INTRODUCTION:

As per WHO greater than one million people depend on herbal medicines and around 21,000 plants are used for medicinal purpose around the world. India has rich flora of 25,000 species, among these around 150 species was commercially used for the preparation of medicines, drug formulation. Medicinal plants are crucial in health care since ancient, modern cultures. Indian system generally uses plant based formulations to treat variety of ailments, as plants contain necessary components of therapeutic value¹which are nutritionally significant. A perfect antioxidant must get readily absorbed and must quench free radicals. Antioxidants of both synthetic, natural are effective in preventing free radical formation by scavenging free radical and promoting their decomposition. It reflects the inhibitory activity in the complex auto-oxidation system.

Received on 09.04.2015 Modified on 21.04.2015

Research J. Pharm. and Tech. 8(6): June, 2015; Page 697-701

DOI: 10.5958/0974-360X.2015.00110.9

Antioxidants maintain optimal cellular functions required for systemic health and well-being. The need for safe, economic antioxidants with improved activity from natural resource is a must. Hence, an attempt has been initiated to study the antioxidant activities of plants selected from the experimental site i.e near Omalur bus stand. The following plants were selected for the study: Tectona grandis, Azadirachta indica, Tamarindus indica, Ficus religiosa, Millettia pinnatta, Syzygium cumini, Terminalia catappa, Melia composita, Artocarpus heterophyllus, Ciccia acida, Santalum album, Mangifera indica, Annona squamosa, Morinda pubescens, Wrightia tinctoria.

MATERIALS AND METHODS:

Leaf sample collection:

For the present study, fresh leaves from each plant was collected from the experimental site near road sides of Omalur bus stand, Salem, Tamil Nadu, India during the month of December 2014 – January 2015. Common plants identified were selected from the study area. All the selected plants were identified by Dr. A. Balasubramanian and also by comparing with book named Dictionary of Medicinal Plants written by Dr. A. Balasubramanian, Executive Director, ABS Botanical garden, Salem, Tamil Nadu, India.

Extract Preparation:

Fresh leaves were used according to the standard prescribed methods adopted. 100mg of fresh leaves was ground to a paste in a mortar and pestle using 1ml of distilled water. 0.1ml of clear extract was used for the each experiment assessed.

Quantitative Assays

Secondary Metabolites

Total Phenolics:

To 0.1ml of extract, added 2.8ml of 10% sodium carbonate, 0.1ml of 2N Folin ciocalteu phenol reagent. After 40minutes incubation, the color developed was read at 725nm using UV- Spectrophotometer. Total phenolic contents calculated was expressed as mg of Gallic acid equivalents/g of sample using standard calibration curve constructed.²

Total Flavonoids:

0.1ml of plant extract was mixed with 1.5ml of methanol, 0.1ml of 10% aluminium chloride, 0.1ml of 1M potassium acetate and 2.8ml of distilled water. All the tubes were kept at room temperature for 30minutes. The absorbance of the reaction mixture was measured at 415nm with UV/Visible spectrophotometer. The Total flavonoid content was calculated from a calibration curve obtained using Quercetin as a standard.^3,4

Assay of antioxidants

Total Antioxidant activity by Phosphomolybdenum complex Method:

0.1ml of extract was mixed with 4ml of reagent solution containing 0.6M sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate. The contents in the tube was incubated in a water bath at 95°C for 90minutes. After the samples had been cooled to RT, the absorbance of mixture was measured at 695nm using UV Visible spectrophotometer. Standard calibration plot was prepared using Ascorbic acid.⁵

Reducing Power Assay:

0.1ml of plant extract was mixed with 1ml of phosphate buffer (0.2M, pH6.6), 1% Potassium ferricyanide, the content was shaken well and incubated at 50°C for 20minutes. After incubation, 1ml TCA (10%) was added to stop the reaction. It was centrifuged at 3000rpm for 10minutes. To 1.5ml of supernatant, 1.5ml of distilled water and 0.1ml Ferric chloride (0.1%) was mixed and incubated for 10 minutes, the absorbance was read at 700nm using UV Visible spectrophotometer. Standard calibration curve was plotted using ascorbic acid.⁶

Nitric oxide scavenging activity:

To 0.1ml of extract, 2ml of 10mM sodium nitroprusside, 0.5ml of phosphate buffered saline 1M was added and then incubated at 25°C for 150minutes. After incubation, 1ml of sulphanilic acid reagent (0.33%), 1ml of naphthylene diamine dihydrochloride (1%) was added and mixed, allowed to stand for 30minutes. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions which can be estimated by the use of Griess illsovery reaction at 540nm.^7,8Quercetin was used as a standard.

Metal Chelating Activity:

To 0.1ml of extract add 2.16ml of distilled water, 80µl of 2mM ferric chloride. The reaction was initiated by the addition of 160µl of Ferrozine. The contents in the tube was mixed well and allowed to stand for 10minutes at room temperature. After incubation the absorbance was read at 562nm using UV Visible spectrophotometer. The calibration plot was drawn using ascorbic acid as standard.⁹

Hydrogen Peroxide Scavenging Activity:

To 0.1ml of extract add 0.6ml hydrogen peroxide solution (0.6ml, 40mM). The absorbance of hydrogen peroxide at 230nm was determined after ten minutes against a blank solution containing phosphate buffer without hydrogen peroxide. A solution of hydrogen peroxide (40 mM) was prepared in phosphate solution. The percentage of hydrogen peroxide scavenging by the extract was calculated as follows:

% Scavenged [H2O2] = [(Ao − A1)/Ao] × 100

Where, Ao was the absorbance of the control and A1 was the absorbance in the presence of the sample of extract.^3,10 Readings were taken for all the experiments using Schimadzu model UV 1800.

Statistical Analysis:

Each experiment was carried out in triplicate and the results are given as the Mean ± Standard deviation. The Mean and Standard deviation (S) was calculated by using the following formula: Mean = Sum of x values / n (Number of values),

RESULTS AND DISCUSSION:

Table.1 represents the results of secondary metabolites assessed and Table.2 depicts the results of antioxidant activities of selected plants.

Total Phenolics:

Total phenolics was found to be high in Artocarpus heterophyllus 7.10±0.17, Ciccia acida 6.76±3.34, Melia composite 6.36±0.23, Santalum album 6.30±3.29, Annona squamosa 6.20±0.34, Terminalia catappa 5.13±1.67. Moderate level of phenolics was observed in Wrightia tinctoria 4.70±2.25, Tamarindus indica 4.63±2.19, Morinda pubescens 4.36±0.05, Millettia pinnatta 4.36±0.40, Mangifera indica 3.86±0.11, Syzygium cumini 3.01±0.31, Tectona grandis 3.00±1.38. The phenolics level was low for Azadirachta indica 2.60±0.69. Similar result was reported by Krishnaveni et.al for Mangifera indica,¹¹Annona squamosa,¹² Mangifera indica,¹²Tamarindus indica,¹³ Ficus religiosa.¹³

Table.1. Secondary metabolites of plants studied

S.No	Name of the plants	Total phenolics (mg/g)	Total Flavonoids (mg/g)
1.	Tectona grandis	3.00±1.38	3.96±0.23
2.	Azadirachta indica	2.60±0.69	4.13±0.11
3.	Tamarindus indica	4.63±2.19	4.16±0.05
4.	Ficus religiosa	2.65±0.08	4.13±0.11
5.	Millettia pinnatta	4.36±0.40	4.66±0.75
6.	Syzygium cumini	3.01±0.31	4.63±0.63
7.	Terminalia catappa	5.13±1.67	4.03±0.05
8.	Melia composita	6.36±0.23	3.83±0.11
9.	Artocarpus heterophyllus	7.10±0.17	4.10±0.00
10.	Ciccia acida	6.76±3.34	4.03±0.05
11.	Santalum album	6.30±3.29	4.70±0.69
12.	Mangifera indica	3.86±0.11	4.26±0.40
13.	Annona squamosa	6.20±0.34	4.66±0.57
14	Morinda pubescens	4.36±0.05	4.76±0.57
15	Wrightia tinctoria	4.70±2.25	4.36±0.80

Values are Mean ± SD for three experiments

Table.2. Antioxidant activities of selected plants Values are Mean ± SD for three experiments

S.No	Name of the plants	Total antioxidant activity (mg/g)	Reducing power activity (mg/g)	Nitric oxide scavenging activity (mg/g)	Metal chelating activity (mg/g)	Hydrogen peroxide scavenging activity (%)
1.	Tectona grandis	0.90±0.76	1.20±0.08	2.33±0.02	3.50±0.69	4.02±0.64
2.	Azadirachta indica	1.01±0.84	2.76±0.31	2.70±0.34	2.80±0.34	5.00±0.81
3.	Tamarindus indica	2.10±0.08	3.00±0.86	3.55±0.25	4.03±0.98	4.05±0.66
4.	Ficus religiosa	2.35±0.51	3.10±0.00	2.93±0.49	2.63±0.63	4.24±1.55
5.	Millettia pinnatta	3.06±0.75	2.76±0.31	2.66±0.02	6.03±3.40	4.82±1.69
6.	Syzygium cumini	2.80±0.43	3.48±0.46	3.91±0.49	3.11±1.52	4.44±0.00
7.	Terminalia catappa	2.68±0.28	2.36±0.14	3.53±1.15	2.66±0.92	4.52±1.07
8.	Melia composita	2.80±0.43	2.61±0.83	3.23±0.46	5.13±1.84	5.12±0.59
9.	Artocarpus heterophyllus	2.93±0.37	2.76±1.44	2.58±0.20	2.50±0.17	3.93±0.21
10.	Ciccia acida	4.78±1.58	2.18±0.80	3.50±0.00	3.63±0.28	4.60±1.50
11.	Santalum album	2.61±0.57	3.33±0.20	2.95±0.51	3.26±0.40	4.18±0.43
12.	Mangifera indica	3.03±0.72	3.16±0.31	4.06±0.57	3.96±0.05	5.34±0.78
13.	Annona squamosa	3.60±0.51	3.65±0.60	4.06±0.31	3.50±0.34	5.14±0.00
14	Morinda pubescens	2.63±0.28	3.30±0.51	3.75±1.12	3.86±1.27	3.93±0.21
15	Wrightia tinctoria	2.40±0.08	3.03±1.24	2.41±0.05	4.73±0.98	4.44±0.00

Values are Mean ± SD for three experiments

Phosphomolybdenum Assay:

Total antioxidant activity was measured in terms of phosphomolybdenum activity. Phosphomoly bdenum activity was moderate for almost all the plants studied. Each plant showed its own antioxidant activity. Ciccia acida 4.78±1.58, Annona squamosa 3.60±0.51, Millettia pinnatta 3.06±0.75, Mangifera indica 3.03±0.72, Artocarpus heterophyllus 2.93±0.37, Melia composite 2.80±0.43, Syzygium cumini 2.80±0.43, Terminalia catappa 2.68±0.28, Morinda pubescens 2.63±0.28, Santalum album 2.61±0.57, Wrightia tinctoria 2.40±0.08, Ficus religiosa 2.35±0.51, Tamarindus indica 2.10±0.08. Very low activity was observed with Azadirachta indica 1.01±0.84, Tectona grandis 0.90±0.76. Similar result was reported by Krishnaveni et. al for Ficus religiosa,¹⁵ Mangifera indica.¹⁶

Reducing Power Assay:

The reducing power activity was found to be moderate in the plants studied from the experimental site. The obtained values were as follows: Annona squamosa 3.65±0.60, Syzygium cumini 3.48±0.46, Santalum album 3.33±0.20, Morinda pubescens 3.30±0.51, Mangifera indica 3.16±0.31, Ficus religiosa 3.10±0.00, Wrightia tinctoria 3.03±1.24, Tamarindus indica 3.00±0.86, Millettia pinnatta 2.76±0.31, Artocarpus heterophyllus 2.76±1.44, Azadirachta indica 2.76±0.31, Melia composite 2.61±0.83, Terminalia catappa 2.36±0.14, Ciccia acida 2.18±0.80, Tectona grandis 1.20±0.08. Similar result was reported by Krishnaveni et.al for Azadirachta indica,¹⁴ Ficus religios.¹⁷

Nitric Oxide Scavenging Assay:

Nitric oxide scavenging activity was also moderate. Mangifera indica 4.06±0.57, Annona squamosa 4.06±0.31, Syzygium cumini 3.91±0.49, Morinda pubescens 3.75±1.12, Tamarindus indica 3.55±0.25, Terminalia catappa 3.53±1.15, Ciccia acida 3.50±0.00, Melia composite 3.23±0.46, Santalum album 2.95±0.51, Ficus religiosa 2.93±0.49, Azadirachta indica 2.70±0.34, Millettia pinnatta 2.66±0.02, Artocarpus heterophyllus 2.58±0.20, Wrightia tinctoria 2.41±0.05, Tectona grandis 2.33±0.02.

Metal Chelating Activity:

Millettia pinnatta 6.03±3.40, Melia composite 5.13±1.84, Wrightia tinctoria 4.73±0.98, Tamarindus indica 4.03±0.98, Mangifera indica 3.96±0.05, Morinda pubescens 3.86±1.27, Ciccia acida 3.63±0.28, Tectona grandis 3.50±0.69, Annona squamosa 3.50±0.34, Santalum album 3.26±0.40, Syzygium cumini 3.11±1.52, Azadirachta indica 2.80±0.34, Terminalia catappa 2.66±0.92, Ficus religiosa 2.63±0.63, Artocarpus heterophyllus 2.50±0.17. Similar result was reported by Krishnaveni et.al for Tamarindus indicia,¹⁸ Tectona grandis,¹² Ficus religiosa,¹³ Azadirachta indica,¹⁹ Tamarindus indica,¹⁹Mangifera indica,¹⁶ Annona squamosa,^16,18Ficus religiosa,¹⁷ Morinda pubescens.²⁰

Hydrogen Peroxide Scavenging Activity

Mangifera indica 5.34±0.78, Annona squamosa 5.14±0.00, Melia composite 5.12±0.59, Azadirachta indica 5.00±0.81, Millettia pinnatta 4.82±1.69, Ciccia acida 4.60±1.50, Terminalia catappa 4.52±1.07, Wrightia tinctoria 4.44±0.00, Syzygium cumini 4.44±0.00, Ficus religiosa 4.24±1.55, Santalum album 4.18±0.43, Tamarindus indica 4.05±0.66, Tectona grandis 4.02±0.64, Artocarpus heterophyllus 3.93±0.21, Morinda pubescens 3.93±0.21. The ability to scavenge hydrogen peroxide might be an efficient assessment method to assess the antioxidant property of water extract.

Phenol an important plant constituent shows high free radical scavenging ability due to their hydroxyl group. Therefore, the phenolic content of plants contribute directly to their antioxidant action.²¹The secondary plant metabolite flavonoid exhibiting significant antioxidant and chelating properties depend on the hydroxyl groups structure, substitution pattern.²² The antioxidant capacity of the Ficus species was reported by Konan et.al.²³Millettia pinatta was reported for its healing property.²⁴ According to our results, the amount of phenolic contents in extracts are significant for their antioxidant activities. Other parts of Terminalia catappa plants were reported by Krishnaveni et.al for its phytochemicals through GC-Ms analysis,²⁵ phytonutrient analysis,²⁶antioxidant potential.^{27, 28}

CONCLUSION:

From the results it is concluded, that the hydrogen peroxide scavenging activity was high compared to all the other antioxidant activities studied. And all the plants showed moderate amount of antioxidant activities suggesting it be a good natural antioxidant and might be used in the treatment of diseases due to its pharmaceutical functions. It is assumed that consumption of plant derived phytochemicals such as phenolics, flavonoids have good antioxidant status. It is necessary that full structural detection of active components, antioxidants of plant, their toxicological properties has to be investigated further.

ACKNOWLEDGEMENT:

The author wishes her thanks to Honorable Vice-Chancellor Dr. C. Swaminathan Avl, and Registrar Dr. K. Angamuthu Avl, Periyar University, Salem for their administrative support and excellent infrastructure facilities provided and also Co-ordinator, School of Bio-Sciences, Periyar University, Salem, Dr. A. Balasubramanian, ABS Botanical garden, Salem for his help in identifying plants. The author would like express her gratitude to her dedicated teachers.

REFERENCES:

1. World health organization regional office for the western pacific. Research guidelines for evaluating the safety and efficacy of herbal medicines. Manila, World Health organization regional office for the western pacific, 1993.

2. Makkar HPS, Bluemmel M, Borowy NK, Becker K. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J Sci Food Agr. 1993; 61: 161-165.

3. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Hamidinia A, Bekhradnia AR. Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica Mey. Pharmacologyonline 2008a; 2: 560-567.

4. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Fazelian M and Eslami B. In vitro antioxidant and free radical scavenging activity of Diospyros lotus and Pyrus boissieriana growing in Iran. Pharmacognosy Magazine 2009a; 4(18): 123-127.

5. Prieto P, Pineda M, Agulia M. Spectrophotometric quantification of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of Vitamin E. Analytical Biochemistry 1999; 269: 337-341.

6. Oyaizu M. Studies on products of browning reaction prepared from glucosamine. Japan Journal of Nutrition 1986; 44: 307-315.

7. Garrat DC. The quantitative analysis of drugs, Champman and Hall, Japan, 1964; 3: 456-458.

8. Sreena KP, Poongothai A, Soundariya SV, Srirekha G, Santhi R, Annapoorani S. Evaluation of invitro free radical scavenging efficacy of different organic extracts of Morinda tinctoria leaves. Int.J Pharm Sci 2011; 3(3): 207-209.

9. Decker EA, Welch B. Role of ferritin as a lipid oxidation catalyst in muscle food. J of Agri. and Food Chem. 1990; 38: 674 - 677.

10. Nabavi SM, Ebrahimzadeh MA, Nabavi SF and Jafari M. Free radical scavenging activity and antioxidant capacity of Eryngium caucasicum Trautv and Froripia subpinnata. Pharmacologyonline 2008b; 3: 19-25.

11. Krishnaveni M, Amsavalli L, Chandrasekar R, Madhaiyan P, Durairaj S., Antioxidant activity of plants at Govt. College of Engineering Campus, Salem, Tamil Nadu, India. International Journal of Pharmaceutical Sciences Review and Research 2013;21(1):160-163.

12. Krishnaveni M, Kalimuthu R, Ponraj K, Lavanya K, Magesh P, Jasbin shyni G. Antioxidant activities of plants studied in yercaud road sides, Salem, Tamil Nadu, India. International Journal of Pharmaceutical Sciences Review and Research 2014; 27(1): 61-65.

13. Krishnaveni M, Ponraj K, Kalimuthu R, Lavanya K, Mahesh P, Jasbin Shyni G. Antioxidant activity of plants studied at Thoppur hill road sides, Dharmapuri, Tamil nadu, India. International Journal of Pharmaceutical Sciences Review and Research 2014; 26(2): 171-176.

14. Krishnaveni M, Chandrasekar R, Amsavalli L, Durairaj S, Madhaiyan P. Free radical scavenging activity of plants at Perumalmalai Hill. International Journal of Pharmaceutical Sciences Review and Research 2013; 21(1); 155-159.

15. Krishnaveni M and Jasbin Shyni G. Comparative study on APTI, antioxidant status of plants and soil health, Lambert Academic Publishing, ISBN: 978- 3-659-52656-5, 2014.

16. Krishnaveni M, Lavanya K, Magesh P, Ponraj K, Kalimuthu R and Jasbin Shyni G. Free radical scavenging activity of selected plants. World Journal of Pharmacy and Pharmaceutical Sciences 2014; 3(5): 765-775.

17. Krishnaveni M, Durairaj S, Madhiyan P, Amsavalli L and Chandrasekar R. Invitro Free Radical Scavenging activity of aqueous leaf extract of plants near thermal power plant, Mettur, Salem. International Journal of Pharmaceutical Sciences and Research 2013; 4(9): 3659-3662.

18. Krishnaveni M, Madhaiyan P, Durairaj S, Amsavalli L, Chandrasekar R. Antioxidant activity of plants at Chinnathirupathi, Salem, Tamil Nadu, India. International Journal of Pharmaceutical Sciences and Research 2013; 4(10): 3917-3919.

19. Krishnaveni M, Mahesh P, Ponraj K, Kalimuthu R, Lavanya K, Jasbin Shyni G. A comparative study on antioxidant activities of selected plants from road sides, Salem, Tamil Nadu, India. International Journal of Pharmaceutical Sciences Review and Research 2014; 26(2): 112-116.

20. Krishnaveni M. Antioxidant activities of selected plants. International Journal of Pharmacy and Pharmaceutical Sciences 2014; 6(2): 126-128.

21. Tosun M, Ercisli S, Sengul M, Ozer H, Polat T. Antioxidant properties and total phenolic content of eight Salvia species from Turkey. Biol. Res 2009; 41: 175-181.

22. Sharififar F, Nudeh- Dehghn G, Mirtajaldini M. Major flavonoids with antioxidant activity from Teucrium polium L. Food Chem 2008; 112: 885-888.

23. Yao Konan, Kone Mamidou witabouna, Bonfoh Bassirou, Kamanzi Kagoyire. Antioxidant activity and total phenolic content of nine plants from Cote d IOvoire (West Africa). Journal of Applied Pharmaceutical Science 2014; 4(8): 036-041.

24. Chowdry Habibur Rahaman, Suman Karmakar. Ethanomedine of Santal tribe living around Susunia hill of Bankura District, West Bengal, India: The quantitative approach. Journal of Applied Pharmaceutical Science 2014; 5(2): 127-136.

25. Krishnaveni M, Krishnakumari G, Raginabanu C, Kalaivani M, GC-MS/MS analysis of phytochemicals in Terminalia catappa L, Antimicrobial assay. Indo American Journal of Pharmaceutical Research 2015; 5(3): 1250-1254.

26. Krishnaveni M, Dhanalakshmi R. Phytonutrient analysis in Terminalia catappa fruit flesh, nut, shell. International Journal of Current Pharmaceutical Review and Research 2015; 6(1): 28-35.

27. Krishnaveni M, Jasbin Shyni G, Dhanalakshmi R, Magesh P, Ponraj K, Lavanya K, Kalimuthu R. A preliminary study on phytoanalysis, antioxidant potential of Terminalia catappa l. fruit flesh. International Journal of Pharmaceutical Sciences Review and Research 2014; 28(1): 83-87.

28. Krishnaveni M. Invitro antioxidant potential of Terminalia catappa nuts. Asian Journal of Pharmaceutical and Clinical Research 2014; 7(4): 33-35.