Basic Informations

C.V

Master Title

Phytochemical Studies and Biological Evaluation of Clerodendrum Species Cultivated in Egypt

Master Abstract

General summary Clerodendrum is a very large and diverse genus and are widely distributed in Asia, Australia, Africa and America. The present study includes the following: Part I: Phytochemical study includes preliminary phytochemical screening of certain Clerodendrum species and investigation of the contents of ethyl acetate, n-butanol fractions and dichloromethane extract of C. chinense (Osbeck) Mabb. . Part II: Biological study of different Clerodendrum species and isolated compounds. Part I: Phytochemical study of certain Clerodendrum species: Chapter I: Preliminary phytochemical screening: Preliminary phytochemical screening of the leaves of C. chinense, C. indicum and C. glabrum revealed the presence of carbohydrates and/or glycosides, sterols and/or triterpenes, tannins and free flavonoids in all the leaves of the plants under investigation. Especially C. chinense contains appreciable amount of free flavonoids. Flavonoid glycosides are present in the leaves of C. chinense and C. glabrum and large amount in C. indicum. Chapter II: Investigation of the contents of dichloromethane extract of C. chinense (Osbeck) Mabb.: The air-dried powdered leaves were macerated in dichloromethane until exhaustion. The extracts were evaporated under reduced pressure at low temperature to yield a residue. The residue was decolorized by dissolving it in methanol and adding activated charcoal followed by warming in water bath then filter. The filtrate was evaporated under reduced pressure at a low temperature to yield a yellow residue. The residue was chromatographed on silica gel column using gradient elution with n-hexane and increasing the polarity with ethyl acetate till 100% ethyl acetate then increasing the polarity by methanol. Two main fractions were collected. First one is almost pure compound and the second subjected for further purification through silica gel column and preparative TLC. Comparing our finding with those cited in the literature, the identified compounds were identified by different spectral techniques as the following: D1: Lupeol D2: Rengyolone (Cleroindicin F) Chapter III: Investigation of the contents of ethyl acetate fraction of C. chinense (Osbeck) Mabb.: The air-dried powdered leaves were macerated in 80% aqueous methanol until exhaustion. The extracts were evaporated under reduced pressure at low temperature to yield a residue. The residue was dissolved in water and left over night in a refrigerator and filtered. The filtrate was extracted with successive portions of chloroform, ethyl acetate and n-butanol till exhaustion. All similar portions were combined and evaporated under reduced pressure to yield semisolid residue from chloroform, ethyl acetate and n-butanol fractions respectively. The ethyl acetate residue was fractionated on chromatographic silica gel column using gradient elution with n-hexane and increasing the polarity with ethyl acetate. The fractions were monitored by TLC using solvent system ethyl acetate : hexane (7.5 : 2.5) and similar fractions were pooled. First fraction was rechromatographed on chromatographic silica gel to get one compound. Second fraction was rechromatographed on polyamide column to get two main fractions which separately purified on RP C18 column. Two compounds were isolated. Comparing our finding with those cited in the literature, the identified compounds were identified by different spectral techniques as the following: L1: Hispidulin L2: Verbascoside L3: Isoverbascoside Chapter IV: Investigation of the contents of n-butanol fraction of C. chinense (Osbeck) Mabb.: The n-butanol residue was fractionated on polyamide column using water with increasing the percent of methanol. The fractions were monitored by TLC using solvent system ethyl acetate : formic acid : acetic acid : water (30 : 1.2 : 0.8 : 8) and similar fractions were pooled. Three main fractions were collected. Both of them were separately purified by RP chromabond C18 ec using water and acetonitrile to isolate two compounds. The remaining fractions subjected to RP chromabond C18 ec using water, RP C18 column using water and acetonitrile and preparative RP TLC plate using water : acetonitrile : acetic acid (94 : 2 : 4) to isolate one compound. Comparing our finding with those cited in the literature, the identified compounds were identified as the following: B1: Cornoside B2: Decaffeoyl verbascoside B3: Icariside B5 Part II: Biological study of Clerodendrum species. Chapter I: In vivo pharmacological studies of alcoholic, chloroformic and aqueous extracts of leaves of C. chinense, C. indicum, C. splendes and C. glabrum as well as of isolated verbascoside: a) Toxicological studies: The tested extracts and purified verbascoside are more or less safe. b) Acute anti-inflammatory effect: The rat groups administrated the methanolic extract of leaves of C. chinense and C. indicum in an oral dose of 100 mg/Kg b. wt. and verbascoside in an oral dose of 25 mg/Kg b. wt. showed highly significant anti-inflammatory effect that was found to be about 77- 89 % of the effect of indomethacin after 4 hours. The rat groups administrated the chloroformic extract of leaves of C. chinense and methanolic extract of leaves of C. glabrum in an oral dose of 100 mg/Kg b. wt. showed significant anti-inflammatory effect that was found to be about 56- 63 % of the effect of indomethacin after 4 hours. Chloroformic extracts of leaves of both C. indicum and C. glabrum showed less anti-inflammatory effect at an oral dose of 100 mg/Kg b. wt. c) Analgesic effect: The rat groups administrated the methanolic extract of leaves of C. chinense in an oral dose of 100 mg/Kg b. wt. and verbascoside in an oral dose of 25 mg/Kg b. wt. showed highly significant analgesic effect that was found to be about 74- 89 % of the effect of novalgin after 1 hour and about 61- 73 % of novalgin activity after 2 hours. All other groups showed less analgesic effect at an oral dose of 100 mg/Kg b. wt. d) Antipyretic effect: The rat groups administrated the methanolic extract of leaves of C. chinense in an oral dose of 100 mg/Kg b. wt. and verbascoside in an oral dose of 25 mg/Kg b. wt. showed highly significant antipyretic effect that was found to be about 58- 73 % of the activity of paracetamol after 1 hour and about 67- 81 % of paracetamol effect after 2 hours. The rat group administrated the methanolic extract of leaves of C. glabrum in an oral dose of 100 mg/Kg b. wt. showed significant anti-inflammatory effect that was found to be about 57 % of the effect of paracetamol after 1 hour and about 55 % of paracetamol effect after 2 hours. The rat group administrated the methanolic extract of leaves of C. indicum in an oral dose of 100 mg/Kg b. wt. showed less antipyretic effect. The rat groups administrated the chloroformic extracts of leaves of both C. indicum and C. glabrum showed non significant antipyretic effect at an oral dose of 100 mg/Kg b. wt. e) Antioxidant activity: The aqueous extracts of the leaves of both C. chinense and C. indicum in an oral dose of 100 mg/Kg b. wt. as well as verbascoside in an oral dose of 25 mg/Kg b. wt. showed highly antioxidant effect comparable with methanolic extract of the same dose. Chapter II: Antimicrobial and cytotoxic effects of alcoholic and chloroformic extracts of different plant organs of C. chinense, C. indicum, C. splendes and C. glabrum as well as some of the isolated compounds: The choroformic extracts of different parts of different Clerodendrum species showed variable extent of antiprotozoal effect. Different chloroformic extract show marginal effect against L. inflatum but more powerful effect against P. falciparum especially chloroformic extracts of flowers of both C. chinense and C. splendes where IC50 < 10 µg/ml. The chloroformic extracts of stem and flower of C. chinense are very active against T. cruzi with marginal cytotoxicity. The chloroformic extracts of leaves of both C. chinense and C. splendes have promising effects against T. cruzi without cytotoxic effect on human cell line and this suggest the selectivity of those two extracts against T. cruzi. The methanolic extracts of different parts of different Clerodendrum species are less active against parasites comparable with the effect of chloroformic extracts. Verbascoside show marginal effect against T. cruzi. Isolated rengyolon shows a broad but non specific effect, related to its general cytotoxicity. Methanolic extract of leaves of C. chinense show marginal effect against Microsporum canis. Other extracts and isolated compounds did not show antibacterial or antifungal effects.

PHD Title

Structural and Biochemical Characterization of the Organomercurial Lyase MerB

PHD Abstract

Abstract Mercury is introduced into the environment from either natural occurrences (volcanoes) or from human activities (combustion of fossil fuels). Once mercury is introduced into the environment, mercury undergoes a complex geochemical cycle and is converted to both inorganic and organic forms. The three inorganic forms of mercury are elemental mercury (Hg0), mercurous ions (HgI), and mercuric ions (HgII), and the most abundant organic form of mercury is methylmercury (MeHg). Organomercurial compounds like MeHg are the most toxic form because of their hydrophobicity and the increased toxicity is believed to be associated with the ability of organomercurials to efficiently permeate membranes and bioaccumulate in organisms. High levels of MeHg have been found in fish in many areas around the world, and therefore human consumption of contaminated seafood represents a serious danger for human health. Bacteria isolated from mercury-contaminated environments have evolved a system that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. This mercury resistance is due to the acquisition of a transferable genetic element known as the mer operon. The mer operon encodes for several proteins including two enzymes, the organomercurial lyase MerB and the mercuric ion reductase MerA. MerB catalyzes the protonolysis of the carbon-mercury bond that results in the formation of two products, a reduced-carbon compound and inorganic ionic mercury HgII. MerA catalyzes the reduction of HgII to elemental mercury Hg0, which is volatile and less toxic. Due to their unique ability to breakdown MeHg, MerA and MerB are considered crucial to bioremediation efforts to clean up MeHg from contaminated waterways. The mechanistic details of how MerB and MerA function together at the atomic level to protect bacteria from mercury toxicity is one of the interests of Omichinski group. A clear understanding of these mechanistic details is crucial for maximizing utilization of the mer system in bioremediation efforts. In particular, we are interested in understanding the unique mechanism by which MerB cleaves carbon-mercury bond. More specifically, we have been using NMR spectroscopy and X-ray crystallography to structurally and mechanistically characterize MerB. Based on our previous structural studies, three important residues have been identified (Cys96, Asp99 and Cys159). They are required for cleavage of the carbon-Hg bond by MerB and we have determined the structure of the mercury bound product in the active site. As a follow up to the earlier studies, my project involves using X-ray crystallography to more precisely define the roles of Cys96, Asp99 and Cys159 in substrate binding and carbon-Hg bond cleavage. Two different approaches were implemented to fulfil this goal. In the first approach MerB mutants were tested for their activity and to help define the exact role for the three catalytic residues. In the second approach, MerB inhibitors and other potential non-organomercurial substrates were used to probe the MerB active site. In the first approach we mutated D99 to serine because in all but four of the known MerB variants, the Cys,-Asp-Cys catalytic triad found in E.Coli MerB is conserved. In the other four MerB variants, the two cysteine residues are conserved but the Asp is changed to Ser. To model this, a MerB D99S variant of E. Coli MerB was expressed and purified for crystallization to compare with wild-type active site in the free and mercury-bound state. Interestingly, we obtained a protein with a pink color during the purification of the MerB D99S variant and the electron density maps obtained during x-ray crystallography studies indicated the presence of a metal-MerB complex. Analysis by ICP-MS and X-ray fluorescence indicated that the D99S mutant of MerB bound copper in the active site. Further, electron paramagnetic resonance (EPR) and NMR studies identified the copper as Cu (II). In contrast, the wild-type MerB protein containing the serine residue in the active stie did not co-purify with copper and the X-ray structure of this MerB in complex with mercury is virtually identical to the structure of the MerB D99S-Hg complex. Our results provide evidence of how MerB may have evolved from the structurally homologous copper-binding protein NosL. In addition, they suggest that the aspartic acid residue is crucial for the cleavage of carbon-Hg bonds of organomercurials without binding other metals such as copper. In the second approach, we probed the active site of MerB through testing its binding to additional organometals other than organomercurial. We have tested the interaction of MerB with organotin and organolead compounds. Trimethyltin (TMT) and trimethylead (TEL) showed binding to D99 , whereas diethyltin (DET) and diethylead (DEL) were cleaved by MerB, In addition, DET and DEL show higher binding affinity than even MeHg for MerB. Furthermore, dimethyltin (DMT) inhibits MerB using an alternative mechanism as seen with TMT and TEL. DMT induces a dramatic change in the active site by disrupting a cation-p interaction between Try95 and Arg155 in the active and it appears that this interaction is essential for catalytic activity. These results suggest that organomercurials may not be the sole substrate for MerB. On the other hand, these observations raiss the question of whether or not MerB could function in bioremediation efforts for other organometal contaminations. These results indicate also that the presence of other metals may have important implications when using MerB in bioremediation systems. The information obtained from this study will be crucial for maximizing the applications of MerB in bioremediation efforts.