The leaves and shoots are boiled and drunk as an antihypertensive remedy [6]. Phytochemical studies have reported the isolation of flavonols, triterpenoids, steroids and tannins; β-sitosterol, stigmasterol, campesterol, lupeol being few of its important constituents [7]. The petroleum ether and benzene extracts inhibit the growth of some human and plant pathogenic bacteria [8]. Previous study on this plant showed that it has hepatoprotective activity [9] and potent nootropic activity [10].

Although extensive studies have been carried out on leaf, the pharmacology of the whole plant of Alternanthera sessilis has still remained unexplored. Therefore, the objective of the present study was to evaluate the antioxidant potential of a 30% ethanolic extract of Alternanthera sessilis. The extract was examined for different reactive oxygen species (ROS) scavenging activities (hydroxyl, superoxide and hydrogen peroxide), phenol content, flavonoid content and chelating capacity of iron.

MATERIALS AND METHODS:

Plant Material

Whole plant of Alternanthera sessilis was collected from Kalyan, District Thane, Maharashtra, India and was authenticated from St. Xavier’s College, Mumbai (India) as Alternanthera sessilis (Amaranthaceae). A voucher specimen was submitted as herbarium to the Department of Botany, Birla College, Kalyan (India).

Chemicals and Reagents

All the drugs and chemicals, used in the study, were of analytical grade. Folin-Ciocalteu reagent was obtained from Hi-Media, India. Ethylene diamine tetra acetic acid (EDTA), trichloroacetic acid (TCA), NBT, NADH, thiobarbituric acid (TBA), 2-deoxy-2-ribose and other chemicals used for evaluation of oxidative stress parameters were obtained from Sisco Research Laboratories, India.

Extraction

Whole plant of Alternanthera sessilis was dried at room temperature for 7 days, finely powdered and used for extraction. The powder (100 g) was mixed with 500 ml of ethanol: water (3:7) using a magnetic stirrer for 15 hours, then the mixture was centrifuged at 2850 × g and the supernatant was decanted. The marc was mixed again with 500 ml of ethanol: water and the entire extraction process were repeated. The filtrate collected from above were mixed in a round bottom flask and concentrated under reduced pressure in a rotary evaporator. The concentrated extract was then lyophilized. The residue was kept at - 20°C for future use and was abbreviated as HAAS.

Scavenging of Hydroxyl Radicals (^•OH)

The reaction between deoxyribose and the extracts was measured as the determinant for hydroxyl ion scavenging activity. The thiobarbituric acid-reactive substances (TBARS) [11]formed due to attack of the hydroxyl radical on deoxyribose were measured by the method given by Ohkawa et al. [12] Briefly, the extracts were added to the reaction mixture containing 2.8 μM deoxyribose, 100 μM FeCl₃, 104 μM EDTA, 100 μM ascorbic acid, 1 mM H₂O₂ and 230 mM phosphate buffer (pH 7.4), making a final volume of 1.0 ml. One millilitre of thiobarbituric acid (TBA, 1%) and 1.0 ml trichloroacetic acid (TCA, 2.8%) were added to the test tube and incubated at 100°C for 20 min. After cooling, absorbance was measured at 532 nm against a blank containing deoxyribose and buffer. Reactions were carried out in triplicate.

Superoxide radical scavenging

This activity was measured by the reduction of NBT according to a previously reported method. [13] The non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH) system generates superoxide radicals, which reduce nitro blue tetrazolium (NBT) to a purple formazan. The reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT (50 μM), PMS (15 μM) and various concentrations (0–20 μg/ml) of sample solution. After incubation for 5 minutes at ambient temperature, the absorbance at 562 nm was measured against an appropriate blank to determine the quantity of formazan generated. All tests were performed six times. Quercetin was used as positive control.

Hydrogen Peroxide Radical Scavenging Activity

Hydrogen peroxide scavenging activity of the extract was estimated by method prescribed [14]. A solution of hydrogen peroxide (20 mM) was prepared in phosphate buffer saline (pH 7.4). Different concentrations of plant extract and standard ascorbic acid solution viz. 10-100 μg/ ml in ethanol were added to hydrogen peroxide solution. Absorbance of hydrogen peroxide at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. For each concentration, a separate blank sample was used for back ground subtraction. The experiment was performed in triplicate.

Metal Chelating Activity

The chelating effect of ferrous ions by Alternanthera sessilis extract was estimated by the method of Dinis and Madeira [15]. Briefly, 200 μl of different concentrations of the extracts and 740 μl of methanol were added to 20 μl of 2 mM FeCl₂. The reaction was initiated by the addition of 40 μl of 5 mM ferrozine to the mixture and shaken vigorously and kept standing at ambient temperature of 10 minutes. The absorbance of the reaction mixture was measured at 562 nm. Three replicates were made for each test sample.

Determination of total phenolic content

Total phenolic content was determined using Folin-Ciocalteu (FC) reagent according to the method given by Singleton and Rossi [16] with a slight modification. Briefly, the plant extract was mixed with 0.75 ml of FC reagent (previously diluted 1000-fold with distilled water) and incubated for 5 minutes at 22°C and then 0.06% Na₂CO₃ solution was added. After incubation at 22°C for 90 minutes, the absorbance was measured at 725 nm. All tests were performed six times. The phenolic content was evaluated from a gallic acid standard curve.

Determination of total flavonoid content

The total flavonoid content was determined with aluminium chloride (AlCl₃) according to a method (Zhishen et al.) [17] using quercetin as a standard. The plant extract (0.1 ml) was added to 0.3 ml distilled water followed by NaNO₂ (0.03 ml, 5%). After 5 min at 25°C, AlCl₃(0.03 ml, 10%) was added. After a further 5 min, the reaction mixture was treated with 0.2 ml 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. All tests were performed six times. The flavonoid content was calculated from a quercetin standard curve.

Statistical Analysis

Results were analyzed statistically using one way analysis of variance (ANOVA) and expressed as mean ± SE of three observations. Values of P<0.05 were considered significant. The statistical analysis was performed on Graph-pad Prism software of version 4. The % inhibition of various radicals was calculated by comparing the results of the test with those of control using the formula.

% Inhibition = Abs (Control) ‑ Abs (Test) /Abs (Control) ×100

Table 1: Scavenging of reactive oxygen species and iron chelating activity (IC₅₀ values) of Alternanthera sessilis and reference compounds

Activity	IC₅₀ (#)
Hydroxyl radical (OH^.) scavenging	112.18 ± 3.27*
Superoxide anion (O2 ^.-) scavenging	13.46 ± 0.66 *
Hydrogen peroxide (H₂O₂) scavenging	44.74 ± 25.61*
Iron Chelating	66.54 ± 0.84 *

# Units of IC₅₀ for all activities are μg/ml, except H₂O₂ scavenging, where the units are mg/ml. Data are expressed as mean ± S.D. Data in parenthesis indicate number of independent assays. * p < 0.05.

RESULTS:

Hydroxyl radical scavenging

This assay shows the abilities of the extract and standard mannitol to inhibit hydroxyl radical-mediated deoxyribose degradation in a Fe³⁺-EDTA-ascorbic acid and H₂O₂ reaction mixture. The results are shown in Figure 1. The IC₅₀ values (Table 1) of the extract and standard in this assay were 112.18 ± 3.27 μg/ml and 571.45 ± 20.12 μg/ ml, respectively. The IC₅₀ value of the extract was less than that of the standard. At 200 μg/ml, the percentage inhibition values were 53.7% and 23% for Alternanthera sessilis and mannitol, respectively.

Superoxide radical scavenging

The superoxide radicals generated from dissolved oxygen by PMS-NADH coupling can be measured by their ability to reduce NBT. The decrease in absorbance at 560 nm with the plant extract and the reference compound quercetin indicates their abilities to quench superoxide radicals in the reaction mixture. As shown in Figure 2, the IC₅₀ values (Table 1) of the plant extract and quercetin on superoxide scavenging activity were 13.46 ± 0.66 μg/ml and 42.06 ± 1.35 μg/ml, respectively. The IC₅₀ value of the extract was less than that of the standard. At 20 μg/ml, the percentage inhibition of the plant extract was 55.2% whereas that of quercetin was 29.6%.

Figure1: Hydroxyl radical scavenging activities of the Alternanthera sessilis extract and the reference compound mannitol. The data represent the percentage inhibition of deoxyribose degradation.

Figure 2: Scavenging effect of Alternanthera sessilis plant extract and the standard quercetin on superoxide radical. The data represent the percentage superoxide radical inhibition. All data are expressed as mean ± S.D. (n = 6). *p < 0.05

Figure 3: H₂O₂ scavenging assay. Effects of Alternanthera sessilisplant extract and the standard sodium pyruvate on the scavenging of H₂O₂. The data represent the percentage H₂O₂ scavenging. All data are expressed as mean ± S.D. *p < 0.05

Hydrogen peroxide scavenging

Figure 3 shows that the plant extract is a very poor scavenger of H₂O₂ (IC₅₀ = 44.74 ± 25.61 mg/ ml) compared to standard sodium pyruvate (IC₅₀ = 3.24 ± 0.3 mg/ml). The IC₅₀ value (Table 1) of the extract was greater than that of the standard. At a concentration of 2 mg/ml, the scavenging percentages were 6.5% and 57.7% for Alternanthera sessilis and sodium pyruvate, respectively.

Figure 4: Effects of Alternanthera sessilisplant extract on ferrozine-Fe²⁺ complex formation. The data are expressed as percentage inhibition of chromogen formation. The results are mean ± S.D. *p < 0.05

Fe²⁺ chelation

Ferrozine produces a violet complex with Fe^2+. In the presence of a chelating agent, complex formation is interrupted and as a result the violet colour of the complex is decreased. The results (Figure 4) demonstrated that formation of the ferrozine-Fe²⁺ complex is inhibited in the presence of the test and reference compounds. The IC₅₀ values (Table 1) of the plant extract and EDTA were 66.54 ± 0.84 μg/ml and 1.27 ± 0.05 μg/ml, respectively. At 120 μg/ml, the percentage inhibition of the plant extract was 51.8% whereas at 45 μg/ml that of EDTA was 99.5%.

Determination of total phenolic content

Phenolic compounds may contribute directly to anti-oxidative action. The total phenolic content was 56.78 ± 1.01μg/mg gallic acid equivalent per 100 mg plant extract.

Determination of total flavonoid content

The total flavonoid content of the 70% methanolic extract of Alternanthera sessilis was 350.5 ± 0.004 mg/ml quercetin equivalent per 100 mg plant extract.

DISCUSSION:

In living systems, free radicals are constantly generated and they can cause extensive damage to tissues and biomolecules leading to various disease conditions, especially degenerative diseases, and extensive lysis [18]. Many synthetic drugs protect against oxidative damage but they have adverse side effects. An alternative solution to the problem is to consume natural antioxidants from food supplements and traditional medicines. [19, 20] Recently, many natural antioxidants have been isolated from different plant materials [21, 22].

Hydroxyl radicals are the major active oxygen species causing lipid peroxidation and enormous biological damage [23]. They were produced in this study by incubating ferric-EDTA with ascorbic acid and H₂O₂ at pH 7.4, and reacted with 2-deoxy-2-ribose to generate a malondialdehyde (MDA)-like product. This compound forms a pink chromogen upon heating with TBA at low pH [24]. When Alternanthera sessilis extract was added to the reaction mixture, it removed the hydroxyl radicals from the sugar and prevented the reaction. The IC₅₀ value indicates that the plant extract is a better hydroxyl radical scavenger than the standard mannitol.

Superoxide anion is also very harmful to cellular components [25]. Robak and Glyglewski [26] reported that flavonoids are effective antioxidants mainly because they scavenge superoxide anions. As shown in Figure 3, the superoxide radical scavenging activities of the plant extract and the reference compound are increased markedly with increasing concentrations. The results suggest that the plant extract is a more potent scavenger of superoxide radical than the standard quercetin.

Hydrogen peroxide is an important reactive oxygen species because of its ability to penetrate biological membranes. However, it may be toxic if converted to hydroxyl radical in the cell [27]. Scavenging of H₂O₂ by the plant extracts may be attributed to their phenolic contents, which donates electron to H₂O₂, thus reducing it to water. The extract was capable of scavenging hydrogen peroxide in a concentration dependent manner.

Metal chelating capacity is significant since it reduces the concentration of the transition metal that catalyzes lipid peroxidation [28]. According to the results, the plant extract is not as good as the standard EDTA; but the decrease in concentration dependent colour formation in the presence of the extract indicates that it has iron chelating activity.

The results indicate that Alternanthera sessilis plant extract contains significant amounts of flavonoids and phenolic compounds. Both these classes of compounds have good antioxidant potential and their effects on human nutrition and health are considerable. The mechanism of action of flavonoids is through scavenging or chelation [29]. Phenolic compounds are also very important plant constituents because their hydroxyl groups confer scavenging ability [30].

CONCLUSION:

On the basis of the results obtained in the present study, it is concluded that a 30% ethanolic extract of Alternanthera sessilis, which contains large amounts of flavonoids and phenolic compounds, exhibits high antioxidant and free radical scavenging activities. It also chelates iron and has reducing power. These in vitro assays indicate that this plant extract is a significant source of natural antioxidant, which might be helpful in preventing the progress of various oxidative stresses. However, the components responsible for the anti-oxidative activity are currently unclear. Therefore, further investigation is needed to isolate and identify the antioxidant compounds present in the plant extract. Furthermore, the in vivo antioxidant activity of this extract needs to be assessed prior to clinical use.

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Received on 28.04.2013

Modified on 30.05.2013

Accepted on 07.06.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(4): July- August 2013, 194-198