In vitro antiproliferative activity of partially purified Trigona laeviceps propolis from Thailand on human cancer cell lines

Abstract

Background: Cancers are some of the leading causes of human deaths worldwide and their relative importance continues to increase. Since an increasing proportion of cancer patients are acquiring resistance to traditional chemotherapeutic agents, it is necessary to search for new compounds that provide suitable specific antiproliferative affects that can be developed as anticancer agents. Propolis from the stingless bee, Trigona laeviceps, is one potential interesting source that is widely available and cultivatable (as bee hives) in Thailand.

Methods: Propolis (90 g) was initially extracted by 95% (v/v) ethanol and then solvent partitioned by sequential extractions of the crude ethanolic extract with 40% (v/v) MeOH, CH₂Cl₂ and hexane. After solvent removal by evaporation, each extract was solvated in DMSO and assayed for antiproliferative activity against five cancer (Chago, KATO-III, SW620, BT474 and Hep-G2) and two normal (HS27 fibroblast and CH-liver) cell lines using the MTT assay. The cell viability (%) and IC₅₀ values were calculated.

Results: The hexane extract provided the highest in vitro antiproliferative activity against the five tested cancer cell lines and the lowest cytotoxicity against the two normal cell lines. Further fractionation of the hexane fraction by quick column chromatography using eight solvents of increasing polarity for elution revealed the two fractions eluted with 30% and 100% (v/v) CH₂Cl₂ in hexane (30DCM and 100DCM, respectively) had a higher anti-proliferative activity. Further fractionation by size exclusion chromatography lead to four fractions for each of 30DCM and 100DCM, with the highest antiproliferative activity on cancer but not normal cell lines being observed in fraction# 3 of 30DCM (IC₅₀ value of 4.09 - 14.7 μg/ml).

Conclusions: T. laeviceps propolis was found to contain compound(s) with antiproliferative activity in vitro on cancer but not normal cell lines in tissue culture. The more enriched propolis fractions typically revealed a higher antiproliferative activity (lower IC₅₀ value). Overall, propolis from Thailand may have the potential to serve as a template for future anticancer-drug development.

Figure 1

Average in vitro IC₅₀ values (μg/ml) of the three different solvent partitioned fractions (see Table 2) on the five cancer and two normal cell lines in tissue culture, as determined by the MTT assay. Hex = hexane extract, DCM = CH₂Cl₂ extract and MeOH = methanol extract. Data came from the mean ± 1 SD of percentage of cell viability which was derived from three replicates.

Figure 2

Average IC₅₀ (μg/ml) values of the eight fractions, obtained after quick column chromatography, on the five cancer and two normal cell lines. 100HEX, 10DCM, 30DCM, 50DCM, 70DCM, 100DCM, 5MET and 10MET stand for the fractions eluted in 0:1, 1:9, 3:7, 1:1, 7:3 and 1:0 (v/v) ratios of CH₂Cl₂: hexane, and 1:19 and 1:9 (v/v) ratios of MeOH: CH₂Cl₂, respectively (see table 3). Data came from the mean ± 1 SD of percentage of cell viability which was derived from three replicates.

Figure 3

The decrease in the in vitro IC₅₀ values (μg/ml) on five cancer cell lines, plus the CH-liver and HS27 fibroblast normal cell lines as a comparative reference control, with increasing purification stages of the propolis extract from T. laeviceps. Data came from the mean ± 1 SD of percentage of cell viability which was derived from three replicates.