Anti-oxidant activity of methanolic extract of aquatic owering plant Nymphaea capensis leaf

Background: Nymphaea capensis is an aquatic owering plant which is included in the family of Nymphaeaceae. Literature review on the plants of Nymphaeaceae family exhibited signicant medicinal activities. Therefore, the objective of the present study is to evaluate possible anti-oxidant activity of crude methanol extract of N. capensis leaf. Method: In antioxidant study, methanolic plant extract was evaluated for 1,1-diphenyl,2-picrylhydrazyl (DPPH) and reducing power capacity. Moreover, total phenolic and total avonoid content of plant extracts were determined and expressed in mg of gallic acid equivalent per gram of dry sample (mg GAE/g dry weight). Result: In the DPPH free radical scavenging assay, methanol extract showed concentration dependent inhibition of the free radicals. IC 50 of Ascorbic acid was 14.84 µg/ml whereas N. capensis was 130.94 µg/ml. In case of reducing capacity, at conc. 62.5, 125, 250, 500, 1000 ug/ml, the absorbances of Ascorbic acid were 0.65, 1.12, 1.45, 1.78 and 1.89 respectively. In case of N. capensis, the absorbances were 0.46, 0.75, 1.04, 1.27 and 1.50 respectively. The extract displayed a concentration dependent increase in reducing power. In the case of total phenolic content, the methanol extract of N. capensis contained a considerable amount of phenolic contents which was 215±7 of GAE/g. In the case of total avonoid content, methanol extract of N. capensis contained signicant amount of avonoid contents which was 184.75±6.78 of GAE/g.


Introduction
Nymphaea capensis is an aquatic owering plant which family is Nymphaeaceae. It occurs in freshwater areas in temperate and tropical climates throughout the world. In Bangladesh, N. capensis is commonly known as Nil Sapla or Blue water-lily. It commonly found in lakes, ponds, canals and swamps of Bangladesh as well as other parts of the world such as Africa, Asia Australia and Northern America [1,2].
In Madagascar, the rootstocks of N. capensis are eaten as food and supposedly are delicious in taste. In the Nymphaeaceae family, other plants such as the boiled or raw inner rhizomes of N. jamesoniana were eaten as vegetables in Argentina. In Asia and Africa, the rhizomes and tubers of N. lotus were also eaten as fruit which sweet in tastes. N. odorata leaves were consumed by North Americans which contains good amount of tannins, 40% crude protein. N. tetragona leaf buds and seeds are consumed as vegetables in Japan [2].
N. capensis also has good medicinal properties. The methanol extract of N. capensis leaves possess signi cant sedative and anxiolytic activities as well as mild cytotoxic activities on mice [3]. In the case of Nymphaeaceae family, other plants also has great medicinal properties and various types of compounds were isolated from them. Such as N. jamesoniana has been used as an astringent, N. lotus has been consumed as a traditional medicine which produce analgesic, anti-in ammatory, astringent, and sedative effect. It also exhibited signi cant antibiotic and antioxidant effect. N. Mexicana also exhibited antioxidant, DNA protective properties and antiproliferative activity. N. ampla owers and other parts possess narcotic properties, which contain aporphine and quinolizidine alkaloids [2,4]. According to this traditional consumption and laboratory experiments, Nymphaeaceae family exhibited great medicinal properties. Therefore, this literature review on Nymphaeaceae family made us curious to nd out the antioxidative properties of methanolic extract of N. capensis leaves.
Nowadays people are prone to in ammation due to imbalance lifestyle and diet which later turn into chronic in ammation. This in ammation occurs mainly because of reactive oxygen species (ROS).
Through autooxidation and lipid peroxidation in ammatory mediators such as cytokines, interleukins, chemokines, bioactive amines, bradykinin, and in ammatory lipids produce reactive oxygen species (ROS) [5]. Reactive oxygen species (ROS) are superoxide anion (O2•-), hydroxyl radicals (OH•), singlet oxygen (1O2) and hydrogen peroxide (H2O2) which causes great harm to the cells and are also responsible for various diseases such as cardiovascular diseases, ischemia, diabetes, in ammation, cancer, anemia, ageing and degenerative diseases [6,7]. Antioxidants plays pivotal role to neutralize the free radicals. Lots of drugs were isolated from natural sources such as anticancer drugs in clinical trials were also isolated from plant sources which contains signi cant amount of antioxidant activity [8,9].
Henceforth, our experiment was designed to investigate the anti-oxidant activity of methanol extract of Nymphaea capensis leaves.

Extract preparation
The leaves were sun dried and ground. The ground (300 g) were soaked in su cient amount of methanol for one week at room temperature with occasional shaking and stirring then ltered through a cotton plug followed by Whitman lter paper No. 1. The solvent was evaporated under vacuum at room temperature to yield semisolid. The extract was then preserved in a refrigerator till further use. Antioxidant Activity Antioxidant activity of N. capensis is determined by DPPH radical scavenging assay, reducing power capacity assay, total phenolic content and total avonoid content.

DPPH radical scavenging assay
Free radical scavenging activity of methanolic extract of N. capensis leaf was determined according to the Brand-Willium [10] method with a slight modi cation. This activity was determined spectrophotometrically by taking absorbance of DPPH at 517 nm. Then the % of free radical inhibition was calculated using following equation:

Percentage of inhibition = [(Abs of control − Abs of sample) ÷ Abs of control] × 100
Lower the absorbance with high concentration of extract indicates potential antioxidant activity of test sample. Ascorbic acid was used as a reference standard.

Reducing power capacity
Reducing power capacity of the methanolic extract of N. capensis was determined according to Oyaizu (1986) method [11]. The amount of ferrous complex was determined spectrophotometrically by taking absorbance at 700 nm where plant extracts having excellent antioxidant property show greater absorbance with the higher concentration of extract solution. Ascorbic acid was used as a reference standard.
Estimation of total phenolic content TPC in the N. capensis plant extract was determined by following the Folin-ciocalteu method [12]. TPC was determined spectrophotometrically by taking absorbance at 760 nm. Standard Gallic acid solution of different concentrations with same procedure were used to prepare a calibration curve by plotting the absorbance against their respective concentrations from where a standard equation was formulated to determine the unknown concentration of Gallic acid equivalent (GAE) phenolic concentration of the sample by putting the value of absorbance in the equation.
Total phenolic content was determined by following equation: Determination of total avonoid content Total avonoid of N. capensis was determined using Aluminum chloride colorimetric method with slight modi cation [13]. Brie y, 1.0 ml of each of the concentrations of N. capensis was mixed with 3.0 ml of methanol, 0.2 ml of 10% AlCl3, 0.2 ml of 1 M potassium acetate and 5.6 ml of distilled water was added to the resulting mixture which was then incubated at room temperature for 30 min to complete the reaction. The reaction mixture was recorded for absorbance at 420 nm. Gallic acid was used as standard and the results were expressed as milligrams of gallic acid equivalents per gram of dried fraction.

Statistical analysis
The data was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test with GraphPad Prism Data Editor for Windows, Version 6.0 (GraphPad Software Inc., San Diego, CA). Values were expressed as mean ± Standard error of mean (± SEM). P < 0.05 was considered as statistically signi cant.

Antioxidant Activity DPPH Free radical scavenging activity
In the DPPH free radical scavenging assay, methanol extract showed concentration dependent inhibition of the free radicals as shown in Figure 1    Determination of total phenolic content Total phenolic content was estimated by gallic acid (Figure 3) and expressed as milligrams of gallic acid equivalent (GAE). The methanol extracts of N. capensis contained a considerable amount of phenolic contents of 215±7 of GAE/g of extract (Table 4).

Discussion
Free radicals such as Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are undoubtedly produced in our body which is a part of normal physiological process [14].Various degenerative disorders, like mutagenesis, carcinogenesis, cardiovascular disturbances and ageing occurs due to free radicals [15,16]. Antioxidants are the compounds which are accountable to sabotage the free radicals and protect our body from various free radical associated diseases. The mechanism is associated with free radical mediated oxidative process through initiation, propagation and termination [17]. Production of antioxidant can be occurred inside the body as well as naturally in many foods [18]. In this way, endogenous antioxidants such as superoxide dismutase, catalase, glutathione peroxidase or nonenzymatic compounds, such as uric acid, bilirubin, albumin, metallothioneins as well as exogenous antioxidants like vitamin E, vitamin C, β-carotene, vitamin E, avonoids protect physiological system from free radicals by destroying them [19].
DPPH radical scavenging assay is extensively used system for the detection of antioxidant activity in plant extracts. In this assay, at the time of receiving an electron or hydrogen, DPPH radical go through a color change from purple to yellow. At the absorbance of 517 nm, the activity of experimented antioxidant agents can be determined [20]. Degree of color change is proportional to the concentration and potency of antioxidant agents. Scavenging activity of antioxidant agent is determined by the degree of discoloration [21]. In this assay, IC50 of Ascorbic acid was 14.84 µg/ml whereas N. capensis was 130.94 µ g/ml. Lower IC50 value denotes an extract with greater scavenging activity.
In this reducing power assay, due to the presence of the reductants in the solution, the reduction occurs and ferric (Fe3+) form converts to ferrous (Fe2+) form. As a result, at an absorbance of 700 nm, ferrous (Fe2+) ion can be observed. The reduction of ferric ion is an indicator of electron donating activity. It is an important mechanism of antioxidant action. Due to antioxidants, free radicals are destroyed. [22,23].According to the gure 2, N. capensis exhibited increased absorbance's in increased concentrations which showed the considerable antioxidant activity of this methanolic plant extract.
Phenolic compounds have extensive antioxidant activity due to their ability to scavenge free radicals. It contains aromatic ring bearing one or more hydroxyl groups [24]. Phenolic are the largest group of phytochemicals for most of the antioxidant activity in plant or plant extracts [25,26]. As a result, this test is widely used for the determination of antioxidant activity in plant extracts. In this test, Total phenolic content was estimated by gallic acid and expressed as milligrams of gallic acid equivalent (GAE). The methanol extracts of N. capensis contained a substantial amount of phenolic contents of 215±7 of GAE/g of extract.
Flavonoids generally exhibit signi cant antioxidant activity by radical scavenging, ion chelating and lipid peroxidation inhibiting properties [27,28]. Among naturally occurring phenolic compounds, avonoids are the largest group. It contains various biological activities such as ntimicrobial, mitochondrial adhesion inhibition, antiulcer, antiarthritic, antiangiogenic, anticancer and so on [29]. Flavones and avonols are the most extensively found in all the phenolics. Flavonoids have two benzene rings separated by a propane unit [30]. Antioxidant activity of avonoids can sabotage free radicals such as super oxide and hydroxyl radicals [31]. In this test, Total phenolic content was estimated by gallic acid and expressed as milligrams of gallic acid equivalent (GAE). The methanol extract of N. capensis contained a substantial amount of phenolic contents of 184.75±6.78 of GAE/g of extract.

Conclusion
The results presented in this experiment indicated that crude methanol extract of Nymphaea capensis leaf possess considerable amount of anti-oxidant activity. This study also supports the traditional use of this plant as a therapeutic agent. Further studies are required to unfold the underlying mechanism and major constituents of this aquatic plant.  Calibration curve of Gallic acid for total phenol content determination.

Figure 4
Calibration curve of Gallic acid for Total avonoid contents.

Figure 5
Total phenol and Total avonoid