Phytochemical and biological studies of Phyllanthus emblica and Ficus benghalensis on female reproductive function in rats A Thesis submitted by Sally Eid Morsy Khaled Research Assistant Pharmacognosy Department, National Research Centre For the Degree of Doctor of Philosophy in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr. Fatma Abdel-Megeed Hashem Prof. Dr. Mohamed Ali Ali Farag Professor of Pharmacognosy Professor of Pharmacognosy Pharmacognosy Department Pharmacognosy Department National Research Centre Faculty of Pharmacy, Cairo University Ass. Prof. Dr. Manal Hassan Shabana Ass. Prof. Dr. Dalia Adel M. Al-Mahdy Associate Professor of Phytochemistry Associate Professor of Pharmacognosy Phytochemistry and plant systematics Department Pharmacognosy Department National Research Centre Faculty of Pharmacy, Cairo University Pharmacognosy Department Faculty of Pharmacy Cairo University A.R.E. 2019 Thesis Abstract Phytochemical and biological studies of Phyllanthus emblica and Ficus benghalensis on female reproductive function in rats By Sally Eid Morsy Khaled Research Assistant Pharmacognosy Department, National Research Centre Phyllanthus emblica L. fruits and Ficus benghalensis aerial roots have long been used in Ayurvedic medicine for their many health benefits. This study attempts to justify biochemically the traditional use of the aforementioned plants in treatment of female reproductive disorders in relation to their secondary metabolite profile. The effect of the total ethanol extract and successive fractions of these plants on the female reproductive system was evaluated by assessing their estrogenic and gonadotropic activities. Results revealed that the non-polar petroleum ether and chloroform fractions of P. emblica exhibited the strongest estrogenic activity besides gonadotropic activity, while n-butanol fraction exhibited a significant follicle stimulating hormone-like [FSH] activity and luteinizing hormone-like [LH] activity. Regarding F. benghalensis, only non-polar petroleum ether and chloroform fraction exhibited moderate estrogenic and FSH-like activity with no LH-like activity. To pinpoint active agents in these fractions, metabolite profiling was attempted via UPLC-MS and GC-MS. UPLC-MS annotated 92 and 84 metabolites in P. emblica fruit and F. benghalensis aerial root, respectively. Secondary metabolites annotated in P. emblica included ellagitannins, gallic acid derivatives, terpenoids, sterols, phthalates and fatty acids. The active non-polar fractions of F. benghalensis revealed similar metabolites profile being composed of isoflavonoids, triterpenes, sterols, fatty acids and cyclic peptides; likely to mediate for its effects. GC-MS analysis of the non-polar bioactive fractions was further employed revealing the enrichment of non-polar fractions of both plants in fatty acids/fatty acyl esters, terpenes and phenolic compounds. Correlation between extracts/ fractions bioassays and UPLC-MS data of P. emblica was attempted using orthogonal projection to latent structures-discriminant analysis (OPLS- DA) revealing that guaiane-type sesquiterpenes, phthalates, diterpenes and oxygenated fatty acids showed positive correlation with estrogenic and gonadotropic activities. Five compounds were isolated from the active fractions of P. emblica fruit. Finally, this is considered the first report on the estrogenic and gonadotropic activities of P. emblica fruits and F. benghalensis aerial roots in relation to their metabolite fingerprint. Key words: Phyllanthus emblica, Ficus benghalensis, estrogenic activity, gonadotropic activity, metabolomics, UPLC-MS, GC-MS, multivariate data analysis Introduction Female reproductive disorders such as endometriosis, polycystic ovary syndrome, leucorrhea, menstrual disturbance and infertility, are evolving as predominant health care problems among the female population mainly characterized by abnormal hormone production (Adhikari et al., 2018). Though most reproductive or gynecological disorders are not life threatening, nevertheless they may severely affect women’s quality of life and mental well-being. Gynecological disorders may lead to different physiological symptoms as pelvic pain or urinary incontinence that consequently generates psychosocially based symptoms, such as fatigue and insomnia having a negative impact on women’s health and functioning (Rannestad et al., 2000, Rannestad et al., 2001). Moreover, inflammatory, endocrine and metabolic mechanisms associated with gynecological disorders are responsible for an increased incidence of obstetric complications and female infertility (Vannuccini et al., 2015). Female infertility accounts for 35-40% of overall infertility. It is a challenging issue for a lot of females affecting 8-12% couples worldwide causing damaging psychological effects and triggering negative emotions such as anxiety, stress and depression. Ovulatory problems cause about 20-30% of female infertility cases. Ovulation, fertilization and implantation are generally regulated by hormonal balance through the hypothalamic/pituitary/ovarian axis, so keeping this balance is important to treat infertility (Gaware et al., 2009). Hormonal therapy e.g. clomiphene, human chorionic gonadotropin (hCG) and gonadotropin releasing hormone (GnRH) agonists are commonly used for treatment of gynecological disorders and infertility through induction of ovulation (Dalal and Agarwal, 2015). But nowadays, many women refuse hormone replacement therapy, due to adverse effects like vaginal bleeding and increased risk of developing cardiovascular diseases, breast and ovarian cancers. Therefore, much more effort is given to safer natural alternatives, including herbal drugs as soyabean, chasteberry and evening primrose (Jadhav and Bhutani, 2005). Medicinal plants are used worldwide for the treatment of various ailments, as well as for development of novel drugs. Over 20,000 species of medicinal plants are used in traditional systems of medicine with many people resorting to these plants for treatment of various health challenges (Kar et al., 2015). According to a survey by the World Health Organization (WHO), 80% of the population living in developing countries rely almost entirely on traditional medicine for their primary health care needs and the WHO has endorsed their safe and effective use (Ekor, 2014). Nowadays, the need for alternative medication for menopausal and postmenopausal women, based on plant extracts is increasing day by day. Taking all this into consideration, the evaluation of the rich heritage of traditional medicine is deemed essential to identify new drugs or to find new lead structures for development of novel therapeutic agents. Consequently, our goal in this study is to search for natural alternatives for treatment of female reproductive disorders. By surveying the available literature, ethnopharmacological studies demonstrated the wide traditional use of Phyllanthus emblica fruits and Ficus benghalensis aerial roots in alleviating gynecological disorders (Gaware et al., 2009). These two medicinal plants are used alone or in multicomponent herbal preparations for alleviating menstrual disturbances, reducing menstrual pain, curing leucorrhea, gonorrhea, female genital tract diseases and infertility (Jadhav and Bhutani, 2005, Adhikari et al., 2018, Balamurugan et al., 2018). Phyllanthus emblica L. fruit is one of the oldest edible fruits commonly used in Ayurvedic and traditional Chinese medicine for its anti-inflammatory, rejuvenating and health-promoting properties, as well as nutritional qualities (Gaire and Subedi, 2014, Jaiswal et al., 2016). The name Phyllanthus emblica in Sanskrit, an old Indian language means female-supporter fruit referring to its effects on the female reproductive system (Scartezzini and Speroni, 2000). Ficus benghalensis L. is the national tree of India, belongs to family Moraceae that comprises ca. 38 genera and more than 1100 species (Christenhusz and Byng, 2016). F. benghalensis is considered a well reputed Asian plant medicinally, socio-culturally and ethnobotanically with a very long-life span. Almost all parts of the Ficus benghalensis tree e.g. aerial roots, latex, stem, bark, leaves and fruits are used widely in Ayurvedic medicine for the treatment of various ailments like toothache, diarrhea, inflammation, female sterility, leucorrhoea, vaginal diseases, gonorrhea, rheumatism and skin disorders (Manjusha Vinjamury, 2004, Ahmad et al., 2011). F. benghalensis and P. emblica are both safe, well reputed plants in different traditional medicine systems and easily available in Egypt. Taking this into account, in addition to their reported traditional use in treatment of gynecological disorders and the absence of pharmacological evidence supporting this use, it was deemed necessary to carry out detailed pharmacological and phytochemical investigations on Phyllanthus emblica fruit and Ficus benghalensis aerial roots to discover the unexploited potential of these medicinal plants on female reproductive function with the aim of discovering novel, safe and economic natural therapeutic agents to treat gynecological disorders. Herbal drugs have been long used to improve or regulate fertility through several mechanisms including: stimulation of normal pituitary response of gonadotropin releasing hormone (GnRH), enhancing secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), induction of ovulation via estrogenic activity. Therefore, the present study aims to assess the estrogenic