Antioxidative characteristics of ethanol and aqueous extracts of Curcuma amada rhizomes

Ram Kumar Sahu^1*, Harideep Singh¹, Amit Roy²

¹Oriental College of Pharmacy, Raisen Road, Bhopal (M.P.) India

²Columbia Institute of Pharmacy, Tekari, Near Vidhan Sabha, Mandhar Colony, Raipur (C.G.) India

ABSTRACT

The plants belonging to Zingiberaceae family are found to be a rich source of substances of phytochemical interest. Curcuma amada is one member of this family which is traditionally used as carminative and stomachic. The antioxidant activity of ethanol and aqueous extract of Curcuma amada rhizomes was studied. The antioxidant activity in vitro was measured by means of the 1, 1-diphenyl-2-picrylhydrazyl (DPPH), Nitric Oxide and Super oxide free radical scavenging assay. Ascorbic acid, a natural antioxidant, was used as a control. The extracts of ethanol and aqueous were strongly scavenged DPPH radical with the IC₅₀ being 265.33 and 217.90 µg/ml respectively. While the extracts of ethanol and aqueous were moderately scavenged superoxide radical with the IC₅₀ values of 526.87 and 440.89 µg/ml and moderately inhibited nitric oxide with the IC₅₀ values of 386 and 510.67 µg/ml. All the parameters were found to dose dependent.

Keywords: Curcuma amada, DPPH, Superoxide, Nitric Oxide.

INTRODUCTION

Plants produce an extensive range of chemicals, including ‘‘secondary metabolites”, which may exert beneficial health effects when consumed by man. Many of the plant secondary metabolites act as antioxidants in animals. On the other hand, these metabolites of plant origin may be used to prevent food deterioration via inhibition of lipid oxidation. Antioxidant action is a combination of several distinct chemical events such as metal chelation; quenching free radicals by hydrogen donation from phenolic groups; oxidation to a non-propagating radical; redox potential, enzyme inhibition¹.When oxidation caused by free radicals and reactive oxygen species emerges in food or in biological systems, antioxidants can prevent or delay this process via single or combination of aforementioned mechanisms. Hence, antioxidants may help the body to protect itself from various types of oxidative damage which are linked to diseases such as cancer, diabetes, cardiovascular disorders and aging². Consequently, search for food and drinks with high antioxidant content and enhancement of their antioxidant properties for nutritional purposes are currently of major interest. Food such as common vegetables and fruits which are consumed all over the world as well as wild plants that are consumed by local inhabitants are screened for their antioxidant capacity. The assessment of antioxidant capacity of such Curcuma amada rhizomes remains an interesting and useful task for finding new sources of natural antioxidants.

Synthetic antioxidants, like butylated hydroxyanisole, are good free radical scavengers, however, the synthetic antioxidants can be carcinogenic. Therefore, there is an increasing interest in searching for antioxidants of natural origin. We report here the results of a screening for antioxidant activity of ethanol and aqueous extracts of Curcuma amada (Zingibercaeae), known in Hindi as Aama haldi^3,4.

Table 1. Free Radical Scavenging Capacity of various extract of C. amamda

Concentration (µg/ml)	DPPH Scavenging %
Concentration (µg/ml)	Ethanol Extract	Aqueous Extract	Ascorbic Acid
200	42.65±0.58	44.11±1.68	94.35±1.25
400	59.58±1.14	62.41±1.75	-
600	77.45±1.85	80.52±1.96	-
800	85.32±2.45	88.56±1.37	-
1000	91.15±1.32	93.12±0.85	-
IC₅₀	265.33	217.90	-

Values are means ± SEM of six determinations IC₅₀ values, from the data, were calculated by regression analysis.

MATERIALS AND METHODS

Plant material

Curcuma amada rhizomes were collected from the Korba, Chhattisgarh in the month of September. It was identified by the research scientists at the Agriculture University, Raipur (Chhattisgarh), India.

Preparation of extract

The dried and powdered rhizomes (250 gm) were successively extracted on a Soxhlet apparatus, employing petroleum ether, ethanol and distilled water respectively. The extracts were further concentrated under reduced pressure with a rotary evaporator. Rhizomes of C. amada yielded 1%, 5.52% and 4.88% w/w powdered extract with petroleum ether, chloroform, ethanol and distilled water respectively.

Hydrogen-Donating Activity Hydrogen donating activity was quantified in presence of stable DPPH radical on the basis of Blois method. Briefly, to a methanolic solution of DPPH (100m M, 2.95 ml), 0.05 ml of both extracts dissolved in methanol was added at different concentrations (200-1000 µg/ml). Reaction mixture was shaken and after 30 min at room temperature, the absorbance values were measured at 518 nm and converted into percentage of antioxidant activity (% AA). Ascorbic acid was used as standard^5,6. The degree of discoloration indicates the scavenging efficacy of the extract, was calculated by the following equation:

% AA = 100 – {[(Abs_sample – Abs_blank) x 100] / Abs_DPPH}

Fig. 1. %DPPH Scavenging of Ethanol Extract

Nitric Oxide Scavenging Nitric oxide scavenging activity was measured spectrophotometrically. Sodium nitroprusside (5mM) in phosphate buffered saline was mixed with different concentrations of both extracts (200-1000 µg/ml l) dissolved in methanol and incubated at 25 °C for 30 min, then 1.5 ml of the incubation solution were removed and diluted with 1.5 ml of Griess reagent (1% Sulfanilamide, 2% phosphoric acid, and 0.1% naphthyl ethylene diamine dihydrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with naphthylethylene diamine was measured at 546 nm along with a control^7,8.

Table 2. Nitric oxide scavenging capacity of various extract of C. amamda

Concentration (µg/ml)	Nitric oxide Scavenging %
Concentration (µg/ml)	Ethanol Extract	Aqueous Extract	Ascorbic Acid
200	39.32±2.13	31.23±1.15	92.19±1.75
400	48.55±2.45	43.49±1.59	-
600	65.65±1.85	56.68±0.89	-
800	74.42±1.24	64.44±0.69	-
1000	86.45±2.09	79.37±2.42	-
IC₅₀	386	510.67	-

Values are means ± SEM of six determinations. IC₅₀ values, from the data, were calculated by regression analysis.

Superoxide Scavenging Activity Superoxide scavenging was carried out by using alkaline DMSO) Solid potassium superoxide was allowed to stand in contact with dry DMSO for at least 24 h and the solution was filtered immediately before use. Filtrate (200m l) was added to 2.8ml of an aqueous solution containing nitroblue tetrazolium (56 m M), EDTA (10m M) and potassium phophate buffer (10mM, pH 7.4). Sample extract (1 ml) at various concentrations (200-1000 µg/ml) in water was added and the absorbance was recorded at 560 nm against a control in which pure DMSO has been added instead of alkaline DMSO^9,10.

Statistics Analysis

The data were reported as mean values ± standard deviation (SEM). Values representing the concentrations of investigated extracts that cause 50% of neutralization/inhibition (IC₅₀) were determined by the linear regression analysis.

RESULTS

DPPH is stable nitrogen centered free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agents, then losing colour stoichometrically with the number of electrons consumed, which is measured spectrophotometricallty at 517 nm. As shown in Table 1, C. amamda of ethanol and aqueous extract strongly scavenged DPPH radical with the IC₅₀ being 265.33 and 217.90 µg/ml respectively. The scavenging was found to dose dependent.

C. amamda of ethanol and aqueous extracts also moderately inhibited nitric oxide in dose dependent manner (Table 2) with the IC₅₀ being 386 and 510.67 µg/ml respectively. Nitric oxide (NO) is a potent pleiotropic mediator of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity.

It is a diffusible free radical which plays many roles as an effector molecule in diverse biological systems including neuronal messenger, vasodilation and antimicrobial and antitumor activities. Studies in animal models have suggested a role for NO in the pathogenesis of inflammation and pain and NOS inhibitors have been shown to have beneficial effects on some aspects of the inflammation and tissue changes seen in models of inflammatory bowel disease. Thus establishing the usage of the plant in the Indian indigenous system as an anti-inflammatory agent.

Fig. 2. %DPPH Scavenging of Aqueous Extract

Fig. 3 IC₅₀ values, from the data, were calculated by regression analysis.

Fig. 4 IC₅₀ values, from the data, were calculated by regression analysis.

Table 3. Super oxide scavenging capacity of various extract of C. amamda

Concentration (µg/ml)	Superoxide Scavenging %
Concentration (µg/ml)	Ethanol Extract	Aqueous Extract	Ascorbic Acid
200	29.23±0.47	33.75±1.25	89.41±1.52
400	41.39±1.08	47.23±2.36	-
600	55.85±0.96	60.25±2.41	-
800	67.45±1.48	75.45±2.16	-
1000	79.12±1.56	86.32±2.04	-
IC₅₀	526.87	440.89	-

Values are means ± SEM of six determinations. IC₅₀ values, from the data, were calculated by regression analysis.

SOD is an important cellular antioxidant enzyme, which converts superoxide radical into H₂O₂ and O₂. We also looked for the protective effect of the both extract on antioxidant enzyme SOD in mitochondria exposed to H₂O₂. Table 3 gives the changes in the activity of SOD upon treatment with the both extracts. The ethanol and aqueous extracts also moderately scavenged superoxide radical with the IC₅₀ values of 526.87 and 440.89 µg/ml respectively.

Fig. 5 IC₅₀ values, from the data, were calculated by regression analysis.

Fig. 6 IC₅₀ values, from the data, were calculated by regression analysis.

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Received on 01.05.2009

Accepted on 25.05.2009

Research Journal of Pharmacognosy and Phytochemistry. 1(1): July.-Aug. 2009, 41-43