NMR-Based Metabolomic Approaches To Evaluate The Effects Of Eurycoma Longifolia Extracts On Rat Spermatogenesis
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Date
2016-09
Authors
Forough Ebrahimi
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Abstract
Eurycoma longifolia (Tongkat Ali, TA) is known for boosting male fertility and libido. A nuclear magnetic resonance (NMR)-based metabolomics analysis in combination with advanced chemometric methods was developed, validated and applied for the simultaneous identification and quantification of metabolites in E. longifolia and mammalian fluids (rat urine and plasma). A general profile of the aqueous extract derived from 30 samples in Perak, Malaysia was established for metabolites especially for the bioactive quassinoids of eurycomanone, eurycomanol, 13,21-dihydroeurycomanone and eurycomanol-2-O-β-D-glycopyranoside. The reference profile was then compared with other aqueous extracts from Selangor, Kedah and Terengganu to investigate any discriminatory metabolites with respect to location and variations such as environmental temperature and soil pH. Another study dealt with the NMR analysis of rat urine to investigate any correlation of the discriminated metabolites with the sperm count (SC) status following the treatment of rats with varying quassinoid content in water (TAW, 125 mg/kg), quassinoid-poor Tongkat Ali (TAQP, 125 mg/kg) and quassinoid-rich Tongkat Ali (TAQR, 21 mg/kg) extracts. A 6-fold increase in dose of TAW, equal with TAQP in concentration of quassinoids, was administered to investigate whether the compounds in the two extracts were responsible for the increase in rat SC. The rats were then categorized into normal- and high-SC groups following the
reference median value of normal rat SC. The urine NMR profiles of the normal- and high-SC groups were next examined for metabolomic discrimination. In addition, the results of the same treatment on the rat plasma NMR profiles were investigated. The last part of study aimed at the fertility biomarker discovery of the urine and plasma metabolites. The E. longifolia aqueous extract general profile contained α- glucose, alanine, phenylalanine, tyrosine, choline, formic, succinic, methylsuccinic, fumaric, syringic, lactic and acetic acids and also eurycomanone, eurycomanol, 13,21-dihydroeurycomanone and eurycomanol-2-O-β-D-glycopyranoside. The quantitative metabolomic profiles of E. longifolia roots were not different with respect to temperature and soil pH. The validated quantitative NMR (qNMR) reliably determined the quassinoid levels in E. longifolia aqueous extracts, showing eurycomanone (% w/w ± SD) with the highest quassinoid concentration at a range of 5.984 ± 1.949 to 7.752 ± 2.892 and not statistically different in the samples at the four locations. In contrast, the samples from different locations were statistically different in concentration of choline, eurycomanol, eurycomanol-2-O-β-D-glycopyranoside, lactic and succinic acids. TAW- and TAQR-treated animals had significantly higher sperm number compared to the other two groups. The SC values (x106/mL/g testis) were 25.35 ± 6.42, 65.9 ± 26.11, 28.73 ± 17.42 and 68.27 ± 14.96 for control, TAW-treated, TAQP-treated and TAQR-treated groups, respectively. From the urine analysis, trigonelline, benzoic acid and alanine levels significantly increased in high-SC group compared to the normal-SC one, whereas the ethanol level statistically decreased in high-SC group. Based on the literature data, the altered level of discriminatory metabolites correlated with the different levels of testosterone in urine samples, following the treatment with E.
longifolia extracts containing different quassinoid concentration. Further evidence was obtained by measuring testosterone level in rat urine. The organic-phase (org) urine, rich in testosterone, was obtained from the diethylether extracted urine. The organic and aqueous phase urine were analyzed by LC-MS/MS for testosterone and eurycomanone (as the most abundant quassinoid), respectively. The presence of eurycomanone (only in high-SC group) was proven in aqueous-phase (aq) urine by the isotope peak [M+H]+ at m/z 409.02. Testosterone was identified in normal- and high-SC groups displaying the molecular ion at m/z 289.2 [M+H]+ and the fragmented ions at m/z 96.8 and 108.8. The testosterone amount (% w/v) in the high-SC groups was 2.2 fold higher than that of the normal-SC. The statistical analysis of rat plasma also revealed the increased levels of alanine, lactate and histidine in the high-SC group compared to the normal-SC group. However, the ethanol level significantly decreased in the high-SC group. Similar discriminatory plasma metabolites as those of urine were found. In conclusion, a standardized metabolomic profile of Perak samples was obtained as a reference. The samples in Perak and those in three other locations were discriminated according to their metabolite levels. The efficacy on the increase in sperm count of the quassinoids was confirmed by the increase in urinary quassinoid and testosterone levels, together with some of the above-mentioned primary metabolites. The NMR-based metabolomic approach has provided urine and plasma biomarkers associated with an increase in sperm count, and has potential for the evaluation of male fertility status without requiring semen sample.
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NMR analysis of rat urine to investigate any correlation , of the discriminated metabolites