Quassinoids, the major secondary metabolites of roots, improve male fertility. Hence, it is crucial to investigate their quantitative level in extracts. A profile was established to identify the primary metabolites and major quassinoids, and quantify quassinoids using external calibration curves. Furthermore, the metabolic discrimination of roots from different regions was investigated. TheH-NMR spectra of the quassinoids, eurycomanone, eurycomanol, 13,21-dihydroeurycomanone, and eurycomanol-2---D-glycopyranoside were obtained. The H-NMR profiles of root aqueous extracts from Perak (n = 30) were obtained and used to identify primary metabolites and the quassinoids. Selangor, Kedah, Terengganu (n = 5 for each), and Perak samples were checked for metabolic discrimination. Hotelling's T plot was used to check for outliers. Orthogonal partial least-squares discriminant analysis was run to reveal the discriminatory metabolites. Perak samples contained formic, succinic, methylsuccinic, fumaric, lactic, acetic and syringic acids as well as choline, alanine, phenylalanine, tyrosine, -glucose, eurycomanone, eurycomanol, 13,21-dihydroeurycomanone, and eurycomanol-2---D-glycopyranoside. The extracts from other locations contained the same metabolites. The limit of quantification values were 1.96 (eurycomanone), 15.62 (eurycomanol), 3.91 (13,21-dihydroeurycomanone), and 31.25 (eurycomanol-2---D-glycopyranoside) ppm. The Hotelling's T plot revealed no outlier. The orthogonal partial least-squares discriminant analysis model showed that choline, eurycomanol, eurycomanol-2---D-glycopyranoside, and lactic and succinic acid levels were different among regions. Terengganu and Perak samples contained higher amounts of eurycomanol and eurycomanol-2---D-glycopyranoside, respectively. The current approach efficiently detected root metabolites, quantified the quassinoids, and discriminated roots from different locations. These findings could be applicable to future research on where the higher content of quassinoids is required.
TA: Tongkat Ali; LOD: limit of detection; LOQ: limit of quantification; HPLC-UV: high performance liquid chromatography-ultrviolet; PDA: photodiode array; NMR: nuclear magnetic resonance; FID: free induction decay; LC-MS: liquid chromatography-mass spectrometry; GC-MS: gas chromatography-mass spectrometry; HSQC: heteronuclear single quantum coherence; CPMG: Carr-Purcell-Meibum-Gill; VLDL: very low density lipoprotein; HDL: high density lipoprotein; EDTA: ethylenediaminetetraacetic acid; ANOVA: analysis of variance; AMIX: analysis of mixtures; SIMCA: soft independent modeling of class analogy; PCA: principal components analysis; OPLS-DA: orthogonal partial least-squares discriminant analysis; VIP: variable importance plot; AUROC: area under the receiver operating characteristic; TSP: sodium 3-(trimethylsilyl) propionate- 2,2,3,3- d4; DSA: deuterated 4-dimethyl-4-silapentane-1-ammonium trifluoroacetate; ESI: electrospray ionization; TCA: trichloroacetic acid; ACN: acetonitrile; dd HO: distilled deionized water; FSH: follicle-stimulating hormone; LH: luteinizing hormone; OECD: Organisation for Economic Co-operation and Development.
Eurycoma longifolia Jack is popularly sought in Southeast Asian countries for traditional remedies to improve sexual performance and fertility. 13α(21)-Epoxyeurycomanone and eurycomanone, two major quassinoids in a root extract (TAF2) were reported to improve rat spermatogenesis and fertility. Unfortunately, these quassinoids possess low bioavailability because of high aqueous solubility and low lipid membrane permeability. Often, other possible barriers may be P-glycoprotein (P-gp) efflux in the gut and presystemic hepatic metabolism. The present study attempted to solve these problems by formulating a lipid-based solid dispersion (TAF2-SD) of optimized mixture of TAF2 and emulsifiers, which was then orally administered to rats prior to sperm count analysis. The TAF2-SD-treated rats showed significantly twofold (p < 0.001) and fourfold (p < 0.001) higher sperm count than did TAF2-treated and vehicle-treated (control) rats, respectively. The study also demonstrated no significant in vitro ileal absorption changes of the quassinoids by P-gp efflux inhibitors and concentration change or secondary metabolite formation upon in vitro incubation with rat liver homogenates, suggesting that P-gp-mediated efflux and presystemic metabolism were not limiting their bioavailability. Further study on orally TAF2-treated rats confirmed that the area under the curve and bioavailability curve of each quassinoid in the absence and presence of ketoconazole were unchanged. Copyright © 2017 John Wiley & Sons, Ltd.
The Nuclear Magnetic Resonance (NMR)-based plant metabolomic method was used for the analysis of 29 Eurycoma longifolia Jack (Tongkat Ali) roots, harvested from the same source in Perak. The objective of study was to establish a standardised profile of the plant metabolites for comparison with those of E. longifolia root samples derived from other sources. The age of the plant, the climatic conditions, soil pH and geographical locations were considered as probable variables. The roots were extracted with distilled-diionized water. The 1 H-NMR profile of each extract and the major secondary quassinoid metabolites, eurycomanone, eurycomanol, and eurycomanol-2-O-β-D-glycopyranoside were obtained. The NMR spectra were binned to 0.04 ppm and the data were analysed using the AMIX-TOOLS and SIMCA-P. Preliminary identification of the secondary metabolites confirmed the presence of the quassinoids of study in all the extracts. The Principle Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) were performed. The PCA results at 95 % confidence level showed that no outliers were observed among the samples, indicating that the plants were similar in NMR profile. However, from OPLS-DA, five groups were identified to be different in terms of age and were statistically significant at 95 % confidence level. The differences among the groups strongly reflected the changes in the type and level of the metabolites. The identification and quantification of the discriminating metabolites among the groups will be discussed. Further studies on the profiling of the root organic extract and the biomarker quantification are ongoing.
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