International Journal of Drug Development and Research

Keywords

Andrographis paniculata, Antioxidant Activity, DPPH, Total Reducing Power, -Carotene Bleaching Assay, Hydrogen Peroxide Radical Scavenging Assay, Phytochemistry

Introduction

Andrographis paniculata, known as Kalmeghin the Indian system of Ayurveda, is a widely used home remedy for the common cold. The plant is native to India and Sri Lanka and belongs to the family Acanthaceae. The effectiveness of this herb has been widely recognized and its demand is on the rise. [1,2]The major active compound in the plant is Andrographolide. [3] This is often called a wondrous compound which possesses antiinflammatory, anti-cancer, antiviral and even antimalarial properties. [4-8]The present study is aimed at understanding the plant’s role and value as an anti-oxidant. This study is a continuation of our earlier work. [9]Anti-oxidants are gaining importance due to the increase in the reactive oxygen species generated in our cells due to modern lifestyle. [10]Along with being an antidiabetic drug, if the extracts are found to be

Materials and methods

Chemicals and reagents: All the chemicals, reagents, kits and solvents used in this study were of analytical grade and procured locally.

Plant material and extract powder: Fresh leaves of the plant were collected from Gudiyattham District, Tamil Nadu, India. Plant material was shade dried and ground into a powder. Sequencing extracts were prepared by successive maceration of the powder (10g) at room temperature with 100ml of various solvents on an orbital shaker for 2 days. The solvents used were n- Hexane, Ethyl Acetate, Chloroform and Methanol respectively. The final extracts obtained were filtered and the filtrates were subjected to lyophilisation (Shimadzu Analytical (India) Pvt Ltd, Mumbai, India) to obtain powdered extracts which were used for the following assays.

Antioxidant Activity: 1ml of four concentrations (25, 50, 75 and 100mg/ml) of each extract of the plant dissolved in methanol was added to 1ml of DPPH (0.16mM in methanol). 1ml DPPH in methanol with 1ml pure methanol was used as a control. The reaction mixtures were mixed and incubated in the dark at room temperature for 30minutes. Their absorbance was then read at 517nm. [11]The DPPH scavenging ability of plant extracts was calculated using the following equation:

Total Phenolic Content: 1ml aliquots of extracts (100 mg/l) were added to a volumetric flask containing 9 ml of water. 1 ml of Folin-Ciocalteu’s reagent was added to the mixture and vortexed. After 5 min, 10 ml of 7% sodium carbonate was added to the mixture and incubated for 90 min at 25°C. The absorbance against reagent blank was determined at 750 nm. A reagent blank was prepared and the amount of phenolic compound in the extract was determined from the standard curve. The total phenolic content of the plant was then calculated based on the equation below and expressed as mg gallic acid equivalent (GAE)/g fresh weight.[12]

Where C = total content of phenolic compound in gallic acid equivalent (GAE)/g, c = the concentration of gallic acid established from the calibration curve (μg/ml), V= volume of extract (ml), and m = weight of the crude plant extract (g).

Hydrogen Peroxide Radical Scavenging Activity:

The hydrogen peroxide (H2O2) radical scavenging activity was determined by the Ruch et al method.1 ml of extract at four concentrations 25, 50, 75,100 mg/ml was taken in 4 test tubes. 0.6 ml of hydrogen peroxide (40mM) was added with phosphate buffer. The samples were incubated at 300C for 10 minutes. After incubation, absorbance was determined at 230 nm against phosphate buffer as a blank. [13]This was done in replicate. The percentage H2O2scavenged was calculated using the formula:

Where A1= absorbance of the H2O2 without extract.A2= absorbance of the sample with extract.

- carotene bleaching activity: 3ml of -carotene solution (5mg of carotene/50ml of chloroform) was added to 40mg of linoleic acid and 400mg of Tween 20. Then, the mixture was mixed well and dried. To the dried mixture, 100ml of distilled water was added in order to form -carotene linoleic acid emulsion. 1ml of solvent extract was taken in 4 test tubes and 1.5ml of emulsion was added. Then, the mixture was incubated in water bath at 50ºC for 60 minutes. This was done in replicate. Finally, the absorbance was read at 470nm.[14]This was done in triplicates. Antioxidant activity (AA %) of A. paniculata extract was calculated by using the formula:

Where DRC= Degradation rate of the control, DRS= degradation rate of the sample [(a/b)/60] a=initial absorbance of the sample. b=absorbance after 60 min of incubation.

Reducing power: The ability of extracts to reduce iron (III) was determined. Four concentrations (25, 50, 75 and 100 mg/ml) of A. paniculata solvent extracts were mixed with 2.5ml of phosphate buffer and 2.5 ml of potassium ferricyanide. Then, the mixture was incubated at 500C for 30 minutes. 2.5 ml of trichloroacetic acid was added and the mixture was centrifuged at 3000 rpm for 10 minutes. 2.5 ml of supernatant solution was taken and mixed with 2.5 ml of distilled water and 0.5 ml of ferric chloride. L-ascorbic acid was used as standard. Finally, the absorbance of all samples was measured at 700 nm. This was done in triplicates.[15]

Statistical Analysis

Statistical analyses were carried out using Microsoft Excel 2010. The standard errors in all the analyses were found to be less than or equal to 5% of the mean values.

Results and Discussion

Total Phenolic Content: The results for the total phenolic content are given in Table 1. As can be inferred from the table, the methanolic extract had the highest total phenolic content. This further implies that the methanolic extract contains the maximum number of tannins and flavonoids, since these are phenolic compounds.

Antioxidant Activity: The results for the antioxidant activity are given in Table 2. The antioxidant activity for the methanolic extract was found to be the highest. In all cases except the methanolic extract, the antioxidant activity decreased till a concentration of 75 mg/ml, after which, a sharp increase was seen at 100 mg/ml. The antioxidant activity was found to be highest at a concentration of 100 mg/ml. The antioxidant activity increased with concentration incase of the methanolic extract. These results have been graphically represented in Figure 1. The higher antioxidant activity of methanolic extract can be attributed to its higher phenolic compound content. This is because flavonoids, the primary compounds responsible for antioxidant activity of plants are phenolic compounds. [16]

Hydrogen Peroxide Radical Scavenging Activity: The results of the H2O2 radical scavenging activity are given in Table 3. The scavenging activity of the methanolic extract was found to be the highest, followed by chloroformic and ethyl acetate, respectively. This has special significance due to the fact that H2O2 is a potent oxidant. It has a strong tendency to oxidize DNA in the cells, causing mutations. Reactive agents of H2O2 may sometimes even cause cell death due to the generation of free hydroxyl radicals inside the cell. The H2O2 radical scavenging activity is attributed to primary antioxidants, which in case of plants would be phenolics. [17] Thus, the higher scavenging activity of the methanolic extract is most likely due to this. The results are graphically depicted in Figure 2.

on the hydroperoxides. Again, the methanolic extract showed the lowest amount of carotene, and thus, the highest concentration of antioxidants. This can be correlated to the results of the antioxidant activity and total phenolic content. The results are graphically depicted in Figure 3.carotene bleaching activity: The results of the carotene bleaching activity are given in Table 4. carotene bleaching activity is again an indirect measure of the antioxidant activity of a plant extract. carotene gets bleached by hydroperoxides like H2O2. [18] In the presence of antioxidants, this bleaching is greatly reduced due to the neutralizing effect of the antioxidants on the hydroperoxides. Again, the methanolic extract showed the lowest amount of – carotene, and thus, the highest concentration of antioxidants. This can be correlated to the results of the antioxidant activity and total phenolic content. The results are graphically depicted in Figure 3.

Reducing Power: The ferric reducing assay measures the ability of an antioxidant to reduce a reactive oxygen species against that species’ oxidative power.[19]This is important to make the reactive oxygen species more stable and unreactive. The results are tabulated in Table 5 and graphically represented in Figure 4. As can be observed from the graph, the methanolic extract showed a reducing power nearly equal to that of L–Ascorbic Acid. The chloroformic extract showed a very good reducing power at lower concentrations. However, at higher concentrations, its reducing power was lesser than that of the methanolic extracts.

Conclusion

Based on the above experiments, we can conclude that methanolic extracts of A. paniculata are the most effective as antioxidants. These could find potential application in today’s urban lifestyle which increases our exposure to various harmful oxidants. These findings indicate that in addition to its anti-diabetic effect, A. paniculata has a potent antioxidant activity.

Acknowledgements

We would like to thank the management of VIT University for supporting this study.

Conflict of Interest

The authors would like to declare that they have no conflict of interest.

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4

References

1. 1) Chauhan JS, Tomar YK, Singh NI, Ali S, BadoniA,Debarati, Rana A (2009). Assessment of complete substratum of Andrographispaniculatastandard seed germination testing. Journal of American Science; 5: 70-5.
2. 2) Bhattacharya S, Sunil P, Arti J, Shikha S (2012). Studies on seed germination and seedling growth in kalmegh (AndrographispaniculataWall.Ex. Nees) under abiotic stress conditions.International Journal of Environmental Scienceand Technology; 1(3):197-204.
3. 3) Mishra K, Dash AP,Dey N (2011). Andrographolide: A Novel Antimalarial Diterpene Lactone Compound from Andrographispaniculataand Its Interaction with Curcumin and Artesunate. Journal of Tropical Medicine; doi:10.1155/2011/579518.
4. 4) Coon JT, Ernst E (2004). Andrographispaniculatain the treatment of upper respiratory tract infections: a systematic review of safety and efficacy.PlantaMedica;70(4): 293-8.
5. 5) Habtemariam S (1998). Andrographolide inhibits the tumour necrosis factor-_-induced upregulation of ICAM-1 expression and endothelial-monocyte adhesion.PhytotherapyResearch; 12(1): 37–40.
6. 6) Rahman NNNA, Furuta T, Kojima S, TakaneK,Mohd MA (1999). Antimalarial activity of extracts of Malaysian medicinal plants. Journal of Ethnopharmacology; 64(3): 249–54.
7. 7) Ying H, Bu LIM, Ji X, Liu CY, Wang ZH (2005). Modulation of multidrug resistance by andrographolid in a HCT-8/5-FU multidrugresistantcolorectal cancer cell line.ChineseJournal of Digestive Diseases; 6(2): 82-6.
8. 8) Mishra K, Dash AP, Swain BK, Dey N (2009). Antimalarial activities of AndrographispaniculataandHedyotiscorymbosaextracts and their combination with curcumin.Malaria Journal; 8(1).
9. 9) Arunachalam S, SundaramoorthyS, Rastogi A, Sathiavelu M (2013). InVitroanti-diabetic activity of aqueous extract of themedicinal plants Nigella sativa, Eugenia jambolana,AndrographispaniculataandGymnemasylvestre. International Journal of Drug Development & Research; 5(2): 323-8.
10. 10) Mascio PD, Murphy ME, Sies H (1991). Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. TheAmerican Journal of Clinical Nutrition; 53: 194- 200.
11. 11) Farhan H, Ramma H, Hijazi A, HamadH,Badran B (2012). Phytochemical screening and extraction of polyphenol from stems and leaves of a Lebanese EuphorbiamacroladaschyzocerasBoiss. Annals of Biological Research; 3(1):149-56.
12. 12) Anyasor GN, Ogunwenmo KO, OyelanaOA, Akpofunure BE (2010). Phytochemical constituents and antioxidant activities of aqueous and methanol stem extracts of CostusaferKerGawl. (Costaceae). African Journal of Biotechnology;9(31): 4880-4.
13. 13) Wettasinghe M, Shahidi F (1997). Antioxidant activity of preformed cooked cured-meat pigment in a b carotene/linoleate model system. Food Chemistry; 58: 203-7.
14. 14) Ruch RJ, Cheng SJ, Klaunig JE (1989). Prevention of cytotoxicity and inhibition of ntercellularcommunication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis; 10: 1003-8.
15. 15) Yildirim A, Mavi A, Kara AA (2001). Determination of antioxidant and antimicrobial activities of RumexcrispusL. extracts. Journal of Agricultural and Food Chemistry; 49: 4083–9.
16. 16) Pietta PG (2000). Flavonoids as Antioxidants.Journal of Natural Products; 63(7): 1035-42.
17. 17) Dorman HJD, Kosar M, Kahlos K, Holm Y, Hiltunen R (2003). Antioxidant properties and composition of aqueous extracts from Menthaspecies, hybrids, varieties, and cultivars.Journalof Agricultural and Food Chemistry; 51: 4563-9.
18. 18) KhareDP, Kumar B, Hussain A,Verma S, Mishra M (2012). Evaluation of antioxidant activity of large
19. cardamom(leaves of Amomumsubulatum). International Journal of Drug Development &Research; 4(1): 175-9.
20. 19) Saklani S, Mishra AP, RawatA, Chandra S (2011). Free Radical Scavenging (DPPH) and Ferric
21. Reducing Ability (FRAP) ofAphanamixispolystachya (Wall) Parker. International Journal of Drug Development& Research; 3(4): 271-4.