INTRODUCTION:

Polyalthia longifolia Thw., belonging to the Family Annonaceae, is native to Sri Lanka and is grown in gardens throughout the warmer parts of India. It is known as Mast tree, False Asoka tree, False Devadaru and Cemetry tree in English. It is called Devadaari in Ayurveda. The stem bark contains clerodanediterpenes, polyalthialdoic acid and kolavenic acid. The stem and its bark also contain the cytotoxic aporphine alkaloid, liriodenine, besides nor-oliveroline and oliveroline-beta-N-oxide. Azafluorene alkaloids are also present in the bark and leaves ^[1].Various parts of P. longifolia have shown to possess antimicrobial^[2-4], antioxidant^[4,5], antitumor^[5], antiulcer^[6], antileishmanial^[7], hypotensive^[8], anti-hyperglycemic^[9], anti-inflammatory^[10,11], hepatoprotective^[10], anticataracto- genesis activity^[12] and others. The juice extracted from the fresh stem bark is taken orally to treat indigestion in Uthiramerur taluk, Kancheepuram District, Tamil Nadu, India^[13]. Literatures on biological activities, in particular antioxidant activity of ripe pericarp extract of P. longifolia are very scanty.

Hence, the present study was carried out to screen antioxidant potential of pericarp extracts of ripe fruit of P. longifolia.

MATERIALS AND METHODS:

Collection and Extraction:

The ripe fruits of P. longifolia were collected during May 2012 from the college campus. The fruits were washed well to remove adhering matter, pericarp was separated, dried under shade and powdered using blender. 100 gram of powdered pericarp material was subjected to soxhlet extraction and extracted successively with ethyl acetate, chloroform and ethanol. The extracts were filtered through 4-fold muslin cloth followed by Whatman No. 1 and concentrated in vacuum under reduced pressure and dried in the desiccator^[14].

Antioxidant activity of solvent extracts:

DPPH free radical scavenging assay:

The free radical scavenging efficacy of different concentrations of solvent extracts and ascorbic acid (standard) was evaluated by mixing equal volume (2ml) of DPPH solution (0.002% in methanol) and different concentrations of extracts and standard (5-100µg/ml of methanol) in clean and labelled test tubes. The tubes were incubated in dark at room temperature for 30 minutes and the absorbance of the reaction mixtures was read at 517nm. The absorbance of DPPH control (without extract/standard) was also noted. The scavenging activity (%) of each concentration of extract and standard was calculated using the formula: A₀-A₁/A₀ x 100 where A₀ is absorbance of control and A₁ is absorbance of test (extract/standard). The concentration of extract required to inhibit 50% of free radicals (Inhibitory concentration, IC₅₀) was calculated for each extract ^[15].

Ferric reducing assay:

The reducing power of solvent extracts and tannic acid (standard) was determined by employing the method of Kekuda et al.^[15]. Briefly, different concentrations of solvent extracts and tannic acid (5-100µg/ml of methanol) in 1ml of methanol were mixed with 2.5ml of phosphate buffer (pH 6.6), 2.5ml of potassium ferricyanide (1%) and incubated at 50oC for 20 minutes. Afterwards, 2.5ml of trichloroacetic acid (10%) was added to each tube followed by addition of 0.5ml of ferric chloride (0.1%). The absorbance of the reaction mixtures was read at 700nm after 10 minutes. An increase in the absorbance with increase in concentration of extracts/standard indicated increasing reducing power.

Total phenolic content of solvent extracts:

Total phenolic content in the solvent extracts was estimated by Folin-Ciocalteau method (Kekuda et al., 2011). Here, a dilute concentration of extract (0.5 mL) was mixed with 0.5 ml of 1:1 diluted Folin-Ciocalteu reagent and 4 ml of sodium carbonate (1 M). The mixtures were allowed to stand for 15 minutes and the absorbance was determined colorimetrically at 765nm. A standard curve was plotted using different concentrations of Gallic acid (standard, 0-1000 μg/ml of methanol). The concentration of total phenolic compounds was determined as μg Gallic acid equivalents (GAE) from the graph^[15].

Statistical Analysis:

All data were expressed as mean ± Standard deviation of the number of experiments (n 3). Past software version 1.92 was used. The IC₅₀ values were calculated by Origin 6.0 software.

RESULTS:

DPPH free radical scavenging activity of solvent extracts:

Table 1 shows DPPH free radical scavenging activity of different concentrations of solvent extracts and ascorbic acid. The scavenging efficacy was dose dependent and was high in ethanol extract followed by ethyl acetate and chloroform extracts. The IC₅₀ value for ethyl acetate extract, chloroform extract and ethanol extract was found to be 52.22±0.05, 61.82±0.01 and 06.85±0.05µg/ml respectively. Scavenging efficacy of ascorbic acid was higher than that of solvent extracts.

Ferric reducing activity of solvent extracts:

The reduction of Fe³⁺ to Fe²⁺ was investigated in the presence of extracts and standard (tannic acid) in order to examine the reducing power of extract and the result is shown in Table 2. The absorbance of the reaction mixtures (at 700nm) was found to increase with the concentration of extracts and standard and is indicating reducing power. Among extracts, the reducing activity was found higher in ethanol extract and is followed by ethyl acetate and chloroform extracts.

Total phenolic content of solvent extracts:

The total phenolic content of solvent extracts was estimated by the Folin-Ciocalteu method and expressed as GAE. The phenolic content of ethyl acetate, chloroform and ethanol extracts was found to be 95±0.09, 85±0.01 and 224±0.10 µg GAE/mg extract respectively.

Table 1: DPPH free radical scavenging activity of solvent extracts and ascorbic acid

Concentration (µg/ml)	Radical scavenging activity (%)
Concentration (µg/ml)	Ethyl acetate extract	Chloroform extract	Ethanol extract	Ascorbic acid
2.5	43.11±0.09	39.69±0.03	44.13±0.05	51.98±0.03
5	47.25±0.10	42.85±0.10	48.35±0.09	65.37±0.50
10	48.35±0.05	45.05±0.09	56.54±0.05	69.63±0.09
25	49.45±0.03	47.25±0.10	62.74±0.10	81.56±0.09
50	50.54±0.09	48.35±0.05	68.94±0.09	86.36±0.03
100	52.74±0.05	52.74±0.09	78.35±0.03	91.56±0.03

Table 2: Ferric reducing activity of solvent extracts and tannic acid

Concentration (µg/ml)	Absorbance at 700nm
Concentration (µg/ml)	Ethyl acetate extract	Chloroform extract	Ethanol extract	Tannic acid
2.5	0.29±0.09	0.28±0.05	0.32±0.03	0.68±0.10
5	0.32±0.05	0.31±0.05	0.37±0.05	0.81±0.10
10	0.38±0.09	0.38±0.09	0.42±0.03	0.83±0.05
25	0.43±0.03	0.42±0.03	0.47±0.05	0.91±0.10
50	0.55±0.03	0.47±0.03	0.57±0.03	1.04±0.03
100	0.69±0.05	0.66±0.05	0.79±0.05	1.11±0.03

DISCUSSION:

In a normal healthy individual, a well maintained balance exists between free radical production and antioxidant defense mechanisms. In diseased conditions, this balance shift towards production of more free radicals or a deficit in antioxidant defense which leads to oxidative stress. Oxidative stress is involved in the generation of reactive oxygen species (such as superoxide anion, hydroxyl radical, nitric oxide and others) which have been implicated in the aetiology of over one hundred human diseases such as cardiovascular diseases, Alzheimer disease, Parkinson’s disease, cancer etc. Antioxidants are a group of substances which, when present at low concentrations, in relation to oxidizable substrates, significantly inhibit or delay oxidative processes. Endogenous antioxidants such as ascorbic acid, uric acid, vitamin E and others present in extracellular fluids act as primary defense system that protects against oxidative damage. In pathophysiological conditions, however, there is an extra requirement for exogenous antioxidants from food and medicinal plants. Hence, consumption of natural compounds, with antioxidant efficacy, in the form of fruits, vegetables or medicinal plants has been correlated with a lower incidence of many human ailments associated with oxidative stress^[16-19].

Many methods have been developed for measuring the antioxidant capacity in vitro. One of the most widely used methods, which is based on quenching of stable free radicals, is DPPH assay. This assay uses commercially available and stable free radical 2,2-diphenyl-1-picrylhydrazil which is soluble in methanol. DPPH is relatively stable nitrogen centered free radical that easily accepts an electron or hydrogen radical to become a stable diamagnetic molecule. In its radical form DPPH shows an absorption peak at 517nm, which disappears on reduction by an antioxidant compound. DPPH radicals react with suitable reducing agents as a result of which the electrons become paired off forming the corresponding hydrazine. The solution therefore loses colour stoichometrically depending on the number of electrons taken up^[18,20,21]. In this study, the bleaching of the DPPH solution increased with increasing amount of extract in a given volume of solution. The concentration of extract required to scavenge 50% of DPPH, IC₅₀, was found to be least for ethanol extract followed by ethyl acetate and chloroform extracts. The lower the IC₅₀ the better it is able to scavenge the radicals^[22]. The reducing potential of a compound may serve as a significant indicator of its potential antioxidant activity. The presence of reductants in the antioxidant samples causes the reduction of the Fe³⁺/ferricyanide complex to the ferrous form. Therefore, Fe²⁺ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm ^[23,24]. In this study, reducing potential of ethyl acetate and chloroform extracts was lesser when compared to ethanol extract.

The use of herbal products for improving health has increased in recent years. Phenols including flavonoids are widely distributed in vegetables, fruits and medicinal plants. These phytoconstituents possess different antioxidant capacities, which can be ascribed to a broad range of pharmacological activity. Phenolic contents of plants have been extensively studied for their contribution to antioxidant activity of plants. Phenolic compounds are considered as high level antioxidants due to their ability to scavenge free radicals and active oxygen species such as singlet oxygen, superoxide free radicals and hydroxyl radicals. There are many reports which correlate the total phenolic content of plants and their antioxidant activity^[25-28]. In our study also, the ethanol extract was found to contain high phenolic content. The antioxidant activity of extracts, as observed in this study is directly correlated with the phenolic content of extract.

CONCLUSION:

From the study, it was found that the ripe pericarp of P. longifolia has promising antioxidant activity. The phenolic contents in the pericarp could be responsible for the observed antioxidant activity. To the best of our knowledge, this is the first report on the antioxidant activity of pericarp of P. longifolia.

ACKNOWLEDGEMENTS:

The authors express thanks to HOD, Dept. of Microbiology and Principal, SRNMN College of Applied Sciences and NES, Shivamogga for providing all the facilities to conduct work.

REFERENCES:

1. Khare CP. Indian Medicinal Plants: An Illustrated Dictionary. Springer Verlag, Berlin, 2007, 341.

2. Murthy MM, Subramanyam M, Bindu HM and Annapurna J. Antimicrobial activity of clerodanediterpenoids from Polyalthia longifolia seeds. Fitoterapia76(3-4); 2005: 336-339

3. Faizi S, Khan RA, Mughal NR, Malik MS, Sajjadi KE and Ahmad A. Antimicrobial activity of various parts of Polyalthia longifolia var. pendula: Isolation of active principles from the leaves and the berries. Phytotherapy Research 22(7); 2008: 907-912

4. Bose S, Byahatti VV, D’Souza M and Bose A. Antioxidant and antimicrobial activities of isolated constituents from the bark of Polyalthia longifolia. International Journal of Green Pharmacy 4(2); 2010: 93-97

5. Manjula SN, Kenganora M, Parihar VK, Kumar S, Nayak PG, Kumar N, Pai RKS and Rao CM. Antitumor and antioxidant activity of Polyalthia longifolia stem bark ethanol extract. Pharm Biol 48(6); 2010: 690-696

6. Malairajan P, Gopalakrishnan G, Narasimhan S and Veni KJK. Evaluation of anti-ulcer activity of Polyalthia longifolia (Sonn.)Thwaites in experimental animals. Indian Journal of Pharmacology 40(3); 2008: 126-128

7. Pal D, Bhattacharya S, Baidya P, De BK, Pandey JN and Biswas M. Antileishmanial activity of Polyalthia longifolia leaf extract on the in vitro growth of Leishmaniadonovani promastigotes. Global Journal of Pharmacology 5(2); 2011: 97-100

8. Saleem R, Ahmed M, Ahmed SI, Azeem M, Khan RA, Rasool N, Saleem H, Noor F and Faizi S. Hypotensive activity and toxicology of constituents from root bark of Polyalthia longifolia var. pendula. Phytotherapy Research 19(10); 2005: 881-884

9. Ghosh G, Kar DM, Subudhi BB and Mishra SK. Anti-hyperglycemic and antioxidant activity of stem bark of Polyalthia longifolia var. angustifolia. Der Pharmacia Lettre 2(2); 2010: 206-216

10. Tanna A, Nair R and Chanda S. Assessment of anti-inflammatory and hepatoprotective potency of Polyalthia longifolia var. pendula leaf in Wister albino rats. Journal of Natural Medicines 63(1); 2009: 80-85

11. Sharma RK, Mandal S, Rajani GP, Gupta N, Srivastava DP. Antiulcer and antiinflammatory activity of fresh leave extracts of Polyalthia longifolia in Rats. International Journal of Drug Development and Research 3(1); 2011: 351-359

12. Sivashanmugam AT and Chatterjee TK. Anticataractogenesis activity of Polyalthia longifolia leaves extracts against glucose-induced cataractogenesis using goat lenses in vitro. European Journal of Experimental Biology 2(1); 2012: 105-113

13. Sugumaran M, Bharathi V and Lakshmi HRM. Ethnomedicinal Plants for Indigestion in Uthiramerur Taluk, Kancheepuram District, Tamil Nadu, India. Journal of Chemical and Pharmaceutical Research 2(6); 2010: 463-470

14. Kekuda TRP, Raghavendra HL, Swathi D, Venugopal TM and Vinayaka KS. Antifungal and Cytotoxic Activity of Everniastrum cirrhatum (Fr.) Hale. Chiang Mai Journal of Science. 39(1); 2012: 76-83

15. Kekuda TRP, Vinayaka KS, Swathi D, Suchitha Y, Venugopal TM and Mallikarjun N. Mineral Composition, Total Phenol Content and Antioxidant Activity of a Macrolichen Everniastrum cirrhatum (Fr.) Hale (Parmeliaceae). E-Journal of Chemistry 8(4); 2011: 1886-1894

16. Devasagayam TPA, Baloor KK and Mishra KP. Some new methods for free radical research. SFRR-India Bulletin 2(2); 2003: 20-28

17. Dixit P, Ghaskadbi S, Mohan H and Devasagayam TPA. Antioxidant properties of germinated fenugreek seeds. Phytotherapy Research 19; 2005: 977-983

18. Chatterjee S, Poduval BT, Tilak JC and Devasagayam TPA. A modified, economic, sensitive method for measuring total antioxidant capacities of human plasma and natural compounds using Indian saffron (Crocus sativus).Clinica Chimica Acta 352; 2005: 155-163

19. Shirwaikar A, Prabhu KS and Punitha ISR. In vitro antioxidant studies of Sphaeranthus indicus (Linn). Indian Journal of Experimental Biology 44; 2006: 993-996

20. Blois MS. Antioxidant determinations by the use of stable free radical. Nature 29; 1958: 1199

21. Di Mambro V M, Azzolini A E C S, Valim Y M L and Fonseca J V. Comparison of antioxidant activities of tocopherols alone and in pharmaceutical formulations. International Journal of Pharmaceutics 262; 2003: 93–99

22. Lim YY, Lim TT and Tee JJ. Antioxidant properties of several tropical fruits: A comparative study. Food Chemistry 103; 2007: 1003-1008

23. Meir S, Kanner J, Akiri B, Hadas SP. Determination and involvement of aqueous reducing compounds in Oxidative Defense systems of various senescing Leaves. Journal of Agricultural and Food Chemistry 43; 1995:1813-1817

24. Chung YC, Chang CT, Chao WW, Lin CF, Chou ST. Antioxidative activity and safety of the 50% ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1. Journal of Agricultural and Food Chemistry 50; 2002: 2454-2458

25. Tilak JC, Adhikari S and Devasagayam TPA. Antioxidant properties of Plumbago zeylanica, an Indian medicinal plant and its active ingredient, plumbagin. Redox Report 9(4); 2004: 220-227

26. Coruh N, Celep AGS, Ozgokce F and Iscan M. Antioxidant capacities of Gundeliatournefortii L. extracts and inhibition on glutathione-S-transferase activity. Food Chemistry 100; 2007: 1249-1253

27. Behera BC, Verma N, Sonone A, Makhija U. Determination of antioxidative potential of lichen Usneaghattensisin vitro. LWT 39; 2006: 80-85

28. Rekha C, Poornima G, Manasa M, Abhipsa V, Devi PJ, Kumar VHT and Kekuda TRP. Ascorbic acid, Total phenol content and antioxidant activity of fresh juices of four ripe and unripe citrus fruits. Chemical Science Transactions 1(2); 2012: 303-310.

Received on 09.09.2012 Modified on 17.09.2012

Research J. Pharm. and Tech. 5(10): October 2012; Page 1312-1315