Investigational New Drugs

, Volume 37, Issue 4, pp 602–615 | Cite as

Hexane partition from Annona crassiflora Mart. promotes cytotoxity and apoptosis on human cervical cancer cell lines

  • Viviane A. O. Silva
  • Ana Laura V. Alves
  • Marcela N. Rosa
  • Larissa R. V. Silva
  • Matias E. Melendez
  • Fernanda P. Cury
  • Izabela N. F. Gomes
  • Aline Tansini
  • Giovanna B. Longato
  • Olga Martinho
  • Bruno G. Oliveira
  • Fernanda E. Pinto
  • Wanderson Romão
  • Rosy I. M. A. Ribeiro
  • Rui M. ReisEmail author


Cervical cancer is the third most commonly diagnosed tumor type and the fourth cause of cancer-related death in females. Therapeutic options for cervical cancer patients remain very limited. Annona crassiflora Mart. is used in traditional medicine as antimicrobial and antineoplastic agent. However, little is known about its antitumoral properties. In this study the antineoplastic effect of crude extract and derived partitions from A. crassiflora Mart in cervical cancer cell lines was evaluated. The crude extract significantly alters cell viability of cervical cancer cell lines as well as proliferation and migration, and induces cell death in SiHa cells. Yet, the combination of the crude extract with cisplatin leads to antagonistic effect. Importantly, the hexane partition derived from the crude extract presented cytotoxic effect both in vitro and in vivo, and initiates cell responses, such as DNA damage (H2AX activity), apoptosis via intrinsic pathway (cleavage of caspase-9, caspase-3, poly (ADP-ribose) polymerase (PARP) and mitochondrial membrane depolarization) and decreased p21 expression by ubiquitin proteasome pathway. Concluding, this work shows that hexane partition triggers several biological responses such as DNA damage and apoptosis, by intrinsic pathways, and was also able to promote a direct decrease in tumor perimeter in vivo providing a basis for further investigation on its antineoplastic activity on cervical cancer.


Annona crassiflora Mart Natural compounds Hexane partition apoptosis Cytotoxity and cervical cancer cell lines 


Author contributions

Viviane A O Silva designed all the experiments as well as participated of data acquisition, its interpretation and drafted the manuscript. Viviane A O Silva, Ana Laura V Alves and Marcela N Rosa carried out the studies of cell culture including cytotoxicity and proliferation assay, wound healing migration assay, colony formation assay, and statistical analysis from Hexane partition. Viviane A O Silva, Marcela N Rosa and Larissa R V Silva carried out the studies of cell culture including cytotoxicity and proliferation assay, wound healing migration assay, drug combination studies and statistical analysis from crude extract. Aline Tansini helped to carry out flow cytometry assays and its interpretation. Matias Melendez, Giovanna Longato and Olga Martinho contributed to design some experiments, interpretation of data and involved in revising critically the manuscript. Ana Laura V Alves and Fernanda Cury helped to design and performed the in vivo experiments. Bruno G. Oliveira, Fernanda E. Pinto and Wanderson Romãohas been responsible for the preparation of extracts. Izabela Faria Gomes contributed to design some experiments and analysed about the profile of secondary compounds identified in the hexane partition. Rosy Ribeiro conceived the extracts and partition, participated in its design, interpretation of data. Rui Manuel Reis conceived the study, participated in its design and coordination, interpretation of data, drafted the manuscript and have been involved in revising it critically for important intellectual content. All authors read and approved the final manuscript.


This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT), CAPES, FAPEMIG, UFSJ and Barretos Cancer Hospital, all from Brazil.

Compliance with ethical standards

Conflict of interest

The authors confirm that this article content has no conflicts of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

10637_2018_657_MOESM1_ESM.docx (23.2 mb)
ESM 1 (DOCX 23754 kb)


  1. 1.
    Siegel RL, Miller KD, Jemal A (2017) Cancer statistics, 2017. CA Cancer J Clin 67(1):7–30. Google Scholar
  2. 2.
    Catarino R, Petignat P, Dongui G, Vassilakos P (2015) Cervical cancer screening in developing countries at a crossroad: emerging technologies and policy choices. World J Clin Oncol 6(6):281–290. Google Scholar
  3. 3.
    Florea AM, Busselberg D (2011) Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers 3(1):1351–1371. Google Scholar
  4. 4.
    Newman DJ, Cragg GM (2016) Natural products as sources of new drugs from 1981 to 2014. J Nat Prod 79(3):629–661. Google Scholar
  5. 5.
    Butler MS, Robertson AA, Cooper MA (2014) Natural product and natural product derived drugs in clinical trials. Nat Prod Rep 31(11):1612–1661. Google Scholar
  6. 6.
    Dutra RC, Campos MM, Santos AR, Calixto JB (2016) Medicinal plants in Brazil: pharmacological studies, drug discovery, challenges and perspectives. Pharmacol Res 112:4–29. Google Scholar
  7. 7.
    de Melo JG, Santos AG, de Amorim EL, do Nascimento SC, de Albuquerque UP (2011) Medicinal plants used as antitumor agents in Brazil: an ethnobotanical approach. Evid Based Complement Alternat Med 2011:365359. Google Scholar
  8. 8.
    Lopes JC, Mello-Silva R (2014) Diversity and characterization of Annonaceae from Brazil. Rev Bras Frutic 36:125–131. Google Scholar
  9. 9.
    Tundis R, Xiao J, Loizzo MR (2017) Annona species (Annonaceae): a rich source of potential antitumor agents? Ann N Y Acad Sci 1398(1):30–36. Google Scholar
  10. 10.
    Chatrou LWP, D M, Erkens RHJ, Couvreur TLP, Neubig KM, Abbott JR, Mols JB, Maas JW, Saunders RMK, Chase MW (2012) A new subfamilial and tribal classification of the pantropical flowering plant family Annonaceae informed by molecular phylogenetics. Bot J Linn Soc 169:5–40Google Scholar
  11. 11.
    Pimenta LP, Garcia GM, Goncalves SG, Dionisio BL, Braga EM, Mosqueira VC (2014) In vivo antimalarial efficacy of acetogenins, alkaloids and flavonoids enriched fractions from Annona crassiflora Mart. Nat Prod Res 28(16):1254–1259. Google Scholar
  12. 12.
    Rocha RS, Kassuya CAL, Formagio ASN, Mauro MO, Andrade-Silva M, Monreal ACD, Cunha-Laura AL, Vieira MC, Oliveira RJ (2016) Analysis of the anti-inflammatory and chemopreventive potential and description of the antimutagenic mode of action of the Annona crassiflora methanolic extract. Pharm Biol 54(1):35–47Google Scholar
  13. 13.
    Formagio AS, Vieira MC, Volobuff CR, Silva MS, Matos AI, Cardoso CA, Foglio MA, Carvalho JE (2015) In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae. Braz J Med Biol Res 48(4):308–315. Google Scholar
  14. 14.
    Pimenta L, Mendonça D, Pretti D, Cruz B, Leite E, De Oliveira M (2011) Evaluation of in vivo antitumor activity of Annona crassiflora wood extract. IJPSDR 3(3):270–273Google Scholar
  15. 15.
    Silva-Oliveira RJ, Silva VA, Martinho O, Cruvinel-Carloni A, Melendez ME, Rosa MN, de Paula FE, de Souza Viana L, Carvalho AL, Reis RM (2016) Cytotoxicity of allitinib, an irreversible anti-EGFR agent, in a large panel of human cancer-derived cell lines: KRAS mutation status as a predictive biomarker. Cell Oncol (Dordr) 39(3):253–263. Google Scholar
  16. 16.
    Silva VAO, Rosa MN, Miranda-Goncalves V, Costa AM, Tansini A, Evangelista AF, Martinho O, Carloni AC, Jones C, Lima JP, Pianowski LF, Reis RM (2018) Euphol, a tetracyclic triterpene, from Euphorbia tirucalli induces autophagy and sensitizes temozolomide cytotoxicity on glioblastoma cells. Investig New Drugs.
  17. 17.
    Moniz S, Martinho O, Pinto F, Sousa B, Loureiro C, Oliveira MJ, Moita LF, Honavar M, Pinheiro C, Pires M, Lopes JM, Jones C, Costello JF, Paredes J, Reis RM, Jordan P (2013) Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasiveness. Hum Mol Genet 22(1):84–95. Google Scholar
  18. 18.
    Martinho O, Silva-Oliveira R, Miranda-Goncalves V, Clara C, Almeida JR, Carvalho AL, Barata JT, Reis RM (2013) In vitro and in vivo analysis of RTK inhibitor efficacy and identification of its novel targets in Glioblastomas. Transl Oncol 6(2):187–196Google Scholar
  19. 19.
    Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzym Regul 22:27–55Google Scholar
  20. 20.
    Liu J, Cooks RG, Ouyang Z (2011) Biological tissue diagnostics using needle biopsy and spray ionization mass spectrometry. Anal Chem 83(24):9221–9225. Google Scholar
  21. 21.
    Pinheiro MLB, Xavier CM, ADLd S, Rabelo DM, Batista CL, Batista RL, Costa EV, Campos FR, Barison A, Valdez RH, Ueda-Nakamura T, Nakamura CV (2009) Acanthoic acid and other constituents from the stem of Annona amazonica (Annonaceae). J Braz Chem Soc 20:1095–1102Google Scholar
  22. 22.
    Alali FQ, Liu XX, McLaughlin JL (1999) Annonaceous acetogenins: recent progress. J Nat Prod 62(3):504–540. Google Scholar
  23. 23.
    FMAd S, Koolen HHF, RAd A, ADLd S, Pinheiro MLB, Costa EV (2012) Desreplicação de alcaloides aporfínicos e oxoaporfínicos de Unonopsis guatterioides por ESI-IT-MS. Quim Nova 35:944–947Google Scholar
  24. 24.
    Etcheverry S, Saphaz S, Fall D, Laurens A, Cavé A (1995) Annoglaucin, an acetogenin from Annona glauca. Phytochemistry 35(6):1423–1426.
  25. 25.
    FdCd N, Boaventura MAD, Assunção ACS, Pimenta LPS (2003) Acetogeninas de anonáceas isoladas de folhas de Rollinia laurifolia. Quím Nova 26:319–322Google Scholar
  26. 26.
    Lin RJ, Wu MH, Ma YH, Chung LY, Chen CY, Yen CM (2014) Anthelmintic activities of aporphine from Nelumbo nucifera Gaertn. cv. Rosa-plena against Hymenolepis nana. Int J Mol Sci 15(3):3624–3639. Google Scholar
  27. 27.
    TJ CVCO, Queiroga CS, MVS C-B, MFFM D, CUGBd L, BVdO S, JCLR P, MSd S, IMF S (2012) Chemical constituents of the leaves from Rollinia leptopetala R. E. Fries. Quím Nova 35:138–142. Google Scholar
  28. 28.
    Silva FMA, Silva Filho FA, Lima BR, Almeida RA, Soares ER, Koolen HHF, Souza ADL, Pinheiro MLB (2016) Chemotaxonomy of the Amazonian Unonopsis species based on leaf alkaloid fingerprint direct infusion ESI-MS and chemometric analysis. J Braz Chem Soc 27:599–604Google Scholar
  29. 29.
    Alali FQ, Kaakeh W, Bennett GW, McLaughlin JL (1998) Annonaceous acetogenins as natural pesticides: potent toxicity against insecticide-susceptible and -resistant German cockroaches (Dictyoptera: Blattellidae). J Econ Entomol 91(3):641–649Google Scholar
  30. 30.
    Mo S, Dong L, Hurst WJ, van Breemen RB (2013) Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry. Lipids 48(9):949–956. Google Scholar
  31. 31.
    FMAd S, BRd L, Soares ER, RAd A, FAd SF, Corrêa WR, Salvador MJ, AQLd S, Koolen HHF, ADLd S, Pinheiro MLB (2015) Polycarpol in Unonopsis, Bocageopsis and Onychopetalum Amazonian species: chemosystematical implications and antimicrobial evaluation. Rev Bras 25:11–15Google Scholar
  32. 32.
    Wele A, Zhang Y, Caux C, Brouard J-P, Dubost L, Guette C, Pousset J-L, Badiane M, Bodo B (2002) Isolation and structure of cyclosenegalins a and B, novel cyclopeptides from the seeds of Annona senegalensis. J Chem Soc Perkin Trans 1(23):2712–2718. Google Scholar
  33. 33.
    Hisham A, Sunitha C, Sreekala U, Pieters L, De Bruyne T, Van den Heuvel H, Claeys M (1994) Reticulacinone, an acetogenin from Annona reticulata. Phytochemistry 35(5):1325–1329. Google Scholar
  34. 34.
    Wiart C, Martin MT, Awang K, Hue N, Serani L, Laprevote O, Pais M, Rhamani M (2001) Sesquiterpenes and alkaloids from Scorodocarpus borneensis. Phytochemistry 58(4):653–656Google Scholar
  35. 35.
    Karapandzova M, Stefkov G, Cvetkovikj I, Stanoeva JP, Stefova M, Kulevanova S (2015) Flavonoids and other phenolic compounds in needles of Pinus peuce and other pine species from the Macedonian Flora. Nat Prod Commun 10(6):987–990Google Scholar
  36. 36.
    Da Silva ELM, Roblot F, Laprévote O, Sérani L, Cavé A (1997) Coriaheptocins A and B, the first Heptahydroxylated Acetogenins, isolated from the roots of Annona coriacea. J Nat Prod 60(2):162–167. Google Scholar
  37. 37.
    Rockenbach II, Jungfer E, Ritter C, Santiago-Schübel B, Thiele B, Fett R, Galensa R (2012) Characterization of flavan-3-ols in seeds of grape pomace by CE, HPLC-DAD-MSn and LC-ESI-FTICR-MS. Food Res Int 48(2):848–855. Google Scholar
  38. 38.
    Etse JT, Gray AI, Lavaud C, Massiot G, Nuzillard J-M, Waterman PG (1991) Chemistry of the annonaceae, part 29. Structure of mezzettiaside-2, −4, −5, −6 and −7, new partially esterified 1-O-octyl tri- and tetra-rhamnosyl derivatives from Mezzettia leptopoda. J Chem Soc Perkin Trans 1(4):861–864. Google Scholar
  39. 39.
    Gartel AL, Tyner AL (2002) The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 1(8):639–649Google Scholar
  40. 40.
    Bendjennat M, Boulaire J, Jascur T, Brickner H, Barbier V, Sarasin A, Fotedar A, Fotedar R (2003) UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell 114(5):599–610Google Scholar
  41. 41.
    Fragkos M, Jurvansuu J, Beard P (2009) H2AX is required for cell cycle arrest via the p53/p21 pathway. Mol Cell Biol 29(10):2828–2840Google Scholar
  42. 42.
    Martin-Cordero C, Leon-Gonzalez AJ, Calderon-Montano JM, Burgos-Moron E, Lopez-Lazaro M (2012) Pro-oxidant natural products as anticancer agents. Curr Drug Targets 13(8):1006–1028Google Scholar
  43. 43.
    Kuete V, Seo EJ, Krusche B, Oswald M, Wiench B, Schroder S, Greten HJ, Lee IS, Efferth T (2013) Cytotoxicity and pharmacogenomics of medicinal plants from traditional korean medicine. Evid Based Complement Alternat Med 2013:341724. Google Scholar
  44. 44.
    JM SMAP (1990) Assays related to cancer drug discovery. In: HOSTETTMANN K (ed) Methods in plant biochemistry: assays for bioactivity. Academic Press, London, pp 71–133Google Scholar
  45. 45.
    Matsushige A, Kotake Y, Matsunami K, Otsuka H, Ohta S, Takeda Y (2012) Annonamine, a new aporphine alkaloid from the leaves of Annona muricata. Chem Pharm Bull 60(2):257–259Google Scholar
  46. 46.
    Xu L, Li K, Sun N, Kong J (1992) Alkaloids of Annona reticulata L. Zhongguo Zhong Yao Za Zhi 17(5):295–296 inside backcoverGoogle Scholar
  47. 47.
    Wang X, Martindale JL, Holbrook NJ (2000) Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem 275(50):39435–39443. Google Scholar
  48. 48.
    Brozovic A, Osmak M (2007) Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance. Cancer Lett 251(1):1–16. Google Scholar
  49. 49.
    Abbas T, Dutta A (2009) p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 9(6):400–414. Google Scholar
  50. 50.
    Cagnol S, Chambard JC (2010) ERK and cell death: mechanisms of ERK-induced cell death--apoptosis, autophagy and senescence. FEBS J 277(1):2–21. Google Scholar
  51. 51.
    Gartel AL, Radhakrishnan SK (2005) Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Res 65(10):3980–3985. Google Scholar
  52. 52.
    Liu G, Lozano G (2005) p21 stability: linking chaperones to a cell cycle checkpoint. Cancer Cell 7(2):113–114Google Scholar
  53. 53.
    Atoui AK, Mansouri A, Boskou G, Kefalas P (2005) Tea and herbal infusions: their antioxidant activity and phenolic profile. Food Chem 89(1):27–36Google Scholar
  54. 54.
    Sznarkowska A, Kostecka A, Meller K, Bielawski KP (2017) Inhibition of cancer antioxidant defense by natural compounds. Oncotarget 8(9):15996–16016. Google Scholar
  55. 55.
    Liu S, Bishop WR, Liu M (2003) Differential effects of cell cycle regulatory protein p21(WAF1/Cip1) on apoptosis and sensitivity to cancer chemotherapy. Drug Resist Updat 6(4):183–195. Google Scholar
  56. 56.
    Martinho O, Pinto F, Granja S, Miranda-Goncalves V, Moreira MA, Ribeiro LF, di Loreto C, Rosner MR, Longatto-Filho A, Reis RM (2013) RKIP inhibition in cervical cancer is associated with higher tumor aggressive behavior and resistance to cisplatin therapy. PLoS One 8(3):e59104. Google Scholar
  57. 57.
    Mojzis J, Varinska L, Mojzisova G, Kostova I, Mirossay L (2008) Antiangiogenic effects of flavonoids and chalcones. Pharmacol Res 57(4):259–265. Google Scholar
  58. 58.
    Staton CA, Reed MW, Brown NJ (2009) A critical analysis of current in vitro and in vivo angiogenesis assays. Int J Exp Pathol 90(3):195–221. Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Viviane A. O. Silva
    • 1
  • Ana Laura V. Alves
    • 1
  • Marcela N. Rosa
    • 1
  • Larissa R. V. Silva
    • 1
  • Matias E. Melendez
    • 1
  • Fernanda P. Cury
    • 1
  • Izabela N. F. Gomes
    • 1
  • Aline Tansini
    • 1
  • Giovanna B. Longato
    • 2
  • Olga Martinho
    • 1
    • 3
    • 4
  • Bruno G. Oliveira
    • 5
  • Fernanda E. Pinto
    • 5
  • Wanderson Romão
    • 5
  • Rosy I. M. A. Ribeiro
    • 6
  • Rui M. Reis
    • 1
    • 3
    • 4
    Email author
  1. 1.Molecular Oncology Research CenterBarretos Cancer HospitalBarretosBrazil
  2. 2.Research Laboratory in Cellular and Molecular Biology of Tumors and Bioactive CompoundsSan Francisco UniversityBragança PaulistaBrazil
  3. 3.Life and Health Sciences Research Institute (ICVS), School of MedicineUniversity of MinhoBragaPortugal
  4. 4.ICVS/3B’s - PT Government Associate LaboratoryBraga/GuimarãesPortugal
  5. 5.Petroleomic and Forensic Laboratory, Chemistry DepartmentFederal University of Espírito SantoVitóriaBrazil
  6. 6.Laboratory of Experimental PathologyFederal University of São João del Rei—CCO/UFSJDivinópolisBrazil

Personalised recommendations