EVALUATION OF IN VITRO ANTIOXIDANT ACTIVITY OF FLOWERS OF BLEPHARIS MOLLUGINIFOLIA

EVALUATION OF IN VITRO ANTIOXIDANT ACTIVITY OF FLOWERS OF BLEPHARIS MOLLUGINIFOLIA

S. Deepika* and S.V. Rajagopal

Department of Biotechnology, GITAM Institute of science, GITAM University, Visakhapatnam, Andhra Pradesh, India

ABSTRACT: The objective of this study was to characterize the non-enzymatic and enzymatic antioxidants and Scavenging activities of different extracts (Petroleum ether, Benzene, Chloroform, Acetone, Ethanol, Methanol and Water) of dried flowers powder of Blepharis molluginifolia (Acanthaceae). Enzymatic antioxidant activity such as Superoxide Dismutase, Glutathione –S-transferase, peroxidase and Catalase of B. molluginifolia flowers were high in methanol extract. Non enzymatic antioxidants like Tocopherol, Ascorbic acid, Phenols, Carotenoids and Lycopene were analysed. Acetone extract showed highest Total Phenol content and Vitamin-C were 100.66±1.5 mg GAE/gm & 52.08±2.42 mg/gm respectively. Scavenging activities like DPPH, ABTS and H₂O₂ were high in Methanol extract. In this study, the non-enzymatic antioxidant activity was found to be significant, which prove to be a better scavenger of free radical in comparison to enzymatic extracts in the shade dried extracts.

Blepharis Molluginifolia, Reactive oxygen species, In vitro Antioxidant activity, free radical scavenging activities

INTRODUCTION:Reactive oxygen species(ROS)such as Superoxide (O₂^-), Hydrogenradicals (OH^-), and Hydrogen peroxide (H₂O₂^-) are considered as important factors causing many diseases like cardiovascular, diabetes, inflammation, cancer and neurogenerative diseases ^{1, 2}. ROS are degraded to non-reactive forms by Enzymatic and Non-Enzymatic defence mechanisms. Free radicals react with known biological molecule and damage protein, breakdown of DNA strands and initiates peroxidation of various molecules

Antioxidants act as a major defence against radical-mediated toxicity, by protecting the damages caused by free radicals 3.
Antioxidative components of natural origin have attracted special interest because they can protect human body from free radicals.Enzymatic antioxidants include primary enzymes like Superoxide Dismutase, Catalase, Glutathione peroxidase and secondary enzymes include Glutathione reductase etc 4.
Non-Enzymatic antioxidants include either water-soluble (vitamin C and phenolic compounds) or lipid-soluble (vitamin E and carotenoids) compounds which act as defence against oxidative stress 5, 6. However antioxidant compounds like Phenols, Flavonoids scavenge these free radicals and protect the system from the Oxidative mechanisms.
Blepharis molluginifolia belongs to the family Acanthaceae, is a threatened medicinal herb. This plant is used for Urinary discharges and also equated with Uttangana. Traditionally the plant is used for bone fractures, skin diseases and allergies.7Abundant occurrence of Phenols in seed samples has been reported. Steroids and Cardiac glycosides were found in seed samples of Blepharis genus.8Theaim of the present studywas to determine non-enzymatic and enzymatic antioxidants and scavenging activitiesof different flower extracts of Blepharis molluginifolia.
MATERIALS AND METHODS:
Chemicals and reagents: Folin-Ciocalteau Reagent, Nitroblue Tetrazolium (NBT), DL-Alpha-Tocopherols, Hydrogen Peroxide, Xylene,2,2`-Dipyridyl, Ferric Chloride, Pyrogallol, 2,2-Diphenyl-1-Picrylhydrazyl, Gallic acid, Riboflavin,Chloro-2,4-dinitrobenzene (CDNB), Standard ascorbate, Reduced glutathione were purchased from Merck.
Plant materials: The flowers of Blepharis Molluginifolia were collected from its natural habitat at Koyathanda in Nallamala forest region, Andhra Pradesh, in the month of December and January. The plants were authenticated by the Dr. N. Balahussaini, Agricultural College, Kadapa.
Preparation of extracts: The flowers of Blepharis molluginifolia (100g) were dried under shade, and undergone crushing in electric blender to form powder. After that this powder was used for extraction by using various polar and nonpolar solvents like Petroleum ether, Benzene, Chloroform, Acetone, Water, Ethanol and methanol by using Soxhlet extractor. These are dried and preserved for the further tests.
Enzymatic antioxidants
1) Assay of Superoxide Dismutase (SOD) 9, 10: The activity of superoxide dismutase was assayed spectrophotometrically by the method of Misra and Fridovich (1972) in the flower extracts of the plant.
2) Assay of Catalase 10, 11 (CAT): Catalase activity was determined by adopting the method of Luck (1974).
3) Assay of Peroxidase 10, 12 (POX): The activity of peroxidase was determined by the method of Reddy et al (1995).
4) Assay of Glutathione s-transferase 10, 13 (GST): The activity of glutathione S-transferase activity was performed by the method of Habig et al (1974).
NON-ENZYMIC ANTIOXIDANTS:
1) Estimation of Ascorbic Acid 14: The amount of ascorbic acid present in the flower extracts, of this plant was estimated by the method of Roe and Keuther (1943).
2) Estimation of Tocopherol 15: The level of tocopherol was estimated Spectrophoto-metrically by the method reported by Rosenberg (1992).
3) Estimation of total Carotenoids and Lycopene 16: The estimation of total carotenoids and lycopene was done by the method described by Zakaria et al (1979).
4) Determination of total Phenols 17: Total phenols was determined by the method (Folin-Ciocalteu) proposed by Mallick and Singh (1980).
EVALUATION OF RADICAL SCAVENGING ASSAYS:
1) DPPH Radical Scavenging Assay 18: The ability of the plant extracts to scavenge the stable free radical DPPH was assayed by the method of Mensor et al (2001).
2) ABTS Radical Scavenging Assay 19: The ability of all the plants flower extracts to scavenge the free radical ABTS (2, 2-azino-bis 3-ethyl benz thiazoline-6-sulfonic acid) was studied using the method adopted by Shirwaikar et al (2006).

3) Hydrogen Peroxide Radical Scavenging Effect 20: The scavenging activity of hydrogen peroxide by the plant extracts was determined by the method of Ruch et al (1989).
RESULTS & DISCUSSION:
Assay of Enzymatic Antioxidants: The levels of enzymatic antioxidants assessed in B. molluginifolia flowers in different extracts (petroleum ether, benzene, chloroform, acetone, ethanol, methanol and water) were collectively represented in Fig. 1. The activity of GST, CAT, SOD and POX was high in methanol extract. Reports showed Methanolic extracts of Nerium indicum flowers have more Cellular antioxidant enzymes 21.
Least activity was shown by chloroform extract for GST and CAT. Acetone extract showed least activity for SOD and Benzene extract for POX. In our experiment, there is a correlation between catalase activities and scavenging of hydrogen radical and superoxide dismutase and glutathione-s-transferase, peroxidase activity, which are indicators OH radical scavengers. This indicates this medicinal plant is highly potential antioxidant.

FIG. 1: ENZYMATIC ANTIOXIDANT ACTIVITY OF BLEPHARIS MOLLUGINIFOLIA FLOWERS. Values are mean ± SD of three parallel measurements
Assay of non-enzymatic antioxidants: In the non-enzymatic activity, the total phenol content, vitamin-C, vitamin-E and carotenoids and lycopene were analysed in different extracts, collectively represented in Table 1.
Total phenol compounds, as determined by Folin Ciocalteu method, are reported as Gallic acid equivalents by reference to standard curve (y = 19.473x-17.582, r2 = 0.9979). Acetone extract showed highest activity for total phenol content with; chloroform extract showed least activity for total phenol content with. Similarly Acetone extract showed highest activity in Bombax malabaricum flowers 22.
Vitamin‐C content was expressed as ascorbic acid equivalents per gm. Highest Ascorbic acid were present in Acetone extract and least is in chloroform extract with. Similarly Acetone extract showed highest activity in Bauhinia tomentosa flowers 23. In plant cells, the most important reducing substrate for H2O2 removal is ascorbic acid 24.
Carotenoids have capacity for quenching singlet oxygen and free radicals 25. Highest activity was in Benzene and least value in water extract. Same activity was seen in Tagetes erecta dried flowers 26. Carotenoids are therefore efficient free-radical scavengers.
Lycopene is a bright red carotene and carotenoid pigment and phytochemical found in tomatoes and other red fruits and vegetable. Lycopene's eleven conjugated double bonds give it its deep red colour and are responsible for its antioxidant activity. Lycopene may also interact with reactive oxygen species, such as hydrogen peroxide and nitrogen dioxide 27. Lycopenes and vitamin- E) showed highest activity with methanol extract.
α–tocopherol interact with the polyunsaturated acyl groups of lipids, stabilize membranes, scavenge and various reactive oxygen species (ROS) and lipid soluble by products of oxidative stress 28.
The strong relationship between the total phenol content and antioxidant activity in sweet basil was also reported 29. The high amount of phenols in extracts may explain their high antioxidative activities.

TABLE 1: NON-ENZYMATIC ANTIOXIDANT ACTIVITY OF BLEPHARIS MOLLUGINIFOLIA FLOWERS
Extract Phenol (mg/g) Vitamin-C (mg/g) Vitamin-E (mg/g) Carotenoids (mg/g) Lycopene (mg/g)
Petroleum ether 24.09±0.57
6.16±0.74
0.01±0.00004
26.7±3.06
2.62±0.09
Benzene 5.61±+0.57
16.14±1.26
0.00±0.0003
67.4±2.95
1.72±0.04
Chloroform 4.62±0.57
3.31±0.16
0.01±0.0001
24.7±4.62
1.23±0.03
Acetone 100.66±1.51
52.08+±2.42
0.02±0.0001
16.7±1.43
2.31±0.02
Ethanol 10.89±0.99
7.02±0.43
0.02±0.0001
36.7±1.15
1.49±0.01
Methanol 98.02±0.99
51.58±2.98
0.04±0.0007
60.00±2.00
5.69±0.01
Water 11.22±1.51
15.97±1.34
0.01±0.0002
3.3±1.15
1.34±0.03
Values are mean ± SD of three parallel measurements

Radical Scavenging activities:
1. DPPH radical-scavenging activity: DPPH stable free radical method is an easy, rapid and sensitive way to survey the antioxidant activity of a specific compound or plant extracts30. From Fig. 2, highest % of inhibition was shown by Methanol extract and least by ethanol extract. Usually, higher total phenol and flavonoids contents lead to better DPPH-scavenging activity.
2. Hydrogen Peroxide Radical Scavenging activity: H2O2 also forms OH.in the presence of metal ions and oxygen facilitates this reaction. Hence, metal chelating and H2O2 scavenging processes are important for living organisms 31. The scavenging ability of different extracts on hydrogen peroxide was shown in Fig. 2. The radical scavenging capacity may be attributed to phenolic compounds in methanol extract with the ability to accept electrons, which can combine with free radical moiety to decrease hydroxyl radical compared with DPPH assay.
3. ABTS Radical Scavenging activity: ABTS assay is an excellent tool for determining the antioxidant activity of hydrogen‐donating antioxidants and of chain‐breaking antioxidants 32. Different artificial free radical species, such as ABTS + radical cation, has been used to assess radical scavenging ability and antioxidant activity. From Fig. 2, highest % of inhibition was shown by methanol extract and least by Benzene.
All tested extracts for DPPH, H2O2 and ABTS scavenging activities of this plant can inhibit the presence and production of free radicals but lesser when compared to reference standards Ascorbic acid and Butylated Hydroxy Toulene (BHT).

FIG. 2: RADICAL SCAVENGING ACTIVITIES OF BLEPHARIS MOLLUGINIFOLIA FLOWERS. Values are mean ± SD of three parallel measurements
Assay of standards:
Antioxidant assays Ascorbic acid (100µg/ml) BHT(100µg/ml)
DPPH 59.33 88.67
ABTS 68.5 68.8
H2O2 50.8 60.7
CONCLUSION: It could be concluded from the results of the present investigation that some oxidation parameters of assessing in vitro antioxidant effectiveness might be rapid and convenient depending on storage conditions, nature of oil and extent of oxidation. A number of different methods may be necessary to adequately assess the in vitro antioxidant activity of a specific plant material. By combining the knowledge of different antioxidant assays and assessment of oxidation parameters in the present study, it can be asserted that the investigated plant materials are a viable source of natural antioxidants and might have potential as “nutraceuticals” for the preparation of functional foods.
As India is rich in medicinally important flora, the meaningful exploitation of more indigenous plant materials and agricultural wastes is thus further recommended. An assessment of the toxicity and kinetic studies, as well as the function of these extracts in food and biological systems also needs to be investigated.
ACKNOWLEDGEMENT: My gratitude goes to Gitam Institutes, for giving me an opportunity to conduct this research.
REFERENCES:
1. Halliwell B: Establishing the significance and optimal intake of dietary antioxidants; The biomarker concept. Nutr. Rev 1999; 57: 104‐113
2. Hogg N: Free radicals in diseases. Semin. In Reprod .Endocrin., 1998, 16, 241-88.
3. Shinde:, Effect of Free Radicals & Antioxidants on Oxidative Stress: A Review Journal of Dental & Allied Sciences 2012;1(2):63-6
4. Ratnam, D.V. Ankola, D.D. Bhardwaj, V.; Sahana, D.K.; Kumar: M.N.V.R. Role of antioxidants in prophylaxis and therapy: A pharmeumaceutical perspective. J. Control Release. 2006,113, 189-207.
5. Hazra B, Biswas S, Mandal N: Antioxidant and free radical scavenging activity of Spondiaspinnata, BMC Comp Alt Med, 8, 2008, 63-64.
6. Podsędek, A. Natural antioxidants and antioxidant activity of Brassica vegetables: A review.LWT-Food Sci. Technol. 2007, 40, 1-11.
7. Pattar, Pramod V.; Jayaraj, M.; Arunkumar, B. S.; Ananth, B.:Pharmacognostical and Preliminary Phytochemical Investigation of Blepharismolluginifolia, Pers. -A Threatened Medicinal Herb. Pharmacognosy Journal; 1/24/2011, Vol. 3 Issue 19, p29
8. Kiran Kumar Mundla:comparative study of phytochemical, antimicrobial, Cytotoxic and antioxidant activities in blepharis genus plant seeds IJSIT, 2013, 2(1), 07-20
9. Misra HP, Fridovich I (1972) :The role of superoxide anion in the antioxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247: 3170-3171.
10. Sathishkumar P.T.V. lakshmi1, and A. Annamalai: Comparative analyses of non enzymatic and enzymatic Antioxidants of enicostemma littorale blume., International Journal of Pharma and Bio Sciences V1(2)2010; p 1-16.
11. Luck H (1974): Methods in enzymatic analysis. Academic Press, New York, 885
12. Reddy KP, Subhani SM, Khan PA, Kumar KB (1995): Effect of light and benzyl adenine on dark treated growing rice (Oryzasativa) leaves-changes in peroxidase activity. Plant Cell. Physiol. 26: 987-994.
13. Habig WH, Pabst MJ, Jokoby WB (1974) Glutathione transferase: A first enzymatic step in mercapturic acid III formation, J. Biol. Chem. 249: 7130-7139.
14. Roe JH, Heather CA: The determination of Ascorbic acid in whole blood and urine through 2,4 Dinitro phenyl hydrazine derivative of dehydro Ascorbic acid.J.Biol.Chem.1953;147:399-407.
15. Rosenberg HR.Chemistry and physiology of the vitamins. Interscience Publishers, Inc. NewYork. 1992;452-453
16. Zakaria H, Sinpson K, Brown PR, Krotatin A: Use of reversed phase high performance liquid chromatographic analysis for the determination pro vitamin A in tomatoes.J.Chromatography.1979;176:109-117
17. Cheruth Abdul Jaleel: Alterations in non-enzymatic antioxidant components of Catharanthusroseus exposed to paclobutrazol, gibberellic acid and Pseudomonas fluorescens Plant Omics Journal Southern Cross Journals©2009 2(1):30-40 (2009) ISSN: 1836-3644
18. D.Satheeshkumar: In vitro Antioxidant Activity of Various Extracts of whole Plant of Mucunapruriens (Linn) JPRIF ISSN : 0974-4304 Vol.2, No.3, pp 2063-2070, July-Sept 2010
19. Shirwaikar A, Prabhu K and Punitha ISR: In-vitro antioxidant studies of Sphaeranthusindicus, Indian Journal of Experimental Biology. 2006;44:993-998.
20. Ruch, R.J.; Chug, S.U.; Klaunig,:J.E. Methods Enzymol. 1984, 105, 198–209.20. A. Vinayagam and P. N. Sudha : Antioxidant activity of methanolic extracts of leaves and flowers of Neriumindicum IJPSR, 2011; Vol. 2(6): 1548-1553
21. Yu YG, In vitro antioxidant activity of Bombaxmalabaricum flower extracts.Pharm Biol. 2011 Jun;49(6):569-76.
22. K Thenmozhi1 Preliminary Phytochemical Screening from Different Parts of Bauhinia tomentosa L. And BauhiniamalabaricaRoxb. (Caesalpiniaceae); (2012) Research and Reviews: Journal of Botanical Sciences.
23. Jaleel,P.Manivannan,P.V.Murali,M.Gomothinayagam and R.Panneerselvam,2008c. Antioxidant potential and indole alkaloid profile variations with water deficits along different parts of two varieties of Catharanthusroseus.Colloids and SurfacesB Biointerfaces, 62:312-318.
24. Jannat M. Rolda´n-Gutie´rrez Lycopene: The need for better methods for characterization and determination Trends in Analytical Chemistry, Vol. 26, No. 2, 2007.
25. Miglena Valyova1*, Stanimir Stoyanov2, Yuliana Markovska3,Yordanka Ganeva Evaluation of in vitro antioxidant activity and free radical scavenging potential of variety of Tagete serecta L.flowers growing in Bulgaria , International Journal of Applied Research in Natural Products 2012 Vol. 5 (2), pp. 19-25.
26. S.K. Clinton, Nutr. Rev. 56 (1998) 35.
27. Cheruth Abdul Jaleel Non-enzymatic antioxidant changes in withaniasomnifera with varying drought stress levels -american-eurasian journal of scientific research 4 (2): 64-67, 2009.
28. D.BhaskarRao,Ch.RaviKiran,Y.Madhavi,P.KoteswaraRao and T.RaghavaRao: Evaluation of antioxidant potential osClitoria ternate L and Eclipta prostrate L,Indian Journal of Biochemistry and Biophysics vol46,June2009,pp.247-252.
29. F.Pourmorad,S.J.Hosseinimehr,N.Shahabimajd: Antioxidant activity,phenol and flavonoid contents of selected Iranian plants,African Journal of Biotechnology vol.5(11),pp.1142-1145,2June2006.
30. Gulcin I, Buyukokuroglu M E, Oktay M, Kufrevioglu I O, (2003). Antioxidant and analgesic activities of turpentine of PinusnigraArn. Subsp. Pallsiana (Lamb.) Holmboe. Journal of Ethnopharmacology, 86, 51-58.
31. Yogamaya Dhal1, BanditaDeo comparative antioxidant activity of non-enzymatic and enzymatic extracts of curcuma zedoaria, curcuma angustifolia and curcuma caesia International Journal of Plant, Animal and Environmental Sciences 2012 vol-2231-4490
32. Sathishkumar comparative analyses of non- enzymatic and enzymatic antioxidants of Enicostemmalittoraleblume, International Journal of Pharma and Bio Sciences V1(2)2010.

How to cite this article:

Deepika S and Rajagopal SV: Evaluation of in vitro antioxidant activity of flowers of Blepharis molluginifolia. Int J Pharm Sci Res2014; 5(6): 2225-29.doi: 10.13040/IJPSR.0975-8232.5(6).2225-29

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.