Free radical scavenging and antioxidant activities of Glinus oppositifolius (carpet weed) using different in vitro assay systems

Abstract

Glinus oppositifolius (L.) Aug. DC. (Aizoceae), commonly called slender carpet weed, is a prostrate or diffuse herb which acts as stomachic, uterine stimulant, aperient and lochia. It is used traditionally in the treatment of earache, itch and skin diseases. Glinus oppositifolius was extracted with ethanol (70%) and used for the evaluation of various in vitro antioxidant assays which includes H-donor activity, nitric oxide scavenging, superoxide anion scavenging, reducing ability, hydroxyl radical, hydrogen peroxide scavenging, total phenolic content, total flavonoid content, total antioxidant activity by thiocyanate and phosphomolybdenum method, metal chelating, β-carotene bleaching, total peroxy radical assays. The pro-oxidant activity was measured using bleomycin-dependent DNA damage. Ex vivo models such as lipid peroxidation were used to study the antioxidant property of the extract. The various antioxidant activities were compared with suitable standard antioxidants such as ascorbic acid, butylated hydroxyl toluene (BHT), α-tocopherol, curcumin, quercetin and Trolox. The generation of free radicals, viz., O₂^·−, OH^·, H₂O₂, NO^· and peroxyl radicals, were effectively scavenged by the ethanol extract of Glinus oppositifolius. The antioxidant activity depends on concentration and increases with increasing amounts of the extract. The total phenolic content, flavonoid content and total antioxidant activity in Glinus oppositifolius were determined as microgram (μg) pyrocatechol, quercetin and α-tocopherol equivalent/mg, respectively. The extract did not exhibit any pro-oxidant activity when compared with ascorbic acid. The results obtained in this study indicate that Glinus oppositifolius scavenges free radicals and reduces lipid peroxidation, ameliorating the damage imposed by oxidative stress in different disease conditions and serve as a potential source of natural antioxidant.

Keywords::

• Lipid peroxidation
• reactive oxygen species
• scavenging activity

Introduction

Free radicals are continuously produced in our body and they are important for the maintenence of normal physiological function (Singal & Kirshenbaum, 1990). They are generally highly reactive and participate in hydrogen abstraction, radical addition, bond scission and annihilation reactions damaging the macromolecules such as proteins, DNA and lipids (Evans & Halliwell, 1999) The reactive oxygen species (ROS) include free radical such as superoxide anion (O₂·⁻), hydroxyl radical (OH^·), hydroperoxyl (HO₂·), peroxyl (ROO·), alkoxyl (RO^·) and non-radicals hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), ozone (O₃) and singlet oxygen (¹O₂·). Similarly, reactive nitrogen species (RNS) include nitric oxide (NO·), peroxy nitrite (ONOO^·), nitrogen dioxide (NO₂⁻) and dinitrogen trioxide (N₂O₃) (Halliwell & Gutteridge, 1999). Oxidative stress is a condition in which there is an increased production of oxygen species and diminished levels of antioxidant system resulting in cell damage leading to the pathogenesis of a variety of human diseases (Frei, 1994). Antioxidants prevent the oxidative reactions that occur naturally in tissues by scavenging free radicals, chelating metal ions and acting as electron donors. A search of naturally occurring antioxidant compounds from plant sources might provide leads for the development of novel drugs, which may reduce the risk of chronic diseases caused by free radicals (Kim et al., 2003).

Glinus oppositifolius (L.) Aug. DC. (Aizoaceae), is a pan-tropical weed commonly found in moist shady places throughout India, Malaysia, Ceylon, tropical Africa, and Australia. The plant is considered as stomachic, aperient, antiseptic, uterine stimulant and given to promote mensus and lochia. The fresh juice of the plant (made warm) is dropped into the ear for earache; it is also applied to itch and other skin diseases (Kirtikar & Basu, 1981; Khory & Katrak, 1985). The plant is also reported to relieve joint pain, inflammation, fever, malaria and wounds (Inngjerdingen et al., 2005). Chemical constitutents present in G. oppositifolius are triterpenoids, saponins, glinoside A and B which possess antiprotozoal activity (Traore et al., 2000), immunomodulating action and also contain pectin polysaccharides (Inngjerdingen et al., 2007). The present study was carried out to investigate the free radical scavenging activity of G. oppositifolius using in vitro and ex vivo models.

Materials and methods

Plant materials

G. oppositifolius was collected from Coimbatore District, Tamil Nadu, India, during the month of August 2006, and was shade-dried. The plant was identified and authenticated by G.V.S. Murthy, Joint Director, Botanical Survey of India, Tamil Nadu Agriculture University Campus, Coimbatore. (Ref No BSI/SC/5/23/05-06/Tech 304). A voucher specimen is available in the herbarium file of our department.

Preparation of extract

The whole plant was pulverized into fine powder using a grinder and was sieved through a No. 22 mesh sieve and stored in an air-tight container. About 750 mL of 70% ethanol were added to 75 g of powder and kept on a mechanical shaker for 72 h. The content was filtered and concentrated under reduced pressure under controlled temperature to yield a dark gummy residue. The concentrated extract was stored dry at 4°C in amber-coloured jars with Teflon lined caps. The percentage yield of the G. oppositifolius ethanol extract (GOEE) was found to be 7.2% w/v.

Drugs and chemicals

2,2-Diphenyl-1-picryl hydrazyl hydrate (DPPH) was procured from Himedia, Mumbai. Xanthine oxidase, quercetin, hypoxanthine, pyrocatechol were purchased from SRL, Mumbai. Folin-Ciocalteu reagent, from SD Fine, Mumbai, calf thymus DNA from Genei Chemicals, Bangalore, ferrozine, 29, 29-azobis(2-amidinopropane)dihydrochloride (AAPH), Trolox from Sigma Aldrich, USA, and 2,7-dichloroflurescein diacetate from Fluka, Deisenhofen, Germany. All other chemicals used in the study were of analytical grade and procured from local suppliers.

Experimental animals

Wistar albino rats of either sex (150-200 g) were used for the ex vivo study. They were housed in standard polypropylene cages and kept under controlled room temperature (24° ± 20°C, relative humidity 45–55%) in a 12 h light-dark cycle. The rats were given a standard laboratory diet and water ad libitum. The study was conducted after ethical clearance from the institutional animal ethics committee bearing the reference number 817/04/ac/CPCSEA.

In vitro antioxidant activity

DPPH radical scavenging assay

The hydrogen donating ability of GOEE was examined in the presence of DPPH stable radical (Mensor et al., 2001). An aliquot of 1 mL 0.3 mM DPPH ethanol solution was added to 2.5 mL sample solution of different concentrations and allowed to react at room temperature. After 30 min the absorbance values were measured at 517 nm. Ethanol (1 mL) plus plant extract solution (2.5 mL) was used as a blank. DPPH solution (1 mL, 0.3 mM) plus ethanol (2.5 mL) served as negative control. Ascorbic acid was used as positive control.

Nitric oxide radical scavenging assay

Various concentrations of the GOEE and sodium nitroprusside (10 mM) in phosphate buffer saline (0.025 M, pH 7.4) in a final volume of 3 mL was incubated at 25°C for 150 min. Control experiments without the test compounds but with the equivalent amount of buffer were prepared in the same manner as done for the test. Thereafter, 0.5 mL of incubation solution was removed and diluted with 0.5 mL Griess’ reagent (1% sulphanilamide, 2% o-phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride) and allowed to react for 30 min. The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylene diamine dihydrochloride was read at 546 nm. The percentage inhibition was calculated. The experiment was done in triplicate using curcumin (50-800 μg/mL) as positive control (Sreejayan & Rao, 1997).

Deoxyribose degradation assay

The decomposing effect of GOEE on hydroxyl radicals was determined by the assay of malondialdehyde chromogen formation due to 2-deoxyribose degradation (Halliwell et al., 1987). The assay mixture contained in a final volume of 1 mL:100 μL of 28 mM 2-deoxyribose dissolved in phosphate buffer, pH 7.4, 500 μL of the plant extract of various concentrations in buffer, 200 μL of 200 mM ferric chloride (1:1 v/v) and 1.04 mM EDTA and 100 μL of 1 mM hydrogen peroxide and 100 μL of 1 μM ascorbic acid. After incubation of the test sample at 37°C for 1 h the extent of free radical damage imposed on the substrate deoxyribose was measured using thiobarbituric acid (TBA) test. Percentage inhibition of deoxyribose degradation was calculated. Quercetin was used as standard.

NBT reduction assay

A reaction mixture with a final volume of 3 mL per tube was prepared with 1.4 mL of 50 mM KH₂PO₄-KOH, pH 7.4, containing 1 mM EDTA, 0.5 mL of 100 μm hypoxanthine, and 0.5 mL of 100 μM NBT (Guzman et al., 2001). The reaction was started by adding 0.066 units per tube of xanthine oxidase freshly diluted in 100 μL of phosphate buffer and 0.5 mL of test extract in saline. The xanthine oxidase was added last. The subsequent rate of NBT reduction was determined on the basis of spectrophotometric determinations of absorbance at 560 nm. Ascorbic acid was used as standard. The results are expressed as the percentage inhibition of NBT reduction rate with respect to the reaction mixture without test compound (saline only).

Reducing power ability

Reducing power ability was measured by mixing 1 mL extract of various concentrations prepared with distilled water to 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide and incubated at 50°C for 30 min. Next an aliquot of 2.5 mL of trichloroacetic acid (10%) was added to the mixture and centrifuged for 10 min at 3000 g, 2.5 mL from the upper part was diluted with 2.5 mL water and shaken with 0.5 mL fresh 0.1% ferric chloride. The absorbance was measured at 700 nm. The reference solution was prepared as above, but contained water instead of the samples. Increased absorbance of the reaction mixture indicates increased reducing power. All experiments were done in triplicate using butylated hydroxyltoluene (BHT) as a positive control (Oyaizu, 1986).

Estimation of total phenolic component

Total soluble phenolics of the extract was determined with Folin-Ciocalteu reagent using pyrocatechol as a standard following the method of Slinkard and Singleton (1977). An aliquot of 1 mL of extract solution in a test tube was added to 0.2 mL of Folin-Ciocalteu reagent (1:2 in distilled water) and, after 20 min, 2 mL of purified water and 1 mL of sodium carbonate (15%) was added. After 30 min, the absorbance was measured at 765 nm. The concentration of total phenolic component in the extract was determined as micrograms of pyrocatechol equivalent.

Total flavonoid content

Total soluble flavonoid of the extract was determined with aluminum nitrate using quercetin as standard (Hsu, 2006). Plant extract (1000 μg) was added to 1 mL of 80% ethanol. An aliquot of 0.5 mL was added to test tubes containing 0.1 mL of 10% aluminium nitrate, 0.1 mL of 1 M potassium acetate and 4.3 mL of 80% ethanol. The absorbance of the supernatant was measured at 415 nm after 40 min at room temperature.

Phosphomolybdate method

The total antioxidant capacity of the extract was determined with phosphomolybdenum using α-tocopherol as the standard. An aliquot of 0.1 mL of GOEE (100 μg) solution was combined with 1 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against blank in a UV spectrophotometer. The blank solution contained 1 mL of reagent solution and the appropriate volume of the same solvent used for the sample and it was incubated under the same conditions as the rest of the sample. The total antioxidant capacity was expressed as equivalents of α-tocopherol (Baydar, et al., 2007).

Bleomycin-dependent DNA damage

The reaction mixture contained 0.5 mL calf thymus DNA (10 μg/mL), 50 μg of 1 mL bleomycin sulfate, 1 mL of 5 mM magnesium chloride, 1 mL of 50 μM ferric chloride and 1 mL of different concentrations of GOEE. The mixture was incubated at 37°C for 1 h. The reaction was terminated by addition of 0.05 mL EDTA (0.1 M). The color was developed by adding 0.5 mL thiobarbituric acid (TBA) (1% w/v) and 0.5 mL hydrochloric acid (25% v/v) followed by incubation at 37°C for 15 min. After centrifugation the extent of DNA damage was measured at 532 nm employing ascorbic acid as a positive control. Each determination was done in triplicate (Ng et al., 2003).

Thiocyanate method

The peroxy radical was determined by thiocyanate method using α-tocopherol as standard (Gutierrez et al., 2006). Increasing concentration of the samples (25-400 μg/mL) in 0.5 mL of distilled water was mixed with 2.5 mL of linoleic acid emulsion (0.02 M, in 0.04 M pH 7.0 phosphate buffer) and 2 mL phosphate buffer (0.04 M, pH 7) in a test tube and incubated in darkness at 37°C. At intervals during incubation, the amount of peroxide formed was determined by reading absorbance of red color developed at 500 nm by the addition of 0.1 mL of 30% ammonium thiocyanate solution and 0.1 mL of 20 mM ferrous chloride in 3.5% hydrochloric acid to the reaction mixture.

Hydrogen peroxide scavenging activity

Hydrogen peroxide solution (2 mM) was prepared with standard phosphate buffer (pH, 7.4). Extract samples (25-400 μg/mL) in distilled water were added to hydrogen peroxide solution (0.6 mL). Absorbance of hydrogen peroxide at 230 nm was determined spectrophotometrically after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging of hydrogen peroxide of both plant extract and standard compound (α-tocopherol) was determined (Oktay et al., 2003).

Metal chelating complex

The ferrous level was monitored by measuring the formation of the ferrous ion-ferrozine complex. The reaction mixture containing different concentrations of GOEE (1 mL) were added to 2 mM ferrous chloride (0.1 mL) and 5 mM ferrozine (0.2 mL) to initiate the reaction, and the mixture was shaken vigorously and left to stand at room temperature for 10 min. The absorbance of the solution was measured at 562 nm. The positive controls were those using ascorbic acid, and all tests and analysis were run in triplicate. The percentage chelating effect of Ferrozine-Fe²⁺ complex formation was calculated (Huang & Kuo, 2000).

β-Carotene linoleic acid assay

A solution of β-carotene was prepared by dissolving 2 mg of β-carotene in 10 mL chloroform and 1 mL of this solution was then pipetted into a flask containing 20 mg of linoleic acid and 200 mg of Tween 40 emulsifier. Chloroform was completely evaporated using a vacuum evaporator. Aliquots of 5 mL of this emulsion were transferred into a series of tubes containing various concentrations of GOEE (25–400 μg/mL) or α-tocopherol for comparison. Optical density (OD) at 470 nm was taken for GOEE and standard immediately (t = 0) and at the end of 90 min (t = 90). The tubes were incubated at 50°C in a water bath during the test. Measurement of OD was continued until the colour of β-carotene disappeared in the control (Jayaprakasha et al., 2002).

Total peroxy radical trapping potential (TRAP)

A water soluble azo initiator 2,29-azo bis(2-amidino propane)dihydrochloride (AAPH) produced the peroxyl radicals while a spectrophotometric analysis of 2,7-dichlorofluresecin-diacetate (DCF) monitored the scavenging activity of the plant extracts. A 350 μL of 1 mM stock of DCF in ethanol was mixed with 1.75 mL of 0.01 N sodium hydroxide and allowed to stand for 20 min before the addition of 17.9 mL of 25 mM sodium phosphate buffer (pH 7.2). The reaction mixture contained 0.5 mL of various concentrations of GOEE in ethanol, 150 μL of activated DCF solution and 25 μL of AAPH (56 mM). The reaction was initiated with the addition of the AAPH. Absorbance was read at 490 nm. Trolox (6-hydroxy 2,5,7-8 tetra methyl chroman 2 carboxylic acid) was used as standard and all the determinations were done in triplicate (McCune & Johns, 2002).

Ex vivo studies

Assay of lipid peroxidation

Lipid peroxidation induced by the Fe²⁺-ascorbate system in rat liver homogenate was estimated by the TBA reaction method (Ohkawa et al., 1979). The reaction mixture consisted of rat liver homogenate 0.1 mL (25% w/v) in Tris-HCl buffer (20 mM, pH 7.0), potassium chloride (30 mM), ferrous ammonium sulfate (0.16 mM), ascorbate (0.06 mM), and various concentrations of the GOEE in a final volume of 0.5 mL. The reaction mixture was incubated for 1 h at 37°C. After the incubation time, 0.4 mL was removed and treated with 0.2 mL sodium dodecyl sulfate (SDS) (8.1%), 1.5 mL TBA (0.8%), and 1.5 mL glacial acetic acid (20%, pH 3.5). The total volume was made up to 4 mL by distilled water and then kept in a water bath at 95-100°C for 1 h. After cooling, 1 mL of distilled water and 0.5 mL of n-butanol and pyridine mixture (15:1, v/v) were added to the reaction mixture, shaken vigorously and centrifuged at 4000 rpm for 10 min. The organic layer was removed and its absorbance at 532 nm was measured. Inhibition of lipid peroxidation was determined by comparing the OD of the treatments with that of control. Ascorbic acid was used as standard.

Calculation of 50% inhibitory concentration (IC₅₀)

The concentration (mg/mL) of the extract required to scavenge 50% of the radicals was calculated by using the percentage scavenging activities at five different concentrations of the extract. Percentage inhibition (I%) was calculated using the formula

where Ac is the absorbance of the control and As is the absorbance of the sample.

Results

DPPH assay

GOEE demonstrated DPPH radical scavenging activity in a concentration-dependent manner and the IC₅₀ was found to be 1.013 mg/mL. A positive DPPH test suggests that the extract is a potential free radical scavenger. However, the activity was less when compared with the standard, ascorbic acid (Table 1).

Table 1.  Antioxidant activity of Glinus oppositifolius.

Test Substance	In vitro methods IC50 (mg/mL)								Ex vivo method IC50 (mg/mL)
	DPPH	NO^·	OH^·	O2^·−	H2O2	Thiocyanate method	Metal chelating	TRAP assay	Lipid peroxidation
GOEE	1.013 ± 8.8	0.601 ± 15.8	0.436 ± 73.1	0.093 ± 4.41	0.048 ± 0.33	0.146 ± 26.5	0.121 ± 2.9	0.265 ± 49.2	0.315 ± 8.66
Ascorbic acid	0.003 ± 0.06			0.072 ± 12.6			0.044 ± 26.8		0.081 ± 4.41
Quercetin			0.112 ± 11.2
Curcumin		0.076 ± 9.33
α-Tocopherol					0.066 ± 1.66	0.093 ± 3.33
Trolox								0.099 ± 2.33

Nitric oxide scavenging assay

Nitric oxide was generated by incubation of a solution of sodium nitroprusside in phosphate buffered saline at 25°C for 150 min. The GOEE effectively decreased the amount of nitrite generated from the decomposition of sodium nitroprusside in vitro at 546 nm. The extract exhibited strong NO^· scavenging activity with an IC₅₀ of 0.601 mg/mL, and that for standard, curcumin, was found to be 0.076 mg/mL (Table 1).

Hydroxyl radical scavenging assay

The hydroxyl (OH·) scavenging activity of GOEE was quantified by measuring the effect on 2-deoxyribose degradation in the presence of EDTA. The OH^· radical scavenging activity was evident at all the concentrations of GOEE and correlated well with increasing concentrations. The IC₅₀ value of GOEE was 0.436 mg/mL and that of standard, quercetin, was 0.112 mg/mL (Table 1).

Superoxide radical scavenging activity

GOEE was found to be an efficient scavenger of superoxide anion radical generated from hypoxanthine/xanthine oxidase system in vitro and the activity was comparable to that of positive control, BHT. Inhibition of NBT reduction by superoxide in the presence of the test preparation increased with increasing concentrations. The decrease of absorbance at 560 nm with GOEE and BHT thus indicates the consumption of O₂^·- in the reaction mixture. The IC₅₀ value of GOEE was 0.093 mg/mL and it is apparently higher than that of ascorbic acid, 0.072 mg/mL (Table 1).

Reducing power

Table 2 shows the reductive capability of the extract when compared to the standard, BHT, and serves as a significant indicator of its potential antioxidant activity. The reducing power increased with increasing amount of the GOEE and it was higher than the standard, BHT.

Table 2.  Reducing power of Glinus oppositifolius.

Test substance	Concentration (μg/mL)*
	50	100	200	400	800
GOEE	0.436 ± 0.002	0.454 ± 0.001	0.495 ± 0.002	0.568 ± 0.002	0.785 ± 0.001
BHT	0.092 ± 0.002	0.214 ± 0.004	0.314 ± 0.004	0.640 ± 0.001	1.092 ± 0.008

*Results are given as absorbance at 700 nm.

Total phenolic flavonoid content and total antioxidant capacity

The content of total phenolics in GOEE was determined using the Folin-Ciocalteu assay, calculated from the regression equation of the calibration curve of pyrocatechol. Phenolic content of GOEE was found to be 40 μg pyrocatechol equivalent/mg. The total flavonoid content of GOEE was found to be 168 μg quercetin equivalent/mg. The total antioxidant capacity (phosphomolybdenum method) of GOEE was found to be 6 μg α-tocopherol equivalent/mg.

Bleomycin-dependent DNA damage

The pro-oxidant activity of GOEE and the standard, ascorbic acid, were assessed by their effects on damage to DNA in the presence of bleomycin-Fe²⁺ complex and represented in Table 4. GOEE and ascorbic acid were tested at concentrations ranging from 25-400 μg/mL. The absorbance decreased with increasing concentrations of GOEE proving that the extract is devoid of pro-oxidant activity and this effect was comparable with that of ascorbic acid.

Table 3.  β-Carotene bleaching inhibitory activity.

Fractions	Time of incubation (min)	Concentration (μg/mL)*					IC50(mg/mL)
		25	50	100	200	400
GOEE	0	0.105	0.135	0.167	0.198	0.257	200 ± 10.00
	90	0.078	0.105	0.134	0.160	0.210
α-Tocopherol	0	0.098	0.126	0.189	0.285	0.725	0.100 ± 0.01
	90	0.058	0.082	0.138	0.230	0.664

*Results are given as absorbance at 470 nm.

Table 4.  Pro-oxidant activity of Glinus oppositifolius.

Test substance	Concentration (μg/mL)*
	25	50	100	200	400
GOEE	0.180 ± 0.01	0.170 ± 0.009	0.144 ± 0.004	0.105 ± 0.004	0.025 ± 0.009
Ascorbic acid	0.805 ± 0.016	0.655 ± 0.009	0.425 ± 0.008	0.150 ± 0.005	0.035 ± 0.001

*Results are given as absorbance at 532 nm.

Thiocyanate method

The total antioxidant activity of the GOEE was determined by the thiocyanate method and compared with that of standard, α-tocopherol. The effects of various amounts of GOEE (from 25 to 400 μg/mL) on peroxidation of linoleic acid emulsion exhibited effective antioxidant activity. The antioxidant activity of GOEE increased concentration dependently. The IC₅₀ value was found to be 0.146 mg/mL. The standard, α-tocopherol, showed the IC₅₀ value of 0.093 mg/mL (Table 1).

Scavenging of hydrogen peroxide

GOEE was capable of scavenging H₂O₂ in a concentration-dependent manner. The scavenging ability of the extract and standard, α-tocopherol are shown in Table 1. This indicates that GOEE mediates effective H₂O₂ scavenging activity, relative to that of α-tocopherol.

Metal chelating ability

In this assay system, the extract and standard compound, ascorbic acid, interfered with the formation of ferrous and ferrozine complex, suggesting its chelating effect. The formation of ferrozine-ferrous complex was not complete in the presence of GOEE. The ability of chelating ferrous ions also increased with increase in GOEE concentrations. The absorbance of Fe²⁺-ferrozine complex linearly decreased dose-dependently. The values shown in Table 1 demonstrate the action of GOEE as peroxidation protector.

β-Carotene bleaching method

The antioxidant activity of GOEE and the standard drug α-tocopherol were evaluated by the β-carotene bleaching method and the results are presented in Table 3. Increasing concentrations of GOEE and standard, α-tocopherol, decreased the absorbance, and this was due to the inhibition of bleaching of the color of β-carotene. The 50% inhibition value for GOEE was 200 mg/mL and for α-tocopherol was 0.1 mg/mL. GOEE exhibited equivalent β-carotene bleaching activity when compared with α-tocopherol.

Total radical antioxidant potential (TRAP)

The peroxyl radical scavenging activity was determined for GOEE and the results were compared with Trolox (Table 1). Addition of graded increasing concentrations of GOEE to solution containing AAPH-derived peroxyl radical decreased the luminescence produced by DCF and the absorbance decreased in a linear fashion. GOEE and Trolox exhibited IC₅₀ values of 0.265 and 0.099 mg/mL, respectively.

Lipid peroxidation

The lipid peroxidation induced by Fe²⁺-ascorbate system in rat liver homogenate was effectively inhibited by GOEE. The MDA generated as a result of lipid peroxidation reacts with thiobarbituric acid and was found to be inhibited in the presence of the extract. The IC₅₀ value was found to be 0.315 mg/mL for GOEE while for standard ascorbic acid it was 0.081 mg/mL (Table 1).

Discussion

Antioxidants exert their mode of action by suppressing the formation of reactive oxygen species either by inhibition of enzymes or by chelating trace elements which are involved in free radical generation, scavenging reactive species and augmenting the activity of the antioxidant enzymes (Cioffi et al., 2002).

DPPH is a proton free radical that shows a maximum absoption at 517 nm. When DPPH encounters proton radical scavengers, its purple color fades rapidly. Antioxidants, by providing a hydrogen atom or by donation of electrons, can quench DPPH· free radicals and convert them to a colorless bleached product resulting in a reduction in absorbance. The antioxidant activity was confirmed by a decrease in absorbance band upon increasing concentrations of the GOEE (Illavarasan et al., 2005; Oyaizu, 1986).

The reductive ability of the GOEE was compared with BHT. The presence of reductants in the extract causes the reduction of Fe³⁺-ferricyanide to the ferrous form which was monitored by measuring the formation of Perl’s prussian blue at 700 nm. The antioxidant activity exerted by the extract was by breaking the free radical chain by contributing a hydrogen atom. The reducing power of GOEE increased with increasing levels of the sample like the antioxidant activity (Meir et al., 1995).

Folin-Ciocalteu method and aluminium chloride coloration are currently used to determine the total polyphenol and flavonoid contents as they are important in various food materials. Phenolic compounds present in herbs and other plant materials are reported as important antioxidative components because of their scavenging ability (Cao et al., 1997). The high flavonoid content in the ethanol extract of Glinus oppositifolius may be responsible for its free radical scavenging activity.

The antioxidant capacity of GOEE was measured spectrophotometrically at 695 nm which was based on the reduction of MO(VI) to MO(V) by the extract and subsequent formation of green phosphate/MO(V) complex at acid pH. The phosphomolybdenum method is quantitative since the antioxidant activity is expressed as α-tocopherol equivalent. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which play an important role in neutralizing free radicals, quenching singlet and triplet oxygen (Prieto et al., 1999; Javanmardi et al., 2003).

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH, spontaneously interacts with oxygen to produce nitrite ions, which is estimated by the use of Griess reagent. Nitric oxide has an unpaired electron and acts as a free radical which plays an important role in the pathogenesis of pain, inflammation, cancer, etc. Scavengers of nitric oxide compete with oxygen leading to the reduced production of nitric oxide (Toda et al., 1988; Marcocci et al., 1994). Our finding suggests that the phenolic components present in the GOEE might be responsible for nitric oxide scavenging effect.

Superoxide anions generated in vitro by the system was determined spectrophotometrically by NBT photoreduction method. The amount of O₂^·- formed reduced the electrophilic NBT to form a chromophore (diformazan) which can be calculated by measuring the absorbance at 560 nm. Superoxide radicals initiate lipid oxidation and serve as precursors for many toxic ROS like hydroxyl radicals (OH·), hypochlorous acid (HOCl), singlet oxygen (¹O₂) and hydrogen peroxoide (H₂O₂) (Taubert et al., 2003). The decrease of absorbance by GOEE indicates the consumption of superoxide anion and thus its antioxidant activity.

Hydroxyl radicals are highly reactive oxygen centered radicals causing lipid oxidation and enormous biological damage. They attack all proteins, DNA, polyunsaturated fatty acids in membranes and almost any biological molecules it touches (Koppenol, 1993). In this assay, ferric-EDTA was incubated with hydrogen peroxide and ascorbic acid at pH 7.4. Hydroxyl radicals produced in free solution were detected by their ability to degrade 2-deoxy-2-ribose into fragments, which forms a pink chromogen (Fenton reaction) upon heating with TBA at low pH. When GOEE and reference compound quercetin were added to the reaction mixture they removed hydroxyl radicals from the sugar and prevented their degradation.

Metal chelating activity is based on the chelating effect of Fe²⁺ ions by the reagent ferrozine, which is a quantitative formation of a complex with Fe²⁺ ions. The formation of complex is disrupted by other chelating agent, which results in the reduction of the formation of red color in the complex (Dinis et al., 1994). Determining the rate of reduction of the color therefore allows estimation of the chelating activity of the co-existing chelator. In this method, both extract and standard compound (ascorbic acid) interfered with the formation of ferrous complex with the reagent ferrozine, suggesting that it has chelating effect and captures the ferrous ion before ferrozine. The absorbance of Fe²⁺-ferrozine complex decreased linearly in a dose-dependent manner. It is reported that certain phenolic compounds have properly oriented functional groups, which can chelate metal ions (Thompson et al., 1976). The ion chelating activity of the extract may be attributed due to the presence of endogenous chelating agents, mainly phenolics.

The discoloration of β-carotene is widely used because β-carotene is extremely susceptible to free radical mediated oxidation of linoleic acid. In the absence of antioxidant, oxidation products such as lipid hydroperoxides, conjugated dienes and volatile byproducts of linoleic acid simultaneously attack β-carotene, resulting in bleaching of its characteristic yellow color (Elzaawely et al., 2005). GOEE inhibited β-carotene oxidation, suggesting that the antioxidant activity could be related to the high levels of phenolic compounds.

Hydrogen peroxide itself is not very reactive, but it can sometimes be toxic to cells because it can give rise to hydroxyl radicals. Thus, the removal of H₂O₂ as well as O₂^·- is very important for antioxidant defense in cell or food systems. H₂O₂ can cross membranes and may oxidize a number of compounds (Gulcin et al., 2004). Our extract (GOEE) scavenged H₂O_2, which may be attributed to the phenolics, which could donate electrons to H₂O₂, thereby neutralizing it into water.

The antioxidant activity by the use of the ammonium thiocyanate method measures the amount of peroxides produced at the initial stage of lipid peroxidation which is depicted by a decrease in absorbance, indicating an increased level of antioxidant activity. The antioxidant activity might be due to hydroperoxide inhibition, inactivation of free radicals or complex formation with metal ions or combinations thereof (Gutierrez et al., 2006). The good antioxidant activity exhibited by GOEE might be attributed to the presence of flavonoid like phytoconstituents.

TRAP assay is based upon the potential of antioxidants in extract to scavenge peroxyl radicals generated by thermal decomposition of a water-soluble azo initiator AAPH. Detection of oxidation products was based upon colorimetric absorption of the oxidation product, dichloroflurescein at 490 nm (Khanam et al., 2004). GOEE decreased the absorbance upon increasing concentrations of the sample, which is similar to that of the standard, Trolox.

Oxidative stress can lead to peroxidation of cellular lipids and can be measured by the determining the levels of thiobarbituric acid-reactive substances. Lipid peroxidation has been thought of for a long time in the biological system as a toxicological phenomenon, as it resulted in many pathological consequences. Quantification of MDA, one of the products of lipid peroxidation, with TBA at low pH and high temperature (100°C), resulted in the formation of a red complex, which is measured at 532 nm. This is a most common assay used for the determination of the rate of extent of lipid peroxidation and the concentration of lipid peroxidation product may reflect the degree of oxidative stress in any disorder (Yokozawa et al., 2000; Zin et al., 2002). Our extract GOEE inhibited the rate of lipid peroxidation by a reduction in the red color complex formed reflecting its anti-lipid peroxidative potential.

Based on the various in vitro and ex vivo assays, it can be concluded that the ethanol extract of Glinus oppositifolius possesses strong antioxidant activity, evidenced by the free radical scavenging property, which may be due to the presence of phenolic components in the extract. Further isolation of bioactive constituents in the extract would certainly help to ascertain its potency, which could be further exploited by in vivo study systems to increase the overall antioxidant activity by protecting against various ailments that are induced by oxidative stress.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.