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.

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.

UPLC/Tandem mass and antimicrobial activity of Khaya senegalensis (A. Juss.) / UPLC/Tándem masa y la actividad antimicrobiana de Khaya senegalensis

Characterization of flavonoids and limonoids in the defatted acetone extract of Khaya senegalensis flowers (A. Juss.) contents was performed using ultra performance liquid chromatography (UPLC) coupled with ultraviolet (UV) and electrospray ionization (ESI) mass spectrometry, furthermore, tandem mass spectrometry (MS/MS) was performed to assist in the structural elucidation. The antimicrobial effect was tested against representative gram positive and negative bacteria and candida. Cytotoxicity of extract was evaluated using the mitochondrial- dependent reduction of MTT. The method used enabled identification of five flavonoid glycosides (di and mono- sugar) and twelve limonoids of different types viz: mexicanolides, phragmalins and angolensate were tentatively identified. The extract was effective against tested microorganism revealing potent growth inhibitory effect on Salmonella typhimurium ATCC 25566, Escherichia coli NRRN 3008 , Pseudomonas aeruginosa ATCC 10145 and fungus Candida albicans EMCC105, MIC ≤ 25µg/µl while MIC ≤ 50 µg/µl for Bacillus cereus, staphylococcus aureus ATCC 6538. Extract showed cytotoxicity against MCF7 (Breast carcinoma cell line) compared to doxorubicin, IC50=88.1(µg/mL) but no activity on HCT 116 (Colon carcinoma cell line) and HepG2 (liver cell carcinoma) was observed. Bioactive compounds in K senegalensis flowers acetone extract possesses promising antimicrobial activity with low cytotoxic effect warrants further investigation for their therapeutic and prophylactic roles

Se ha procedido a la caracterización de los flavonoides y limonoides del extracto acetónico de las flores de la especie Khaya senegalensis (A. Juss.) El análisis de sus componentes se realizó mediante cromatografía líquida de ultra resolución (UPLC) con detección ultravioleta (UV) y de espectrometría de masas de ionización por electrospray (ESI), así como espectrometría de masas (MS / MS) para ayudar en la elucidación estructural de los compuestos. Se comprobó el efecto antimicrobiano en bacterias gram positivas y negativas y levaduras como el género Candida. La citotoxicidad del extracto se evaluó mediante la reducción mitocondrial dependiente de MTT. El método de análisis permitió la identificación de cinco glucósidos flavonoides (di y mono-azúcar) y doce limonoides de diferentes tipos:se identificaron tentativamente mexicanolidos, phragmalinas y angolensato. El extracto fue efectivo contra microorganismos (≤ MIC 25µg / l ), revelando potente efecto inhibidor del crecimiento de Salmonella typhimurium (ATCC 25566), Escherichia coli (NRRN 3008), Pseudomonas aeruginosa (ATCC 10145) y el hongo Candida albicans (EMCC105). Igualmente fue eficaz a MIC ≤ 50 mg / l sobre Bacillus cereus, Staphylococcus aureus (ATCC 6538). Igualmente, el extracto mostró citotoxicidad contra línea celular de carcinoma de mama (MCF7) en comparación con la doxorrubicina, (IC50 = 88.1 (µg/ mL), pero no modificó la actividad en una línea celular de carcinoma colon (HCT 116) y en células de carcinoma del hígado (HepG2). Los compuestos bioactivos en extracto acetónico de flores de K. senegalensis poseen una actividad antimicrobiana prometedora con bajos efectos citotóxicos que garantizan la necesidad de una mayor investigación para conocer completamente sus papeles terapéuticos y profilácticos