Elsevier

Journal of Ethnopharmacology

Volume 144, Issue 3, 18 December 2012, Pages 747-753
Journal of Ethnopharmacology

Croton lechleri sap and isolated alkaloid taspine exhibit inhibition against human melanoma SK23 and colon cancer HT29 cell lines

https://doi.org/10.1016/j.jep.2012.10.032Get rights and content

Abstract

Ethnopharmacological relevance

Croton lechleri Mull. Arg. (Euphorbiaceae) is a traditional medicinal plant which produces a red sap, traditionally known as “Sangre de Drago”; it is used in folk medicine externally for wounds, fractures, and haemorrhoids, internally for intestinal and stomach ulcers and also for the empirical cure of cancers.

Materials and methods

We investigated the effects of Croton lechleri sap and taspine in comparison with taxol and vinblastine on the growth of human cancer cell lines of SK23 (melanoma), LoVo and HT29 (colorectal cancer) using MTT and Trypan blue assays. Further, we studied cell cycle by flow cytometry and detected acetylated-α-tubulin by confocal microscope.

Results

Croton lechleri inhibited cell proliferation starting from 1 μg/mL in SK23 cells, whereas 10 times higher concentrations were required for growth inhibition of HT-29 and LoVo cell lines. Also taspine (0.1 μg/mL) inhibited the SK23 and HT29 cell proliferation. Further, assay was assessed on SK23 and HT29 cell lines with 24–48 h treatment with sap and taspine. Both sap and taspine inhibited cancer cell proliferation; taspine showed higher activity on SK23 cells, which was significantly increased after 48 h of SK23 treatment. Using confocal microscopy we observed that Croton lechleri (1 μg/mL) caused a loss of microtubule structure, whereas taspine (0.5 μg/mL) caused an increase in acetylated α-tubulin and a modification of cellular morphology, mainly in SK23 cells. Croton lechleri sap 10 and 50 μg/mL influence cell cycle; 50 μg/mL sap caused a dramatic reduction of cells in G1/G0 and S phases with a great increase of subG0 cells.

Conclusions

The data showed that Croton lechleri and taspine could inhibit cell proliferation with higher potency against melanoma SK23 cells, supporting the empirical use of the sap as anticancer in ethnomedicine and taspine as a possible anticancer agent.

Introduction

Croton lechleri Müll.Arg. (fam. Euphorbiaceae) is a traditional medicinal plant which produces a red sap, traditionally known as “Sangre de Drago” that is obtained carving the bark of plants having two–three or more years. Amazonic natives use this sap to treat several diseases as wound healing, gastrointestinal illness and also cancers (Jones, 2003). Furthermore, the dried sap is marketed in health products in Western countries. The phytochemical characterization of the sap has lead to the finding that the oligomeric proanthocyanidins and flavonols constitute almost the 90% of the dry weight (Cai et al., 1991). But various minor compounds also have been found; one is the alkaloid taspine found in the sap of mature trees (Perdue et al., 1979). Others are the lignan 3′,4-O-dimethylcedrusin (Cai et al., 1993a, Pieters et al., 1993), various diterpenoids (Cai et al., 1993b) and nor-isoprenoid blumenol derivatives (De Marino et al., 2008).

Proanthocyanidins and other condensed tannins are known to be protective against various pathologies, including cancer (Bobe et al., 2009, De Bruyne et al., 1999, Hanausek et al., 2011). Croton lechleri has been evaluated in cell lines such as KB, V-79, K562 and HeLa for anticancer activity showing low activity (Alfonso-Castro et al., 2012, Chen et al., 1994, Itokawa et al., 1991, Rossi et al., 2003, Vaisberg et al., 1989). This activity was mainly ascribed to the presence of the alkaloid taspine (Fig. 1) which has also been reported as the active compound with wound healing properties (Porras-Reyes et al., 1993, Vaisberg et al., 1989). Taspine is the main alkaloid which characterizes Croton lechleri sap, but it is also present in Leontice eversmanii Bge (Berberidaceae), Caulophyllum thalictroides (L.) Michx. and Calophyllum robustum Maxim. (Berberidaceae), Magnolia x soulangiana Soul.-Bod. (Magnoliaceae). Recently, interest is growing on the Croton lechleri sap and on its isolated alkaloid taspine (Fayad et al., 2009, Rollinger et al., 2006, Zhang et al., 2010).

The incidence rate of cutaneous malignant melanoma and colorectal cancer is increasing over the years and classical chemotherapy agents are still partly unsuccessful, therefore there exists a huge need of new therapeutic approaches against these cancers (Goffin et al., 2005). Further, malignant melanoma is the most serious of all skin cancers and strongly resistant to chemo- and radiotherapy. Epidemiological studies have shown that its incidence is rapidly increasing (Kruijff and Hoekstra, 2012).

In this framework of renewed researches, we have considered the activities of Croton lechleri and taspine on epithelial cancer in vitro also in relation with its traditional use, which is mainly for topical application. In this context, Croton lechleri sap and isolated taspine were studied in comparison with taxol and vinblastine, two natural anticancer agents, on the cell viability and the cell cycle, and on the α-tubulin structure of melanoma and colon cancer cell lines in culture.

Section snippets

Plant material

The red sap of Croton lechleri was collected by traditional way, carving the bark from trees growing in the province of Napo, Ecuador. The voucher code number (SdD 007) for the crude drug was deposited in the Department of Pharmaceutical and Pharmacological Sciences of Padua University.

The sap was freeze-dried in a lyophilizer and stored at −20 °C. For cell culture experiments, the freeze-dried SdD was solubilised in water.

Croton lechleri sap and taspine

Before lyophilisation, pH and density of the sap were determined. Dry

MTT assay

The pH of the sap from Croton lechleri was 3.9 and the density was 1.08 g/mL according with the literature data (Risco et al., 2003). The chemical characterization has been performed through the determination of the alkaloid taspine as previously reported (Froldi et al., 2009). To allow the conservation, we freeze-dried the fresh sap obtaining a crystal brown powder with a dry residue of 27.5±0.6%.

We studied the activity of Croton lechleri sap and taspine on cellular proliferation using MTT

Discussion

In this research, we studied the potential anticancer activity of Croton lechleri sap and its isolated alkaloid taspine in comparison with taxol and vinblastine on the growth of melanoma and colon cancer cell lines, studying cell proliferation, α-tubulin structure and cell-cycle. The sap (1–100 μg/mL) showed antiproliferative activity against all cell lines with a higher inhibition of SK23 cells growth, determined by MTT assay. Also taspine showed antiproliferative activity from 0.1 μg/mL against

Acknowledgements

This work was supported by the Italian Ministry for University and Research (MIUR) n. 60A04-5521/11 and 60A04-8432/12.

The authors acknowledge the traditional healers of the western Amazon basin of South America who use the latex of the Croton lechleri tree to treat many diseases. The authors also thank “Il Crisolito”s.r.l. (Spresiano, Treviso, Italy), Padri Giuseppini and Dr. Francesco Farnesi for providing the Croton lechleri sap from Ecuador.

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