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 1 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), 2 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. 3 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