Characteristics, chemical compositions and biological activities of propolis from Al-Bahah, Saudi Arabia

Abstract

Propolis has been used to treat several diseases since ancient times, and is an important source of bioactive natural compounds and drug derivatives. These properties have kept the interest of investigators around the world, leading to the investigation of the chemical and biological properties and application of propolis. In this report, the chemical constituents that are responsible for the anticancer activities of propolis were analyzed. The propolis was sourced from Al-Baha in the southern part of the Kingdom of Saudi Arabia. Standard protocols for chemical fractionation and bioactivity-guided chemical analysis were used to identify the bio-active ethyl acetate fraction. The extraction was performed in methanol and then analyzed by gas chromatography-mass spectrometry (GC-MS). The major compounds are triterpenoids, with a relative concentration of 74.0%; steroids, with a relative concentration of 9.8%; and diterpenoids, with a relative concentration of 7.9%. The biological activity was characterized using different approaches and cell-based assays. Propolis was found to inhibit the proliferation of cancer cells in a concentration-dependent manner through apoptosis. Immunofluorescence staining with anti-α-tubulin antibodies and cell cycle analysis indicated that tubulin and/or microtubules are the cellular targets of the L-acetate fraction. This study demonstrates the importance of Saudi propolis as anti-cancer drug candidates.

Figure 1. Hierarchical cluster analysis of data from impedance curves obtained with L929 cells that were incubated with L-acetate fraction and as a set of reference compounds L-acetate fraction found in close proximity to tubulysin B, griseofulvin, and nocodazol.

The three compounds are known to interact with tubulin as a cellular target.

Figure 2. Effect of L-acetate fraction on the microtubules of PtK2 cells as shown by immunofluorescence technique using anti-α-tubulin antibodies (red).

Nuclei stained with DAPI (blue). (A) Control Cell, (B), and (C) treated cells (100 μg/ml, 24 h).

Figure 3. L-acetate fraction of Saudi proplis induced cell cycle arrest at G₂M-phase in Jurkat cells.

(A) Control cells, (B) Treated cells (100 μg/ml, 24 hours).

Figure 4. Induction of apoptosis in Jurkat cells after treatment the cells with L-acetate fraction of saudi propolis.

After incubation times cells were stained with anexinV-FITC and PI and analysed with flow cytometry. (A) control (mehanol), (B) treated (100 μg/ml, 3 h), (C) treated, (100 μg/ml, 24 h), and (D) treated (100 μg/ml, 24 h).

Figure 5. Representative photomicrograph of the histopathological examinations of both kidney and liver.

(A,B) Control and treated renal tissues stained with Masson Trichrome (X 400). (C,D) Control and treated hepatic tissues stained with Masson Trichrome (X 400). (E,F) Control and treated hepatic tissues stained with Reticulin (X 400).

Figure 6. GC-MS analysis of L-Acetate fraction.

(A) Total ion current (TIC) traces showing the major organic compounds. (B) Examples of typical GC-MS key ion plots (m/z 189/218) for triterpenoids (U = unknown).