A Pharmacognostical Study of Melilotus Indicus (L.) All. Growing in Egypt

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

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)

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

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 HO O

OH O 3-Flavanones Liquiritigenin OH M. messanensis (L.) All. Whole plant Macías et al. (1999) HO O

O Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus

Compound Species Part used Author (year) 4-Isoflavones HO O

O OCH3 Formononetin R= H M. italic (L.) Lam. Leaf diffusate Ingham (1977)

M. messanensis (L.) All. Whole plant Macías et al. (1999) Cyclosin R=OH 5-Isoflavanones

Vestitone M. messanensis (L.) All. Whole plant Macías et al. (1999) HO O

O HO OCH3 Vestitol M. alba Desr. Leaf Ingham (1976) HO O

HO OCH3

Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus

Compound Species Part used Author (year) 6-Pterocarpans R 1 5 4 RO O 6 3

2 7 1 8 O R2 9 10 OR3 (3R,4R)- Medicarpin R= H R1=H M. alba Desr. Leaf Ingham (1976) R =H R =CH 2 3 3 M. messanensis (L.) All. Whole plant Macías et al. (1999) M. alba Desr. Aerial parts Al-Hazimi and Al-Andis 3-Hydroxy-8,9 R=H R1=H (2000) dimethoxypterocarpan R2= OCH3 R3=CH3

3,8-Dihydroxy-9- R=H R1=H methoxypterocarpan R2= OH R3=CH3 3,9-Dihydroxy- R= H R1=H M. alba Desr. Leaf Ingham (1976) pterocarpan R2=H R3=H

3,4,9-Trihydroxy- R= H R1=OH pterocarpan R2=H R3=H Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus

Compound Species Part used Author (year) 3,4-Dihydroxy-9- R=H R1=OH M. alba Desr. Leaf Ingham (1976) methoxypterocarpan R2= H R3=CH3 3,9-Dimethoxy-4- R= CH3 R1=OH hydroxypterocarpan R2=H R3= CH3 Melilotocarpane A R= CH3 R1=OH M. alba Desr. Aerial parts Miyase et al. (1982) R2=H R3= CH3 Melilotocarpane B R= CH3 R1=OH R2=H R3= H M. messanensis (L.) All. Whole plant Macías et al. (1999)

H3CO O

OR1 R2O Melilotocarpane C R1=CH3 R2= CH3 M. alba Desr. Aerial parts Miyase et al. (1982)

Melilotocarpane D R1=CH3 R2= H

Melilotocarpane E R1=H R2= CH3

Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus

Compound Species Part used Author (year) 6a-Hydroxymedicarpin M. alba Desr. Leaf Ingham (1976) HO O

H M. messanensis (L.) All. Whole plant Macías et al. O (1999)

OCH3

6a-Hydroxyisomedicarpin M. alba Desr. Leaf Ingham (1976) H3CO O OH

H O

Review of literature Table 1(cont.): Reported phenolic compounds in genus Melilotus

Compound Species Part used Author (year) 7-Coumestans 8 1 R1O 7 O O 2 6 3 1' 6' R2 4 5 5' 2' O R4 4' 3' OR3 Coumestrol R1=H R2=H M. messanensis (L.) All. Whole plant Macías et al. R3=H R4=H (1999)

7-Hydroxy -4',5'- R1=H R2=H dimethoxy coumestan R3=CH3 R4=OCH3

Melimessanol A R1=CH3 R2=OH R3=H R4=H

4'-O-Methylcoumestrol R1=H R2= H R3= H R4= CH3 Review of literature

General summary

Melilotus indicus (L.) All. belongs to family Fabaceae which is one of the largest families of flowering plants, consisting of about 19500 species.Numerous plants from family Fabaceae have medicinal properties. Melilotus is a genus in the family Fabaceae including about 20–25 species that are widely distributed all over the world. 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. It is known for its medicinal activities such as antibacterial, anticoagulant, astringent, emollient, laxative and narcotic.

Reviewing the available literature, a few reports were traced concerning M.indicus (L.) All. Our study aimed to investigate M. indicus(L.) All.growing in Egypt regarding its botanical features, phytoconstituents andpossible biological activities.

This study includes:

Part I: Botanical study of Melilotus indicus (L.) All.

This included macromorphological study of the roots, stems,leaves, flowers, fruits and seeds followed by a detailed micromorphological investigation of the roots,stems and leaves. Then, micromorphological investigation of the powder of flowers and fruits.

Chapter I: Macromorphological studyof Melilotus indicus (L.) All. Melilotus indicus is an annual herbs, 20-130cm high. The herb has a branched stem which carries compound leaves and small, yellow, papilionaceous flowers. The flowers develop to short, small one seeded fruits.

The root

Melilotus indicushas a tap root system. The root is yellowish-brown in colour and measures 4-15 cm. in length and 2-5 mm. in diameter. It bears several lateral rootlets. The outer surface shows fine longitudinal wrinkles on drying. The root is tough to break. Review of literature

The stem

The stem is erect, cylindrical, longitudinally striated. It is green in colour in the upper part and pink in colour at the lower part.Attains from 21 to 110 cm. in length and 2 to 5 mm. in diameter. It shows somewhat long internodes in the lower part up to 5cm and shorter internodes at the upper part up to 3cm.

The leaf:

The leaves are compound, green in colour. They are ternate and stipulate.The leaflet has an oval lamina, symmetric base and truncate apiculate apex with small apiculus. The margin is serrate towards the apex entire near the base.

The inflorescence:

The inflorescence is a simple, axillary raceme which carries 13 to 30 yellow flowers. Its axis is cylindrical and attain 2.5-5cm.

The flower:

The floweris zygomorphic and hermaphrodite. The flower measures 2-3.5mm. in length.it is yellow in colour. The calyx is formed of 5 united, green sepals. Thecorolla is irregular and is formed of five petals. The posterior odd petal which is called standard. The two lateral petals form the wings.The two anterior petals are adherent by the neighbouring edges of their limbs forming the keel enclosing the androecium and gynaecium.The androecium is formed of 10 diadelphous stamens,9 being united by their filaments while the tenth is free. The gynaecium, the ovary is superior, mono-carpellary, containing one ovule attached to a marginal placenta.

The fruit:

The fruitis a legumewith persistent calyx.It contains one seed carried on a marginal placenta.The pericarp is green becoming yellowish-brown in colour on ripening.

The seed:

The seed is yellow or greenish yellowturns to brownish when ripe, sub-reniform or ovoid in shape. Review of literature

Chapter II: Micromorphological studyof Melilotus indicus (L.)All.

I-The root

A transverse section in the root shows a cork, narrow cortex, phloem and xylem.The cork is formed from up to 5 rows of radially arranged tabular cells.The cortex is formed from a few rows of thin-walled parenchymatous cells.The phloem is composed of thin- walled elements.The xylem vessels are moderately wide and lignified mostly of the pitted type. They are accompanied by tracheids, wood fibers and wood parenchyma.

II-The stem

A transverse section in the stem consists of epidermis, a single layer of polygonal cells. Stomata are anomocytic rarely paracytic.Trichomes of the non-glandular and glandular types are occasionally present. The cortex isformedof few parenchymatous cells sometimes showing 2-3 layers of collenchyma followed by up to 5 layers of thin-walled parenchyma filled with small starch granules.The pericycle is formed of groups of non-lignified to slightly lignified pericyclic fibersinterrupted by parenchyma. The fibers have thin walls and tapering apices. The pericyclic fibers are accompanied by a crystal sheath. The vascular system,The vascular bundles form together with the narrow primary medullary rays a continuous ring surrounding the pith. The phloemformed of thin-walled elements.The xylem is formed of lignified vessels.They are accompanied by fibers and wood parenchyma.

III-The leaf

A.The lamina

A transverse section in the the leaf lamina shows inner and an outer epidermises enclosing in between a dorsiventral mesophyll. The leaflet is traversed in the midrib region by a vascular bundle.The epidermis;both upper and lower epidermis consist of polygonal cells.Lower epidermis having more wavy walls and higher number of stomata. Stomata are present on both surfaces, but are more frequent on the lower surface. They are usually of the anomocytic and the paracytic types. Trichomes of both glandular and non-glandular types are present. The leafis dorsiventral showing a layer composed of two to three rows of columnar palisade cells. The rest of the mesophyll tissue is formed of 4-5 rows of small rounded or irregular spongy cells. Review of literature

Conclusion

1. Botanical features of Melilotus indicus (L.) All. growing in Egypt were established to help in its authentication and identification ensuring its quality.

2. 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.

3. Seven compounds have been isolated and identified via physical, chemical, 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 a phenolic acid(o-coumaric acid). All were isolated for the first time from Melilotus indicus (L.) All.n- Nonacosane and trifolin were isolated for the first time from genus Melilotus.

4. UPLC analysis led to the identification of 14 compounds from which 5 compounds were identified for the first time and confirmed the presence of the previously isolated compounds.

5. Defatted 80% methanol extract and its subfractions have weak antioxidant activity. 6. Methylene chloride fraction showed cytotoxic activity on liver and colon human cell lines which may be due to coumarin, kaempferol and o-coumaric acid .