Antioxidant and antigenotoxic activities of Ginkgo biloba L. leaf extract are retained after in vitro gastrointestinal digestive conditions

  • Daniela Oliveira
  • Cheryl Latimer
  • Pier Parpot
  • Chris I. R. Gill
  • Rui OliveiraEmail author
Original Contribution



The recognized biological properties of Ginkgo biloba extracts potentiate their utilization as an ingredient for functional foods. However, the digestive conditions can affect the chemical composition of the extracts and consequently their biological properties, which can lead to food safety problems. Thus, the impact of in vitro-simulated upper gastrointestinal tract digestion on the chemical composition and bioactivity of Ginkgo biloba leaf extract (GBE) was evaluated.


Physicochemical conditions of human digestion were simulated in vitro, and its impact on the chemical composition of GBE was investigated by electrospray ionization-mass spectrometry. The persistence of bioactivity was investigated by subjecting GBE and the in vitro digested extract (DGBE) to the same methodology. Antioxidant properties were assessed using 2′,7′-dichlorofluorescein diacetate to measure the intracellular oxidation of Schizosaccharomyces pombe cells pre-incubated with GBE or DGBE and exposed to H2O2. Antigenotoxicity was tested by comet assay in HT-29 colon cancer cells pre-incubated with GBE or DGBE and challenged with H2O2.


The chemical analysis revealed a considerable change in chemical composition upon digestion. Pre-incubation with GBE or DGBE attenuated the H2O2-imposed intracellular oxidation in wild-type S. pombe cells, unlike the oxidative stress response-affected mutants sty1 and pap1, and decreased H2O2-induced DNA damage in HT-29 cells. The extracts did not induce toxicity in these eukaryotic models.


The chemical composition of GBE was affected by in vitro digestion, but the antioxidant and antigenotoxic activities persisted. Therefore, G. biloba extract may be suitable for use as food additive and contribute to a healthy colon.


Antioxidant Antigenotoxic DNA damage Comet assay Schizosaccharomyces pombe Colonocytes 



In vitro digested Ginkgo biloba leaf extract


Gallic acid equivalents


Ginkgo biloba leaf extract


2′,7′-Dichlorofluorescein diacetate


Mass spectrometry


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Ethylenediaminetetraacetic acid, disodium salt


Phosphate-buffered saline


Propidium iodide


Reactive oxygen species


Total phenolic content





This work is supported by European Investment Funds by FEDER/COMPETE/POCI—Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT—Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019. This work has also been funded by national funds through FCT—Fundação para a Ciência e a Tecnologia for Centre of Chemistry (UID/QUI/00686/2013 and UID/QUI/0686/2016).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical standards

The manuscript does not contain clinical studies or patient data.

Supplementary material

394_2019_1915_MOESM1_ESM.docx (38.3 mb)
Supplementary material 1 (DOCX 39225 KB)


  1. 1.
    Dolatabadi JEN, Kashanian S (2010) A review on DNA interaction with synthetic phenolic food additives. Food Res Int 43(5):1223–1230. Google Scholar
  2. 2.
    Lugasi A, Dworschak E, Horvatovich P (1999) Additional information to the in vitro antioxidant activity of Ginkgo biloba L. Phytother Res 13(2):160–162Google Scholar
  3. 3.
    Boghdady NAE (2013) Antioxidant and antiapoptotic effects of proanthocyanidin and Ginkgo biloba extract against doxorubicin-induced cardiac injury in rats. Cell Biochem Funct 31(4):344–351. Google Scholar
  4. 4.
    Mahadevan S, Park Y (2008) Multifaceted therapeutic benefits of Ginkgo biloba L. chemistry, efficacy, safety, and uses. J Food Sci 73(1):R14–R19. Google Scholar
  5. 5.
    He J, Lin J, Li J, Zhang JH, Sun XM, Zeng CM (2009) Dual effects of Ginkgo biloba leaf extract on human red blood cells. Basic Clin Pharmacol Toxicol 104(2):138–144. Google Scholar
  6. 6.
    Ude C, Schubert-Zsilavecz M, Wurglics M (2013) Ginkgo biloba extracts: a review of the pharmacokinetics of the active ingredients. Clin Pharmacokinet 52(9):727–749. Google Scholar
  7. 7.
    Smith JV, Luo Y (2004) Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol Biotechnol 64(4):465–472. Google Scholar
  8. 8.
    Marques F, Azevedo F, Johansson B, Oliveira R (2011) Stimulation of DNA repair in Saccharomyces cerevisiae by Ginkgo biloba leaf extract. Food Chem Toxicol 49(6):1361–1366. Google Scholar
  9. 9.
    Vilar JB, Leite KR, Chen Chen L (2009) Antimutagenicity protection of Ginkgo biloba extract (Egb 761) against mitomycin C and cyclophosphamide in mouse bone marrow. Genet Mol Res 8(1):328–333Google Scholar
  10. 10.
    Gohil K, Moy RK, Farzin S, Maguire JJ, Packer L (2000) mRNA expression profile of a human cancer cell line in response to Ginkgo biloba extract: induction of antioxidant response and the golgi system. Free Radic Res 33(6):831–849. Google Scholar
  11. 11.
    Jeyakumar A, Dissabandara L, Gopalan V (2017) A critical overview on the biological and molecular features of red and processed meat in colorectal carcinogenesis. J Gastroenterol 52(4):407–418. Google Scholar
  12. 12.
    Coates EM, Popa G, Gill CI, McCann MJ, McDougall GJ, Stewart D, Rowland I (2007) Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models of colon cancer. J Carcinog 6:4. Google Scholar
  13. 13.
    Demeyer D, Mertens B, De Smet S, Ulens M (2016) Mechanisms linking colorectal cancer to the consumption of (processed) red meat: a review. Crit Rev Food Sci Nutr 56(16):2747–2766. Google Scholar
  14. 14.
    Araújo JR, Goncalves P, Martel F (2011) Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutr Res 31(2):77–87. Google Scholar
  15. 15.
    Singleton VL, Orthofer R, Lamuela-Raventós RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. In: Methods in enzymology, vol 299. Academic Press, New York, pp 152–178. Google Scholar
  16. 16.
    Vivancos AP, Jara M, Zuin A, Sanso M, Hidalgo E (2006) Oxidative stress in Schizosaccharomyces pombe: different H2O2 levels, different response pathways. Mol Genet Genom 276(6):495–502. Google Scholar
  17. 17.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63. Google Scholar
  18. 18.
    Brown EM, Latimer C, Allsopp P, Ternan NG, McMullan G, McDougall GJ, Stewart D, Crozier A, Rowland I, Gill CI (2014) In vitro and in vivo models of colorectal cancer: antigenotoxic activity of berries. J Agric Food Chem 62(18):3852–3866. Google Scholar
  19. 19.
    Collins AR (2004) The comet assay for DNA damage and repair. Mol Biotechnol 26(3):249. Google Scholar
  20. 20.
    Lee H, Lim H, Yang J, Hong J (2013) Rapid determination of ginkgolic acids in Ginkgo biloba leaf using online column switching high-performance liquid chromatography-diode array detection and confirmation by liquid chromatography-tandem mass spectrometry. Bull Korean Chem Soc 34(12):3629–3634. Google Scholar
  21. 21.
    Lin LZ, Chen P, Ozcan M, Harnly JM (2008) Chromatographic profiles and identification of new phenolic components of Ginkgo biloba leaves and selected products. J Agric Food Chem 56(15):6671–6679. Google Scholar
  22. 22.
    He X-g, Bernart MW, Nolan GS, Lin L-z, Lindenmaier MP (2000) High-performance liquid chromatography—electrospray ionization-mass spectrometry study of ginkgolic acid in the leaves and fruits of the ginkgo tree (Ginkgo biloba). J Chromatogr Sci 38(4):169–173. Google Scholar
  23. 23.
    Pereira E, Barros L, Dueñas M, Antonio AL, Santos-Buelga C, Ferreira ICFR (2015) Gamma irradiation improves the extractability of phenolic compounds in Ginkgo biloba L. Ind Crops Prod 74:144–149. Google Scholar
  24. 24.
    Marín L, Miguélez EM, Villar CJ, Lombó F (2015) Bioavailability of dietary polyphenols and gut microbiota metabolism: antimicrobial properties. BioMed Res Int 2015:18. Google Scholar
  25. 25.
    Linoleic acid (2016) MassBank Accessed 4 Jan 2018
  26. 26.
    Karaś M, Jakubczyk A, Szymanowska U, Złotek U, Zielińska E (2017) Digestion and bioavailability of bioactive phytochemicals. Int J Food Sci Technol 52(2):291–305. Google Scholar
  27. 27.
    McDougall GJ, Dobson P, Smith P, Blake A, Stewart D (2005) Assessing potential bioavailability of raspberry anthocyanins using an in vitro digestion system. J Agric Food Chem 53(15):5896–5904. Google Scholar
  28. 28.
    Goh LML, Barlow PJ (2004) Flavonoid recovery and stability from Ginkgo biloba subjected to a simulated digestion process. Food Chem 86(2):195–202. Google Scholar
  29. 29.
    Yokomizo A, Moriwaki M (2005) Myricitrin degraded by simulated digestion inhibits oxidation of human low-density lipoprotein. Biosci Biotechnol Biochem 69(4):693–699. Google Scholar
  30. 30.
    Bermúdez-Soto MJ, Tomás-Barberán FA, García-Conesa MT (2007) Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion. Food Chem 102(3):865–874. Google Scholar
  31. 31.
    Huang D, Ou B, Prior RL (2005) The chemistry behind antioxidant capacity assays. J Agric Food Chem 53(6):1841–1856. Google Scholar
  32. 32.
    Yen G-C, Chen H-Y, Peng H-H (1997) Antioxidant and pro-oxidant effects of various tea extracts. J Agric Food Chem 45(1):30–34. Google Scholar
  33. 33.
    Llópiz N, Puiggròs F, Céspedes E, Arola L, Ardévol A, Bladé C, Salvadó MJ (2004) Antigenotoxic effect of grape seed procyanidin extract in Fao cells submitted to oxidative stress. J Agric Food Chem 52(5):1083–1087. Google Scholar
  34. 34.
    Jang I-C, Jo E-K, Bae M-S, Lee H-J, Jeon G-I, Park E, Yuk H-G, Ahn G-H, Lee S-C (2010) Antioxidant and antigenotoxic activities of different parts of persimmon (Diospyros kaki cv. Fuyu) fruit. J Med Plant Res 4(2):155–160Google Scholar
  35. 35.
    He Y-T, Xing S-S, Gao L, Wang J, Xing Q-C, Zhang W (2014) Ginkgo biloba attenuates oxidative DNA damage of human umbilical vein endothelial cells induced by intermittent high glucose. Pharmazie 69(3):203–207. Google Scholar
  36. 36.
    Križková L, Chovanová Z, Ďuračková Z, Krajčovič J (2008) Antimutagenic in vitro activity of plant polyphenols: Pycnogenol® and Ginkgo biloba extract (EGb 761). Phytother Res 22(3):384–388. Google Scholar
  37. 37.
    Kahle K, Kraus M, Scheppach W, Richling E (2005) Colonic availability of apple polyphenols—a study in ileostomy subjects. Mol Nutr Food Res 49(12):1143–1150. Google Scholar
  38. 38.
    Borges G, Lean ME, Roberts SA, Crozier A (2013) Bioavailability of dietary (poly) phenols: a study with ileostomists to discriminate between absorption in small and large intestine. Food Funct 4(5):754–762. Google Scholar
  39. 39.
    Stalmach A, Edwards CA, Wightman JD, Crozier A (2012) Gastrointestinal stability and bioavailability of (poly)phenolic compounds following ingestion of Concord grape juice by humans. Mol Nutr Food Res 56(3):497–509. Google Scholar
  40. 40.
    Brown EM, McDougall GJ, Stewart D, Pereira-Caro G, Gonzalez-Barrio R, Allsopp P, Magee P, Crozier A, Rowland I, Gill CI (2012) Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation. PLoS One 7(11):e49740. Google Scholar
  41. 41.
    Del Pino-García R, Rivero-Pérez MD, González-SanJosé ML, Ortega-Heras M, García Lomillo J, Muñiz P (2017) Chemopreventive potential of powdered red wine pomace seasonings against colorectal cancer in HT-29 Cells. J Agric Food Chem 65(1):66–73. Google Scholar
  42. 42.
    El Mesallamy HO, Metwally NS, Soliman MS, Ahmed KA, Abdel Moaty MM (2011) The chemopreventive effect of Ginkgo biloba and Silybum marianum extracts on hepatocarcinogenesis in rats. Cancer Cell Int 11(1):38. Google Scholar
  43. 43.
    Keles MS, Demirci N, Yildirim A, Atamanalp SS, Altinkaynak K (2008) Protective effects of N-acetylcysteine and Ginkgo biloba extract on ischaemia-reperfusion-induced hepatic DNA damage in rats. Clin Exp Med 8(4):193–198. Google Scholar
  44. 44.
    Abdel-Wahab BA, Abd El-Aziz SM (2012) Ginkgo biloba protects against intermittent hypoxia-induced memory deficits and hippocampal DNA damage in rats. Phytomedicine 19(5):444–450. Google Scholar
  45. 45.
    Fang L, Neutzner A, Turtschi S, Flammer J, Mozaffarieh M (2015) The effect of Ginkgo biloba and Nifedipine on DNA breaks in circulating leukocytes of glaucoma patients. Expert Rev Ophthalmol 10(3):313–318. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biology, Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)University of MinhoBragaPortugal
  2. 2.Nutrition Innovation Centre for Food and Health, Centre for Molecular BiosciencesUniversity of UlsterColeraineUK
  3. 3.Centre of ChemistryUniversity of MinhoBragaPortugal
  4. 4.Centre of Biological EngineeringUniversity of MinhoBragaPortugal

Personalised recommendations