, Volume 70, Issue 2, pp 713–728 | Cite as

Phytochemical study and evaluation of cytotoxicity, mutagenicity, cell cycle kinetics and gene expression of Bauhinia holophylla (Bong.) Steud. in HepG2 cells in vitro

  • Diego Luis RibeiroEmail author
  • Heloísa Lizotti Cilião
  • Ana Flávia Leal Specian
  • Juliana Mara Serpeloni
  • Marcelo Tempesta De Oliveira
  • Eliana Aparecida Varanda
  • Wagner Vilegas
  • Luiz Leonardo Saldanha
  • Wilner Martínez-López
  • Anne Lígia Dokkedal
  • Ilce Mara Syllos Cólus
Original Article


Bauhinia holophylla (Bong.) Steud. (Fabaceae) is a plant used in Brazilian folk medicine to treat diabetes and inflammation. This study evaluated the phytochemical properties, cytotoxic, apoptotic, mutagenic/antimutagenic effects and alterations in gene expression (RNAm) in HepG2 cells treated with the B. holophylla extract. The phytochemical profile highlight the presence of flavonoids isorhamentin and quercetin derivates. The MTT assay was used to evaluate the cytotoxicity of different concentrations for different treatment times. Three concentrations (7.5, 15, 30 µg/mL) were chosen for assessment of apoptosis (AO/EB), mutagenicity (micronucleus), and cell cycle kinetics (flow cytometry). Thereafter, the concentration of 7.5 µg/mL was chosen to evaluate the protective effects against DNA damage induced by benzo[a]pyrene (B[a]P). At concentrations higher than 7.5 µg/mL (between 10 and 50 µg/mL), the extract was cytotoxic, induced apoptosis, and caused antiproliferative effects. However, it did not induce micronucleus and a reduction of apoptotic and micronucleated cells was observed in treatments that included the extract and B[a]P. The protective effect is attributable to the presence of flavonoids, described as antioxidants, inhibitors of DNA adduct and activators of detoxifying enzymes. The results of the present study such as absence of cytotoxic and mutagenic effects and protective effects against known carcinogens suggest that B. holophylla has potential for use soon as herbal medicine.


Antimutagenic Micronucleus Flavonoids Flow cytometry RT-qPCR 



The authors also wish to thank Dr. E. N. Itano for the use of the spectrophotometer; the Service of Flow Cytometry and Cell Classification of the Institute of Biological Research Clemente Estable (Uruguay) for the use of the FACSVantage Flow Cytometer.


The authors thank the financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) in the BIOTA Program (2009/52237-9; 2009/16147-5; 2012/01996-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CAPES-PROAP); the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a scholarship granted to masters student Ribeiro, D.L and for productivity grant to Cólus, I.M.S, Varanda, E.A. and Vilegas, W.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10616_2017_173_MOESM1_ESM.docx (493 kb)
Supplementary material 1 (DOCX 493 kb)


  1. Agrawal RC, Pandey S (2009) Evaluation of anticarcinogenic and antimutagenic potential of Bauhinia variegata extract in Swiss albino mice. Asian Pac J Cancer Prev 10:913–916Google Scholar
  2. Andersen OM, Markhan KR (2006) Flavonoides: chemistry, biochemistry and applications. Taylor & Francis group, New YorkGoogle Scholar
  3. Aranda PS, LaJoie DM, Jorcyk CL (2012) Bleach gel: a simple agarose gel for analyzing RNA quality. Electrophoresis 33:366–369CrossRefGoogle Scholar
  4. Atienzar FA, Novik EI, Gerets HH, Parekh A, Delatour C, Cardenas A, MacDonald J, Yarmush ML, Dhalluin S (2014) Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans. Toxicol Appl Pharmacol 275:44–61CrossRefGoogle Scholar
  5. Bresolin S, Vargas VMF (1993) Mutagenic potencies of medicinal plants screened in the Ames test. Phytother Res 7:260–262CrossRefGoogle Scholar
  6. Burczynski ME, Penning TM (2000) Genotoxic polycyclic aromatic hydrocarbon ortho-quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor. Cancer Res 60:908–915Google Scholar
  7. Caiment F, Gaj S, Claessen S, Kleinjans J (2015) High-throughput data integration of RNA–miRNA–circRNA reveals novel insights into mechanisms of benzo[a]pyrene-induced carcinogenicity. Nucleic Acids Res 43:2525–2534CrossRefGoogle Scholar
  8. Cardozo TA, Rosa DP, Feiden IR, Rocha JAV, D’Avila de Oliveira NC, Pereira TS, Pastoriza TF, Marques DM, Lemos CT, Terra NR, Vargas VMF (2006) Genotoxicity and toxicity assessment in urban hydrographic basins. Mutat Res 603:83–96CrossRefGoogle Scholar
  9. Cechinel Filho V (2009) Chemical composition and biological potential of plants from the genus Bauhinia. Phytother Res 23:1347–1354CrossRefGoogle Scholar
  10. Chen Z, Zheng S, Li L, Jiang H (2014) Metabolism of flavonoids in human: a comprehensive review. Curr Drug Metab 15:48–61CrossRefGoogle Scholar
  11. Cuyckens F, Claeys M (2004) Mass spectrometry in the structural analysis of flavonoids. J Mass Spectrom 39:1–15CrossRefGoogle Scholar
  12. Da Justa Neves DB, Caldas ED (2015) Dietary supplements: International legal framework and adulteration profiles, and characteristics of products on the Brazilian clandestine market. Regul Toxicol Pharm 73:93–104CrossRefGoogle Scholar
  13. Da Silva KL, Cechinel Filho VC (2002) Plantas do gênero Bauhinia: composição química e potencial farmacológico. Quim Nova 25:449–454CrossRefGoogle Scholar
  14. David B, Wolfender JL, Dias DA (2015) The pharmaceutical industry and natural products: historical status and new trends. Phytochem Rev 14:299–315CrossRefGoogle Scholar
  15. Delgado ME, Haza AI, Arranz N, García A, Morales P (2008) Dietary polyphenols protect against N-nitrosamines and benzo(a)pyrene induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 human hepatoma cells. Eur J Nutr 47:479–490CrossRefGoogle Scholar
  16. Eastmond DA, Tucker JD (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environ Mol Mutagen 13:34–43CrossRefGoogle Scholar
  17. Estrada O, Hasegawa M, Gonzalez-Mujíca F, Motta N, Perdomo E, Solorzano A, Mendéz J, Mendéz B, Zea EG (2005) Evaluation of flavonoids from Bauhinia megalandra leaves as inhibitors of glucose-6-phosphatase system. Phytother Res 19:859–863CrossRefGoogle Scholar
  18. European Food Safety Authority (EFSA) (2008) Guidance document on the submission of a dossier on a substance to be used in Food Contact Materials for evaluation by EFSA by the Panel on food additives, flavourings, processing aids and materials in contact with food (AFC). Accessed 8 Jan 2017
  19. FDA (Food and Drug Administration) (2006) Guidance for industry and review staff recommended approaches to integration of genetic toxicology study results. Accessed 18 Dec 2016
  20. Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Prot 2:1084–1104CrossRefGoogle Scholar
  21. Geller FC, Teixeira MR, Pereira ABD, Dourado LPA, Souza DG, Braga FC, Simões CMO (2015) Evaluation of the wound healing properties of Hancornia speciosa leaves. Phytother Res 29:1887–1893CrossRefGoogle Scholar
  22. Gonzalez-Mujica F, Motta N, Márquez AH, Capote-Zulueta J (2003) Effects of Bauhinia megalandra aqueous leaf extract on intestinal glucose absorption and uptake by enterocyte brush border membrane vesicles. Fitoterapia 74:84–90CrossRefGoogle Scholar
  23. Gonzalez-Mujica F, Motta N, Estrada O, Perdomo E, Méndez J, Hasegawa M (2005) Inhibition of hepatic neoglucogenesis and glucose-6-phosphatase by quercetin 3-o-α (2″-galloyl) rhamnoside isolated from Bauhinia megalandra leaves. Phytother Res 19:624–627CrossRefGoogle Scholar
  24. Hatia S, Septembre-Malaterre A, Le Sage F, Badiou-Bénéteau A, Baret P, Payet B, Lefebvre d’hellencourt C, Gonthier MP (2014) Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress. Free Radic Res 48:387–401CrossRefGoogle Scholar
  25. Kim KC, Lee IK, Kang KA, Kim HS, Kang SS, Hyun JW (2012) Baicalein (5,6,7-trihydroxyflavone) reduces oxidative stress-induced DNA damage by upregulating the DNA repair system. Cell Biol Toxicol 28:421–433CrossRefGoogle Scholar
  26. Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M, Lorge E, Norppa H, Surrales J, Von der Hude W, Wataka A (2000) Report from the in vitro micronucleus assay working group. Environ Mol Mutagen 35:167–172CrossRefGoogle Scholar
  27. Lee SH, Yumnam S, Hong GE, Raha S, Venkatarame Gowda Saralamma V, Lee HJ, Heo JD, Lee SJ, Lee W-S, Kim E-H, Park HS, Kim GS (2015) Flavonoids of Korean Citrus aurantium L. Induce apoptosis via intrinsic pathway in human hepatoblastoma HepG2 cells. Phytother Res 29:1940–1949CrossRefGoogle Scholar
  28. Lewis GP, Schrire BD, Mackinder BA, Lock JM (2005) Legumes of the world. Royal Botanic Gardens, Kew, LondonGoogle Scholar
  29. Llana-Ruiz-Cabello M, Pichardo S, Maisanaba S, Puerto M, Prieto AI, Gutiérrez-Praena D, Jos A, Cameán AM (2015) In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: a review. Food Chem Toxicol 81:9–27CrossRefGoogle Scholar
  30. McGahon AJ, Martin SJ, Bissonnette RP, Mahboudi A, Shi Y, Mogil RJ, Nishioka WK, Green DR (1995) The end of the (cell) line: methods for the study of apoptosis in vitro. In: Schurartz LM, Osbourne BA (eds) Methods in cell biology. Academic Press, San Diego, pp 153–184Google Scholar
  31. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  32. Obici S, Otobone FJ, Sela VRS, Ishida K, Silva JC, Nakamura CV, Cortez DAG, Audi EA (2008) Preliminary toxicity study of dichloromethane extract of Kielmeyera coriacea stems inmiceandrats. J Ethnopharmacol 115:131–139CrossRefGoogle Scholar
  33. OECD (Organization for Economic Co-operation and Development) (2010) Series on testing and assessment, No. 129. Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic toxicity, ParisGoogle Scholar
  34. Ormerod MG (2000) Analysis of DNA—general methods, in flow cytometry. In: Ormerod MG (ed) A practical approach. IRL Press, Oxford, pp 83–97Google Scholar
  35. Pandey S, Agrawal RC (2010) Clastogenic analysis of Bauhinia variegata bark extract using micronucleus assay in mouse bone marrow cells. Am-Eurasian J Toxicol Sci 2:112–114Google Scholar
  36. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefGoogle Scholar
  37. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36CrossRefGoogle Scholar
  38. Rajkapoor B, Jayakar B, Murugesh N, Sakthisekaran D (2006) Chemoprevention and cytotoxic effect of Bauhinia variegata against N-nitrosodiethylamine induced liver tumors and human cancer cell lines. J Ethnopharmacol 104:407–409CrossRefGoogle Scholar
  39. Ramos AA, Lima CF, Pereira ML, Fernandes-Ferreira M, Pereira-Wilson C (2008) Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells: evalution by the comet assay. Toxicol Lett 177:66–73CrossRefGoogle Scholar
  40. Ribeiro DL, Cilião HL, Specian AFL, Serpeloni JM, De Souza MF, Tangerina MMP, Vilegas W, Boldrin PK, Resende FA, Varanda EA, Martínez-López W, Cólus IMS (2015) Chemical and biological characterisation of Machaerium hirtum (Vell.) Stellfeld: absence of cytotoxicity and mutagenicity and possible chemopreventive potential. Mutagenesis 31:147–160CrossRefGoogle Scholar
  41. Rodeiro I, Delgado R, Garrido G (2014) Effects of a Mangifera indica L. stem bark extract and mangiferin on radiation-induced DNA damage in human lymphocytes and lymphoblastoid cells. Cell Prolif 47:48–55CrossRefGoogle Scholar
  42. Roleira FM, Tavares-da-Silva EJ, Varela CL, Costa SC, Silva T, Garrido J, Borges F (2015) Plant derived and dietary phenolic antioxidants: anticancer properties. Food Chem 183:235–258CrossRefGoogle Scholar
  43. Rozza AL, Cesar DAS, Pieroni LG, Saldanha LL, Dokkedal AL, De-Faria FM, Souza-Brito ARM, Vilegas W, Takariha RK, Pellizzon CH (2015) Antiulcerogenic activity and toxicity of Bauhinia holophylla hydroalcoholic extract. Evid Based Complement Alternat Med 2015:1–9CrossRefGoogle Scholar
  44. Rupasinghe HV, Nair SV, Robinson RA (2014) Chemopreventive properties of fruit phenolic compounds and their possible mode of actions. Stud Nat Prod Chem 42:229CrossRefGoogle Scholar
  45. Santos FJBD, Moura DJ, Péres VF, Sperotto ARM, Camarão EB, Cavalcante AACM, Saffi J (2012) Genotoxic and mutagenic properties of Bauhinia platypetala extract, a traditional Brazilian medicinal plant. J Ethnopharmacol 144:474–482CrossRefGoogle Scholar
  46. Serpeloni JM, Specian AFL, Ribeiro DL, Tuttis K, Vilegas W, Martínez-López W, Dokkedal AL, Saldanha LL, Cólus IMS, Varanda EA (2015) Antimutagenicity and induction of antioxidant defense by flavonoid rich extract of Myrcia bella Cambess. in normal and tumor gastric cells. J Ethnopharmacol 176:345–355CrossRefGoogle Scholar
  47. Shiizaki K, Kawanishi M, Yagi T (2013) Dioxin suppresses benzo[a]pyrene-induced mutations and DNA adduct formation through cytochrome P450 1A1 induction and (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide inactivation in human hepatoma cells. Mutat Res 750:77–85CrossRefGoogle Scholar
  48. Sisenando HAAACN, Macedo MFS, Saturnino ACRD, Coelho LCBB, Medeiros SRB (2009) Evaluation of the genotoxic potential of Bauhinia monandra leaf lectin (BmoLL). Food Chem Toxicol 47:303–308CrossRefGoogle Scholar
  49. Sison-Young RL, Mitsa D, Jenkins RE, Mottram D, Alexandre E, Richert L, Aerts H, Weaver RJ, Jones RP, Johann E, Hewitt PG, Ingelman-Sundberg M, Goldring CEP, Kitteringham NR, Park K (2015) Comparative proteomic characterization of 4 human liver-derived single cell culture models reveals significant variation in the capacity for drug disposition, bioactivation, and detoxication. Toxicol Sci 147:412–424CrossRefGoogle Scholar
  50. Verschaeve L, Kestens V, Taylor JLS, Elgorashi EE, Maes A, Van Puyvelde L, DeKimpe N, Van Staden J (2004) Investigation of the antimutagenic effects of selected South African medicinal plant extracts. Toxicol In Vitro 18:29–35CrossRefGoogle Scholar
  51. Waizenegger J, Lenze D, Luckert C, Seidel A, Lampen A, Hessel S (2015) Dose-dependent induction of signaling pathways by the flavonoid quercetin in human primary hepatocytes: a transcriptomic study. Mol Nutr Food Res 59:1117–1129CrossRefGoogle Scholar
  52. Waters MD, Brady AL, Stack HF, Brockman HE (1990) Antimutagenicity profiles for some model compounds. Mutat Res 238:57–85CrossRefGoogle Scholar
  53. Wu XJ, Lu WQ, Mersch-Sundermann V (2003) Benzo(a)pyrene induced micronucleus formation was modulated by persistent organic pollutants (POPs) in metabolically competent human HepG2 cells. Toxicol Lett 144:143–150CrossRefGoogle Scholar
  54. Zhang H, Zhang M, Yu L, Zhao Y, He N, Yang X (2012) Antitumor activities of quercetin and quercetin-5′,8-disulfonate in human colon and breast cancer cell lines. Food Chem Toxicol 50:1589–1599CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Diego Luis Ribeiro
    • 1
    Email author
  • Heloísa Lizotti Cilião
    • 1
  • Ana Flávia Leal Specian
    • 1
  • Juliana Mara Serpeloni
    • 1
    • 2
  • Marcelo Tempesta De Oliveira
    • 1
  • Eliana Aparecida Varanda
    • 2
  • Wagner Vilegas
    • 3
  • Luiz Leonardo Saldanha
    • 4
    • 5
  • Wilner Martínez-López
    • 6
  • Anne Lígia Dokkedal
    • 5
  • Ilce Mara Syllos Cólus
    • 1
  1. 1.Department of General Biology, Biological Sciences CenterState University of Londrina - UELLondrinaBrazil
  2. 2.Department of Biological Sciences, Faculty of Pharmaceutical Sciences of AraraquaraSão Paulo State University - UNESPAraraquaraBrazil
  3. 3.Campus Litoral Paulista, São Paulo State University - UNESPSão VicenteBrazil
  4. 4.Department of Biological Sciences, Faculty of Sciences of BauruSão Paulo State University – UNESPBauruBrazil
  5. 5.Department of Botany, Biosciences InstituteSão Paulo State University - UNESPBotucatuBrazil
  6. 6.Institute of Biological Investigation Clemente EstableMontevideoUruguay

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