• Manisha Singh Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector 62, Noida - 201 307, Uttar Pradesh, India
  • Surya Pratap Singh Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector 62, Noida - 201 307, Uttar Pradesh, India
  • Rachana R Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector 62, Noida - 201 307, Uttar Pradesh, India


Objective: This study is aimed to evaluate the antioxidant (AO) potential, cytotoxicity, and stability of preformulated Ginkgo biloba standard extractmicroemulsion (GBME), to investigate if, it retains the therapeutic potential of EGB761 and remains safe and stable for a longer period.

Method and Results: GBME has shown enhanced AO (85.2±0.78%, IC50=31.3±0.45 μg/ml) in comparison to EGB761 (74.1±0.51%,IC50=49.4±0.05 μg/ml) using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay. Similarly, 2,2-diphenyl-1-picryl-hydrazyl-hydrate(DPPH) assay has also shown that AO for GBME (94.6±0.04%, IC50=11.4±1.03 μg/ml) was higher than EGB761 (78.6±1.20%, IC50=34.6±0.81 μg/ml).Further, IC50 value of antiradical unit of GBME was much lesser (ABTS=14.3±1.05 μg/ml and DPPH=17.03±1.8 μg/ml) in comparison to EGB761(ABTS=34.1±1.62 μg/ml and DPPH=37.5±0.08 μg/ml). Equivalently, both, hydrogen peroxide scavenging activity, and nitric oxide activity wereappreciably higher for GBME than the pure extract. The in vitro cytotoxicity assessment showed that GBME is quite safe (98.68±0.76% cell viability) incomparison to EGB761 (83.29±1.02%). Thereafter, these samples were tested for stability by evaluating their AO activity along with high-performanceliquid chromatography analysis, for the major phytocompounds, after 1 year, and results suggested that AO of GBME remained stable while comparingwith the freshly prepared GBME, whereas AO of EGB761 reduced significantly as compared to freshly taken EGB761 extract implying the degradationof phytocompounds supporting decrease in AO activity.

Conclusion: Therefore, the observed results suggest that GBME maintained AO and scavenging activity along with enhanced shelf life with no observedtoxicity, which can be explored further for its potential therapeutic implications in various oxidative stress-induced central nervous system disorders.

Keywords: Phytoconstituents, Flavonoid, Nitric oxide, 2, Nil, radical scavenging activity, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide assay.

Author Biography

Manisha Singh, Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector 62, Noida - 201 307, Uttar Pradesh, India
Assistant Professor in department of biotechnology


1. Hsieh CL, Lao L, Lin YW, Litscher G. Complementary and alternative medicine for the treatment of central nervous system disorders. Evid Based Complement Alternat Med 2014;2014:175152.
2. Avigan MI, Mozersky RP, Seeff LB. Scientific and regulatory perspectives in herbal and dietary supplement associated hepatotoxicity in the United States. Int J Mol Sci 2016;17(3):331.
3. Nieoullon A. Neurodegenerative diseases and neuroprotection: Current views and prospects. J Appl Biomed 2011;9(4):173-83.
4. Upadhyay RK. Drug delivery systems, CNS protection, and the blood brain barrier. Biomed Res Int 2014;2014:869269.
5. Hotel N, Rico P. Universidad Metropolitana (UMET). New Jersey: New Jersey Institute of Technology; 2006.
6. Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: A review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol 2009;7(1):65-74.
7. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5(1):9-19.
8. Zuo L, Hemmelgarn BT, Chuang CC, Best TM. The role of oxidative stress-induced epigenetic alterations in amyloid-ß production in Alzheimer’s disease. Oxid Med Cell Longev 2015;2015:604658.
9. Sharma P, Jha AB, Dubey RS, Pessarakli M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012;2012:1-26.
10. Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012;24(5):981-90.
11. Chan PC, Xia Q, Fu PP. Ginkgo biloba leave extract: Biological, medicinal, and toxicological effects. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 2007;25(3):211-44.
12. Drago F, Floriddia ML, Cro M, Giuffrida S. Pharmacokinetics and bioavailability of a Ginkgo biloba extract. J Ocul Pharmacol Ther 2002;18(2):197-202. 13. Nikam PH, Kareparamban J, Jadhav A, Kadam V. Future trends in standardization of herbal drugs. J Appl Pharm Sci 2012;2(6):38-44.
14. Ude C, Schubert-Zsilavecz M, Wurglics M. Ginkgo biloba extracts: A review of the pharmacokinetics of the active ingredients. Clin Pharmacokinet 2013;52(9):727-49.
15. van Beek TA, Montoro P. Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals. J Chromatogr A 2009;1216(11):2002-32.
16. Bilia AR, Isacchi B, Righeschi C, Guccione C, Bergonzi MC. Flavonoids loaded in nanocarriers: An opportunity to increase oral bioavailability and bioefficacy. J Food Nutr Sci 2014;2014:1212-27.
17. Singh M, Singh SP, Rachana R. Development, characterization and cytotoxicity evaluation of Gingko biloba extract (EGB761) loaded microemulsion for intra-nasal application. J Appl Pharm Sci 2017;7:24-34.
18. Sarkhejiya N, Nakum M, Patel V, Atara S, Desai T. Emerging trend of microemulsion in formulation and research. Int Bull Drug Res 2012;1(1):54-83.
19. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 1999;26(9-10):1231-7.
20. Tirzitis G, Bartosz G. Determination of antiradical and antioxidant activity: Basic principles and new insights. Acta Biochim Pol 2010;57(2):139-42.
21. Moon JK, Shibamoto T. Antioxidant assays for plant and food components. J Agric Food Chem 2009;57(5):1655-66.
22. Hamed MM, Mohamed MA, Ahmed WS. Chemical constituents, in vitro antioxidant activity, oral acute toxicity and LD50 determination of Moringa oleifera leaves. Int J Pharm Pharm Sci 2017;9:240-7.
23. Emad A. Antiradical and antioxidant activities of different Spirulina platensis extracts against DPPH and ABTS radical assays. J Mar Biol Oceanogr 2013;2:1-14.
24. Wojtasik S, Sroka Z, Zbikowska B, Drys A. The measurement of antiradical activity of some plant raw materials and extracts with use of TAU734 (total antiradical unit). Herba Pol 2011;57(4):16-24.
25. El Darra N, Tannous J, Mouncef PB, Palge J, Yaghi J, Vorobiev E, et al. A comparative study on antiradical and antimicrobial properties of red grapes extracts obtained from different Vitis vinifera varieties. J Food Nutr Sci 2012;3(1):1420.
26. Malakyan M, Vardevanyan L, Yeghiazaryan D, Badzhinyan S, Agababyan A, Gevorgyan G. Antioxidant and antiradical properties of new amino-acid derivatives of aminoalcohols. J Pharm Chem 2010;44:421-3.
27. Nimmi O, George P. Evaluation of the antioxidant potential of a newly developed polyherbal formulation for antiobesity. Int J Pharm Pharm Sci 2012;4(3):505-10.
28. Badgujar N, Mistry K, Patel J. V. negundo, L. camara and B. variegata plants leaf extract exhibit considerable in vitro antioxidant and anticancer activities. Int J Pharm Pharm Sci 2017;9(5):227-32.
29. Keser S, Celik S, Turkoglu S, Yilmaz O, Turkoglu I. Hydrogen peroxide radical scavenging and total antioxidant activity of hawthorn. Chem J 2012;2:9-12.
30. Hazra B, Biswas S, Mandal N. Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complement Altern Med 2008;8:63.
31. Omoya F, Akharaiyi F. Mixture of honey and ginger extract for antibacterial assessment on some clinical isolates. Int J Pharm Biomed Res 2011;2(1):39-47.
32. Sreeramulu D, Reddy CV, Chauhan A, Balakrishna N, Raghunath M. Natural antioxidant activity of commonly consumed plant foods in India: Effect of domestic processing. Oxid Med Cell Longev 2013;2013:369479.
33. Cai H, Dikalov S, Griendling KK, Harrison DG. Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: Comparison of sensitivity and specificity. Methods Mol Med 2007;139:293-311.
34. Chang CL, Lin CS, Lai GH. Phytochemical characteristics, free radical scavenging activities, and neuroprotection of five medicinal plant extracts. Evid Based Complement Alternat Med 2012;2012:984295.
35. Tsai PJ, Tsai TH, Yu CH, Ho SC. Evaluation of NO-suppressing activity of several Mediterranean culinary spices. Food Chem Toxicol 2007;45(3):440-7.
36. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65(1-2):55-63.
37. Basheer HS, Noordin MI, Ghareeb MM. Characterization of microemulsions prepared using isopropyl palmitate with various surfactants and cosurfactants. Trop J Pharm Res 2013;12(3):305-10.
38. Croom E, Pace R, Paletti A, Sardone N, Gray D. Single-laboratory validation for the determination of terpene lactones in Ginkgo biloba dietary supplement crude materials and finished products by high-performance liquid chromatography with evaporative light-scattering detection. J AOAC Int 2007;90(3):647-58.
39. Yao X, Shang E, Zhou G, Tang Y, Guo S, Su S, et al. Comparative characterization of total flavonol glycosides and terpene lactones at different ages, from different cultivation sources and genders of Ginkgo biloba leaves. Int J Mol Sci 2012;13(8):10305-15.
40. Zhang Q, Chen LJ, Ye HY, Gao L, Hou W, Tang M, et al. Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography. J Sep Sci 2007;30(13):2153-9.
41. Mandal S, Mandal SS. Research paper microemulsion drug delivery system: A platform for improving dissolution rate of poorly water soluble drug. Int J Pharm Sci Nanotechnol 2011;3(4):1214-9.
42. Syed HK, Peh KK. Identification of phases of various oil, surfactant/co-surfactants and water system by ternary phase diagram. Acta Pol Pharm 2014;71(2):301-9.
43. Koga K, Nishimon Y, Ueta H, Matsuno K, Takada K. Utility of nano-sized, water-in-oil emulsion as a sustained release formulation of glycyrrhizin. Biol Pharm Bull 2011;34(2):300-5.
44. Saumya S, Basha P. In vitro evaluation of free radical scavenging activities of Panax ginseng and Lagerstroemia speciosa: A comparative analysis. Int J Pharm Pharm Sci 2011;3:165-9.
45. Vicentini FT, Vaz MM, Fonseca YM, Bentley MV, Fonseca MJ. Characterization and stability study of a water-in-oil microemulsion incorporating quercetin. Drug Dev Ind Pharm 2011;37(1):47-55.
46. Plapied L, Duhem N, des Rieux A, Préat V. Fate of polymeric nanocarriers for oral drug delivery. Curr Opin Colloid Interface Sci 2011;16:228-37.
47. Hicks CR. Fundamental Concepts in the Design of Experiments. New York: Holt, Rinehart and Winston; 1964.
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How to Cite
Singh, M., S. P. Singh, and R. R. “ANTIOXIDANT, CYTOTOXICITY, AND STABILITY EVALUATION OF GINKGO BILOBA EXTRACT-BASED MICROEMULSIONS FOR ENHANCED THERAPEUTIC ACTIVITY”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 10, no. 8, Aug. 2017, pp. 335-40, doi:10.22159/ajpcr.2017.v10i8.19537.
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