Eurycoma Longifolia Jack Aqueous Extract - Bio Active Compound And Its Effects Toward Hormone Production And Spermatogenesis

Author

NORHANIZA AMINUDIN

Date

2003

Keyword

Eurycoma longifolia Jack, bioactive compounds, biological activity screening, hormone production, sperm analysis, testosterone production up

Abstract

Isolation and purification of the freeze-dried boiled aqueous extract (FDBAE) of Eurycoma longifolia was initially done using BioGel P2 size exclusion column chromatography. This enables separation of the FDBAE into a major brown colored fraction and a series of fluorescent colored fractions. All fractions were pooled accordingly and freeze-dried. Dried brown colored fraction was then subjected to BioGel P4 size exclusion column chromatography for further separation process. Five different fractions were obtained; each was pooled and freeze-dried, yielding F1AE, F2AE, F3AE, F4AE and F5AE. However, only F2AE, F3AE, F4AE and F5AE provide sufficient quantity to enable further work. These freeze-dried aqueous extract fractions (F2AE - F5AE) were then subjected to biological activity screening involving hormone production and sperm analysis. The effect of the aqueous extract fractions toward hormone production was monitored using capillary gas chromatography (GC). Preliminary GC analysis showed that the presence of aqueous extract fractions help to elevate testosterone hormone production. Testosterone concentration was found to be highest upon incubation of the Sprague Dawley rat testicular homogenate with F3AE i.e. 155% higher than the control. Besides testosterone, the aqueous extract fractions also influenced the production of pheromone (an a). The concentration of the pheromone was shown to be highest with the presence of F4AE i.e. 436% higher than the control. The effect of the aqueous extract fractions toward sperm characteristics was analyzed using the Hamilton-Thorne Motility Analyzer (HTMA). Aqueous extract fractions were administered to male mice either intra-peritoneal or orally. Analyzed results showed that despite the type of treatment, F3AE constantly demonstrated an increase in sperm concentration, the percentage of sperm with progressive movement and the percentage of motile sperm compared to the control. Sperm velocity distribution analysis showed that F3AE increased the percentage of sperm moving rapidly, thus reduced the percentage of static sperm. Analysis on sperm movement characteristics showed that the aqueous extract fractions, particularly F3AE, positively influenced four parameters that are important in predicting male fertility. Average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were shown to be higher with the presence of F3AE compared to the control. Surface-enhanced laser desorbtion/ionization (SELD1) mass spectrometry screening demonstrated the presence of a peptide compound with the molecular weight approximately 4,300 in F3AE. The aqueous extract was subjected to purification methods such as high performance liquid chromatography using hydrophilic interaction chromatography (HILIC) column, amino solid phase extraction (SPE) column, cation exchange spin column and trypsin digestion. Eluted fractions from each method were collected and screened with SELDI. Peptide sequencing analysis performed on each sample containing the 4,300 Da compound has identified a novel peptide (ELP1) containing 32 amino acid residues. The identified sequence is as follows: 1I1e-Pro-Gly-Thr-Ala-Thr-Phe-Tyr-Thr-Thr-Tyr-Val-Pro- 14Ser-Ala-[Gln]-Tyr-Gly-Phe-Gln-Asp-Gln-Gly-Val-Met- 26I1e-Val-Ala-Val-Ser-Asp-32Pro ELPl along with the aqueous extract were subjected to testosterone enzyme immunoassay. Results showed that ELPl and the aqueous extract have the ability to increase testosterone production up to 120% higher than the control. CYP17 and CYP19 gene expression analyses were conducted to observe the effect of ELPl and the aqueous extract. CYP17 is the gene encoding the enzymes 17α-hydroxylase /17,20 lyase that are involved in the conversion of pregnenolone to 17-hydroxypregnenolone and dehydroepiandrosterone (As pathway) as well as the conversion of progesterone to 17-hydroxyprogesterone and 4-androstenedione (A4 pathway). CYP17 gene expression was obviously up-regulated upon incubation with ELPl and the aqueous extract. Relative values for the expression level were two to eight times higher compared to the control. CYP19 is the gene encoding the enzyme aromatase, which directly converts testosterone and 4-androstenedione to oestrogen. An increase was also observed in CYP19 gene expression level following the incubation with ELPl and the aqueous extract. Relative values were four to 12 times higher compared to the control.