A Pharmacognostical Study of Certain Sterculia Species (Family: Sterculiaceae)

A Thesis Submitted by Alia Yassin Ragheb Abd Al-Rahman Research assistant National Research Centre

For the Degree of Doctor of Philosophy in Pharmaceutical Sciences [Pharmacognosy]

Under the Supervision of

Prof. Dr. Moshera Mohamed El-Sherei Professor of Pharmacognosy Department of Pharmacognosy Faculty of Pharmacy Cairo University

Prof. Dr. Mona El-Said Ahmed Kassem Professor of Chemistry of Natural Products Department of Phytochemistry and Plant Systematics National Research Centre

Pharmacognosy Department Faculty of Pharmacy Cairo University 2017

Abstract

Four Sterculia species (Sterculia alata Roxb., Sterculia diversifolia G. Don, Sterculia platanifolia L.f. and Sterculia foetida L.) grown in Egypt were selected for studying from a pharmacognostical point of view. On the basis of some morphological features, these species were reclassified under different genera and revised as Pterygota alata (Roxb.) R. Br., Brachychiton populneus (Schott & Endl.) R. Br. and Firmiana simplex (L.) W. Wight, respectively. Sterculia foetida L. remained belonging to genus Sterculia. In the present study, genetic, botanical, phytochemical and biological studies were performed in order to find the infrageneric relationships among the four taxa. Genetic analysis through DNA (RAPD-PCR fingerprinting of the fresh leaves) and protein (SDS-PAGE analysis of the seeds) profilings showed great genetic variations between the studied species. The botanical study included the investigation of macromorphological (vegetative and floral morphology) and micromorphological (leaves) characters. Macromorphological characters displayed considerable variation in leaves, inflorescence, fruits and seeds, while the micromorphological ones showed various similarities among the four species. Non- glandular and glandular trichomes were the main differentiating characters among the four species. The phytochemical investigation included proximate analysis, phytochemical screening, chromatographic investigation, isolation and structural elucidation of the flavonoid compounds, as well as qualitative and quantitative estimations of total flavonoid and phenolic contents by HPLC. From aqueous methanol extracts of leaves and stems of P. alata and B. populneus, 37 flavonoidal compounds (8 of which are common in both plants) in addition to 4 non-alkaloidal nitrogenous bases (from P. alata only) were isolated and identified using chromatographic and spectroscopic techniques. Their structures were established on the basis of detailed chemical degradation (acid hydrolysis, ferric chloride degradation and alkaline hydrolysis) as well as spectral analyses (UV, NMR and EIMS). Also, the flavonoid profiles of the four species were investigated by HPLC using the flavonoid aglycones and glycosides isolated and identified from P. alata and B. populneus in addition to the standard quercetin 3-O-β-glucopyranoside, through which thirteen flavonoid compounds were detected in F. simplex and fifteen ones in S. foetida. Twenty one phenolic compounds were detected through HPLC analysis. Additionally, the total flavonoids and phenolics were quantitatively perceived through HPLC analysis and were represented as 53.036, 150.863, 12.257 and 40.256 (mg AE/100g dry weight) and 10.731, 0.322, 2.933 and 27.228 (mg GAE/100 g dry weight) for P. alata, B. populneus, F. simplex and S. foetida, respectively. In vitro biological screening of the antioxidant activity revealed pronounced effects of B. populneus and S. foetida, moderate effect of P. alata and weak activities of F. simplex as well as the major pure isolated flavonoid compounds; P5 (apigenin 6-C-α-arabinopyranoside 8-C-β-galactopyranoside), P7 (apigenin 6-C-β-glucopyranoside 8-C-α-rhamnopyranoside) and P12 (apigenin 7-O-β- glucopyranoside), on scavenging DPPH free radicals. Moderate cytotoxic activities of the four studied species were exhibited against six carcinoma cell lines HEPG2 (liver), HELA (cervix),

MCF7 (breast), HCT (colon) HEP2 (larynx) and PC3 (prostate). P5, P7 and P12 pure compounds were also found to have moderate activity against HEP2 and HELA, compared to the standard Doxorubicin. The acute lethal toxicity determination of the four species indicated that those plants were safe and non toxic. In vivo biological studies were also included acute anti-hyperglycemic activity and anti-oxidative stress in diabetic rats. P. alata, B. populneus and S. foetida extracts (500 mg/kg) possessed significant acute anti-hyperglycemic effect and restored the body weight values of diabetic rats after 24 hrs of treatment compared to alloxan-induced diabetic rats and standard Diamicron group, while F. simplex exhibited no change. In addition, the four studied plants extracts counteracted the effect of the oxidative stress induced by alloxan causing significantly increase in the GSH level and relative decrease in the MDA and NO contents in serum after 24 hrs of treatment compared to alloxan-induced diabetic rats. From our point of view, the four species had significant differences and might be treated as separate genera and not under the genus Sterculia.

Key words: Sterculiaceae, Sterculia, Pterygota, Brachychiton, Firmiana, genetics, macromorphology, micromorphology, phenolics, flavonoids, chemotaxonomy, antioxidant, cytotoxicity, LD50, anti- hyperglycemic.

Introduction For over 300 thousand plant species, early man had to put science for naming, identifying, describing and classifying the basic essential questions on his mind; can we eat these plants? Are those poisons? Years later plants became the source for remedy; for pain management, fever, skin rashes, diarrhea and stomach disorders, fractures….etc. Still in the 21st century, plant is considered the source or reference for 75% of medicine, and hence showing the rising importance of being as accurate as possible in the science of grouping/classification; helping to pass the right accurate information to the following scientific generation of what had been achieved and known about different species. It is clear to us that biological monitoring studies yield the greatest benefits using genus- or species-level taxonomy. With further accumulation of knowledge, however, species-level data may show far greater differences relative to family-level data. No single character has gained broad acceptance as diagnostic of the genus because of the lack of strongly confirmatory characters. The biggest change in plant classification occurred after the highly informative results produced by molecular analysis through the chromosomal structure, DNA sequence and genome structure. Comparative biochemists have continued to present useful information for phylogenists through chemotaxonomy. Secondary metabolites; phenolics and flavonoids, are stable compounds and have definite synthesis pathway beside their wide structural diversity. Sterculiaceae is one of the questionable families that was treated together with Bombacaceae, Tiliaceae and Malvaceae as a single monophyletic family Malvaceae. On the other hand, some scholars recognized Sterculiaceae as a separate family within the order Malvales. Moreover, the classification of species of genus Sterculia has been controversial for a long time. Some species had been revised and reclassified under different genera based on different morphological features. These genera were Pterygota Schott & Endl., Brachychiton Schott & Endl., Firmiana Marsili, Hildegardia Schott & Endl., Pterocymbium R.Br. and Scaphium Schott & Endl. Four taxa of Sterculia species grown in Egypt (viz: Sterculia alata Roxb., Sterculia diversifolia G. Don, Sterculia platanifolia L.f. and Sterculia foetida L.) were selected for this study. These species had been revised and renamed as belonging to the genera Pterygota, Brachychiton and Firmiana, respectively. Conversely, Sterculia foetida L. has remained belonging to genus Sterculia. Their accepted names were Pterygota alata (Roxb.) R. Br., Brachychiton populneus (Schott & Endl.) R. Br. and Firmiana simplex (L.) W. Wight, respectively. Some scholars insist that those plants should belong to the genus Sterculia. Pterygota alata (Roxb.) R. Br. and Firmiana simplex (L.) W. Wight are the only species revised to genera Pterygota and Firmiana, respectively, cultivated in Egypt. The availability of Brachychiton populneus (Schott & Endl.) R. Br. together with the little mentioned

4 information concerning it was the reason for selection of this species. The detailed studied Sterculia foetida L. which is still belonging to genus Sterculia was selected to be a model of comparison. Accordingly, it was deemed of interest to dig in depth in all informative modern tools and must not forget morphological characters for assessing phylogenetic relationships at higher levels in the genus Sterculia. Reviewing the current literature, little information was traced dealing with the taxonomical, genetical, botanical and chemical characters as well as the biological activities of the selected plant species; S. alata, S. diversifolia and S. platanifolia. While S. foetida was found to be previously studied. Therefore, the aim of this thesis is to carry a pharmacognostical study to revise the genetic, macro and micro morphological features to find the infrageneric relationship within the selected species, as well as, carrying out comparative investigations of their active constituents and some of biological activities which could play a role in supporting the classification status.

The present work includes: Studying the infrageneric relationship between the four selected species mentioned above based on: Part I. Genetic profiling 1. DNA profiling. 2. Protein profiling. Part II. Botanical study 1. Macro-morphological study. 2. Micro-morphological study (leaf). Part III. Phytochemical study 1. Proximate analysis. 2. Preliminary phytochemical screening. 3. Chromatographic investigation. 4. Isolation and structural elucidation of the isolated compounds. 5. Flavonoid and phenolic profiles and quantitative estimations by HPLC. Part IV. Biological study 1. In vitro study: 1.1. Antioxidant activity assay. 1.2. Cytotoxic activity assay. 2. In vivo study: 2.1. Determination of the Median Lethal Dose. 2.2. Acute anti-hyperglycemic activity. Part V. General discussion

Review of Literature

Sterculiaceae is a family of flowering plants, and its name was based on the genus Sterculia. The generic name was based on the Latin word, "stercus", meaning "manure", which is referred to the smell of the flowers of some species (Prakash et al., 2012).

The family has a wide economic importance. The most famous products are cacao and chocolate from the seeds of Theobroma cacao L. Cola was produced from the seeds of Cola nitida Schott & Endl.; the caffeine-rich seeds of which are used commercially in soft drinks and medicines. Karaya or Indian gum; from Sterculia urens Roxb., was an inexpensive substitute for tragacanth. Moreover, Sterculia included some species used for the production of timber and mark fibers. The family also included several species cultivated as ornamentals; e.g. the flannel bush in the United States, the kurrajong in Australia and the Chinese parasol tree (Chattaway, 1932).

A survey of the family showed a wide range of flavonoid compounds and other phenolics, many of them were reported in the genus Sterculia (Harborne, 1975).

The following chronological literature survey was achieved aiming to provide helpful guidelines during performance of the current study. In this respect data on isolation and identification of different types of constituents from plants of the genus Sterculia and its related taxa (Pterygota Schott & Endl., Brachychiton Schott & Endl., Firmiana Marsili, Hildegardia Schott & Endl., Pterocymbium R.Br. and Scaphium Schott & Endl.) were gathered and reported in addition to those concerned with the uses and/or biological activities of those plants.

A-Phytoconstituents

1. Phenolic constituents

The classes of the reported phenolics of the genus Sterculia and its related taxa are collected in Table (1) based on their chemical structure according to Harborne, 1975.

Table 1. Classes of the phenolics reported from the genus Sterculia and its related taxa Compound Organ Species References 1. Flavonoids A. Flavones Apigenin L Sterculia Nair et al., 1976 colorata Roxb. 3' OH F Firmiana Ding et al., 1986 2' 4' platanifolia* (L. 8 1 HO O 1' 5' 9 F.) Marsili 7 2 6' L S. foetida* Xia, 2009 6 3 10 St, L P. alata* Lin et al., 2010 5 4 L Brachychiton Farag et al., 2015 OH O

acerifolius (A. Cunn. ex G. Don) Macarthur Apigenin 7-O-β-D-glucoside L B. acerifolius Farag et al., 2015 Apigenin 7-O-β-D-glucuronide L S. colorata Nair et al., 1976 L B. acerifolius Farag et al., 2015 Apigenin 7-O-β-D-glucuronide 6"-ethyl ester L S. foetida* Xia, 2009 Apigenin 7-O-(2"-α-rhamnoside) -β- L B. acerifolius Farag et al., 2015 glucuronide 6-Hydroxyapigenin (Scutellarein) L S. colorata Nair et al., 1976 L S. foetida* Nair et al., 1977 Scutellarin 6-O-β-D-glucuronide L S. foetida* Nair et al., 1977 8-Hydroxyapigenin 8-O-glucuronide L S. colorata. Shi, 2005 (Isoscutellarein 8-O-glucuronide) Isoscutellarein 8-O-β-D-glucoside L S. foetida* Xia, 2009 & Xia Isoscutellarein 8-O-β-D-glucuronide et al., 2009a Isoscutellarein 8-O-β-D-glucuronide 6"- methyl ester Isoscutellarein 8-O-β-D-glucuronide 6"-ethyl ester Isoscutellarein 4`-methyl ether (Takakin) Takakin 7-O-β-D-glucoside Takakin 8-O-β-D-glucoside Takakin 8-O-β-D-glucuronide Takakin 8-O-β-D-glucuronide 6"-methyl ester

Luteolin L S. colorata Nair et al., 1976 OH L Sterculia acerifolia De Laurentis et al., 3' OH 2' 4' A. Cunn. Ex G. Don 2003 and Farag et 8 1 HO O 1' 9 5' al., 2015 7 2 6' L S. foetida* Xia, 2009 6 3 10 5 4 OH O Luteolin 7-O-β-D-glucoside L S. foetida* Xia, 2009 Luteolin 7-O-β-D-glucuronide L S. colorata Nair et al., 1976 L S. foetida* Xia, 2009 L B. acerifolius Farag et al., 2015 Luteolin 7-O-β-D-glucuronide 6"-methyl ester L S. foetida* Xia, 2009 Luteolin 7-O-β-D-glucuronide 6"-ethyl ester Luteolin 3'-methyl ether (Chrysoeriol) L Sterculia villosa Seetharaman, Roxb. 1990 L S. foetida* Xia, 2009 Chrysoeriol 7-O-β-D-glucoside L S. villosa Seetharaman,

1990 Chrysoeriol 7-O-β-D-glucuronide L S. foetida* Xia, 2009 Chrysoeriol 7-O-β-D- glucuronide 6"-methyl ester Chrysoeriol 7-O-β-D-glucuronide 6"-ethyl ester Luteolin 4`-methyl ether (Diosmetin) L S. villosa Seetharaman, Diosmetin 7-O-β-D-glucoside 1990 6-Hydroxyluteolin L S. colorata Nair et al., 1976 6-Hydroxyluteolin 6-O-β-D-glucuronide L S. foetida* Nair et al., 1977 8-hydroxyluteolin 8-O-β-D-glucuronide L S. foetida* Xia, 2009 and Xia (Hypolaetin 8-O-β-D-glucuronide) et al., 2009a Hypolaetin 8-O-β-D-glucuronide 6''-methyl ester Hypolaetin 8-O-β-D-glucuronide 6"-ethyl ester Hypolaetin 3'-methyl ether 8-O-β-D- glucuronide 6''-methyl ester Hypolaetin 4'-methyl ether 8-O-β-D- glucuronide 2''-sulfate Hypolaetin 4'-methyl ether 3'-O-β-D- glucoside 5,7,8,3'-Tetrahydroxy 4'-methoxy flavone L S. foetida* Xia et al., 2009b 5,7,8-Trihydroxy 3',4'-dimethoxy flavone B. Flavonols Kaempferol L F. simplex* Seetharaman,1990 3' OH L Brachychiton Desoky & 2' 4' rupestris (Lindl.) Youssef, 1997 8 1 HO O 1' 9 5' K. Schum 7 2 6' SB S. diversifolia* 6 10 3 5 4 OH L B. acerifolius De Laurentis et al., OH O 2003 and Farag et al., 2015 Kaempferol 3-O-β-D-glucoside SB S. diversifolia* Desoky & Youssef, 1997 Sd Sterculia Wang et al., 2003a lychnophora Hance Fr Scaphium Petchlert et al., scaphigerum (G. 2012 Don) Guib. & Planch.) Kaempferol 3-O-β-D-rutinoside L F. simplex* Seetharaman, 1990 L B. rupestris Desoky &

SB S. diversifolia* Youssef, 1997 Sd S. lychnophora Wang et al., 2003a Fr S. scaphigerum Petchlert et al., 2012 Kaempferol 3-O-(2",6"-dirhamnosyl)-β- L B. rupestris Desoky & glucoside SB S. diversifolia* Youssef, 1997 [K 3-O-(2"-rhamnosylrutinoside)] Kaempferol 3-O-(2",6"-dirhamnosyl)-β- L B. rupestris Desoky & galactoside Youssef, 1997 [K 3-O-(2"-rhamnosylrobinoside)] Quercetin L Sterculia pallens Ranganathan & OH Wall. Ex Hochr. Nagarajan, 1980 3' OH L F. simplex* Seetharaman, 2' 4' 1990 1 8 HO O 1' L B. rupestris Desoky & 7 9 5' 2 6' SB S. diversifolia* Youssef, 1997 6 L Brachychiton Kassem et al., 10 3 5 4 OH australis (Schott & 2002 OH O Endl.) A. Terrac

Quercetin L B. acerifolius De Laurentis et al., 2003 and Farag et al., 2015 L Brachychiton Kassem, 2007 discolor F.j. Muell. L S. foetida* Xia, 2009 Quercetin 3-O-arabinoside SB S. diversifolia* Desoky & Youssef, 1997 Quercetin monorhamnoside R S. foetida* Dubey & Tiwari, 1991 Quercetin 3-O-rhamnoside (Quercitrin) SB F. platanifolia* Ogihara et al., 1975 St F. simplex* Son et al., 2005 SB Kim et al., 2015 L B. discolor Kassem, 2007 Quercetin 3-O-β-D-glucoside L S. pallens Ranganathan & Nagarajan, 1980 L B. australis Kassem et al., 2002 L S. foetida* Xia et al., 2009b Quercetin 3-O-galactoside (Hyperoside) L F. simplex * Seetharaman, 1990 L B. acerifolius De Laurentis et al., 2003 Quercetin 3-O-β-D-rutinoside (Rutin) L F. simplex* Nakaoki et al., [Q 3-O-(6"-α-rhamnosyl)-β-D-glucoside] 1957 L B. australis Kassem et al., 2002 L B. acerifolius De Laurentis et al.,

2003 and Farag et al., 2015 Quercetin 3-O-β-D-neohesperidoside L F. simplex* Seetharaman, [Q 3-O-(2"-α-rhamnosyl)-β-D-glucoside] 1990 Quercetin 3-O-diglucoside (Meratin) L S. pallens Ranganathan & Nagarajan, 1980 Quercetin 7-methyl ether (Rhamnetin) L B. discolor Kassem, 2007 Quercetin 3'-methyl ether (Isorhamnetin) L B. rupestris Desoky & SB S. diversifolia* Youssef, 1997 L B. acerifolius De Laurentis et al., 2003 Isorhamnetin 3-O-β-D-rutinoside L B. rupestris Desoky & SB S. diversifolia* Youssef, 1997 L B. australis Kassem et al., 2002 Sd S. lychnophora Wang et al., 2003a Isorhamnetin 3-O-β-D-rutinoside Fr Sc. Scaphigerum Petchlert et al., 2012 Isorhamnetin 3-O-(2",6"-dirhamnosyl)-β-D- L B. rupestris Desoky & galactoside Youssef, 1997 Quercetin 4'-methyl ether-3-O-rhamnoside St F. simplex* Son et al., 2005 (Tamarixetin 3-O-rhamnoside) SB Kim et al., 2015 Quercetin 3,7,3',4'-tetramethyl ether (Retusin) SB S. foetida* Anjaneyulu & Quercetin 5,7,3',4'-tetramethyl ether Murty, 1981 C. Flavan

5,7-Dihydroxy-2-(4-hydroxyphenyl)-6,8- R Hildegardia Meragelman et al., dimethylchroman-4-one (Farrerol) barteri (Mast.) 2005 Kosterm. D. C-Glycosylflavonoids Apigenin 8-C-β-D-glucoside (Vitexin) L S. colorata Nair et al., 1976 Apigenin 6,8-di-C-β-D-glucoside L S. foetida* Xia et al., 2009b E. Isoflavone 8 1 O 9 7 2 2' 6 10 3 3' 5 4 1'

O 6' 4' 5' 8-C-glucoside-7,4'-dihydroxyisoflavone L S. foetida* Xia et al., 2009b (Puerarin) F. Isoflavans

(3 R)-6, 2'-dihydroxy-7-methoxy-4', 5'- R H. barteri Meragelman et al., methylenedioxyisoflavan (Hildegardiol) 2005 2-Hydroxymaackiain

G. Anthocyanins

Pelargonidin Fo Sterculia Lowry, 1971 Pelargonidin 3-O-arabinoside parviflora Roxb. Pelargonidin 3-O-galactoside In S. kunstleri Pelargonidin 3-O-glucoside King F B. acerifolius Farag et al., 2015 Cyanidin 3-O-arabinoside Fo S. parviflora Lowry, 1971 Cyanidin 3-O-galactoside

Cyanidin-3-O-glucoside F, L S. foetida* Lowry, 1971 L F. platanifolia* Yoshitama et al.,1972 L S. foetida* Nair et al., 1977 Cyanidin-O-rutinoside F B. acerifolius Farag et al., 2015 Leucoanthocyanidin-3-O-α-L- R S. foetida* Dubey &Tiwari, rhamnopyranoside 1991 Procyanidin-β-D-glucuronide L S. foetida* Nair et al., 1977 2. Coumarins 8 1 O O 9 7 2

6 10 3 5 4 7-Hydroxy-6-methoxycoumarin R S.urens Dubey & (scopoletin) Tiwari,1991 L B. australis Kassem et al., 2002 St F. simplex* Son et al., 2005 L Firmiana Kang & Liu, 2007 hainanensis Kosterm. Scopoletin 7-O-β-D-glucoside (scopolin) L S. foetida* Xia, 2009 5,7-Dihydroxy-6-methoxy-7-O-β-D-glucosyl

11 coumarin Fraxetin 7-O-β-D-glucoside (7,8-dihydroxy-6-methoxychromen-2-one 7- O-β-D-glucoside) Isofraxidin 7-O-β-D-glucoside (7-hydroxy-6,8-dimethoxychromen-2-one 7- O-β-D-glucoside) Aquillochin [(2S, 3S)-2-(4-Hydroxy-3,5- St F. simplex* Son et al., 2005 dimethoxy-phenyl)-3-(hydroxymethyl)-5- methoxy-2,3-dihydro-9H [1,4]dioxino[2,3-h] coumarin] 3. Phenolic acids and aldehydes 2,4-Dihydroxy benzoic acid Sd S. lychnophora Wang et al., 2003a p-Hydroxybenzoic acid L S. foetida* Xia, 2009 and Xia 3,4- Dihydroxybenzoic acid et al., 2009a 4-Hydoxy-3,5-dimethoxy-benzoic acid 4-O- β-D-glucopyranosyloxy 4-Hydoxy-3,5-dimethoxyl benzaldehyde L F. hainanensis Kang & Liu, 2007 4. Phenylpropanoids p-Coumaric acid L S. foetida* Xia, 2009 and Xia cis-p-Coumaric acid β-glucoside et al., 2009a trans-Ferulic acid β-glucoside 1,6-Diferuloyl glucose Cinnamic acid St, L P. alata.* Lin et al., 2010 p-Methoxy cinnamic acid 1,6-O-Dicinnamoyl glucose 1-O-p-Coumaroyl 6-O-cinnamoyl-β-D- galactoside 5. Lignans and lignins Dioxane lignin L Pterygota Lal et al., 1977 macrocarpa K. Schum. Simplidin St F. simplex* Son et al., 2005 Syrigaresinol Nitidanin Thespesone St, L P. alata.* Lin et al., 2010 Epieudesmin Diayangambin F = flower; Fo = Follicles; L = Leaves; R = roots; SB = stem bark; Sd = seeds; St = stem; * selected species for investigation S. alata (= P. alata) S. diversifolia (= B. populneus) S. platanifolia (= F. platanifolia = F. simplex) S. foetida

Summary

Sterculiaceae is a family of trees, shrubs and herbs consisting of about 72 genera and 1500 species distributed mainly in tropical and subtropical regions. The family also goes by the name of Sterculia family and Cacao family. The family is of wide economic importance as its most famous products are chocolate, cocoa, cola, Karaya or indian gum. Some species are used for the production of timber and also cultivated as ornamentals. Based on molecular sequence data; particularly from the chloroplast DNA rbcL gene, and morphological data, some scholars circumscribed the tropical and woody groups of Sterculiaceae, Bombacaceae and Tiliaceae as a single monophyletic family Malvaceae (s.l.). Others propose for considering them as three separate families within the order Malvales.

The genus Sterculia belongs to the subfamily Sterculioideae of family Sterculiaceae. It comprised approximately 200 species distributed mainly in tropical and subtropical regions. Some of the Sterculia species are classified under different genera based on distinct morphological features. Four taxa of Sterculia species grown in Egypt (viz: Sterculia alata Roxb., Sterculia diversifolia G. Don, Sterculia platanifolia L. f. and Sterculia foetida L.) were selected for this study. They are cultivated widely in gardens and along roads in Egypt. These species had been revised and renamed as belonging to the genera Pterygota Schott & Endl., Brachychiton Schott & Endl. and Firmiana Marsili, respectively. Conversely, Sterculia foetida L. has remained belonging to genus Sterculia. Their accepted names were Pterygota alata (Roxb.) R. Br., Brachychiton populneus (Schott & Endl.) R. Br. and Firmiana simplex (L.) W. Wight, respectively. Some scholars insist that those plants should belong to the genus Sterculia.

Reviewing the current literature, little information was traced dealing with the taxonomical, genetical, botanical and chemical characters as well as the biological activities for the selected plant species except for S. foetida which was previously studied.

Accordingly, it was deemed of interest to go through all informative modern tools; molecular analysis and chemotaxonomy together with macro and micromorphological characters for assessing phylogenetic relationships between the selected species at higher levels.

On the other hand, several biological activities have been reported for some species of genus Sterculia, viz; antidiabetic , antimicrobial, antioxidant, anticancer, anti-inflammatory and others.

In this thesis, a pharmacognostical study was carried out to find the infrageneric relationship in the genus Sterculia.

The present study includes:

Part I. Genetic profiling 1- DNA profiling. 2- Protein profiling.

Part II. Botanical study 1- Macro-morphological study. 2- Micro-morphological study (leaf).

Part III. Phytochemical study 1- Proximate analysis. 2- Preliminary phytochemical screening. 3. Chromatographic investigation. 4- Isolation and structural elucidation of the isolated compounds. 5- Flavonoid and phenolic profiles and quantitative estimations by HPLC.

Part IV. Biological study 1. In vitro study: 1.1. Antioxidant activity assay. 1.2. Cytotoxic activity assay. 2. In vivo study: 2.1. Determination of the Median Lethal Dose. 2.2. Acute anti-hyperglycemic activity. Part V. General discussion

Literature survey

The different phytoconstituents reported in genus Sterculia and the other related genera Pterygota, Brachychiton and Firmiana in addition to their reported biological activities and uses have been reviewed.

Going through the genetic profiling (DNA and protein profiling), botanical (macro- and micromorphological characters), chemical data as well as biological activities, the main lines in this study were subsequently presented.

Part I. Genetic profiling

1. DNA profiling (fresh leaves)

RAPD-PCR fingerprinting technique was employed to assess the polymorphism among the species under study grown in Egypt. Six RAPD primers (C1, N8, B12, P8, H5 and P13) produced 114 scorable amplified DNA fragments ranging from 170 to 3000 bp, whereas 107 fragments were polymorphic. The six primers revealed mean polymorphic percentage of

92.87% designated high genetic dissimilarity. Similarity coefficient ranged from 0.6 to 0.7 for the RAPD markers.

2. Protein profiling (seeds)

SDS-PAGE molecular technique was applied to establish the level of genetic relationships and polymorphism across the four species under investigation grown in Egypt. The molecular weights of detected bands for all samples were ranged from 25 to 193 KDa. The percentage of polymorphism between the four species was 100% which indicated high variation in their SDS-PAGE profiles. Similarity coefficient was ranged from 0.5 to 0.6.

Part II. Botanical study

1. Macromorphological study

Different plant organs (leaf, inflorescence, flower, fruit and seed) were morphologically examined in order to explore the main diagnostic features for identification and comparing the studied species.

2. Micromorphological study

Surface preparations, transverse sections of lamina and petiole and their powdered forms of the four studied species were carried out to differentiate between Sterculia species.

Part III. Phytochemical study

1. Proximate analysis

The pharmacopoeial constants of P. alata, B. populneus, F. simplex and S. foetida powdered leaves were determined.

2. Preliminary phytochemical screening

The preliminary phytochemical screening of the powdered air dried samples of the leaves and stems of the species under investigation revealed the presence of carbohydrates and/or glycosides, sterols and/or triterpens, phenolics and/or flavonoids (in variable concentrations) and tannins in leaves and stems of all species under investigation.

Alkaloids and/or nitrogenous bases, saponins and coumarins were also present in all species except in F. simplex for alkaloids and/or nitrogenous bases, in S. foetida for saponins and in B. populneus for coumarins. Anthraquinones were found in trace amounts in both P. alata and S. foetida while were absent from the other two species.

3. Chromatographic investigation of P. alata, B. populneus, F. simplex and S. foetida

The comparative chromatographic investigation of 70% methanol extracts of leaves and stems (ME) of the four species revealed the presence of higher concentration of flavonoids in

P. alata and B. populneus with a relative lower one in the extract of F. simplex. The results were compared with that of S. foetida which was selected to be a model of comparison.

The prepared dry residues of P. alata (125 g.) and B. populneus (70 g.) were defatted with petroleum ether (40-60 ºC). The crude defatted extracts were subjected to chromatographic fractionation using polyamide columns with gradient elution (starting with 100% water and ending with 100% methanol) for each plant extract. Then, the fractions were subjected to repeated PPC (3MM) using different solvents as eluents or CC using polyamide and/or Sephadex LH-20 subcolumns.

4. Isolation and structural elucidation of compounds from P. alata and B. populneus

For the structural elucidation of the isolated compounds, chemical degradation methods (acid hydrolysis, ferric chloride degradation and alkaline hydrolysis), spectroscopic methods (UV, 1D and 2D NMR experiments) and spectrometric techniques of analysis (EI/MS) were applied. Further confirmation was carried out through comparison of their spectroscopic data with previously reported values and/or authentic samples.

4.1. Compounds isolated from P. alata

Twenty four compounds; 20 flavonoids (P1 - P20) and 4 non-alkaloidal nitrogenous bases (a, b, c and d) were isolated and identified from the 70% methanol extract of leaves and stems of P. alata. The flavonoids were identified as: luteolin 6-C-β-glucopyranoside-7-O-- glucopyranoside (lutonarin; P1), luteolin 6-C-α-arabinopyranoside-8-C-β-glucopyranoside (isocarlinoside; P2), luteolin 6-C-β-glucopyranoside-8-C-α-arabinopyranoside (carlinoside; P3), apigenin 6,8-di-C-β-glucopyranoside (vicenin; P4), apigenin 6-C-α-arabinopyranoside-8- C-β-galactopyranoside (corymboside; P5), apigenin 6-C-α-rhamnopyranoside-8-C-β- glucopyranoside (isoviolanthin; P6), apigenin 6-C-β-glucopyranoside-8-C-α- rhamnopyranoside (violanthin; P7), apigenin 6-C-β-glucopyranoside-7-O--glucopyranoside (saponarin; P8), luteolin 7-O-β-glucuronide (P9), apigenin 7-O-β-glucuronide (P10), luteolin 7-O-β-glucopyranoside (P11), apigenin 7-O-β-glucopyranoside (P12), kaempferol 3-O-β-(4"- p-coumaryl)-glucopyranoside (P13), kaempferol 3-O-β-(p-coumaryl)-glucopyranoside (P14), diosmetin (P15), apigenin 7,4'-dimethyl ether (P16), apigenin (P17), acacetin (P18), luteolin (P19) and kaempferol (P20). The non alkaloidal nitrogenous bases: uracil (a), 1-methyl uracil (b), 3-methyl uracil (c) and adenine (d). All compounds were isolated for the first time from P. alata with exception of apigenin (P17).

4.2. Compounds isolated from B. populneus

Seventeen flavonoid compounds (B1 – B17) were isolated and identified from the 70% methanol extract of leaves and stems of B. populneus. The flavonoids were identified as: apigenin 8-C-β-glucopyranoside (vitexin; B1), apigenin 6-C-β-glucopyranoside (isovitexin; B2), apigenin 6, 8-di-C-β-glucopyranoside (vicenin; B3), isorhamnetin 3-O-β-rutinoside (B4),

Quercetin 3-O-β-rutinoside (rutin; B5), kaempferol 3-O-β-rutinoside (B6), luteolin 7-O-β- glucuronide (B7), apigenin 7-O-β-glucuronide (B8), quercetin 3-O-α-rhamnopyranoside (quercetrin; B9), kaempferol 3-O-β-glucopyranoside (astralagin; B10), luteolin 7-O-β- glucopyranoside (B11), apigenin 7-O-β-glucopyranoside (B12), isorhamnetin (B13), quercetin (B14), apigenin (B15), lutoelin (B16) and kaempferol (B17). Eleven compounds of which; B1, B2, B3, B5, B7, B8, B9, B11, B12, B15 and B16, were isolated for the first time from the plant.

5. Flavonoid and phenolic profiles and quantitative estimations by HPLC

5.1. Flavonoid profiles of P. alata, B. populneus, F. simplex and S. foetida and their quantitative estimations by HPLC

5.1.1. Flavonoid profiles

Apigenin 7-O-β-glucopyranoside, lutoelin, kaempferol and apigenin were identified in the four studied species with variable concentrations. Quercetin was estimated with high concentrations in the studied species in spite of its absence in P. alata. The C- glycosylflavonoids are characterizing P. alata flavonoidal profile. B. populneus profile was differentiated by detection of quercetin, kaempferol and isorhamnetin glycosylated with rutinosides or rhamnosides. Apigenin 7-O-β-glucuronide was identified as the major flavonoid of S. foetida. F. simplex was recognized to be the poorest plant with fewer concentrations of flavonoids.

5.1.1.1. Flavonoid profile of Firmiana simplex (L.) W. Wight

Thirteen flavonoid compounds were detected in the 70% methanol extract of leaves and stems of F. simplex. The estimated flavonoids were: apigenin 6-C-α-rhamnopyranoside 8-C- β-glucopyranoside (isoviolanthin; P6), apigenin 6-C-β-glucopyranoside 8-C-α- rhamnopyranoside (violanthin; P7), quercetin 3-O-β-rutinoside (rutin; B5), kaempferol 3-O- β-rutinoside (B6), apigenin 7-O-β-glucopyranoside (P12, B12), quercetin 3-O-α- rhamnopyranoside (quercetrin; B9), kaempferol 3-O-β-glucopyranoside (astralagin; B10), quercetin (B14), kaempferol 3-O-β-(4"-p-coumaryl)-glucopyranoside (P13), kaempferol 3-O- β-(p-coumaryl)-glucopyranoside (P14), lutoelin (P19, B16), kaempferol (P20, B17) and apigenin (P17, B15). Eight compounds of which; P6, P7, P12, B10, B14, P13, P14 and P19 were established for the first time from the plant.

5.1.1.2. Flavonoid profile of Sterculia foetida L.

Fifteen flavonoid compounds were detected in the 70% methanol extract of leaves and stems S. foetida. The recorded flavonoids were: apigenin 8-C-β-glucopyranoside (vitexin; B1), apigenin 6-C-β-glucopyranoside (isovitexin; B2), luteolin 6-C-β-glucopyranoside 8-C-α- arabinopyranoside(carlinoside; P3), apigenin 6, 8-di-C-β-glucopyranoside (vicenin; P4),

17 apigenin 6-C-α-rhamnopyranoside 8-C-β-glucopyranoside (isoviolanthin; P6), luteolin 7-O-β- glucopyranoside (P11), luteolin 7-O-β-glucuronide (P9), quercetin 3-O-β-glucopyranoside (St1), apigenin 7-O-β-glucopyranoside (P12), quercetin 3-O-α-rhamnopyranoside (quercetrin; B9), apigenin 7-O-β-glucuronide (P10), quercetin (B14), lutoelin (P19), Kaempferol (P20) and apigenin (P17). Eight compounds of which; B1, B2, P3, P6, P12, B9, P10 and P20, were assessed for the first time from the plant.

5.1.2. Quantitative estimation of the total flavonoid contents

The percentage of total flavonoids calculated as apigenin equivalent (AE) was found highest in the leaves and stems of B. populneus (150.863 mg AE/g), followed by that detected in the leaves and stems of P. alata (53.036 mg AE/g). The lowest concentration of the flavonoid content (12.257 mg AE/g) was detected in F. simplex.

5.2. Phenolic profiles of P. alata, B. populneus, F. simplex and S. foetida and their quantitative estimations by HPLC

5.2.1. Phenolic profiles

Phenolic profiles of the 70% methanol extracts of leaves and stems of each plant of the four selected species; P. alata, B. populneus, F. simplex and S. foetida (0.1 g) were determined by HPLC according to the method of Goupy et al. (1999).

Twenty one phenolic compounds were detected along side with the available authentic phenolic acids. Catechol, ferulic, iso-ferulic, vanillic, ellagic, o- and p-coumaric, p-hydroxy benzoic acids, syringic acid, catechein, chlorogenic acid, epi-catechin and gallic acid were determined with variable concentrations in all studied species. O- and p-coumaric acids were the main phenolic compounds in the leaves of B. populneus, P. alata, and F. simplex. Catechol was identified as the major constituent of S. foetida.

5.2.2. Quantitative estimation of the total phenolic contents

The percentage of total phenolics calculated as gallic acid equivalent (GAE) was observed highest in the leaves and stems of S. foetida (27.228 mg GAE/g) followed by P. alata (10.731 mg GAE/g). Whereas the other two studied species; F. simplex and B. populneus showed relatively lower contents of phenolics (2.933 and 0.322 mg GAE/g; respectively).

Part IV. Biological study Bioactivity studies were carried out on 70% methanol leaves and stems extracts of P. alata, B. populenus, F. simplex and S. foetida, as well as, some pure isolated flavonoid compounds from P. alata as follows:

1. In vitro study

1.1. Antioxidant activity assay

A pronounced antioxidant effect; scavenging DPPH free radical, was produced by 70% methanol extracts of leaves and stems of B. populneus (95.9% inhibition) and S. foetida

(82.2% inhibition) with IC50 of 31.9 µg/ml and 35.7 µg/ml, respectively, compared to vitamin C reference standard (98.4% inhibition). P. alata showed a moderate inhibition effect of

64.3% (IC50 = 45.7 µg/ml) all at 77μl concentration level. F. simplex extract as well as the major pure isolated flavonoid compounds (P5, P7 and P12) revealed weak activities on scavenging DPPH free radicals at different concentrations.

1.2. Cytotoxic activity assay

The crude extracts of the four studied species; P. alata, B. populneus, F. simplex and S. foetida demonstrated moderate antitumour activity (Sulforodamine B assay) against the cell lines HEP2, HELA, MCF7, PC3, HEPG2 and HCT. In addition to, the isolated compounds; P5, P7 and P12 exerted moderate cytotoxic activity against HEP2 and HELA, compared to the standard Doxorubicin.

2. In vivo study

2.1. Determination of the Median Lethal Dose

Non toxicity and safety of P. alata, B. populneus, F. simplex and S. foetida were designated by absence of the signs of toxcity and their high LD50; 6.250, 7.211, 5.623 and 4.375 g/kg, respectively.

2.2. Acute anti-hyperglycemic activity

Alloxan induced-diabetes model in rats was employed to investigate the anti- hyperglycemic activity of the selected plant species. Evaluation was performed through assessment of blood glucose level, estimation of the body weight of rats and determination of the anti-oxidative stress biomarkers.

2.2.1. Determination of blood glucose level

Determination of the blood glucose level was done by the glucose-oxidase principle using the OK glucometer instrument. Three days after being injected with alloxan (150 mg/kg), rats showed an elevated blood glucose level. The effect of the 70% methanol extracts of leaves and stems of P. alata, B. populneus, F. simplex and S. foetida (500 mg/kg bw, i.p.) on blood glucose level of alloxan-induced diabetic rats was determined at 0, 4 and 24 hrs time intervals. Diamicron (5 mg\kg, p.o.) served as standard drug.

Throughout 24 hrs, significant reduction in blood glucose levels was observed in case of P. alata (63%), B. populneus (59%) and S. foetida (55%) 70% methanol leaves and stems extracts, as compared to diabetic control group. Those results are comparable to that of the standard drug (Diamicrone); 68%. F. simplex exhibited no change of blood glucose level at 24 hours time interval.

2.2.2. Effect of the plants extracts on body weight

The 70% methanol extracts of leaves and stems of P. alata, S. foetida and B. populneus (500 mg/kg bw, i.p.) restored the body weight reduction caused by diabetes by 7.22%, 4.76% and 1.43%, respectively, compared to Diamicron (9.97%). F. simplex showed significant reduction in weights.

2.2.3. Anti-Oxidative stress biomarkers evaluation

The hyperglycemia induced to the experimental rats after three days of being injected with alloxan (150 mg\kg bw, i.p.) was associated with a decrease of serum reduced glutathione content (GSH), an elevation of serum MDA and NO levels. The results of the acute effect of 70% methanol extracts of leaves and stems of P. alata, B. populneus, F. simplex and S. foetida (500 mg/kg bw, i.p.) on the oxidative stress biomarkers (GSH, MDA and NO) in serum of alloxan-induced diabetic rats were evaluated after 24 hrs. P. alata, B. populneus, F. simplex and S. foetida extracts (500 mg/kg) counteracted the effect of the oxidative stress induced by alloxan causing relative decrease in the MDA and NO levels in serum and relative increase in the GSH level.

Conclusion

As was observed in the present study, it could be concluded that the four studied species, represented as four different genera, had significant dissimilarity and suggested to be treated as separate genera and not under the genus Sterculia. These findings are on the basis of the obtained data:  Genetic analysis through DNA and protein profiling.

 Macromorphological characters displayed considerable distinction in such as leaf, inflorescence, fruit and seed.  The micromorphological features illustrated resemblance between the species. Only, characters of the non-glandular and glandular trichomes and those of the epidermal cells were discriminated.  It could be stated that flavonoid profiles played a significant role in differentiation between Sterculia species.

 In vitro biological investigation provided background for the foundation of studying the diversity of the species.

 The in vivo biological studies of the species under investigation treated P. alata, B. populneus and S. foetida as potent acute anti-hyperglycemic species, which could be attributed to their phenolic and flavonoid contents.