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In-vitro evaluation of antioxidant activity of Nymphaea stellata Willd.


The plant Nymphaea stellata Willd. (Nymphaceae), a medicinal herb has reported for its use in treatment of liver disorders in Ayurvedic literature. The leaves root and flowers have a wide range of pharmacological activities and are used for diabetes eruptive fevers and as cardiotonic, emollient, diuretic, narcotic, and as aphrodisiac [1]. The flowers of plant contain 6 flavonoid, gallic acid, astragalin, quercetin and kaempferol. And the seeds also contain 7 protiens, pentason, mucilage etc. But yet the plant has not been subjected to systematic scientific investigation to assets its antioxidant activity of this plant. There are many evidences indicates that free radical are responsible for birth of many disorders like inflammation, atherosclerosis, aging and hepatic toxicity [2].

Antioxidants or inhibitors of oxidation are the compounds which retard or prevent the oxidation in general and prolong the life of oxidizable matter [3]. Antioxidants can interfere with the oxidation process by reacting with free radicals, chelating catalytic metals and also by acting as reactive species scavenger. The antioxidants activities of the individual compounds may depends on structural factors, such as number of phenolic, hydroxyl or methoxyl groups and other structural features [4]. Among the antioxidative compounds vitamin C, E, A, Selenium, Carotenoids, Ascorbic acid shows very strong intensity of antioxidative activities [5]. Antioxidant determinations by the use of stable free radical were earliest being done in 1958 by Blois [6].

A free radical is a compound with one or more unpaired electrons in its outer orbital [7]. Such unpaired electrons make these species very unstable and therefore quite reactive with other molecules due to the presence of unpaired electrons and try to pair their electrons and generate a more stable compound [8].

The molecule of DPPH is characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that molecules do not dimerise, as would be the case with most other free radicals. The delocalization also gives rise to the deep violet color, characterized by an absorption band in ethanol solution centered at about 517 nm.

When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of this violet color (although there would be expected to be a residual pale yellow color from the picryl group still present). Representing the DPPH radical by Z and the donor molecule by AH, the primary reaction is;

Z* + AH = ZH + A* (1)

Where, ZH is the reduced form and A* is free radical produced in this first step. This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorized) by one molecule of the reductant. The reaction [1] is therefore intended to provide the link with the reactions taking place in an oxidising system, such as the autoxidation of a lipid or other unsaturated substance; the DPPH molecule Z* is thus intended to represent the free radicals formed in the system whose activity is to suppressed by the substance AH [9].

Materials and methods

Collection of Plant Material:

The flower of Nymphaea stellata plant was purchased from Khari-Bawoli market, Old Delhi.The plant was identified as Nymphaea stellata. F-Nymphaceae by the Dr. E Roshini Nayar (Principal Scientist), National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi. A voucher specimen (Specimen No: NHCP/NBPGR/2010-7/6652) is preserved in herbarium section of taxonomic department of NBPGR, New Delhi

Preparation of Extract:

The flower of plant after washing with distilled water was air dried at room temperature. The dried flowers were coarsely powdered and successfully extracted with methanol using Soxhlet apparatus at a temperature of 60-70[degrees]C for a period of 6-8 hrs. The extract was then concentrated over water bath and finally dried to a constant weight.

Preparation of Reagents:

The 500liM solution of $DPPH was made by 23 mg of DPPH (Assay 85%) of Hi Media Laboratories Pvt. Ltd. CAS No. 1898-66-4. TRIS [2-amino-2 (hydroxy methyl) propane 1-3di-ol] Buffer pH 7.4 was prepared by adding 0.605g of TRIS Buffer 7.4 of Qualigens Fine Chemicals in 30 ml of water and adding 0.33 ml of concentrated hydrochloric acid, diluted to 100 ml with distilled water. TRIS buffer prevents the sudden pH change during the preparation of test dilutions [10].

Preparation of Reference Standard Solution:

Various dilutions of butylated hydroxytoluene were made with concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45 & 50 ng per 0.5 ml of methanolic solution of butylated hydroxytoluene.

Preparation of Sample Solution and Dilutions:

Prepared the stock solution by dissolving 250 mg methanolic extract of Nymphaea stellata and made up the volume to 25 ml with methanol. Prepared the initial dilutions from stock solution using volume 0.25 ml, 0.5 ml, 0.75 ml, 1.0 ml, 1.25 ml,1.50 ml 1.75 ml 2.0 ml, 2.25 ml, and 2.5 ml and dilute up to 10 ml with methanol. The final concentrations used for taking the absorbance are 0.25 mg, 0.50 mg, 0.75 mg, 1.00 mg, 1.25 g, 1.5 mg, 1.75 mg, 2.00 mg, 2.25 mg, and 2.5mg per ml.

Measurement of in Vitro Antioxidant Activity:

The antioxidant activity of the flower of Nymphaea stellata was determined by using a method based on the reduction of methanolic solution of colored-free radical 1, 1 di-phenyl-1-2 picryl hydrazyl (DPPH). The radical scavenging activity of tested sample was expressed as an inhibition percentage. Butylated hydroxyl toluene was used as reference standard. In 5 ml volumetric flasks added 1 ml of DPPH solution, 0.5 ml of TRIS Buffer and 0.5 ml of final dilutions of different concentrations range prepared from N. stellata stock solution and made up the volume to 5 ml with methanol. In same way, prepared the control dilutions of DPPH, replacing 0.5 ml of prepared dilutions (the drug solution under investigation) with methanol. The absorbances of all the dilutions were taken after 30 minutes at [lambda] max 517nm using methanol as blank.

Statistical Analysis:

The percentage inhibition was calculated using: Percent Inhibition = ([A.sub.C] - [A.sub.s]/[A.sub.C]) x 100

Where, Ac is absorbance of control, As is the absorbance of sample. [IC.sub.50] value (a concentration at 50% inhibition) was determined from the curve between percentage inhibition and concentration. All determinations were done in triplicate and the [IC.sub.50] value was calculated by using the equation of line [11].

Results and discussion

The methanolic extract of Nymphaea stellata tested for in vitro using DPPH showed moderate free radical scavenging activity, as evidenced by low [IC.sub.50] values. Fig-1 depicted that the [IC.sub.50] value of Nymphaea stellata was 1.68291.



The absorbance of sample (methanolic extract of Nymphaea stellata and standard Butylated hydroxytoluene) were taken in triplicate.

With the increase of concentration, the decrease of absorbance value and the calculated percentage inhibition has shown with the help of tables.


Polyphenolic compounds like flavonoids and phenolic acids, commonly found in plants, have been reported to have multiple biological effects, including antioxidant activity [12]. In vitro study shows that Nymphaea stellata has moderate free radical scavenging action. Antioxidant property of Nymphaea stellata can be attributed to the presence of flavonoids and polyphenols and which in turn may be responsible for its anti-stress effect.


[1.] Nadakarni, K.M., 1954. India Materia Medica: Popular Book Depot: Dhoot Papeshwar Bombay, 1: 860.

[2.] Marx, J.L., 1987. Oxygen free radicals linked to many disease., 235.

[3.] Kalia, AN., 2005. A Text Book of Industrial Pharmacognosy: CBS Publisher and Distributors, Edi 1, pp: 204-205.

[4.] Patt, D.E., B.J.F. Hudson, 1990. Natural antioxidants not exploited commercially: In: food antioxidants, Elsevior Applied Science, 171-191.

[5.] Dekkers, J.C., L.J.P. Doornen, C.G. Han, 1996. The Role of Antioxidant Vitamins and Enzymes in the Prevention of Exercose-Induced Muscle Damage: Sports Medicine, 21: 213-238.

[6.] Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical: Nature, 181: 1199-1200.

[7.] Jesberger, J.A., J.S. Richardson, 1991. Oxygen free radicals in brain dysfunction; International journal of Neuroscience, 57: 1-17.

[8.] Kaczmarski, M., J. Wojicicki, L. Samochowiee, T. Dutkiewicz, Z. Sych, 1999. The influence of exogenous antioxidants and physical exercise on some parameters associated with production and removal of free radicals: Pharmazie, 54: 303306.

[9.] Molyneux, P., 2004. The use of stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity: Songklanakarin Journal of science and Technology, 26(2): 211219.

[10.] Indian, Pharmacopoeia, 2006. The controller of publication, Delhi, 2: A-25.

[11.] Papuc, C., C. Diaconescu, V. Nicorescu, 2008. Antioxidant activity of sea buckthorn (Hippophae rhamnoides) extracts compared with common food additives: Roumamian Biotechnological Letters, 13(6): 4049-4053.

[12.] Paramapojn, S., W. Gritsanapan, 2009. Free radical scavenging activity determination and quantitative analiysis of curcuminoids in Curcumaa zedoaria rhizome extracts by HPLC method: Current Science, 97(7): 1069-1073.


(1) University Institute of Pharmacy, C.S.J.M. University, Kanpur, India

(2) Institute of pharmacy, Dayanand Dinannath College, Ramaipur, Kanpur, India

(3) Department of Pharmacognosy and Phytochemistry, Jamia Hamdard, New Delhi, India

Corresponding Author

Nikhil K Sachan, University Institute of Pharmacy, Chhatrapati Shahu Ji Maharaj University, Kanpur, India.

Tel: +91-9307755497; Fax: +91-512-2573231

Table 1: Values of absorbance and percentage inhibition with
increase in concentration of methanolig extract of Nymphaea

Concentration([micro]gm)   Absorbance    %Inhibition

0.250                      1.623         10.97
0.500                      1.425         21.83
0.750                      1.377         24.46
1.000                      1.264         30.66
1.250                      1.062         41.74
1.500                      0.922         49.42
1.750                      0.816         55.24
2.00                       0.806         55.78
2.250                      0.699         61.65
2.500                      0.547         69.99

Table 2: Values of absorbance and percentage inhibition with
increase in concentration of methanolic solution of butylated
hydroxytoluene (standard antioxidant).

Concentration([micro]g)   Absorbance       Percentage Inhibition

5                         2.163 + 0.01     7.78
10                        1.996 + 0.04     14.91
15                        1.792 + 0.06     23.58
20                        1-601 + 0.02     31.75
25                        1.531 + 0.09     34.72
30                        1.375 + 0.00     41.36
35                        1.211 + 0.01     48.36
40                        1.111 + 0.00     52.62
45                        0.945 + 0.01     59.69
50                        0.892 + 0.01     61.94
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Article Details
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Title Annotation:Original Article
Author:Sachan, Nikhil K.; Das, Doli Rani; Sachan, Anupam K.; Shuaib, Mohd.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9INDI
Date:Jun 1, 2011
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