydrogen peroxide, the decay or loss of hydrogen peroxide can be measured spectrophotometrically at 230 nm. To 1 ml of various concentrations of extracts or standard in methanol was added to 2 ml of hydrogen peroxide (20 mM) in phosphate buffer saline. After 10 min the absorbance was measured at 230 nm25.

RESULTS

Preliminary Qualitative Analysis

Organoleptic characters and extractive yields of successive solvent extracts of I. astragalina are presented in Table 1. Ethanol and water extracts showed highest extractive yield when compared to other solvents. Ethyl acetate gives moderate extractive yield. Preliminary phytochemical screening of the plant extracts showed the presence of various phytochemical constituents. Terpenoids, phytosterols, alkaloids were present in hexane and chloroform extracts. Ethyl acetate extract contains terpenoids, amino acids, flavonoids and saponins whereas phytosterols, carbohydrates, glycosides, alkaloids, terpenoids, tannins, saponins, proteins and amino acids were present in ethanol extract. Aqueous extract consist of many polar constituents like carbohydrates, glycosides, tannins, flavonoids and saponins. Gums and mucilage were absent in all the prepared extracts. The results clearly showed that the presence of each phytochemicals depends upon the solubility of phytochemical constituents in the particular solvents. Many of the constituents were extracted by ethanol. The preliminary qualitative analysis report data is displayed in Table 2.

Table 1: Color, nature and extractive yields of successive solvent extracts of  I. astragalina

Name of the Extract Colour Nature Yield (%)
Hexane Dark green Solid mass 2.3
Chloroform Greenish yellow Solid mass 1.8
Ethyl acetate Yellowish green Solid mass 6.7
Ethanol Brownish yellow Semi- solid mass 10.3
water Brown Dried powder 8.6

Table 2. Preliminary Phytochemical Studies of successive solvent extracts of I. astragalina

Phytochemical constituents Name of the Extract
Hexane Chloroform Ethyl acetate Ethanol Water
Carbohydrates Absent Absent Absent Present Present
Glycosides Absent Absent Present Present Present
Alkaloids Absent Present Present Present Absent
Terpenoids Present Present Absent Present Absent
Phytosterols Present Present Absent Present Absent
Flavonoids Absent Absent Present Present Present
Phenolics Absent Absent Present Present Present
Tannins Absent Absent Absent Present Present
Saponins Absent Absent Absent Present Present
Proteins & amino acids Absent Absent Absent Present Present
Fixed oils & fats Present Present Absent Absent Absent
Gums & mucilages Absent Absent Absent Absent Absent


Radical scavenging and Antioxidant Activities of successive solvent extracts of  I. astragalina

ABTS and DPPH Radical Scavenging Assay

ABTS and DPPH radical scavenging activity of various solvent extracts of I. astragalina are shown in Table 3. The extracts showed potent radical scavenging activity in concentration dependent manner. The successive solvent extracts of I. astragalina exhibited good radical scavenging activity against the tested models. Among the extracts tested ethyl acetate and ethanol extract showed potent activity when compared to other extracts. Aqueous and chloroform solvent extract showed moderate activity. Hexane extract did not show any activity against the radical scavenging and antioxidant assays. The order of activity is: Ethyl acetate>Ethanol>Aqueous>Chloroform>Hexane. Ethyl acetate extract exhibit equally potent activity which is produced in the standard antioxidant ascorbic acid, however the results obtained were comparable with the standards used.

Table 3: Effect of the leaf extracts of Indigofera astragalina on ABTS and DPPH Method
Extracts/

Standards

IC50 (µg/ml)* by method
ABTS DPPH
Hexane >1000 >1000
Chloroform 102.4 ± 3.28 153.2± 10.7
Ethyl acetate 12.34 ± 0.26 21.1 ± 0.79
Ethanol 37.41 ± 1.23 52.7 ± 1.86
Water 84.2 ± 2.61 91.2 ± 2.32
Ascorbic Acid 13.21 ± 0.49 5.21 ± 0.45
Quercetin 1.22 ± 0.04 6.21 ± 0.63

*Average of three determinations; Data are expressed as mean ± SEM

Hydroxyl radical Scavenging Assay

Hydroxyl radical scavenging activity of successive solvent extracts of I. astragalina was measured by p-NDA and Deoxyribose method. In both methods, the ethyl acetate and ethanol extracts showed potent activity when compared to standards used (Table 4). Hexane extract did not show any activity against the hydroxyl radical scavenging assay. The order of activity is: Ethyl acetate>Ethanol>Aqueous>Chloroform>Hexane.

Table 4: Effect of successive extracts of Indigofera astragalina on Hydroxyl Radical Scavenging Assay
Extracts/

Standards

IC50 (µg/ml)* by method
Hydroxyl Radical Scavenging
p-NDA

Method

Deoxyribose

Method

Hexane >1000 >1000
Chloroform 389.1± 12.47 403.3 ±14.6
Ethyl acetate 174.13 ± 7.43 222.4 ± 14.6
Ethanol 242.3 ± 11.3 267.2 ± 6.91
Water 256.6 ± 14.2 372.2 ± 12.3
Ascorbic Acid >1000
Quercetin >1000 27.4 ± 1.72

*Average of three determinations; Data are expressed as mean ± SEM

 

Superoxide and Hydrogen peroxide radical Scavenging Activity

Superoxide radical scavenging activity of successive solvent extracts of I. astragalina was assessed by alkaline DMSO method. The plant extracts moderately inhibit the superoxide radical generation. In hydrogen peroxide radical scavenging assay, the extracts were found to be equipotent with ascorbic acid but less potent when compared to rutin. In both methods hexane extract did not show any activity. The order of activity is: Ethyl acetate>Ethanol>Aqueous>Chloroform>Hexane. The values were tabulated in Table 5.

Table 5. Effect of Successive Extracts of Indigofera astragalina on Superoxide Radical Scavenging and Hydrogen peroxide Scavenging Methods
Extracts/

Standards

IC50 (µg/ml)* by method
Superoxide radical scavenging H2O2 radical scavenging
Hexane >1000 >1000
Chloroform 396.1 ± 14.2 276.8± 12.4
Ethyl acetate 198.6± 10.2 112.8 ± 2.42
Ethanol 267.37± 8.78 129.1 ± 6.82
Water 298.4 ± 11.9 214.3  ± 13.8
Ascorbic Acid >1000 167.4 ± 3.42
Quercetin >1000 21.53 ± 1.32

*Average of three determinations; Data are expressed as mean ± SEM

DISCUSSION

In the last two decades there has been an explosive interest in the role of oxygen free radicals, more generally known as “reactive oxygen species” (ROS) and of “reactive nitrogen species” (RNS) in experimental and clinical medicine1. ROS/RNS are known to play a dual role in biological systems, since they can be either harmful or beneficial to living systems26. Beneficial effects of ROS involve physiological roles in cellular responses to noxia, as for example in defense against infectious agents and in the function of a number of cellular signaling systems. One further beneficial example of ROS at low concentrations is the induction of a mitogenic response. In contrast, at high concentrations, ROS can be important mediators of damage to cell structures, including lipids and membranes, proteins and nucleic acids termed as oxidative stress27. Oxidative stress has been implicated in the pathology of many diseases such as inflammation, cancer, diabetes, neurodegenerative disorders and aging. Reactive oxygen species and reactive nitrogen species such as superoxide anions, hydroxyl radical and nitric oxide inactivate enzymes and damage intracellular components causing injury through covalent binding and lipid peroxidation. Antioxidants are compounds that hinder the oxidative processes and thereby delay or prevent oxidative stress28.

The harmful effects of ROS are balanced by the antioxidant enzymes. Despite the presence of the cell’s antioxidant defense system to counter act oxidative damage from ROS, oxidative damage accumulates during the life cycle, and radical-related damage to DNA, to proteins and to lipids has been proposed to play a key role in the development of agedependent diseases such as cancer, arteriosclerosis, arthritis, neurodegenerative disorders and other conditions1. Antioxidants are an inhibitor of the process of oxidation, even at relatively small concentration and thus have diverse physiological role in the body. Antioxidant phytoconstituents from the plant materials act as radical scavengers. They help in converting the reactive free radicals to less reactive species. Natural antioxidants occur in all parts of plants. These antioxidants include carotenoids, vitamins, phenols, flavonoids, dietary glutathione, and endogenous metabolites. Plant-derived antioxidants have been shown to function as singlet and triplet oxygen quenchers, free radical scavengers, peroxide decomposers, enzyme inhibitors, and synergists. The most current research on antioxidant action focuses on phenolic compounds such as flavonoids. Fruits and vegetables contain different antioxidant compounds, such as vitamin C, vitamin E and carotenoids, whose activities have been established in recent years.

The antioxidant assays used in this study measured the oxidative products at the early and final stages of oxidation. The antioxidant and free radical scavenging activity of successive solvent extracts of I. astragalina was investigated against various in vitro models. Since, free radicals are of different chemical entities, it is essential to test the extracts against many free radicals to prove their antioxidant activity. Hence, a large number of in vitro methods were used for the screening. IC50 values obtained were compared with the standards used, that is, ascorbic acid and quercetin.

ABTS radical scavenging activity is relatively recent one, which involves a more drastic radical, chemically produced and is often used for screening complex antioxidant mixtures such as plant extracts, beverages and biological fluids. The ability in both the organic and aqueous media and the stability in a wide pH range raised the interest in the use of ABTS・+ for the estimation of antioxidant activity29. The ethyl acetate and ethanol extracts showed potent antioxidant activity in ABTS method which is comparable to the standard used. Here, the extracts radical scavenging activity showed a direct role of its phenolic compounds in free radical scavenging.

The electron donation ability of natural products can be measured by 2, 2 -diphenyl-1- picrylhydrazyl radical (DPPH) purple-coloured solution bleaching. The method is based on scavenging of DPPH through the addition of a radical species or antioxidant that decolourizes the DPPH solution. The degree of colour change is proportional to the concentration and potency of the antioxidants. A large decrease in the absorbance of the reaction mixture indicates significant free radical scavenging activity of the compound under test30. The experimental data of the extracts revealed that the extracts are likely to have the effects of scavenging free radicals. From the result we observe that a dose dependent relationship in the DPPH radical scavenging activity. The involvement of free radicals, especially their increased production, appears to be a feature of most of the human diseases including cardiovascular diseases and cancer31. It has been found that cysteine, glutathione, ascorbic acid, tocopherols, flavonoids, tannins and aromatic amines reduce and decolorize the DPPH by their hydrogen donating ability. Flavonoids and phenolic compounds of successive solvent extracts of I. astragalina are possibly involved in its radical scavenging activity.

Among the oxygen radicals, hydroxyl radical is the most reactive and induces severe damage to adjacent biomolecules32. In the present study, the hydroxyl radical scavenging activity of successive solvent extracts of I. astragalina was assessed by the inhibition of p-NDA bleaching method and deoxyribose degradation method. In p-NDA method, the hydroxyl radical is generated through Fenton reaction. In this reaction, iron-EDTA complex reacts with hydrogen peroxide in presence of ascorbic acid to generate hydroxyl radical which can bleach p-NDA specifically. The extracts show potent scavenging activity by inhibition of bleaching of p-NDA. The scavenging activity may be due to the presence of various phytochemicals including polyphenols and flavonoids in the extracts. In deoxyribose method, the sugar is degraded on exposure to hydroxyl radical generated Fenton reaction. The resulting complex mixture of products is heated under acid condition; malondialdehyde (MDA) is formed and detected by its ability to react with thiobarbituric acid to form a pink chromogen. In the deoxyribose method, the plant extracts shows potent hydroxyl radical scavenging activity which can be comparable with the standards used. The scavenging activity may be due to the presence of various phytochemicals including polyphenols and flavonoids17.

Superoxide radical is known to be a very harmful species to cellular components as a precursor of more reactive species33. The superoxide radical is known to be produced in vivo and can result in the formation of hydrogen peroxide via dismutation reaction. Moreover, the conversion of superoxide and hydrogen peroxide into more reactive species. The extracts are found to be an efficient scavenger of superoxide radical generated in alkaline DMSO system. The result clearly indicates that the plant extracts have a noticeable effect as scavenging superoxide radical.

Hydrogen peroxide itself is not very reactive, but sometimes is toxic to cell because it may give rise to hydroxyl radical in the cells33. Therefore, removing of hydrogen peroxide is very important for antioxidant defense in cell system. Polyphenols have also been shown to protect mammalian cells from damage induced by hydrogen peroxide, especially compounds with the orthohydroxy phenolic compounds like quercetin, gallic acid, caffeic acid and catechin34. Therefore, the phenolic compounds of the successive solvent extracts of Indigofera astragalina may probably be involved in scavenging hydrogen peroxide.

The systemic literature collection, pertaining to this investigation indicates that the plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical scavengers. Flavonoids are the most diverse and widespread group of natural compounds and are proposed to be the most important natural phenolics. These compounds possess a broad spectrum of chemical and biological activities including radical scavenging activity. Qualitative analysis of the ethyl acetate and ethanol extract shows that they contains considerable amount of total flavonoids and phenols. Previous literatures showed that high phenol and flavonoid content increases the antioxidant activity and there is a linear relation between the phenol and flavonoid contents and antioxidant activity35,36.

Phenolic compounds are commonly found in both edible and medicinal plants and they have been reported to have various biological effects including antioxidant activity. The antioxidant activities of phenolic compounds is mainly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides37. The successive solvent extracts of Indigofera astragalina showed strong antioxidant activity in various in vitro systems tested. The antioxidant effect of I. astragalina is may be due to the phenolic compounds present in it. To our knowledge this is the first report on the antioxidant and radical scavenging potential of Indigofera astragalina.

CONCLUSION 

The results from various free radicals scavenging systems reveal that successive solvent extracts of Indigofera astragalina have significant antioxidant activity. The extracts are found to have different levels of antioxidant activity in all the methods tested. IC50 values obtained were comparable with that of the standards used, that is, ascorbic acid and quercetin. Since, free radicals are of different chemical entities, it is essential to test the extracts against many free radicals to prove their antioxidant activity. Hence, a large number of in vitro methods were used for the screening. However, the difference in the activity in extracts may be due to the different chemical entities of the free radicals and the diverse chemical nature of the extracts. According to this study, presence of phenolics compounds and flavonoids in the extracts may be responsible for radical scavenging and antioxidant activity. Further studies are in progress for the isolation and identification of phytochemical compounds and to ensure the most important medicinal properties of the plant in vivo in our laboratory to correlate with its antioxidant activity.

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