The Evaluation of Nitric Oxide Scavenging Activity of Acalypha Indica Linn Root
Balakrishnan N *, Panda A B, Raj N R, Shrivastava A and Prathani R
Technocrats Institute of Technology- Pharmacy, Bhopal-462021, India.
*Corresponding Author E-mail: pharmacobala@yahoo.co.in; pharmacobala@rediffmail.com
ABSTRACT
Acalypha indica (Euphorbiaceae) is commonly known as Indian Copperleaf and Indian Nettle. It is a common annual herb, which grows in the hotter parts of India like Bihar, Assam and southwards of Kerala. The ethanol and aqueous extracts of root were screened for antioxidant activity using Nitric Oxide Scavenging activity method, which showed significant percentage of inhibition in dose dependent manner. As antioxidant therapy is found to be useful in complicated disease status related with free radical activity, the present study might be extended for the formulation and evaluation of different antioxidant herbal dosage forms.
KEY WORDS: Acalypha indica, antioxidant, Nitric Oxide Scavenging activity
INTRODUCTION:
Oxidation is a basic part of the aerobic life and our metabolism. During oxidation, many free radicals are produced which have an unpaired, nascent electron. Atoms of oxygen or nitrogen having central unpaired electron, are called reactive oxygen or nitrogen species1-4. This may be harmful to the body and may cause peroxidation of membrane lipids, aggression of tissue membranes and proteins or damage to DNA and enzyme5. These can be related to some pathology, such as arthritis, haemorrhagic shock, coronary artery diseases, cataract, cancer, AIDS as well as age-related degenerative brain diseases6. Currently, there’s a great interest in the study of antioxidant substances mainly due to the findings of the therapeutic effects of free radical scavengers on the organism. The antioxidant activities of phenolic compounds are mainly due to the redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. In addition, they have a metal chelating potential. The antioxidant activity of phenolics plays an important role in the adsorption or neutralization of free radicals7.
Acalypha indica (Euphorbiaceae)8 known as Khokali or Kuppi in Hindi and grows in Indian gardens, backyards of houses and waste place throughout the plains of India. All the parts of the plants are used in various traditional systems among them root seems to be much useful.
Traditionally the plant used as a laxative, anthelmintic, cathartic and the juice is used as a speedy emetic for children and expectorant. The whole plant is useful in treating chronic bronchitis, asthma, scabies and other skin diseases, rheumatism, congestive headache9, 10.
A.indica is a small erect herb up to 60 cm tall or a little more, with a few ascending branches, these angled and pubescent; leaves broadly ovate sub deltoid rather coarsely toothed, on petioles as long as or longer than 3-5 cm long blades; nerves 3-5 from base, thereafter pinnately arranged; stipulous minute; flowers sessile on erect axillary spikes longer than the leaf; male flowers minute, crowded distally; stamens 8; female flowers scattered along the inflorescence axis, each subtended by a conspicuous semicupular foliaceous toothed green bract nearly 7mm long; capsule hispid, 1 mm broad, 3-locular; seeds ovoid, smooth, pale-brown8,9. Previous phytochemical investigations on this species revealed the presence of acalyphamide(as acetate), aurantiamide and its acetate, succinimide, calypholacetate, 2- methyl anthraquinone, tri-o-methylellagic acid, b- sitosterol and its b-D- glucoside , cyanogenetic glycoside, two alkaloids, viz. Acalyphine and triacetonamine and also n- octasanol, b- sitosterol acetate, kaempferol, quebrachitol, tannin, resin, hydrocyanic acid and essential oil8-12. Therefore, our aim in this study was to evaluate the antioxidant activity of ethanol and aqueous extract of the roots of Acalypha indica by Nitric Oxide Scavenging activity method.
Plant Material:
Acalypha indica was collected from Salem district, TamilNadu, India and taxonomically identified by Prof. P. Jayaraman, Plant Anatomy Research Centre, and Chennai, India. The voucher specimen (PARC/2007/153) has been retained in Department of Pharmacognosy, Technocrats Institute of Technology-Pharmacy, Bhopal and Madhya Pradesh. The collected material was dried under shade and then powdered with mechanical grinder. The dried powder material was extracted with ethanol in a soxhlet and water in a maceration method13.
Chemicals:
The sodium nitroprusside (SNP), griess reagent and ascorbic acid were obtained from
Sisco Research Laboratories Pvt. Ltd., India. All chemicals and solvents were of analytical grade.
The preliminary phytochemical screening (14) of the ethanol and aqueous extracts were carried out to know the different constituents present in the root of A. indica as per the standard procedure.
Nitric oxide generation and assay of Nitric oxide scavenging method:
Nitric oxide (NO) was generated from sodium nitroprusside (SNP) and was measured by the Griess reagent. SNP in aqueous solution at physiological pH spontaneously generates NO (15, 16), which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess Reagent. Scavengers of NO compete with oxygen leading to reduced production of NO (17, 16). SNP (10mM) in phosphate buffer saline (PBS) was mixed with different concentration of extract (100-1000µg/ml) of the drug dissolved in ethanol and water and incubated at 25°C for 180 minutes. The samples from the above were reacted with Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine dichloride and 3% phosphoric acid). The absorbance of the chromophores formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylenediamine dichloride was read at 546 nm and referred to the absorbance of ascorbic Acid, used as a positive control treated in the same way with Griess reagent.
Acontrol - Atest
Nitric Oxide scavenged (%) = --------------------- × 100
Acontrol
Where, Acontrol = Absorbance of control reaction and Atest = Absorbance in the presence of the samples of extracts.
RESULTS AND DISCUSSION:
Nitric oxide (NO) is a potent pleiotropic inhibitor 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 that plays many roles as an effectors molecule in diverse biological systems including neuronal messenger, vasodilatation and antimicrobial and antitumor activities18. Preliminary Phytochemical studies of ethanol and aqueous extract of A. indica root shows the presence of alkaloids, saponin, terpenoids, flavonoids and polyphenols. Suppression of released NO may be partially attributed to direct NO scavenging, as the extracts of A. indica decreased the amount of nitrite generated from the decomposition of SNP in vitro. The scavenging of NO by the extracts was increased in dose dependent manner. Figure-1 illustrates a significant decrease in the NO radical due to the scavenging ability of extracts and ascorbic acid. The ethanol and aqueous extracts showed maximum activity of 64.74% and 59.83% respectively at 1000 µg /ml, where as ascorbic acid at the same concentration exhibited 90.16% inhibition. The Ic 50 values were found to be 103.59µg/ml, 315.46µg/ml and 667.82 µg/ml for ascorbic acid, ethanol and aqueous extracts respectively.
CONCLUSION:
The ethanol and aqueous extracts of root of A. indica exhibited significant antioxidant activity compare to ascorbic acid and the activity may be related to the flavonoids and phenolic compounds in this plant extract. Since reactive oxygen species are important contributors to several serious ailments, in the present study, the observed NO scavenging activity of the extract of Acalypha indica root might be useful for the development of newer and more potent natural antioxidants.
ACKNOWLEDGEMENT:
We are thankful to Mr. R. R. Karsaulya, Chairman, TIT group of institution and Dr. A. Balasubramaniam, Director, TIT Pharmacy, for providing necessary facilities to conduct this study.
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Received on 02.04.2009 Modified on 20.05.2009
Accepted on 11.06.2009 © AJRC All right reserved
Asian J. Research Chem. 2(2): April.-June, 2009 page 148-150