A Pharmacognostical Study of Melilotus indicus (L.) All. Growing in Egypt A thesis submitted By Yassmin Samir Youssef Mahmoud For the Degree of Master In Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr.Amal El-SayedKhaleel Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University Dr. MaieSelmyKhader Lecturer of Pharmacognosy, National Organization for Drug Control and Research Pharmacognosy Department Faculty of Pharmacy Cairo University 2019 Abstract "A Pharmacognostical Study of Melilotus indicus (L.) All. Growing in Egypt" Botanical study including macro- and micro-morphological studies of different organs of Melilotus indicus (L.) All. were achieved for authentication and identification of the plant in the entire and powdered forms. The phytochemical study was established including preliminary screening, investigation of the essential oil, quantitative determination of the active constituents, HPLC fingerprint analysis of different plant extractives with assignment of the isolated compounds, UPLC- Mass profile of secondary metabolites of 80% methanolic extract and investigation of the polysaccharide content.Seven compounds have been isolated and identified via physical, chromatographic and spectral data namely; a hydrocarbon (nonacosane), a fatty acid (palmitic acid), a benzopyrane (coumarin), a flavonoid (kaempferol), flavonoid glycosides (trifolin and robinin) and o-coumaric acid.UPLC/Ms analysis led to the identification of 14 compounds from which 5 compounds were identified for the first timein genus Melilotus. Defatted 80% methanol extract and its subfractions have weak antioxidant activity. Methylene chloride subfraction showed cytotoxic activity on liver and colon human cell lines and ethylacetate subfraction showed a strong anticoagulant activity. Key words: Melilotusindicus, phytochemical study, UPLC/MS, botanical study, antioxidant, cytotoxic, aanticoagulant. Introduction Introduction Family Fabaceae, formerly known as Leguminosae, is one of the largest families of flowering plants afterAsteraceae and Orchidaceae, consisting of about 770 genera and 19500 species (Gomez et al. 2018)distributed under three subfamilies; Papilionodeae, Mimosoideae and Caesalpinoideae. It economically occupies the second place after family Poaceae(Gathercoal and Wirth 1949, Benson 1957, Lawrence 1958, Evans2002). Numerous plants from Fabaceae have medicinal properties and are commonly used in traditional medicine. For instance, soybeans are rich in an anti-cancer isoflavone; genistein (Messina et al. 1994, Graham and Vance 2003) , leaf ash of Cassia absus L. is applied to wounds and cuts, leaves of Crotalaria retusa L. are used to cure scabies and impetigo and roots of Indigofera tinctoria L. are used as anti-poisons remedy (Jeeva et al. 2006). Melilotus is a genus in the family Fabaceae including about 20–25 species that are widely distributed all over the world (Mabberley 1997).Members are known as common grassland plants and as weeds of cultivated ground. This legume is commonly named for its sweet smell, which is due to the presence of coumarin in its tissues. Melilotus indicus is a yellow-flowered herb native to northern Africa, Europe and Asia, but naturalized throughout the rest of the world (Webb et al. 1988). It has three varieties, var. indicus, var. tommasinii and var. bonplandii (Fayza 2018). It is known for its medicinal activities such as antibacterial, anticoagulant, astringent, emollient, laxative and narcotic. The plant is also applied externally as a plaster on swelling (Ahmedet al. 2012). Phytochemical investigations have shown the plant to contain flavonoids and flavonoid glycosides (Yadava and Jain 2005,Ahmed and Al‐Refai 2014). Reviewing the available literature, few reports were traced concerning Melilotus indicus(L.) All.(Yadava and Jain 2005, Rana and Fatma2007, Sigaroodi et al.2012, Ahmed et al.2012, Miri et al.2013,Ahmed and Al‐Refai 2014, Ahmad et al. 2014, Sonjuet al. 2017, Naz et al. 2017, Abd El-Hafeez et al. 2018), that revealed the need for a deeper investigation of Melilotus indicus regarding its phytoconstituents,possible biological activities and botanical features. Introduction The present study includes: Part I: Botanical study of Melilotus indicus(L.) All. A. Macromorphological study of roots, stems,leaves, flowers, fruits and seeds. B. Micromorphological study of the roots, stems, leaves, flowers and fruits including seeds. Part II:Phytochemical study of Melilotus indicus (L.)All. 1. Preliminary phytochemical screening. 2. Investigation of the essential oil of the fresh flowering aerial parts of Melilotus indicus (L.) All. 3. Isolation and structure elucidation of the major constituents of the n-hexane extract of the aerial parts of Melilotusindicus (L.) All. 4. Isolation and structure elucidation of the major constituents of the methylene chloride subfraction of defatted 80% MeOH extract of the aerial parts of Melilotus indicus (L.) All. 5. Isolation and structure elucidation of the major constituents of the ethyl acetate subfraction of defatted 80% MeOH extract of the aerial parts of Melilotusindicus (L.) All. 6. Isolation and structure elucidation of the major constituents of the n-butanol subfraction of defatted 80% MeOH extract of the aerial parts of Melilotusindicus (L.) All. 7. Quantitative determination of the active constituents of the aerial parts of Melilotus indicus (L.) All. a. Spectrophotometric determination of the total phenolic content in the defatted methanolic extract of aerial parts of Melilotus indicus (L.)All. Introduction b. Spectrophotometric determination of the total tannin content in the defatted methanolic extract of aerial parts of Melilotus indicus (L.) All. c. Spectrophotometric determination of the total flavonoid content in the defatted methanolic extract of aerial parts of Melilotus indicus (L.) All. d. HPLC analysis of coumarin in 65% ethanolic extract of the aerial parts of Melilotus indicus (L.) All. 8. HPLC fingerprint analysis of different extractivesof Melilotus indicus (L.) All. 9. UPLC-MS profiling of secondary metabolites of the defatted 80% MeOH extract of the aerial parts of Melilotus indicus (L.) All. 10. Investigation of the polysaccharide content of the aerial parts of Melilotus indicus (L.) All. Part III: Biological study of Melilotus indicus (L.)All aerial parts. i. Comparative in-vitro assessment of the anti-oxidant activity of differernt extractives of the aerial parts of Melilotus indicus (L.) All. ii. In-vitro evaluation of the cytotoxic activity of different extractives of the aerial parts of Melilotus indicus (L.) All. iii. In-vitro evaluation of the anticoagulant activity of different extractives of the aerial parts of Melilotus indicus (L.) All. Introduction Review of literature The literature survey was performed on different Melilotus species as an attempt to shed light on the data reported concerning both the chemical and biological activities. The survey revealed that Melilotus species contain different active constituents and showed several biological activities. I-Chemical constituents Phenolics, volatile constituents, sterols, triterpenes, alkaloids and saponins were the main isolated and/or identified classes across different Melilotus species. Reports concerning the different active constituents of genus Melilotus are mentioned in Tables 1-7. Introduction Table 1: Reported phenolic compounds in genus Melilotus Compound Species Part used Author (year) 1- Flavonols OH R2 O R1 OH O Kaempferol R1=OH M. alba Desr. Flower Nicollier and Thompson (1982) R2=OH Kaempferol-3-O- β- R1=O-glucose M. neapolitana Ten. Whole plant Fiorentino et al. (2007) glucopyranoside R2=OH M. officinalis (L.) Lam. Whole plant Anwer et al. (2008) Kaempferol-7-O- β- R1=OH M. officinalis (L.) Lam. Aerial parts Nasser et al. (2014) glucoside R2=O-glucose Kaempferol-3-O-β- R1=O-rutinose M. neapolitana Ten. Whole plant Fiorentino et al. (2007) rutinoside R2=OH Robinin R1=O- M. alba Desr. Flower Nicollier and Thompson (1982) rhamnogalactose (1→6) M. elegans Ser. Leaves Asres et al. (2000) R2=O-rhamnose Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus Compound Species Part used Author (year) 7-O-β-D- R1=O- rutinose M. neapolitana Ten. Whole plant Fiorentino et al. (2007) Glucopyranosyloxy- R2=O- glucose 4',5-dihydroxy-3-[O-α- L-rhamnopyranosyl- (1→6) -3-O-β-D- glucopyranosyloxy ] flavone Melitin R1=O- M. alba Desr. Flower Nicollier and Thompson (1982) galactoglucose (1→6) R2=O- rhamnorhamnose (1→3) Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus Compound Species Part used Author (year) OH OH R2 O R1 OH O Quercetin R1=OH R2=OH M. officinalis (L.) Pall. Aerial parts Liu et al. (2018) M. officinalis (L.) Lam Quercetin-3-O- R1=O- glucose R2=OH M. neapolitana Ten. Whole plant Fiorentino et al. (2007) glucoside Rutin R1=O-rutinose R2=OH Clovin R1=O- rhamnogalactose M. alba Desr. Flower Nicollier and Thompson (1→6) (1982) R2=O- rhamnose OCH3 OH HO O R OH O Isorhamnetin-3-O- R=O-glucose M. neapolitana Ten. Whole plant Fiorentino et al. (2007) glucoside Isorhamnetin-3-O- R=O-rutinose rutinoside Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus Compound Species Part used Author (year) 2-Flavones OH Glu R O OH O Vitexin R=OH M. siculus (Turra) B.D. Whole plant Selim (1975) Vitexin-7-O-glucoside R=O-glu Jacks Orientin OH OH Glu HO O OH O Luteolin OH M. officinalis (L.) Pall. Aerial parts Liu et al. (2018) OH M. officinalis (L.) Lam