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INTERNATIONAL JOURNAL OF ADVANCES IN PHARMACY, BIOLOGY AND CHEMISTRY Research Article In vitro antioxidant properties of different parts of Gaertn.

Kalpana Shukla and Neelam Chaturvedi Department of Food Science and Nutrition, Banasthali University, Dist. Tonk, Rajasthan, -304022.

ABSTRACT Medicinal constitute the major constituents of most indigenous medicines. Recently, interest has increased considerably in finding naturally occurring antioxidants for use in foods or medicinal materials. The mounting interest in appraisal of antioxidant power of herbal as medicine, the present study was carried out to explore the antioxidant potential of aqueous extracts of Nelumbo nucifera Gaertn (Nelumbonaceae) leaves, , seeds and rhizomes in-vitro. Antioxidant activity of plant parts aqueous extracts were determined by total phenolic, flavonoids and DPPH free radical scavenging assay, content using reference standard ascorbic acid, gallic acid and quercetin respectively. All the analysis was made with the use of UV-Visible Spectrophotometer. The highest total phenolic content (mg GAE/100g) was found to be 270.90±0.33 in seeds and 252.34±0.33 in leave and total flavonoids content (mg QE/100g) was 245.22±1.51 and 193.54±1.94 of aqueous extracts of leaves and flower. The DPPH radical scavenging activity of the extract was increased with the increasing concentration. The highest radical scavenging effect was observed in seeds and leaves extract with IC50, 20 and 25 μg /ml respectively. Among the various extracts in the study, the seeds and leave was found to improve the antioxidant property followed and rhizomes. In the present study, results has capability to scavenge the free radicals and protect against oxidative stress causing diseases and nelumbo nucifera parts could be used as ingredients in food formulation. In future it may serve as a good pharmacotherapeutic agent which could be explored to provide affordable medicines to masses.

KEY WORDS: Antioxidant, Free radical scavenging, Flavonoids, Phenols, 1, 1, diphenly-2-picrylhydrazyl, Nelumbo nucifera.

INTRODUCTION Free radicals are more with one or more unpaired Therefore, medicinal plants are believed to be an electrons which makes them highly reactive1. important source of new chemical substances with Subsequently, the targeted molecule becomes a free potential therapeutic effects and more than 80% of radical itself and initiates a cascade of events that can population of developing countries is dependent on ultimately lead to cellular damage2. However, at traditional folk medicine therapies for treating their physiological levels, free radicals also help preserve ailments. Most of the plants have protective hemostatic by acting as signal transducers3. Free biochemical functions of naturally occurring radicals are the new “buzz word” in pathophysiology antioxidants in the cells5. Several pharmaceutically today. They have a special affinity for lipids, proteins active constituents of plants have been assessed to and nucleic acids (DNA). Most molecules have all defend against oxidative damage by inhibiting or their electrons in pairs and are therefore not free quenching free radicals and reactive oxygen species. radicals. Molecules are held together by pairs of Naturally occurring antioxidants in plant cells include electrons forming stable bonds, but breaking a bond peptide defence mechanisms which are catalases, forms highly reactive free radicals4. peroxidases, superoxide dismutases, glutathione and proteins, phenolic defence compounds which include

196 www.ijapbc.com IJAPBC – Vol. 5(2), Apr - Jun, 2016 ISSN: 2277 - 4688 vitamin E, flavonoids, phenolic acids and other ml of 1:10 g/ ml) in methanol were separately mixed phenols, nitrogen compounds (alkaloids, amino acids with 1.5 ml of methanol, 0.1 ml of 10% aluminum and amines) and cartenoids and chlorophyll chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml derivatives6 . of distilled water. It remained at room temperature Lotus (Nelumbo nucifera) is a perennial aquatic plant for 30 min; the absorbance of the reaction mixture with yellow flowers. It is utilized as a dietary staple was measured at 415 nm with a UV-Visible and also for a variety of medicinal purposes in Spectrophotometer. The calibration curve was Eastern Asia, particularly in China. This plant is prepared by preparing quercetin solutions at belonging to the Family Nelumbonaceae. All parts of concentrations 12.5 to 100 g ml-1 in methanol. N. nucifera are used for various medicinal purposes in oriental medicine7. This plant is very effective to DPPH radical scavenging activity: potential antioxidant activity8, antipyretic9, The ability of the methanolic extracts to scavenge Antiplatelet activity10 and Hypoglycemic activity11. free radicals was determined against a very stable Therefore, an attempt has been made investigate the free radical DPPH (2, 2-diphenyl-1-picrylhydrazyl) antioxidant properties in different parts of N. determined spectrometrically 14. Aliquots of the nucifera. sample extract at different concentrations 0.02, 0.05, 0.1, 0.15, 0.25 mg/ml were added to 1 mM MATERIALS AND METHODS methanolic solutions of DPPH. Each mixture was Sample collection and extract preparation: vortexed vigorously and left for 30 min at room Fresh leaves, flower, seed rhizomes of Nelumbo temperature in the dark. The absorbance was nucifera plant used in the present study were measured at 517 nm and activity was expressed as collected from village Ichoi, Milkipur, Faizabad, percentage DPPH scavenging relative to control . The plant parts were separated from using the following equation: weeds, washed and rinsed with distilled water. They DPPH scavenging activity (%) = (Absorbance of were sun-dried for 5 days, ground in a mortar with a control − Absorbance of sample)/ Absorbance of pestle into coarse powder and packed in an air-tight control × 100 plastic container for further analysis. Methanol IC 50 Value was also calculated. extracts were prepared by soaking 100g each of the dry powdered plant parts in 1 litre of methanol at Statistical Analysis: room temperature for 48 h. The extracts were filtered The results obtained were expressed as mean ±SD first through a Whatmann filter paper No. 42 (125 and analysis of variance of three determinations and mm) and then through cotton wool. The extracts were also statistically analyzed to ascertain its significance. concentrated using a rotary evaporator with the hot The significance was estimated at (p≤0.05level). water bath set at 40°C. The percentage yield of extracts ranged from 5 - 20% (w/w). RESULT AND DISCUSSION The level of total phenol and total flavonoid contents Determination of total phenolic content: are shown in table .1 and Figure. 1 & 2 Total Total phenols were determined by Folin Ciocalteu phenolic content in aqueous extracts were determined reagent 12. A dilute extract of each seed (0.5 ml of according to the Folin-Ciocalteu method and 1:10 g ml-1) or gallic acid (standard phenolic expressed as mg of gallic acid equivalent (GAE) per compound) was mixed with Folin Ciocalteu reagent hundred gram of extract (mg GAE/100g) on dry (5 ml, 1:10 diluted with distilled water) and 4 ml of weight basis. Total phenol content of plant parts aqueous sodium carbonate (1 M). The mixtures were ranged from 220.57±0.26 - 270.90±0.33. Seeds kept at dark ambient condition for 15 min and the aqueous extract showed highest total phenolic total phenols were determined by spectrophotometry content (270.90±0.33) followed by leave at 765 nm. The standard curve was prepared using 0, (252.34±0.33), rhizomes (241.01±0.36) and flowers 50, 100, 150, 200, 250 mg/ L solutions of Gallic acid (220.57±0.26). There were significant differences in in methanol: water (50:50, v/v). Total phenol values the total phenol content between all the parts (leave, are expressed in terms of Gallic acid equivalent flowers, rhizomes and seeds) of N. nucifera at p≤0.05 (mg/g of dry mass), which is a common reference level. Phenolic compounds are a class of antioxidant compound. agents which act as free radical terminator15. It is also an important plant constituent due to their scavenging Determination of total flavonoids content: property and their hydroxyl groups which contributes Aluminum chloride colorimetric method was used for directly to antioxidative action16. Results showed that flavonoids determination13. Each seed extracts (0.5 whole plant contain good amount of phenolic content

197 www.ijapbc.com IJAPBC – Vol. 5(2), Apr - Jun, 2016 ISSN: 2277 - 4688 in which seeds aqueous extract contain highest value flower aqueous extract of Halimium halimifolium . while flowers had the lowest when compared to other This result was also in line with the study of plant parts extract which was comparable with the Bhardwaj et al 26 and Sharma et al27 that Boerhaavia result observed by Sagwan et al 17 in Pongamia diffusa L. and Plumbago zeylanica leave contain pinnata L. Likewise, Sadiq et al18 have also highest amount of total flavonoids content when reiterated that seed was found to be highest phenolic compared to other parts. content as compared to other plant parts of Eruca The DPPH radical scavenging activity of the aqueous sativa. Phenolic compounds are important extracts were evaluated by using the parameter IC50 constituents of plants, vegetables, and fruits because which means the concentration of antioxidant of their scavenging ability due to their redox required for 50% scavenging of DPPH radicals in the properties and hydroxyl groups which allow them to particular time period. In vitro activities of all the act as reducing agents, hydrogen donors and singlet plant part extracts were measured with the standard oxygen quenchers19. The phenolic compounds may antioxidant (Ascorbic acid). The N. nucifera aqueous contribute directly to the antioxidative action. They extract had dose dependent action, i.e. the scavenging are easily degraded (e.g., by oxidation or hydrolysis) activity of DPPH increased proportionate to the and may also form covalent products and non- increase in concentration of the extracts. Here, The covalent complexes with various types of molecules. DPPH free radical scavenging activities of different In addition, it was reported that phenolic compounds plant parts leaves, flower, rhizome and seed extracts were associated with antioxidant activity and play an are shown in (Table 2 and Figure 3). The antioxidant 20 important role in stabilizing lipid peroxidation . activity of Nelumbo nucifera plant parts with IC50 According to present study the appropriate amount of (Inhibitory Concentration) values ranged from (20-60 total phenol content in seeds, leave, rhizomes and µg/ml). Seed extract exhibited highest antioxidant flowers of N. nucifera can explain its high free activity with IC50 Values (20µg/ml) followed by radical scavenging activity. However its phenol rich leave (25µg/ml), rhizomes (43µg/ml) and flowers extract was reported to be effective for the control of (60µg/ml). Leave showed lowest antioxidant activity insulin resistance. with IC50 value (60µg/ml). The DPPH is a stable Total flavonoids content in aqueous extracts of nitrogen centered free radical with absorption Nelumbo nucifera plant parts were determined by maximum band around 515 to 528 nm. In this assay, using Aluminium Chloride method and the amount of the antioxidants reduce the DPPH radial (purple flavonoids was expressed as mg quercetin equivalent color) to a yellow coloured compound, (QE) per hundred gram of extract (QE/100g) dry diphenylpicrylhydrazine. The extent of color change weight. The total flavonoid content in the plant parts depends on hydrogen donating ability of the ranged from 140.75±0.58-245.22±1.51. Leave antioxidants 28. This method illustrates that there was aqueous extract had the highest total flavonoid a decrease in the concentration of DPPH radicals due content (245.22±1.51) followed by flowers to the scavenging ability of the soluble constituents in (193.54±1.94), seeds (161.84±0.32) and rhizomes the aqueous extracts of Nelumbo nucifera leave, (140.75±0.58). Data showed that there were flowers, rhizomes, seeds with their standard ascorbic significant differences among all the parts of acid as a reference compound. DPPH assay is based Nelumbo nucifera at p≤0.05. Flavonoids are the most on the ability of DPPH which is a stable free radical common group of polyphenolic compounds in the which decolorize in the presence of antioxidants. It is human diet and are found ubiquitously in plants, a direct and reliable technique for determining radical characterized by a benzo-γ-pyrone structure21. They scavenging action. The aqueous nucifera seeds and are one class of secondary plant metabolites that are leave extract showed strong antioxidant activity. It also known as Vitamin P which are mostly used in was noticed that seed and leave extract of N. nucifera plants to produce yellow and other pigments which showed strong hydrogen donating abilities to act as play a vital role in the colors of plants. Moreover, an effective antioxidant. Contrary to the present Flavonoids contents are readily ingested by humans study, Artun, et al 29 that the young Carica papaya and they seem to demonstrate important anti- leaves aqueous methanol extract exhibited a higher inflammatory, anti-allergic and anti-cancer properties DPPH scavenging effect than seed aqueous methanol 22. The mechanisms of action of flavonoids are extract. This result was also in line with the study of through scavenging or chelating process 23. It also Sadiq et al30 that maximum DPPH scavenging plays an important role in stabilizing lipid oxidation activity was found in methanolic Eruca sativa seeds and is associated with antioxidant activity 24 . The extracts than leaf, stem and flower extracts of plants. study conducted by Rebaya et al25 that the total flavonoids content in leave extract was higher than

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Table 1 The antioxidant activity of different plant parts of Nelumbo nucifera (G.) on dry weight basis. Total Phenolic Total Flavonoids Plant Part Content (mg QE/100g) (Aqueous Extract) (mg GAE/100g)

a Leave 252.34 ± 0.33 245.22 ± 1.51a

b Flowers 220.57 ± 0.26 193.54 ± 1.94b

c Rhizomes 241.01 ± 0.36 140.75 ± 0.58 c Seeds 270.90 ± 0.33d 161.84 ± 0.32d Values are Mean ± Standard deviation of triplicate determinations. Values sharing same uppercase letter between rows are not significantly different at p < 0.05.

Table 2 The DPPH free radical scavenging activity of different plant parts of Nelumbo nucifera (Gaertn.) on dry weight basis. Concentration Ascorbic Acid Leave Flowers Rhizomes seeds (µg/ml)

20 87.44 45.01 36.80 43.60 48.40 40 88.35 66.80 42.20 47.20 68.50 60 89.72 67.20 48.90 57.50 71.50 80 90.60 69.00 49.10 58.10 72.88

100 91.09 70.40 49.30 62.01 72.90 120 92.46 71.01 50.10 62.70 74.66 140 93.80 72.0 51.20 63.10 75.33 160 94.20 72.70 57.40 63.40 78.66 180 95.20 74.20 58.10 64.01 79.45 200 97.03 77.11 62.40 64.70 79.50

IC50 Values(µg/ml) - 25.00 60.00 43.00 20.00

Figure 1 and 2 The Total phenol and Total flavonoid content of different plant parts of Nelumbo nucifera (G.) on dry weight basis.

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Figure 3 The DPPH activity of different plant parts of Nelumbo nucifera (G.) on dry weight basis.

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