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 , 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 and gonadotropic effects of the total ethanol (TE) extract, as well as subfractions, viz. petroleum ether (PE), chloroform (CH), ethyl acetate (EA) and n-butanol (BU) of both plants under investigation. Furthermore, the ethanol extract and subfractions were subjected to comprehensive metabolites profiling using UPLC-ESI- MS and GC-MS to identify underlying active agents. To achieve such goal, UPLC-MS data was subjected to chemometrics analysis to determine similarities and differences among fractions and to correlate metabolite profiles with the examined biological activities. An illustrative diagram outlining the study design is shown in Fig. (1).

Fig. 1: Illustrative diagram outlining the study design.

Aim and rationale of the study To rationalize the Ayurvedic use of P. emblica and F. benghalensis for the treatment of female reproductive disorders in relation to their secondary metabolite profiles aiming to discover novel natural therapeutic alternatives for the treatment of some gynecological disorders especially female infertility.

Plan of work 1- Collection, identification and authentication of the plants. 2- Reviewing the available literature on the plants under study. 3- Preparation of crude extracts and successive fractions. 4- Biological evaluation of the crude extracts and successive fractions of both plants on female reproductive function in rats via assaying estrogenic and gonadotropic activities. 5- Metabolite profiling of crude extracts and successive fractions of both plants under study via UPLC-ESI-MS. 6- Chemometric analysis using multivariate data analysis techniques; principle component analysis (PCA) and orthogonal projection to latent structures- discriminant analysis (OPLS-DA). 7- Metabolite profiling of non-polar fractions of both plants under investigation using GC-MS. 8- Isolation and structural elucidation of phytoconstituents from the bioactive fractions of P. emblica fruits.

Review of Literature

Genus Phyllanthus I. Traditional uses of genus Phyllanthus Several species of Phyllanthus have long been used as herbal drugs in traditional medicine; particularly in the Ayurvedic medicine system, to treat various kinds of diseases, notably, digestive, genitourinary, respiratory, kidney and skin diseases (Duke, 2019). P. emblica, P. niruri and P. reticulatus are the most widely used species traditionally around the world. P. niruri is used to cure inflammation, fever, malaria, hepatitis, kidney disorders and gonorrhea (Narendra et al., 2012); while P. reticulatus is used in treatment of diarrhea, diabetes, urination disorder and asthma (Sharma and Kumar, 2013). The fruit of P. emblica; the plant under study, has a reputed history in traditional medicine systems (Ayurvedic, Unani, Siddha and Homoeopathic systems). It is used as a tonic in Indian Ayurveda and for treatment of liver diseases and urinary diseases (Summanen, 1999). In Traditional Chinese Medicine (TCM), P. emblica is used for clearing heat from throat and moistening lungs for arresting cough because of its sweet and astringent taste. In Thailand, it is used to treat gastrointestinal diseases (Mao et al., 2016). Fruits are used in treatment of gonorrhea (Balamurugan et al., 2018), menorrhagia, female infertility (Adhikari et al., 2018). In addition, it is used in treatment of dysfunctional uterine bleeding, leucorrhoea, menopausal syndrome, anorexia, anemia and diabetes (Manjusha Vinjamury, 2004, Caldecott, 2006). Moreover, the unripe fruits are made into pickles and given before meals to stimulate the appetite in anorexia. The juice of fresh P. emblica is considered an effective remedy for bleeding piles and non-specific epistaxis as P. emblica is considered a haemostatic agent (Nadkarni, 1954). P. emblica is a highly nutritious edible fruit, so products as candy, jam, sharbat and vinegar are developed (Rai et al., 2018). P. emblica is also used cosmetically for promoting hair growth and preventing premature graying, so P. emblica oils and shampoos are very popular aids for hair care. Most of the "rasayana" (rejuvenation and longevity tonics) of Ayurveda are made with P. emblica as an ingredient (Dharmananda, 2003) and consequently it is used in many Ayurvedic formulations as: Triphala, which is an important Ayurvedic herbal preparation composed of three medicinal fruits namely P. emblica, Terminalia chebula, and T. belerica in equal proportions. Triphala is an antioxidant-rich herbal formulation that possesses different beneficial properties. It is a widely prescribed Ayurvedic drug and is used mainly as a colon cleanser, digestive, diuretic, and laxative. Triphala is also known for its rejuvenating effects. The decoction of Triphala has been found to treat leucorrhea in women (Baliga, 2010). Gyanetone®, Gynaelix ®, Lukol® and M2-Tone® are examples of Ayurvedic herbal products indicated for irregular menstrual cycle, dysfunctional uterine bleeding, menorrhagia, dysmenorrhea, anovulation and premenstrual syndrome. They are also used as general tonics for reenergizing the internal and external genital system in women, as well as, acting as uterine tonics improving uterine circulation and for managing infertility. II. Main phytoconstituents reported for genus Phyllanthus Reviewing the reported literature revealed that genus Phyllanthus is enriched with a wide variety of chemical constituents belonging to several chemical classes ranging from polar phenolic compounds i.e. , flavonoids and lignans to non-polar sterols and terpenoids. A. Tannins: Tannins are the largest group of compounds found in the genus Phyllanthus and are often credited for various bioactivities of these plants. Hydrolyzable tannins are more prevalent in Phyllanthus than condensed tannins. Corilagin and geraniin are most extensively obtained from this genus (Qi et al., 2014). Phyllanthus emblica produces the largest variety of tannins, including mucic acid galloyl derivatives, galloyl derivatives, A, emblicanin B chebulagic acid, corilagin, geraniin, pedunculagin, phyllanemblinin A–F, and prodelphinidins (Mao et al., 2016). B. Flavonoids: Flavonoids are one of the main groups of secondary metabolites produced by Phyllanthus comprising flavanones, flavonols, flavones, flavanonols and flavanols. Flavanols are the basic component of condensed tannins. Flavonoids were found abundantly in leaves of P. emblica and P. niruri (Qi et al., 2014). C. Lignans: Different types of lignans have been reported in Phyllanthus where dibenzylbutane-type lignans represent the major class. P. niruri and P. urinaria are the major lignan-producing species. No lignans were reported for P. emblica (Nahar et al., 2011). D. Other Phenolics: Phenolic compounds as pyrogallol, protocatechuic acid, bergenin and were also found in Phyllanthus species (Zhang et al., 2000, Yang et al., 2007). E. Terpenoids: Terpenoids are prevalent in Phyllanthus comprising mainly sesquiterpenes and triterpenes with few reported diterpenes. Terpenoids were mostly reported in P. flexuosus, P. emblica, P. niruri, and P. polyphyllus. Sesquiterpenes: Sesquiterpenes isolated from genus Phyllanthus comprise mainly bisabolane and guaiane-type (Sutthivaiyakit et al., 2003, Ratnayake et al., 2008). P. emblica roots produces the largest number of highly oxygenated norbisabolane sesquiterpenes i.e. Phyllaemblicin A-G and phyllaembelic acid A-C (Zhang et al., 2000, Zhang et al., 2001, Lv et al., 2014). Diterpenes: Limited number of diterpenoids were isolated mainly from P. oxyphyllus e.g. cleistanthane diterpenoids and oxygenated pimarane diterpenes from P. niruri (Sutthivaiyakit et al., 2003, Qi et al., 2014). Triterpenes: The vast majority of triterpenes isolated from Phyllanthus species possess pentacyclic structures mainly lupane, oleanane and friedelane- type, also tetracyclic and acyclic triterpenes are found. Triterpenes occurred mainly in P. flexuosus, P. niruri and P. polyphyllus (Qi et al., 2014). F. Sterols: Stigmasterol, β-sitosterol, and daucosterol are the three main steroids found in a number of Phyllanthus species (Nahar et al., 2011). G. Phthalates: Genus Phyllanthus, like several medicinal plants and marine algae, was reported to naturally produce phthalates viz., bis (2-ethyloctyl) phthalate and bis (2- ethylicosyl) phthalate, and they often exhibited antimicrobial activities (Saleem et al., 2009, Saeidnia and Abdollahi, 2013) H. Nitrogenous compounds: Alkaloids and other nitrogenous compounds were also reported in genus Phyllanthus. Alkaloids identified in Phyllanthus were predominantly of the securinine/norsecurinine-type. Alkamides are found in P. fraternus (Sittie et al., 1998).

Genus Ficus

I. Traditional uses of genus Ficus Genus Ficus commonly known as figs, family Moraceae constitutes an important group of trees with immense medicinal value. Members of this genus have worldwide use and are one of the earliest sources of cultivated medicines and food for both humans and animals. Several plants of this genus are used in medicine as astringents, laxatives and anthelmintics (Bwalya, 2015) and for the treatment of skin diseases, liver diseases, dysentery, diarrhea, diabetes, lung complaints, leucorrhoea, heart diseases, cough, asthma, piles, ulcers, gonorrhea and rheumatism (Burrows and Burrows, 2003, Sawarkar et al., 2011). Among the varied number of Ficus species, the most important ones are the four trees that constitute the group “Nalpamaram”, namely; F. racemosa, F. microcarpa, F. benghalensis and F. religiosa. Nalpamaram is an important ingredient in most Ayurvedic formulations, used in diabetes, diarrhea, nervous disorders, vaginal diseases, leucorrhea and menorrhagia (Sivarajan and Balachandran, 1994). Ficus benghalensis; the plant under study, is considered a sacred tree in some places. It is used in traditional systems of medicine like Ayurveda, unani and homoeopathy. Pharmacopeias of Indian medicine contain much valuable information about the medicinal uses of various parts of this tree in treatment of many ailments. Leaves are used for treatment of ulcers, asthma and male sexual weakness (Atta-ur-Rahman, 2012), while latex is used in treatment of rheumatism and other inflammatory diseases and fruits are tonics (Mandal et al., 2010). Aerial roots are used in treatment of female infertility and menstrual pain (Atta-ur-Rahman, 2012), it is also used in treatment of gonorrhea (Mandal et al., 2010), genital tract diseases (Jadhav and Bhutani, 2005), metrorrhagia and vaginal complains (Adhikari et al., 2018). F. benghalensis is an important component of several Ayurvedic marketed formulations; the most important of which is M2-tone ® used as a uterine tonic and for improving uterine circulation and managing infertility. II. Main phytoconstituents reported for genus Ficus A phytochemical review shows that plants belonging to genus Ficus are rich sources of distinct classes of secondary metabolites e.g. flavonoids, isoflavonoids, coumarins and terpenoids. A. Isoflavonoids: Isoflavonoids are the major class of phenolic compounds reported in genus Ficus (Bwalya, 2015). Isoflavonoid aglycones i.e. genistein and prunetin were found in some Ficus species as F. carica and F. nervosa (Chen et al., 2010, Prokudina et al., 2011). Prenylated isoflavonoids as well, are predominant in Ficus, for example, alpinumisoflavone and derrone pyranoisoflavones in addition to wighteone and lupiwighteone prenyl chain isoflavones (Dai et al., 2012, Jiang et al., 2012). Isoflavone dimers; namely mucuisoflavone A-C, are found exclusively in F. mucuso (Bankeu et al., 2011). Only genistein and alpinumisoflavone were reported for F. benghalensis (Riaz et al., 2012, Naveed et al., 2015). B. Flavonoids: Flavonoids are one of the main groups of secondary metabolites produced by genus Ficus comprising mainly flavonols, flavones, flavanols and flavanones (Bwalya, 2015). Leucoanthocyanidins are isolated from barks of F. benghalensis (Geetha et al., 1994, Daniel et al., 2003).

C. Coumarins: Different types of coumarins have been reported in Ficus e.g. simple coumarins as umbelliferone and scopoletin alongside bergapten and psoralen furanocoumarins. F. tsiangii and F. carica are considered the major coumarin-producing species (Wang et al., 2014, Wang et al., 2017, Duan et al., 2018). D. Lignans: Few lignans were isolated from Ficus species (Li and Kuo, 2000, Wang et al., 2014). E. Phenolic acids: Benzoic acid derivatives and derivates were reported in aerial root of F. benghalensis (Riaz et al., 2012, Naveed et al., 2015). F. Sterols: Sterols are another interesting class of bioactive compounds found in genus Ficus. β-sitosterol, β-sitosterol glucoside and stigmasterol are the main steroids found in Ficus species especially F. benghalensis aerial roots (Riaz et al., 2012, Naveed et al., 2015). G. Triterpenes: Triterpenes isolated from genus Ficus posses either tetracyclic or pentacyclic rings, where major classes of triterpenes isolated form genus Ficus are tirucallanes, dammaranes, oleananes, ursanes and lupanes. F. microcarpa and F. tsiangii are major sources of triterpenes (Chiang et al., 2001, Chiang et al., 2005, Wang et al., 2014).

Summary

Part A: Biological evaluation and metabolite profiling of P. emblica fruit I. Effect of P. emblica extract and fractions on female reproductive functions. In an attempt to study the effect of P. emblica fruit on female reproductive function, a detailed investigation of the hormonal activity and action mechanism post intraperitoneal and oral administration of the total ethanol (TE) extract and different fractions (PE, CH, EA, BU ) of P. emblica fruit was carried out through evaluation of estrogenic and gonadotropic activities. A. Evaluation of estrogenic activity of P. emblica fruit. The estrogenic activity of P. emblica fruits was evaluated using a four-fold criterion: uterotropic assay, vaginal cornification, estimation of serum estradiol levels and histological examination of uteri in ovariectomized immature female rats resulting in significant activity for the PE and CH fractions. Administration of the PE and CH fractions regulated the estrous cycle of ovariectomized rats through their direct estrogenic activity. The estrogenic activity was manifested by significant increase in uterine weight due to uterine fluid retention with an increase in serum estradiol level. Total cornification of vaginal epithelial cells was recorded which pointed to the onset of the estrous stage for ovariectomized rats. Total ethanol extract showed weak estrogenic activity with significant increase in serum estradiol level and a vaginal cornification score of 2. Polar fractions (EA and BU) showed no significant increase in uterine weight or serum estradiol level with no cornified cells in vaginal epithelium. B. Evaluation of gonadotropic activity of P. emblica fruit. Gonadotropic like action of total ethanol extract and different fractions of P. emblica was determined through assay of FSH and LH like activity. 1. Evaluation of FSH-like activity of P. emblica fruit. FSH-like activity was determined using the traditional Steelman–Pohley assay, which measures ovarian hypertrophy resulting from exogenous FSH in immature female rats.The number of follicles at the different stages of maturation in each ovary was counted to determine the relative potency of the total ethanol extract and fractions according to different concentration of standard PMSG (1, 5, 10 and 20 IU). The PE, CH and BU fractions of Emblica fruit showed an increase in genitalia weight together with an increase in total number of follicles at different maturation stages. Histological examination revealed that the aforementioned fractions increase developing follicles compared to atretic follicles in ovaries. It could be concluded that these fractions induce folliculogenesis with different relative potencies. The total ethanol extract showed significant increase in total follicles with few follicular maturations in ovaries, while the EA fraction showed no FSH-like activity. 2. Evaluation of LH- like activity of P. emblica fruit The number of corpora lutea (C.L.) is considered as an index for total ovulation and LH activity supported by the histological picture of the genitalia. In LH-like action experiment, the PE, CH and BU fractions of Emblica fruit showed profound LH-like action manifested by an increase in the number of corpora lutea compared to the control group and confirmed by histological examination of rats’ uteri showing an increase in uterine activity and increase in the height of lining epithelium. The BU fraction of P. Emblica fruits exerted strong LH-like action (>10 IU hCG) which may be the reason for the loss of its estrogenic activity. Administration of the TE extract and EA fraction showed no significant change in corpora lutea number compared to the negative control. II. Metabolite profiling of P. emblica fruit. In this study, an integrated approach utilizing hyphenated techniques viz. UPLC-MS and GC-MS was adopted for identification and comparison of the phytochemical constituents in total ethanol and different successive fractions of P. emblica fruit that may be responsible for the biological activities. A. UPLC-MS metabolite profiling of P. emblica. 1. Identification of metabolites in P. emblica fruits via UPLC–MS analysis. Chemical constituents of the total ethanol and successive fractions of P. emblica fruit were analyzed via reversed phase UPLC-MS in both positive and negative-ion electrospray ionization (ESI) modes for a comprehensive view of the metabolites within a run of 15 min (ca. 900 sec). A total of 100 chromatographic peaks were detected of which 92 were tentatively annotated belonging to various classes, viz. polyphenolic constituents e.g. hydrolyzable tannins (gallotannins and ellagitannins), gallic acid derivatives, phenolic acids, and flavonoids, as well as, lipid components e.g. phospholipids, fatty acids, terpenes and steroids. The identification was based on assigning the molecular ion, chemical formula, tandem MS fragments and comparing their retention times. Hydrolysable tannins represented the most abundant class of metabolites in the polar fractions (EA and BU) whereas hydroxylated fatty acids, phospholipids, terpenes and sterols were predominant in the non-polar fractions (PE and CH). a. Phenolic components Hydrolysable tannins: Tannins mostly produce a strong deprotonated molecular ion [M– H]- in the negative ion mode. Gallotannins: A total of 29 related gallic acid derivatives and gallotannins were identified based on the characteristic ions at m/z 169 and 125 as well as the neutral loss of a dehydrated galloyl moiety 152 amu. The most abundant gallotannin subclass was galloyl mucic acid detected as 14 different metabolites of mucic acid lactone gallates, mucic acid gallates and their methyl esters. The loss of galloyl moiety (152 amu) resulted in the most characteristic fragment ions at m/z 191 and 209 in mucic acid lactone gallate and mucic acid gallate, respectively. Three galloyl esters of glucose were also detected identified as mono-, di- and tetra-galloyl glucose due to sequential losses of galloyl moieties and a hexose moiety from their parent ions at m/z 331, 483 and 787, respectively, yielding a daughter ion at m/z 169 (deprotonated gallic acid). Other gallic acid derivatives: Gallic acid and its derivatives were also identified by the presence of fragment ions at m/z 169 and 125 characteristic of gallic acid. Derivatives of gallic acid were identified as gallic, digallic acid, methyl gallate, methyl digallate and ethyl gallate. Moreover, bergenin, the C-glycoside of 4-O-methyl gallic acid, was detected together with the O-demethylated derivative, norbergenin and the methylated derivative, methyl bergenin. Ellagitannins: 11 ellagitannins were detected in both negative and positive ion modes, with a higher response exhibited in negative mode for most metabolites. Ellagitannins showed characteristic fragment ion of hexahydroxydiphenoyl (HHDP) at m/z 301 through sequential losses of galloyl or gallate moieties. Major ellagitannins identified were galloyl-HHDP-hexoside, digalloyl-HHDP-hexoside, geraniin, chebulagic acid and mallotusinin. Ellagic acid and ellagic acid hexoside were also detected. Flavonoids: Only two flavonol aglycones, quercetin and kaempferol were detected. Other Phenolics: Trimethoxybenzene-tricarboxylic acid and methoxy-(phenyl methoxy) benzoic acid were also annotated. b. Lipids: In the second half of the chromatographic run (13.5-27 min); particularly in the petroleum ether and chloroform fractions, lipid components were detected. This is the first report for the presence of oxygenated fatty acids and phospholipids in P. emblica suggesting that UPLC-MS represents another platform for lipid profiling in P. emblica. Fatty acids: 14 fatty acid derivatives were annotated as hydroxy fatty acids, methyl esters of fatty acid, fatty acylamides and monoglyceride. Negative ionization MS of the petroleum ether fraction revealed several hydroxy fatty acids assigned as the major peaks. Major hydroxyl fatty acids annotated were hydroxyl octadecatrienoic acid, hydroxyl octadecadienoic acid, dihydroxy octadecadienoic acid, dihydroxy octadecenoic acids and dihydroxy octadecanoic acid. Also, oxo-octadecatrienoic acid and oxo-octadecadienoic acid were identified. Positive ionization MS of petroleum ether fraction showed methyl esters of fatty acids identified from the loss of 32 amu annotated as methyl octadecatrienoate and methyl-hydroxy-octadecadienoate. Two fatty acylamide were revealed from their even mass weight and the loss of an ammonia molecule as oleamide and docosenamide. A single monoglyceride derivative was detected from the loss of glycerol moiety namely octadecatrienoylglycerol. Phospholipids: UPLC-MS/MS analysis of P. emblica fruit resulted in identification of 10 lysophospholipids, including five lysophosphatidylcholines (LPC), three lysophosphatidylethanolamines (LPE), and two lysophosphatidylinositols (LPI). Phospholipids were most abundant in the chloroform fraction of P. emblica with LPC detected as the major class. LPC and LPE can be detected in positive ESI mode showing an even mass weight although LPI was assigned in negative ESI mode. Sterols: Three stigmasterols were detected in positive ion mode of the non-polar fractions (PE and CH), annotated as stigmasta-trienol, secostigmasta-tetraenol and stigmasterol. Terpenes: Terpenoids were the most prevalent chemical class of genus Phyllanthus. Two guaiane-type sesquiterpenes were identified in positive ion mode of the non-polar fractions annotated as epoxy guaiadienolide and cinnamoyl epoxy guaiandiol which are considered major metabolites in the PE and CH fractions. Five triterpenes were detected in positive ion mode MS of Emblica fruit non-polar fractions, four of them were oleanane-type triterpenes. Hydroxy abieta-trienone diterpene was also identified. c. Miscellaneous Organic acids: Seven organic acids could be detected in Emblica fruit. Mucic acid and tuberonic acid (hydroxyl jasmonic acid) were the main organic acid in Emblica fruit petroleum ether fraction. Phthalates: Bis-ethyl hexyl phthalate and dibutyl phthalate were the major metabolites in the non-polar fractions of Emblica. Phyllanthus was previously reported to produce phthalic acid esters as secondary metabolites. 2. Correlation between UPLC-MS metabolite profile of P. emblica and bioassays via multivariate data analysis (OPLS-DA). The UPLC-MS fingerprint was subjected to chemometric modeling to correlate the biological activities to specific chemical constituents in P. emblica extract and/or fractions. Multivariate data analysis tools (OPLS-DA) were applied on a data set comprising both the UPLC-MS profiles of P. emblica extracts and fractions as X variables and the corresponding estrogenic and gonadotropic parameters measured as Y variables. Two OPLS models were established for the estrogenic activity and the gonadotropic activity for both the positive and negative ionization UPLC-MS data sets. The OPLS-DA score plots showed clear separation between both sample groups particularly for the estrogenic activity models with R2 and Q2 values higher than 0.4 and close to 1 indicating reliability and good predictive abilities of the models. Twenty-six metabolites were identified as potential biomarker compounds effectively discriminating between the modeled classes for the estrogenic or gonadotropic activities. Correlation analysis revealed that metabolites predominantly present in the bioactive petroleum ether and chloroform fractions positively correlated with the parameters measured for the estrogenic and the gonadotropic activities’ models. The following metabolites were identified:  Bis (ethyl hexyl) phthalate  Dibutyl phthalate  Cinnamoyl epoxy guaiandiol  Epoxy guaiadienolide  Methyl octadecatrienoate  Octadecenamide  Hydroxy abietatrienone  Labdane triterpene  Tuberonic acid In Phyllanthus, cinnamoyl epoxy guaiandiol and epoxy guaiadienolide are considered major metabolites in the PE and CH fractions, respectively and are more likely to exert an effect in this study. The results showed that mucic acid gallate derivatives and 2 tannins (pedunculagin and ) in addition to 2 organic acids identified principally in the less bioactive polar fractions (EA and BU) showed negative correlations with either the estrogenic or the gonadotropic models or both. B. GC-MS analysis of the silylated metabolites in the non-polar fractions of P. emblica. GC-MS was further adopted for metabolite profiling after silylation to provide a comprehensive overview of the PE and CH metabolites that can be mediating for the potential estrogenic and gonadotropic like activity. A total of 107 metabolites were tentatively identified in the PE and CH fractions of P. emblica fruits via GC-MS classified as organic acids, sugars, aromatic compounds, fatty acids and fatty acyl esters, terpenes and sterols. The major annotated metabolite classes in the PE fraction were free fatty acids/ esters (34.07 %) and phthalates and phthalides (13.91%), while major classes identified in the CH fraction were aromatic compounds (19.67%) followed by sugars (10.99%), organic acids (13.75%), and fatty acids (6.86%). This is the first report for annotating phthalides in Phyllanthus. Phthalides are responsible for the estrogenic activity of Angelica sinensis and Ligusticum striatum. The most discriminative metabolites in the CH fraction was pyroglutamic acid. Pyroglutamic acid is the N-terminal amino acid in gonadotropin releasing hormone (GnRH). C. Isolation and structure elucidation of phytoconstituents from bioactive fractions of P. emblica fruit The petroleum ether and chloroform fractions of P. emblica fruit exhibited strong estrogenic and gonadotropic activities and consequently were subjected to detailed phytochemical investigations in an attempt to isolated and identify their major compounds responsible for the activity. The petroleum ether and chloroform fractions of P. emblica were subjected to different chromatographic separation techniques. Three compounds were isolated. They were identified by their physico-chemical and spectral data (1H-NMR, 13C- NMR and HR-ESI-MS) and by comparison with published data and available authentic samples. The isolated compounds were identified as follows: Compound P1: β-sitosterol Compound P2: (E)-Cinnamic acid Compound P3: β-sitosterol glucoside The n-butanol fraction showed good gonadotropic activity and detailed phytochemical investigations led to the isolation of two pure compounds (B1 and B2). Structural elucidation of these compounds was done using conventional chromatographic properties and spectroscopic methods (1H, 13C-NMR and HR-ESI-MS). The two isolated compounds were identified as follows: Compound B1: 1-O-galloyl-β-glucospyranose Compound B2: 1,6-di-O-galloyl-β-glucospyranose

Part B: Biological evaluation and metabolite profiling of F. benghalensis aerial root: I. Effect of F. benghalensis extract and fractions on female reproductive functions: Detailed in-vivo investigation of the effect of the total ethanol (TE) extract and different fractions (PE+CH, EA and BU) of F. benghalensis aerial root on female reproductive hormones and reproductive organs (ovary and uterus) was attempted to validate the traditional uses of this plant in treatment of female infertility and female reproductive disorders. A. Evaluation of estrogenic activity of F. benghalensis aerial root. The estrogenic activity of F. benghalensis was determined using the uterotropic assay, vaginal cornification method along with the estimation of serum estradiol levels and histological examination of uteri in immature ovariectomized female rats resulting in a good estrogenic activity for the non-polar fractions (combined petroleum ether and chloroform). This estrogenic activity was manifested by a significant increase in serum estradiol level, cornification of vaginal cells accompanied by change in the estrus cycle, and a non- significant 1.8-fold increase in uterine weight compared to the negative control group. These findings were confirmed by histological examination of the uteri which showed moderate endometrial activity compared to the negative control group. On the other hand, the total ethanol extract, EA and BU fractions of F. benghalensis showed no estrogenic activity in terms of uterine weight, serum estradiol level, vaginal cornification and histological examination. B. Evaluation of gonadotropic activity of F. benghalensis aerial root. Gonadotropic activity of the total ethanol extract and different fractions of F. benghalensis was determined through assay of FSH and LH like activity. 1. Evaluation of FSH-like activity of F. benghalensis aerial root FSH-like activity was determined through total follicular count at different developmental stages. The combined PE and CH fraction may exert indirect estrogenic activity through their FSH-like activity. Combined PE and CH fraction exerted moderate FSH-like activity manifested as a significant increase in the follicular population count showing relative potency less than 1 IU PMSG for this group. Histological examination of the ovaries confirmed this result revealing that these fractions produced Graafian mature follicles together with developing follicles (primary and secondary follicles) and hence elicited folliculogenesis. The total ethanol extract, EA and BU fractions of F. benghalensis showed no FSH-like action. 2. Evaluation of LH- like activity of F. benghalensis aerial root The number of corpora lutea is considered as an index for total ovulation and LH activity supported by the histological picture of the genitalia. Results showed that all groups injected with the TE and successive fractions of F. benghalensis showed no LH-like activity which was manifested by a non-significant increase in the number of corpora lutea, in addition to histological examination that showed good follicular activity resulting from injection of PMSG with no detected lutenizing effect. Finally, the PE and CH fraction of F. benghalensis aerial root was considered as the non-polar fraction with potential for estrogenic activity and a positive role in folliculogenesis (FSH-like activity), but lacking LH-like activity. Because of this discrepancy in hormonal activity, caution should be taken when considering the intake of F. benghalensis for treatment of female infertility or to alleviate postmenopausal symptoms. II. Metabolite profiling of F. benghalensis aerial root. To correlate bioactivities with chemical composition, an integrated approach was developed herein, utilizing (UPLC-MS) and GC-MS hyphenated techniques, for metabolite profiling and fingerprinting of F. benghalensis aerial root for the first time. A. UPLC-MS metabolite profiling of F. benghalensis. 1. Identification of metabolites in F. benghalensis aerial root via UPLC-MS analysis The total ethanol extract and different fractions derived from F. benghalensis aerial root were analyzed in both the positive and negative ion electrospray ionization (ESI) UPLC–MS modes. After processing the data by UPLC-MS, a total of 100 chromatographic peaks were detected of which 84 were tentatively annotated in both positive and negative ion modes. The identified metabolites belonged to various classes, viz. phenolic compounds e.g. isoflavonoids, phenolic acids and lignans, as well as, lipid components e.g. fatty acids, fatty acyl amides, phospholipids, terpenes and steroids. Other , such as acids, apocarotenoid sesquiterpenes and cyclopeptides were also observed. Interestingly, isoflavones, fatty acids, cyclopeptides, terpenes and sterols were predominant in the bioactive non-polar fractions whereas phenolic acids, apocarotenoids and lignans were more abundant in the polar fractions. Fatty acyl amides were exclusively detected in the ethyl acetate fraction in positive ion mode. a. Phenolic compounds Phenolic compounds included isoflavonoid as the major class which was more prominent in the chloroform fraction, in addition to less abundant phenolic acids appearing in the polar fractions. Isoflavonoids: UPLC-MS analysis of F. benghalensis allowed the tentative identification of 15 isoflavonoids, including several prenylated forms either prenylchain or pyranoisoflavonoids. Prenylated isoflavonoids assignment was based on different diagnostic fragment ions MSn and neutral losses observed in the PI mode. From a biosynthetic prespective, isoflavonoid constituents characterized in F. benghalensis seemed to be biosynthesized from the two simple isoflavones precursors; genistein and hydroxygenistein, followed by prenylation at A- and B-ring, the prenyl group most probably underwent cyclization with the ortho phenolic hydroxyl group to afford pyranoisoflavones that can show hydration at the pyrano ring double bond. In some cases, diprenylation occurred. Phenolic acids: Negative ion MS revealed the presence of 11 phenolic acids in F. benghalensis belonging to hydroxybenzoic and hydroxycinnamic acids eluted between a period of 1.32 to 11.68 min in the total ethanol extract and the polar fractions. Lignans: In the negative ion mode, two aryltetralin lignan glycosides were observed in the polar fractions. b. Lipids: This is considered the first report for lipid profiling in F. benghalensis and suggesting that UPLC-MS represent a potential platform for lipid profiling. Lipoidal metabolites were more abundant in the bioactive non-polar fractions except for fatty acid amides which predominated in the EA fraction. Fatty acids: Negative ionization MS revealed 10 mono, di and trihydroxy fatty acid conjugates assigned from their high-resolution masses and predicted formulae and loss of water molecule (-18 amu) alongside 5 fatty acids. Fatty acid amide: Primary fatty acid amides (PFAMs) were another abundant class identified in F. benghalensis for the first time, in which14 PFAMs were annotated mainly in the EA fraction. PFAMs are ionized in positive mode following protonation at the carbonyl oxygen of the amide group. Sterol/terpenes: A total of 12 terpenoids and one sterol were detected in positive ion mode and were more prevalent in the CH fraction of F. benghalensis. Five of them were assigned as ursane/oleanane type pentacyclic triterpenoids. c. Miscellaneous Apocarotenoid sesquiterpenes: Three apocarotenoids were identified in the negative ion mode predominantly in the polar ethyl acetate and butanol fractions together with the crude ethanol extract. The three identified apocarotenoids are derivatives of the plant hormone, abscisic acid that can be classified as carotenoid sesquiterpenoid, namely dihydrophaseic acid, phaseic acid and 8'-hydroxyabscisic acid. Cyclic peptides: Two cyclic pentapeptides were abundant in the non-polar fractions and were annotated as cyclo-penta(iso)leucine and cyclo-tetra(iso)leucyl-valine Acids: Negative ionization MS revealed four acids viz., quinic acid, malic acid and nonanedioic acid. 2. Multivariate PCA of F. benghalensis UPLC-MS data. UPLC-MS and multivariate data analysis were further used to identify metabolites in F. benghalensis bioactive fractions likely to mediate for the observed activity. From the four successive fractions, a total of 4808 mass signals were extracted by XCMS data analysis software from the UPLC-MS data set acquired in the negative ionization mode and were used for PCA modeling. The PCA score plot showed clear separation of the samples. On the left side of the plot, the PE and CH fractions were positioned representing negative PC1 values, whereas the BU fraction was segregated on the far right side and the EA fraction was located in between. PCA loading plot revealed that the distant clustering of the bioactive PE and CH fractions in the upper left side was mostly attributed for abundance of MS signals of fatty acids and isoflavonoids in these fractions. In contrast, hydroxyabscisic acid hexoside; an apocarotenoid, contributed positively to PC1 and was found almost exclusively in butanol fraction. B. GC-MS analysis of the silylated metabolites in the non-polar fractions of F. benghalensis. GC-MS analysis after silylation was further adopted for F. benghalensis non-polar (PE and CH) fraction as another hyphenated platform to gain more information about the chemical composition of these biologically active fractions. A total of 89 metabolites were identified via GC-MS classified as organic acids, nitrogenous compounds, sugars, phenolic acids, fatty acids/esters, and sterols/terpenes. The major annotated metabolite class was fatty acids/esters accounting for 54 % and 35.3% of the total detected metabolites in the PE and CH fractions, respectively. Sterols/terpenes amounted for 21.9% of total CH metabolites versus traces present in PE fraction (0.6%). Other well recognized metabolite classes were phenolic acids and organic acids. Saturated fatty acids and esters were predominant in F. benghalensis representing 97.6% and 78.5% in the PE and CH fractions, respectively.

Conclusion This study represents the first attempt to rationalize the ayurvedic use of Phyllanthus emblica L.and Ficus benghalenisis L. for the treatment of female reproductive disorders.  The results indicated that both the petroleum ether and chloroform fractions of P. emblica fruit possessed strong estrogenic and gonadotropic activities, while the n-butanol fraction displayed significant gonadotropic activities exhibiting LH-like effects.  A comprehensive non-targeted metabolites profiling was carried out for the total ethanol extract and different fractions of P. emblica via UPLC–MS and GC-MS analysis revealing a wide range of metabolite classes viz. fatty acids, phthalates, phthalides, terpenes and sterols in the bioactive PE and CH fractions while the polar fractions (EA and BU) showed the prevalence of mucic acid gallate derivatives, phenolics and ellagitannins. This is considered the first report for annotating phthalides in genus Phyllanthus using GC/MS.  UPLC-MS-based multivariate OPLS-DA and correlation analysis suggested that metabolites viz. phthalates, terpenes, fatty acyl amide and monoacylglycerol contribute positively to the biological activitiesof P. emblica fractions.  Our study is considered the first study in Egypt to correlate the biological activity to the chemical profile via UPLC-MS-based multivariate OPLS-DA.  Consequently, this study not only supports the use of P. emblica as a hormonal agent, it also provides a first insight on the nature of compounds accounting for its biological effect.  Regarding F. benghalensis, the petroleum ether and chloroform fractions showed moderate estrogenic activities and FSH-like effects and no LH-like activity.  This is the first comprehensive non-targeted metabolites profiling for F. benghalensis using UPLC-MS, where 84 metabolites were identified belonging to a wide range of chemical classes. Prenylated isoflavonoids were reported for the first time in F. benghalensis.  The presence of isoflavonoids, with known estrogenic activity, in the non-polar fractions of F. benghalensis albeit pharmacological results showing just moderate estrogenic activity introduces a question whether other metabolites in these fractions quench the activity.  The cultivation and climatic conditions may affect F. benghalensis phytochemically and biologically as the studies here showed moderate estrogenic and FSH-like actions, although it is used traditionally in treatment of infertility in India and Asian countries.  The structural diversity found among P. emblica and F. benghalensis metabolites is unique, explaining the extensive traditional uses of these plants. Therefore, these two plants will continue to be among the major plant sources of new chemical entities and possibly new drug molecules.