Int J Pharm Pharm Sci, Vol 6, Issue 11, 509-510Short Communication

CYTOTOXICITY OF BYRSONIMA DUCKEANA W. R. ANDERSON (MALPIGHIACEAE) ON COLON CANCER CELLS

MARIA CHRISTINA DOS SANTOS VERDAM1*, FERNANDA GUILHON-SIMPLICIO2, CRISTIANE DA SILVA PAULA1, VINICIUS BEDNARCZUK DE OLIVEIRA1, MARILIS DALLARMI MIGUEL1, PATRÍCIA MARIA STUELP-CAMPELO3, OBDULIO GOMES MIGUEL1

1Programa de Pós Graduação , Universidade Federal do Paraná, Avenida Prefeito Lothário Meissner, 632, Jardim Botânico, 80210-170, Curitiba, Paraná, Brazil – 55413360-4070, 2Faculdade de Ciências Farmacêuticas, Universidade Federal do Amazonas, Rua Alexandre Amorim, 330, Aparecida, 69010-300, Manaus, Amazonas, Brazil – 55 92 3305-1181, 3Escola de Saúde e Biociência, Pontifícia Universidade Católica do Paraná, Rua Imaculada Conceição, 1155, Prado Velho, 80215901, Curitiba, Paraná, Brazil. 55 41 3221-7560. Email: christinaverdam@hotmail.com

Received: 29 Aug 2014 Revised and Accepted: 03 Oct 2014

ABSTRACT

Objective: The objective of this study was the cytotoxicity evaluation of the leaves extract and fractions of Byrsonima duckeana W. R. Anderson.

Methods: The toxicity on Artemia salina, haemolytic potential and cytotoxicity activities against two different cell lines a U937 cell line human, and HT29 tumor colon cell line of the leaves extract and fractions of Byrsonima duckeana W. R. Anderson were evaluated.

Results: There was no IC50 the Artemia salina assay. The chloroform and ethyl acetate fractions showed haemolytic activity. The results were satisfactory at the most polar fractions, with citotoxicity to the HT29 cell line.

Conclusion: The study provides preliminary evidence of cytotoxicity of the most polar fractions at the HT29 cell line, which can indicate a potencial source to offer substances with cytotoxic activity.

Keywords: Medicinal Plant, Alternative Medicine, Cytotoxicity, Cancer Cells.

Plants and herbs have been used to benefit human health since humans inhabited the earth [1]. Natural products are considered a strategy to block the development of cancer in humans. Many of the drugs used in cancer treatment are derived from natural products [2].

In this context, the Byrsonima genus has attracted the interest of researchers, and we found studies in the literature focusing on its action against microorganisms, including several species of bacteria, enteric bacteria, mycobacteria, protozoa and fungi, most of which report positive results. Other biological activities, such as immunostimulatory, anti-inflammatory, anti-hemorrhagic, antihyperglycaemic, antihyperlipidemic, antiulcer, antidiarrheal and antioxidant activities, were also investigated in different species [3].

We did not find any data in the literature regarding the pharmacological properties of Byrsonima duckeana W. R. Anderson. However, based on preliminary pharmacological results, we decide to investigate the possible cytotoxic effect of an ethanol extract and its fractions of Byrsonima duckeana leaves against the U937 cell line human, and HT29 tumor colon cell line. We also evaluate its haemolytic potential and toxicity on Artemia salina nauplii.

Plant material was collected at the Adolpho Ducke Forest Reserve, Manaus, Amazonas, Brazil, and taxonomically identified by the Herbarium of the National Institute of Amazonia Research (INPA) by Alberto Vicentini and W. R Anderson, in the context of the Flora da Reserva Ducke project. A voucher specimen was deposited under number 179696.

Fig. 1: Cytotoxicity effect of the extract and fractions of B. duckeana on the HT29 cell line: A- Crude extract of B. duckeana, B- Hexane fraction, C- Chloroform fraction, D- Ethyl acetate fraction, E- Remaining fraction

Leaf samples of B. duckeana were dried at in a convection oven. The leaves were crushed and submitted to extraction in a Sohxlet apparatus modified [4]. The material was exhaustively extracted with ethanol, yielding the crude extract (EB), which was partitioned using solvents of increasing polarity, giving rise to the hexane (HEX), chloroform (CLO), ethyl acetate (FAE) and remaining (FREM) fractions which were used in the biological tests.

A preliminary assessment of toxicity was conducted with the Artemia salina assay and evaluation haemolytic activity. Cell viability was then determined with the MTT assay - (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide thiazolyl blue). The toxic potential of the crude extract and the fractions of Byrsonima duckeana was evaluated against Artemia salina, and was determined by counting the dead nauplii after 24 hours of incubation [5]. The ethyl acetate and remaining fractions were not verified. The haemolytic activity was measured using sterile paper discs impregnated with 1000 µg of the samples placed on blood agar plates (Newprov®). After incubation at 36°C for 24 hours, the hemolysis halo was verified and compared with the positive control, a 10% Triton solution that is considered with 100% of activity [6]. After this preliminary evaluation of toxicity, a cytotoxicity assessment was performed with the MTT cell viability at 1, 10, 100 e 1000 µg/mL of each sample using U937 (human monocyte) HT29 (tumor colon) cell lines [7], all the results were compared by the ANOVA, considering values of p ≤ 0.05 statistically significant.

There were no deaths of nauplii at the Artemia salina assay. Haemolytic activity was observed only at the chloroform and ethyl acetate fractions with about 65.6 % and 48.4% haemolysis, respectively. Fig. 1 and 2 show the cytotoxic effects of the samples on the HT29 and U937 cells, respectively. The chloroform and ethyl acetate fractions showed decreasing cell viability on HT29 line at 100 and 1000 µg/mL and 10 to 1000 µg/ml, respectively. The same fractions had hemolytic activity, which suggests that the more polar constituents of ethanol extract are more closely related to their toxicity. Interestingly, there was no toxicity to non-tumor cells, at the MTT assay, indicating some selectivity that may be important in further studies on the anticancer potential of this species.

Fig. 2: Cytotoxicity effect of the extract and fractions of B. duckeana on U937 tumor colon cells. A- Crude extract, B- Hexane fraction, C- Chloroform fraction, D- Ethyl acetate fraction, E- Remaining fraction

CONFLICT OF INTERESTS

The authors declare that is no conflict of interests.

REFERENCES

  1. Kaliora AC, Kaggianou DAA, Kefalas P, Papassideri IS, Kalogeropoulos N. Phenolic profiles and antioxidant and anticarcinogenic activities of Greek herbal infusions, balancing delight and chemoprevention? Food Chem 2014;142:233-41.
  2. Farshori NN, Al-Sheddy ES, Al-Oqail MM, Mussarat J, Al-Khedhairy AA, Siddiqui MA. Anticancer activity of Petroselinum sativum seed extracts on MCF-7 Human Breast cancer cells. Asian Pac J Cancer Prev 2013;14:5719-23.
  3. Guilhon-Simplicio F, Pereira MM. Aspectos Químicos e Farmacológicos de Byrsonima (Malpighiaceae). Quim Nova 2011;34:1032-41.
  4. Carvalho JL de S, Cunico MM, Dias Jde F G, Miguel MD, Miguel OG. Termoestabilidade de processos extrativos de Nastirtium officinale R. BR. Brassicaceae por sistema soxhlet modificado. Quim Nova 2009;32:1031-5.
  5. Lewan L, Anderson M, Morales-Gomez P. The use of Artemia salina in toxicity testing. Altern Lab Anim 1992;20:297-301.
  6. Kalegari M, Miguel MD, Dias JFG, Lordello ALL, Lima CP, Miyazaki CMS, et al. Phytochemical constituents and preliminary toxicity evaluation from leaves of Rourea induta Planch (Connaraceae). RBCF Braz J Pharm Sci 2011;47:635-42.
  7. Sieuwerts AM, Klijn JGM, Peters HA, Foekens JA. The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for assessment of growth characteristics, IC50-values and cell survival. Eur J Clin Chem Clin Biochem 1995;33:813-23.