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

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. 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. 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 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. 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.