In Vitro Antioxidant and Free Radical Scavenging Activity of Nardostachys jatamansi DC.

J Acupunct Meridian Stud 2012;5(3):112e118

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Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com

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In Vitro Antioxidant and Free Radical Scavenging Activity of Nardostachys jatamansi DC. Surendra Kumar Sharma*, Ajay Pal Singh Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Haryana, India Available online Apr 10, 2012 Received: Jul 19, 2011 Accepted: Sep 9, 2011 KEYWORDS antioxidative potential; hydroxyl radicals; Nardostachys jatamansi; nitric oxide; superoxide; Valerianaceae

Abstract In this study, the antioxidative potential of a hydroalcoholic extract of Nardostachys jatamansi (NJE) rhizomes was evaluated by various antioxidant assays, including antioxidant capacity by the phosphomolybdenum method, total antioxidant activity in linoleic acid emulsion systems, 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, hydroxyl radicals, nitric oxide (NO) scavenging, metal chelating and reducing power activity. These various antioxidant activities were compared with standard antioxidants such as butylated hydroxytoluene, tocopherol, catechin, and L-ascorbic acid. Total phenolic and flavonoid content of NJE was also determined by a colorimetric method. The extract exhibited high reduction capability and powerful free radical scavenging, especially against DPPH and superoxide anions as well as a moderate effect on NO. Moreover, the peroxidation inhibiting activity of NJE was demonstrated in the linoleic acid emulsion system. The results obtained in the present study clearly established the antioxidative potency of NJE, which may account for some of the medical claims attributed to this plant.

1. Introduction Reactive oxygen species (ROS), including the free radicals superoxide anion ($O
2 ), hydroxyl radicals ($OH), and singlet oxygen (1O2) and non-free-radical species, such as hydrogen peroxide (H2O2), are various forms of activated oxygen resulting from oxidative biological reactions or exogenous

factors [1,2]. ROS have been shown to exert beneficial as well as deleterious actions. At very low concentrations, they may act as a second messenger in some of the signal transduction pathways [3]. However, when produced in excess, they can cause oxidative damage to many vital components of the cells. It has been shown that a dynamic relationship exists between the extent of ROS production

* Corresponding author. Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India. E-mail: [email protected] Copyright ª 2012, International Pharmacopuncture Institute doi:10.1016/j.jams.2012.03.002

Antioxidant potential of Nardostachys jatamansi and antioxidant capacity of a given cell system [4,5]. Oxidative stress, a result of an imbalance between the antioxidant defence systems and the formation of ROS, may damage essential biomolecules such as proteins, DNA, and lipids. This damage may cause cellular injuries and death and exacerbates several degenerative diseases associated with aging including cancer, cardiovascular disease, diabetes, and neurodegeneration [6,7]. Biological antioxidants are natural molecules, which can prevent the uncontrolled formation of free radicals and activated oxygen species or inhibit their reaction with biological structures [8]. Biological antioxidants include antioxidative enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, and small nonenzymatic antioxidant molecules, such as glutathione and vitamins C and E [9]. The efficiency of the antioxidative defence system is altered under some pathological conditions, and, therefore, ineffective scavenging and/or overproduction of free radicals may play a crucial role in determining the tissue damage [4,7]. Involvement of ROS in lipid peroxidation and many pathological events is firmly established [2,4,6]. Antioxidants exert their action through simple or complex mechanisms including prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and radical scavenging [10]. In recent years there has been an increasing interest in finding natural antioxidants since they can protect the human body from oxidative damage and retard the progress of many chronic diseases as well as lipid rancidity in foods [11]. In the prevention of overproduction of free radicals in biological systems, the reinforcement of endogenous antioxidants via intake of dietary antioxidants (mainly from plant sources) may be of particular importance in decreasing the cumulative effects of the oxidatively damaged molecules [12]. Nardostachys jatamansi DC. (commonly named as jatamansi) belongs to family Valerianaceae and is described in Indian traditional system of medicine (Ayurveda) for its use in mental disorders, insomnia, hyperlipidemia, hypertension and heart diseases [13]. It has protective effect in Parkinsonism, epilepsy, cerebral ischemia, and liver damage [14]. Jatamansi showed marked tranquilizing activity in mice. Jatamansi is capable of lowering norepinephrine as well as serotonin in brain [15]. Various sesquiterpenes (such as jatamansic acid and jatamansone) have been reported to be present in the rhizomes of the plant. In view of its wide use and its chemical composition, the hydroalcoholic extract of N jatamansi rhizomes was determined for its in vitro antioxidative activities.

113 Research Lab (Mumbai, India). The remaining chemicals and solvents used were of standard analytical grade and HPLC grade respectively.

2.2. Plant collection The plant material was collected at its flowering stage from Kullu district, Himachal Pradesh, India in July 2009. The plant was identified and authenticated by the Dr. H. B. Singh, Head, Raw Materials Herbarium & Museum, NISCAIR, New Delhi, India, Ref. No. NISCAIR/ RHMD/ Consult/06/ 741/58. The rhizome was shade dried at room temperature for 15 days and pulverized into coarse powder.

2.3. Extraction The brown coarse powder (200 g) was extracted exhaustively in a Soxhlet apparatus with mixture of ethanol: water (7:3 ratio) for 72 hours. The powder: solvent ratio was maintained as 1:6. The extract was then concentrated in vacuo until the solvent was completely removed. The yield of ethanol extract was found to be 14.32 g.

2.4. Total phenolic content Total phenolic content in the extract was determined using the Folin-Ciocalteu’s reagent (FCR) according to the published method [16]. Each sample (0.5 ml) was mixed with 2.5 ml of FCR (diluted 1:10, v/v), and 2 ml of Na2CO3 (7.5%, w/v) was added. The absorbance was then measured at 765 nm after incubation at 30 C for 90 minutes. Results were expressed as gallic acid equivalents (mg of gallic acid/ g of dried extract).

2.5. Total flavonoid content The total flavonoid content of NJE was determined using a colorimetric method described by Zhishen and colleagues [17]. Briefly, each sample (0.5 ml) was mixed with 2 ml of distilled water and subsequently with 0.15 ml of a NaNO2 solution (15%, w/v). After 6 minutes, 0.15 ml of an AlCl3 solution (10%, w/v) was added and allowed to stand for 6 minutes, then 2 ml of NaOH solution (4%, w/v) was added to the mixture. Immediately, water was added to bring the final volume to 5 ml, and then the mixture was thoroughly mixed and allowed to stand for another 15 minutes. Absorbance of the mixture was determined at 510 nm versus prepared water blank. Results were expressed as catechin equivalent (mg of catechin/g of dried extract).

2. Materials and methods 2.1. Chemicals and reagents

2.6. Antioxidant capacity by the phosphomolybdenum method

1,1-diphenyl-2-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), trichloroacetic acid (TCA), gallic acid, and ascorbic acid were purchased from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Folin-Ciocalteau reagent, nitro blue tetrazolium (NBT), ethylene diamine tetra acetic acid (EDTA), sodium nitroprusside (SNP), and potassium hexa cyano ferrate [K3Fe(CN)6] were procured from Sisco

The antioxidant capacity of NJE was evaluated by the method of Prieto and colleagues [18]. A 0.1 ml aliquot of sample solution (equivalent to 100 mg) was combined with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The blank was 0.1 ml of methanol used in place of sample. The tubes were capped and incubated in a boiling water bath at 95 C

114 for 90 minutes. After the samples were cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm. The antioxidant capacity was expressed as an equivalent of ascorbic acid (mg of ascorbic acid/g of dried extract).

2.7. Antioxidant activity in a linoleic acid emulsion system The total antioxidant activity of NJE was assayed by use of a linoleic acid emulsion system [19]. The linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid, 0.2804 g of Tween 20 and 50 ml of phosphate buffer (0.2 M, pH 7.0). The mixture was then homogenized. Different concentrations of the extract and standard sample (0.5 ml in ethanol) were mixed with linoleic acid emulsion (2.5 ml, 0.2 M, pH 7.0) and phosphate buffer (2 ml, 0.2 M, pH 7.0). The reaction mixture was incubated at 37 C in the dark to accelerate the peroxidation process. The levels of peroxidation were determined according to the thiocyanate method by sequentially adding ethanol (5 ml, 75%), ammonium thiocyanate (0.1 ml, 30%), sample solution (0.1 ml), and ferrous chloride (0.1 ml, 20 mM in 3.5% HCl). After mixing, the mixture was left for 3 minutes. The peroxide value was determined by reading the absorbance at 500 nm.

2.8. DPPH radical scavenging activity Radical scavenging activity of NJE was measured according to the method of Blois [20]. Briefly, 1 ml of variable concentrations of extract (25e250 mg/ml of ethanol) was added to 1 ml of a DPPH solution (0.2 mM in ethanol) as the free radical source and kept for 30 minutes at room temperature. The decrease in the solution absorbance, due to proton donating activity of NJE component(s), was measured at 517 nm. L-Ascorbic acid was used as the positive control. The percentage DPPH radical scavenging activity was calculated using the following formula: DPPH radical scavenging activity ð%Þ Z ½ðA0
A1 Þ=A0  100;

S.K. Sharma, A.P. Singh scavenging activity of the NJE extract. The percentage of superoxide radical scavenging was calculated using the following formula: Superoxide radical scavenging activity ð%Þ Z ½ðA0
A1 Þ=A0  100; where A0 is the absorbance of the control and A1 is the absorbance of NJE or the standard sample.

2.10. Hydroxyl radical scavenging activity assay The scavenging activity for hydroxyl radicals was measured with Fenton reaction. This method was recommended by Yu and colleagues [22]. Reaction mixture contained 60 mL of 1.0 mM FeCl2, 90 ml of 1 mM 1,10-phenanthroline, 2.4 ml of 0.2 M phosphate buffer (pH 7.8), 150 mL of 0.17 M H2O2, and 1.5 ml of extract at various concentrations. Adding H2O2 started the reaction. After incubation at room temperature for 5 minutes, the absorbance of the mixture at 560 nm was measured with a spectrophotometer. The hydroxyl radicals scavenging activity was calculated.

2.11. NO scavenging activity The scavenging effect of NJE on NO was measured according to the method of Marcocci and colleagues [23]. Briefly, sodium nitroprusside (5 mM) in phosphate-buffered saline (PBS) (pH 7.4) was mixed with different concentrations of the test sample (100e1000 mg/ml) and incubated at 25 C for 150 minutes. After incubation, nitrite produced from sodium nitroprusside was measured by Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% 1naphthylethylenediamine dihydrochloride in water). The absorbance of the chromophore that formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with 1-naphthylethyelenediamine dihydrochloride was immediately read at 570 nm. Catechin was used as a positive control. The percentage of NO scavenging was calculated using the following formula: NO scavenging activity ð%Þ Z ½ðA0
A1 Þ=A0  100;

where A0 is the absorbance of the control and A1 is the absorbance of NJE or the standard sample.

where A0 is the absorbance of the control and A1 is the absorbance of NJE or the standard sample.

2.9. Superoxide radical scavenging activity

2.12. Hydrogen peroxide scavenging activity assay

The superoxide anion scavenging activity was measured based on the described method [21]. Superoxide radicals were generated in a PMS-NADH system by oxidation of NADH and assayed through reduction of NBT. In this experiment, the superoxide radicals were generated in 3 ml of sodium phosphate buffer (100 mM, pH 7.4) containing 1 ml of NBT (150 mM) solution, 1 ml of NADH (468 mM) solution, and different concentrations of the CRE (25e250 mg/ml) in water. The reaction started by adding 1 ml of PMS solution (60 mM) to the mixture. The reaction mixture was incubated at 25 C for 5 minutes, and the absorbance was measured against the corresponding blank solution. L-Ascorbic acid was used as the positive control. The decrease in the extent of NBT reduction, measured by the absorbance of the reaction mixture, correlates with the superoxide radical

Hydrogen peroxide scavenging activity of the extract was estimated by replacement titration [24]. Aliquot of 1.0 ml of 0.1 mM H2O2 and 1.0 ml of various concentrations of extracts were mixed, followed by 2 drops of 3% ammonium molybdate, 10 ml of 2 M H2SO4 and 7.0 ml of 1.8 M KI. The mixed solution was titrated with 5.09 mM Sodium thiosulphate (NaS2O3) until yellow colour disappeared. Percentage of scavenging of hydrogen peroxide was calculated as: % Inhibition Z ½ðV0
V1 Þ=V0  100; where V0 was volume of NaS2O3 solution used to titrate the control sample in the presence of hydrogen peroxide (without extract), V1 was the volume of NaS2O3 solution used in the presence of the extracts.

Antioxidant potential of Nardostachys jatamansi

115

2.13. Metal chelating activity assay

3.2. Antioxidant capacity

The chelating activity of the extracts for ferrous ions Fe2þ was measured according to the method of Dinis and colleagues [25]. To 0.5 ml of extract, 1.6 ml of deionized water and 0.05 ml of FeCl2 (2 mM) was added. After 30 seconds, 0.1 ml ferrozine (5 mM) was added. Ferrozine reacted with the divalent iron to form stable magenta complex species that were very soluble in water. After 10 minutes at room temperature, the absorbance of the Fe2þFerrozine complex was measured at 562 nm. The chelating activity of the extract for Fe2þ was calculated as:

The phosphomolybdenum method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compounds and formation of green Mo(V) complex with a maximal absorption at 695 nm [18].The antioxidant capacity of NJE was estimated to be equivalent to 109.48  11.72 mg of ascorbic acid/g of the dried extract.

Chelating rate ð%Þ Z ½ðA0
A1 Þ=A0  100; where A0 was the absorbance of the control (blank, without extract) and A1 was the absorbance in the presence of the extract.

2.14. Reducing power assay The Fe3þ reducing power of the extract was determined by the method of Oyaizu [26]. The extract (0.75 ml) at various concentrations was mixed with 0.75 ml of phosphate buffer (0.2 M, pH 6.6) and 0.75 ml of potassium hexacyanoferrate [K3Fe(CN)6] (1%, w/v), followed by incubating at 50 C in a water bath for 20 minutes. The reaction was stopped by adding 0.75 ml of trichloroacetic acid (TCA) solution (10%) and then centrifuged at 3000 r/min for 10 minutes. 1.5 ml of the supernatant was mixed with 1.5 ml of distilled water and 0.1 ml of ferric chloride (FeCl3) solution (0.1%, w/v) for 10 minutes. The absorbance at 700 nm was measured as the reducing power. Higher absorbance of the reaction mixture indicated greater reducing power.

2.15. Statistical analysis All data are presented as mean  standard deviation values. The mean values were calculated from data taken from at least three independent experiments conducted on separate days using freshly prepared reagents. Statistical analyses were performed using Student’s t-test. The statistical significances were achieved at p < 0.05.

3. Results and discussion 3.1. Amount of total phenolic and total flavonoid contents

3.3. Inhibitory effect on linoleic acid oxidation The inhibitory effect of NJE on linoleic acid oxidation was determined by the ferric thiocyanate method. During linoleic acid oxidation, peroxides are formed, and these compounds oxidize Fe2þ too Fe3þ. The complex formed between Fe3þ and SCNˉ ions was quantified by measuring the absorbance at 500 nm. Lower absorbance indicates a higher level of antioxidant activity. Fig. 1 shows the oxidation of linoleic acid in the absence or the presence of NJE at variable time intervals. According to Fig. 1, the antioxidative effect of NJE at low doses (200 mg/ml) is not significant. However, at 800 mg/ml, the antioxidative effect of NJE almost matches that of butylated hydroxy toluene (BHT) at a dose of 100 mg/ml, thus reflecting the typical characteristics of a chain-breaking antioxidant.

3.4. DPPH radical scavenging activity The DPPH radical is a stable organic free radical with an absorption band at 515e528 nm, and thus it is a useful reagent for investigating the free radical scavenging activities of different compounds. The method is based on the reduction of alcoholic DPPH solution in the presence of hydrogen donating antioxidant [29,30]. The extract was capable of neutralizing the DPPH free radicals via hydrogen donating activity by 16.46%, 29.27%, 52.10%, 70.24%, and 84.35 at concentrations of 25, 50, 100, 250, and 500 mg/ml, respectively. As shown in Table 1, DPPH scavenging was 2 Control

1.8

BHT

1.6

NJE 200

1.4

NJE 400

1.2

NJE 800

1 0.8 0.6

Phenolics are the most wide spread secondary metabolite in plant kingdom. These diverse groups of compounds have received much attention as potential natural antioxidant in terms of their ability to act as both efficient radical scavengers and metal chelator. It has been reported that the antioxidant activity of phenol is mainly due to their redox properties, hydrogen donors and singlet oxygen quenchers [27]. Flavonoids are a class of secondary plant phenolics with powerful antioxidant properties [28]. Total phenolic and flavonoid contents were estimated to be equivalents to 41.34  2.30 mg of gallic acid and 32.15  1.27 mg of catechin/g of dried rhizomes extract, respectively.

0.4 0.2 0 0

20

40

60

80

100

Figure 1 Total antioxidant activity of different concentrations (200, 400, and 800 mg/ml) of NJE and BHT (100 mg/ml) in a linoleic acid emulsion determined by the thiocyanate method. Data are mean  SD values (n Z 3). *p < 0.05. **p < 0.01 versus control. BHT: Butylated hydroxy toluene; NJE: Nardostachys jatamansi.

116

S.K. Sharma, A.P. Singh

Table 1 DPPH and superoxide anions radical scavenging activity of NJE and ascorbic acid.

70

NJE concentration (mg/ml)

DPPH scavenging (%)

Superoxide scavenging (%)

50

25 50 100 250 500 Ascorbic acid (18)

16.46  0.82* 29.27  0.97* 52.10  1.51** 70.24  1.56** 84.35  1.87** 94.12  1.21**

09.25  0.91* 16.62  1.15* 36.75  1.11** 48.21  1.23** 62.32  1.82** 81.25  2.11**

Data are mean  standard deviation values (n Z 3). *p < 0.005. **p < 0.01 versus control. DPPH: 1,1-diphenyl-2-picrylhydrazyl; NJE: Nardostachys jatamansi.

increased in a concentration dependent manner compared to ascorbic acid, used as the positive antioxidant control in this investigation.

3.5. Superoxide anion scavenging activity It is well known that superoxide anions damage biomolecules directly or indirectly by forming H2O2, ˉOH, peroxy nitrite or singlet oxygen during aging and pathological events such as ischemic reperfusion injury. Superoxide has also been observed to directly initiate lipid peroxidation [31,32]. The superoxide anion radical scavenging activity of NJE rhizome extract assayed by the PMS-NADH system is shown in Table 1. The superoxide scavenging activity of NJE was increased markedly with the increase in concentrations. Thus, higher inhibitory effects of the rhizomes extracts on superoxide anion formation noted herein possibly render them as a promising antioxidant. These results suggested that NJE has a potent superoxide radical scavenging effects.

3.6. Hydroxyl radical scavenging activity Activity of the rhizomes extract on hydroxyl radical has been shown in Fig. 2. Hydroxyl radical is highly reactive 100 90 80 70 60 50 40 30 20 10 0 0

0.1

0.2

0.3

0.4

0.5

Catechin

60

NJE

40 30 20 10 0 100

250

500

1000

Figure 3 NO scavenging of NJE and catechin as the standard flavonoid. Data are mean  SD values (n Z 3). *p < 0.05. **p < .001 versus control. NJE: Nardostachys jatamansi; NO Z nitric oxide.

oxygen centered radical formed from the reaction of various hydroperoxides with transition metal ions. It attacks proteins, DNA, polyunsaturated fatty acid in membranes, and most biological molecule it contacts [33] and is known to be capable of abstracting hydrogen atoms from membrane lipids [31] and brings about peroxidic reaction of lipids. NJE exhibited concentration dependent scavenging activity against hydroxyl radical generated in the Fenton reaction system.

3.7. NO radical NO is a potentially toxic agent with a free radical character and therefore it is responsible for many physiologic and pathologic events [23]. The moderate NO scavenging activity was observed by NJE (12.2%, 20.6%, 35.3%, and 46.8% at NJE concentrations of 100, 250, 500, and 1000 mg/ml, respectively). This is shown in Fig. 3. Incubation of a sodium nitroprusside solution in PBS at 25 C for 150 minutes resulted in linear time-dependent nitrite production, which was reduced by NJE in a concentration-dependent manner.

3.8. Hydrogen peroxide scavenging activity assay As shown in Fig. 4, NJE also demonstrated hydrogen peroxide decomposition activity in a concentration dependent manner. Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H2O2 can probably react with Fe2þ, and possibly Cu2þ ions to form hydroxyl radical and this may be the origin of many of its toxic effects [34,35]. It is, therefore, biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. The decomposition of H2O2 by NJE may at least partly result from its antioxidant and free radical scavenging activity.

3.9. Metal chelating activity Figure 2 Hydroxyl scavenging activity of Nardostachys jatamansi rhizomes extract at different concentrations. Each value represents means  SD (n Z 3).

Ferrous ion can initiate lipid peroxidation by the Fenton reaction as well as accelerating peroxidation by

Antioxidant potential of Nardostachys jatamansi

117

100 90 80 70 60 50 40 30 20 10 0

Absorbance (700 nm)

1.4 1.2

BHT

1

NJE

0.8

**

0.6

*

0.4 0.2

0

0.2

0.4

0.6

0.8

1

0 25

Figure 4 H2O2 scavenging activity of Nardostachys jatamansi rhizomes extract at different concentrations. Each value represents means  standard deviation (n Z 3).

decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals [36,37]. Ferrozine can make complexes with ferrous ions. In the presence of chelating agents, complex (red colored) formation is interrupted and as a result the red color of the complex is decreased. Thus, the chelating effect of the coexisting chelator can be determined by measuring the rate of color reduction. The formation of the ferrozine-Fe2þ complex is interrupted in the NJE, indicating its chelating activity (Fig. 5). Chelating agents that form bonds with a metal are effective as secondary antioxidants because they reduce the redox potential, and thereby stabilize the oxidized form of the metal ion [38]. This study shows that NJE has a marked capacity for iron binding, suggesting the presence of polyphenols that have potent iron chelating capacity.

3.10. Reducing power activity For the measurements of the reducing ability, the Fe3þFe2þ transformation was investigated in the presence of NJE. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the activity of antioxidants has been assigned to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity, and radical scavenging [10,39]. Fig. 6 100 90 80 70 60 50 40 30 20 10 0

**

0

0.1

0.2

0.3

0.4

0.5

Figure 5 Ferrous ion chelating activity of Nardostachys jatamansi rhizomes ethanol extract at different concentrations. Each value represents means  standard deviation (n Z 3).

50 100 Cocentration (µg/ml)

250

Figure 6 Reducing power of NJE and BHT. Data are mean  standard deviation (n Z 3). *p < 0.05. **p < 0.01 versus control. BHT: Butylated hydroxy toluene; NJE: Nardostachys jatamansi.

depicts the reductive effect NJE. Similar to the antioxidant activity, the reducing power of NJE increased with increasing dosage. The result shows that NJE consists of hydrophilic polyphenolic compounds that cause the greater reducing power.

4. Conclusion The results obtained in the present study indicate that NJE rhizomes exhibit free radical scavenging, reducing power and metal chelating activity. The overall antioxidant activity of NJE might be attributed to its polyphenolic contents and phytochemical constituents. The findings of present study suggest that NJE rhizomes might act as a potential source of natural antioxidant that could have great importance as therapeutic agents for biological system susceptible to free radical mediated reactions.

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