A metabolomics approach for the evaluation of Ficus benghalensis female in vivo reproductive effects relative to its metabolite fingerprint as determined via UPLC-MS and GC-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Ficus benghalensis, commonly known as Banyan Fig, is the national tree of India and its aerial roots are used traditionally to treat female reproductive disorders. However, despite this traditional use, no pharmacological evidence could be traced supporting this use. Additionally, no comprehensive metabolite profiling was reported for F. benghalensis aerial roots. AIM OF THE STUDY: This study attempts to justify biochemically the traditional use of F. benghalensis aerial roots in treatment of female reproductive disorders and in relation to its secondary metabolite profile. MATERIALS AND METHODS: Total ethanol extract (TEE) and subfractions [petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and n-butanol (BUF] were prepared from air-dried powdered aerial roots of F. benghalensis. Detailed in-vivo investigation of the hormonal activity and action mechanism of the total ethanol extract and subfractions was carried out through evaluation of estrogenic and gonadotropic activities. The estrogenic activity was evaluated on ovariectomized immature female rats through estimating uterine weight, vaginal cornification and serum estradiol level along with histological examination of uteri. The gonadotropic activity was measured by assay of follicle stimulating hormone (FSH) and luteinizing hormone (LH) like activities. Total follicular and corpora lutea counts in immature female rats were used to determine FSH and LH like activities, respectively in addition to histological picture of the genitalia. Comprehensive non-targeted metabolite profiling was carried out for the TEE and subfractions using UPLC-HRMS in negative and positive ionization modes. UPLC-MS fingerprint was subjected to principal component analysis (PCA) and partial least squares analyses to correlate the bioactivities to specific chemical constituents in F. benghalensis different subfractions. GC-MS was further used for non-polar silylated fractions. RESULTS: Results revealed that only the non-polar PEF and CHF displayed moderate estrogenic and FSH-like activities but with no LH-like activity. Metabolites profiling via (UPLC-HRMS) and multivariate PCA analysis enabled identification and comparison of various chemical classes in F. benghalensis extract and fractions. The active non-polar fractions revealed nearly similar metabolites profile being composed of isoflavonoids, triterpenes, sterols, fatty acids and cyclic peptides. In contrast, polar fractions were more abundant in apocarotenoids, fatty acyl amides, hydroxybenzoates and hydroxycinnamates in addition to two lignans. PLS analysis revealed strong correlation between hydroxylated fatty acids and pyranoisoflavones with estrogenic and FSH-like activities. GC-MS analysis was further employed for non-polar fractions profiling revealing for their enrichment in fatty acids/esters, terpenes, organic acids and phenolics. CONCLUSION: This is the first study to rationalize the use of F. benghalensis aerial root traditionally in treatment of gynecological disorders, revealing that the petroleum ether and chloroform non-polar subfractions of F. benghalensis showed estrogenic and FSH-like activity with absence of LH-like activity. This biological activity could possibly be attributed to its metabolites profile of isoflavonoids, fatty acids, triterpenes, sterols and cyclic peptides identified via UPLC-MS and GC-MS techniques. Consequently, F. benghalensis aerial roots should be used with caution in traditional treatment of female infertility or other reproductive disorders.

Comparative Metabolite Profiling and Fingerprinting of Medicinal Cinnamon Bark and Its Commercial Preparations via a Multiplex Approach of GC-MS, UV, and NMR Techniques.

Various species of cinnamon (Cinnamomum sp.) are consumed as traditional medicine and popular spice worldwide. The current research aimed to provide the first comparative metabolomics study in nine cinnamon drugs and their different commercial preparations based on three analytical platforms, i.e., solid-phase microextraction coupled to gas chromatography-mass spectrometry method (SPME/GC-MS), nuclear magnetic resonance (NMR), and ultraviolet-visible spectrophotometry (UV/Vis) targeting its metabolome. SPME/GC-MS of cinnamon aroma compounds showed a total of 126 peaks, where (E)-cinnamaldehyde was the major volatile detected at 4.2-60.9% and 6.3-64.5% in authenticated and commercial preparations, respectively. Asides, modeling of the GC/MS dataset could relate the commercial products CP-1 and CP-3 to C. cassia attributed to their higher coumarin and low (E)-cinnamaldehyde content. In contrast, NMR fingerprinting identified (E)-methoxy cinnamaldehyde and coumarin as alternative markers for C. verum and C. iners, respectively. Additionally, quantitative NMR (qNMR) standardized cinnamon extracts based on major metabolites. UV/Vis showed to be of low discrimination power, but its orthogonal projections to latent structures discriminant analysis (OPLS-DA) S-plot showed that C. iners was more abundant in cinnamic acid compared to other samples. Results of this study provide potential insights into cinnamon drugs QC analysis and identify alternative markers for their discrimination.

A biochemometric approach for the assessment of Phyllanthus emblica female fertility effects as determined via UPLC-ESI-qTOF-MS and GC-MS.

Phyllanthus emblica L. fruits have long been used in Ayurvedic medicine for their many health benefits. In this study, we present P. emblica fruit crude extract and fractions' effects on the female reproductive system by assessing its estrogenic and gonadotropic activities. Results revealed that the non-polar petroleum ether and chloroform fractions exhibited the strongest estrogenic and follicle-stimulating hormone-like [FSH] activity, while the n-butanol fraction exhibited a significant luteinizing hormone-like [LH] activity. The ethyl acetate fraction showed neither estrogenic nor gonadotropic activities and in contrast it may impair female fertility suggesting that different metabolite classes contribute to the plant's overall effect on female fertility. To pinpoint active agents in these fractions, UPLC/ESI-qTOF-MS- was employed for secondary metabolite profiling with 100 metabolites annotated including ellagitannins, gallic acid derivatives, terpeneoids, sterols, phthalates and fatty acids. Correlation between extracts/fraction bioassays and UPLC/MS data was attempted using orthogonal partial least squares-discriminant analysis (OPLS-DA) revealing that guaiane-type sesquiterpenes, phthalates, diterpenes and oxygenated fatty acids showed positive correlation with estrogenic and gonadotropic activities. In contrast, mucic acid gallates, gallic acid derivatives and ellagitannins correlated negatively. GC/MS analysis of the non-polar bioactive fractions viz. petroleum ether and chloroform was also attempted which revealed its enrichment in fatty acids/fatty acyl esters (34%) and phenolic compounds (19.6%). This study provides the first report on the estrogenic and gonadotropic activities of P. emblica fruits in relation to their metabolite fingerprint.