Antiproliferative activity of the Netherlands propolis and its active principles in cancer cell lines

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Abstract

The MeOH extract of the Netherlands propolis showed promising antiproliferative activity toward highly liver-metastatic murine colon 26-L5 carcinoma with an EC50 value of 3.5 μg/ml. Further, antiproliferative activity-guided purification of the MeOH extract led us to isolate four flavonoids (14), seven cinnamic acid derivatives (511) and two new glycerol derivatives (12, 13), whose structures were elucidated on the basis of spectral analysis. The isolated compounds were tested for their antiproliferative activity against murine colon 26-L5, murine B16-BL6 melanoma, human HT-1080 fibrosarcoma and human lung A549 adenocarcinoma cell lines. The benzyl (9), phenethyl (10) and cinnamyl caffeates (11) possessed potent antiproliferative activities with EC50 values of 0.288, 1.76 and 0.114 μM, respectively, toward colon 26-L5 carcinoma. These caffeates were considered to be active constituents of the Netherlands propolis in their antiproliferative activity. The antioxidative activity of these caffeates may play an important role in their antiproliferative activities.

Introduction

Propolis is a resinous hive product collected by honeybees from various plant sources. It has a pleasant aromatic odor and yellow–green to dark brown color depending on its source and age (Ghisalberti, 1979). Propolis has a long history of being used in traditional medicine dating back at least to 300 BC (Ghisalberti, 1979) and has been reported to have a broad spectrum of biological activities, viz. anticancer, antioxidant, antiinflammatory, antibiotic, and antifungal activities (Banskota et al., 2001a; Burdock, 1998; Marcucci, 1995). It has recently gained popularity as a health drink and is used extensively in food and beverages in various parts of the world including Japan, the USA and Europe, where it is claimed to improve health and prevent diseases such as inflammation, heart disease, diabetes and even cancer. Due to these facts there is renewed interest in the composition and biological properties of propolis.

Propolis mainly contains sticky plant substances, collected by honeybees and mixed with bees wax and other bee secretions, thus, the composition of propolis other than wax absolutely depends on the vegetation of the area from where it was collected. Propolis from temperate zones contains predominantly phenolic compounds including flavonoids and cinnamic acid derivatives (Marcucci, 1995). Diterpenes and prenylated compounds, which are virtually absent in temperate propolis, on the other hand, were reported from the tropical propolis of the South-American continent together with lignans, flavonoids and other classes of compounds (Bankova et al., 2000). The difference in the composition of propolis from temperate and tropical zones is mainly due to their different vegetations. Even given this difference in their composition, propolis from both regions possessed similar biological properties (Burdock, 1998; Banskota et al., 2001a). In our previous work, we reported the isolation of 31 different constituents from Brazilian propolis, of which three were new and 15 were isolated for the first time from propolis together with either their antiproliferative activity or hepatoprotective activity (Banskota et al., 2001b, Banskota et al., 2000a, Banskota et al., 1998; Basnet et al., 1996). Moreover, we also made a comparative study of propolis from different continents for their antiproliferative, antihepatotoxic and DPPH free radical scavenging activities (Banskota et al., 2000b). In the study, the MeOH extract of the Netherlands propolis showed promising antiproliferative activity. Thus, we conducted a chemical investigation of the MeOH extract of the Netherlands propolis. In this paper, we report the active principle of the Netherlands propolis together with its antiproliferative activities against different tumor cells.

Section snippets

Chemicals

RPMI and Eagle's minimum essential medium (EMEM) were purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) was purchased from Sigma Chemicals (St. Louis, MO). Heat-inactivated fetal calf serum (FCS) was from Gibco BRL Products (Gaitherburg, MD). Coster polystyrene 96-well polystyrene plates (Corning Incorporated, Corning, NY) were used for the antiproliferative assay. Phenyl ethanol and cinnamyl alcohol were from

Extraction and isolation

The crude propolis was successively extracted with MeOH and water under reflux. The MeOH extract, having an interesting antiproliferative activity, was further fractionated into ten fractions by silica gel column chromatography. Chrysin (1, Wagner et al., 1976) and galangin 7-methyl ether (2) were obtained as precipitates from fractions 2 and 3, respectively. Fractions 6–8, having the strongest antiproliferative activity, were further subjected to chemical investigation. Three flavonoids,

Discussion

Propolis, a complex mixture of plant metabolites, possesses a broad spectrum of biological activities including antibiotic, antioxidative, antiinflammatory and anticancer activities (Banskota et al., 2001a; Burdock, 1998; Marcucci, 1995). In our previous work, we found that the MeOH extract of the Netherlands propolis had interesting antiproliferative activity against highly metastatic liver murine colon 26-L5 carcinoma cells with an EC50 value of 3.5 μg/ml (Banskota et al., 2000b). Chemical

Acknowledgements

We are thankful to Nihon Propolis Co. Ltd., Tokyo, Japan for continues support for our propolis research. Marieke Mutsaers, Honeybee Husbandry, the Netherlands is also acknowledged for supplying the Netherlands Propolis.

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