Open Access Repository of Indian Theses

Prasad, Bablu

ABSTRACT 
Biodegradation of phthalate esters (PAEs), namely dimethyl phthalate (DMP), diethyl 
phthalate (DEP), dibutyl phthalate (DBP) and their mixture by a bacterial strain (BS1) is 
reported. This bacterium was isolated from soil that was contaminated with plastic wastes and 
shown in laboratory assays to have the potential to utilize PAEs as the sole source of carbon 
and energy. The isolate was identified as Variovorax sp. based on 16S r-DNA sequence 
analysis. The extent of biodegradation of DMP, DEP, DBP and their mixture (300 mg L-1) by 
Variovorax sp. BS1 were: >97%, >99%, >92% and >99%, respectively after 24 h of 
incubation at 30°C. The Variovorax sp. BS1 was able to biodegrade 200 to 600 mg L-1 of 
individual PAEs and their mixture within 30 h of incubation. The specific growth rate of 
Variovorax sp. BS1 increased with increasing concentrations of PAEs up to 200 mg L-1 and 
further increase in concentrations did not change the specific growth rate. The transient 
intermediates and persistent end product formed due to biodegradation of DMP, DEP, and 
DBP by Variovorax sp. BS1 were not detectable by either HPLC or LC-MS analyses in liquid 
culture medium. However, using GC-MS, trace amounts of phthalic acid and its derivatives 
were observed. Total organic carbon (TOC) analysis suggested complete mineralization of 
DMP, DEP, DBP and their mixture by Variovorax sp. BS1. 
In order identify the enzymes involved in the biodegradation of PAEs, cell free crude extracts 
were prepared and assayed for the presence of enzymes using PAEs and para-nitophenyl 
acetate (pNPA) as the substrates. The extent of biodegradation of 1 mM of DMP using cell 
free extracts derived from Variovorax sp. BS1 grown on DMP was observed to be >80% at 
the end of 60 min of incubation at 30°C. Results suggested that the cell-free extracts 
contained the mixture of enzymes responsible for the biodegradation of DMP. Further it was 
observed that the cell free enzymes were more efficient in biodegrading various 
concentrations of DMP (25 to 1000 μM). The enzyme(s) responsible for the biodegradation of 
DMP exhibited highest activity in freshly prepared cell-free crude extracts while the activity 
declined rapidly upon storage. Zinc ions (Zn+2) inhibited the phthalate dioxygenase activity in 
the cell free extracts which resulted in the generation and accumulation of phthalic acid. Cell 
free crude extracts were also prepared from Variovorax sp. BS1 grown on a mixture of DMP, 
DEP and DBP and used for biodegradation of individual PAEs as well as mixture. The extent 
v
of removal of 1 mM of DMP or DEP or DBP and mixture of PAEs (0.33 mM of each) were: 
59%, 67%, 65% and 53% after 60 min of incubation using cell free enzyme preparations.  
Variovorax sp. BS1 cells were immobilized in calcium alginate beads and used for the 
biodegradation of PAEs in batch experiments. Degradation efficiency of immobilized cells in 
mineral salts medium (MSM) and growth non supportive medium, de-ionized water was 
compared. Results showed higher degradation rate in MSM as compared to de-ionized water. 
Higher rate of degradation in MSM was due to the growth of cells in/on beads as well as in 
spent medium. Therefore, MSM was replaced with de-ionized water and used for repeat batch 
experiments. The results suggested that the immobilized cells of Variovorax sp. BS1 may be 
re-used upto ten cycles with diminutive loss of DMP degradation activity. The results 
presented suggest the bacterial isolate, Variovorax sp. BS1 has the potential for the 
bioremediation of water contaminated with PAEs. 
Keywords: Biodegradation, Dimethyl phthalate, Diethyl phthalate, Dibutyl phthalate, 
Phthalate esters, Variovorax, Enzyme, Immobilization 


English

BIODEGRADATION OF MIXTURE OF PHTHALATE ESTERS BY VARIOVORAX SP. BS1

R, Beema Shafreen

Abstract
Identification and exploration of antibacterial and antibiofilm compounds against different structural targets of Streptococcus pyogenes through in silico and in vitro approaches.
S. pyogenes is a human pathogen capable of causing pharyngitis and strep-throat related infections. Owing to the upsurge of multidrug resistance in S. pyogenes, there is a considerable interest in understanding the pathogenicity of the organism. The first initiative step was to identify proteins that have essential role during survival and regulation of virulence factors in S. pyogenes. The second step is to screen potential antimicrobial compounds against these targets. Due to increased advancement in pharmaceutical sciences, which are yet to be fully explored for therapeutic usage against bacterial pathogens, a large dataset of compounds are available in the public domains. Antibiotics play a crucial part to combat with infectious diseases. Numerous antimicrobial targets are described in the near past to identify lead molecules. In the current study, targets from S. pyogenes that are unexplored are classified into antibacterial and antibiofilm targets. Targets such as SPFabG, SPGyrA, SPGtfs, SPLuxS playing a significant role during survival and pathogenicity of S. pyogenes have been delineated. Screening of the compounds available in the public domains against these targets has culminated in the identification of few potential lead molecules. Therefore, from the in vitro studies, it was confirmed that, these compounds can combat resistance and when fully explored can be used as therapeutic against S. pyogenes related infections. In the current scenario, a user has to necessarily visit all data resources related to the problem in order to get the relevant information. SPEIDS, an annotation database finds utility in the scientific community for a quick view of the collection of data for the inhibitors classified based on various sources and pharmacological actions. Developing database and making it publicly accessible enables a large number of scientists/clinicians to perform further investigation on the drugs essential for their field of research. This classification in SPEIDS will assist the scientific users to choose the ligands for their studies related to S. pyogenes infections.



Identification and exploration of antibacterial and antibiofilm compounds against different structural targets of Streptococcus pyogenes through in silico and in vitro approaches.

Pande, Babita

Undetermined

Time Estimation (Short Intervals)in Human Subjects under Free Living and Constant Routine Conditions

BANERJEE, SOUMYAJIT

Abstract
Numbers of studies on conjugated polymers relating to their synthesis,
characterization, morphology and applications in various fields have been reported in
literature. In spite of these interesting developments, there is a scope to improve the
properties of conjugated polymers and their applications in various fields.
The objectives of the present thesis are:
(i) Synthesis and characterization of the soluble conjugated polymers such as
poly(N-heptylindole), oligoindole and oligo-p-xyleme by chemical
polymerization technique for PLED application with easy protocol.
(ii) Synthesis of processable polyaniline (PANI) salt like PANI-HBF4-DHS by
emulsion polymerization technique and its spectral and thermal
characterization.
(iii) Synthesis of organo soluble PANI salts by using plastdopants such as didecyl
ester of 4-sulfophthalic acid (DESPA) and di-2-ethylhexyl ester of
sulfosuccinic acid (DESSA)and the polymeric materials were characterized by
spectral, electrical, physical and thermal methods.
(iv) PANI-PVC blends were prepared by using processable PANI salts by both
solution and thermal process and their electrical and mechanical behaviors were
studied. In this study corrosive chemicals such as m-cresol , trichloroacetic acid
are completely avoided.
(v) Synthesis and characterization of PANI salts, complexes, substituted PANI salts
and evaluation as recyclable polymer based mild acid catalysts for various
organic transformations.
19
(vi) Polyindole salt was synthesized by chemical oxidative polymerization pathway
and used as reusable catalyst for bis(indolyl methanes) synthesis.



Effects of environmental factors on the larval development and life history traits of Aedes aegypti in Kolkata, India

D'Souza, Pratibha



SYNOPSIS



	SYNOPSIS	

The thesis entitled “ Stereoselective Total Synthesis of (+) Membrenone C And Studies Directed Towards The Total Synthesis Of Siphonarin B” is divided into three chapters.
CHAPTER I : Chapter I is further classified into two sections.

SECTION A: This section of thesis contains the general introduction and the earlier synthetic approaches to  Membrenone C.

SECTION B: This section concerns with the stereoselective total synthesis of (+) Membrenone C and its 7-epimer. 

CHAPTER II : This chapter is further organized into two sections.

SECTION A:  This sections deals with the introduction and earlier synthetic approaches to Siphonarin B.

SECTION B: This section deals with the present work done for the stereoselective synthesis of C3 – C8 and C9 – C21 fragments of Siphonarin B.

CHAPTER III: This chapter describes the development of novel methodologies.

SECTION A : LPDE catalyzed intramolecular Aza – Diels – Alder reaction : A Facile synthesis of Tetrahydrochromanoquinolines. 

SECTION B : Synthesis of 4-Chlorotetrahydropyrans under microwave irradiation.


CHAPTER I :  

STEREOSELECTIVE TOTAL SYNTHESIS (+) MEMBRENONE C :
Naturally occurring polyketides and polypropionates have attracted a lot of attention during the past 50 years, and a great deal of research has been concerned with their isolation, biological activity, biogenesis and total synthesis.  Much is known about these compounds from a biological perspective including a detailed understanding of their biosynthesis, which gives rise to endless chemical diversity and results in rich biological activity.
	Opisthobranchs are marine molluscs scarcely protected by a shell that, generally is reduced fragile and sometimes, completely absent.  Many studies have strongly supported that opisthobranchs are frequently protected against predators by chemicals which are either concentrated in anatomical parts more exposed to predators or released into defensive mucous secretions. Three compounds Membrenones A, B & C were isolated in 1993 from the skin of the marine mollusc pleurobranchus membrane.ceus.  There is an apparent error in the sign of the optical rotation reported for natural Membrenone C in the original isolation paper as concluded by recent reports.



	
Authors envisaged that the total synthesis of natural membrenone C not only would help to solve the sign of the optical rotation ambiguity but also add a route to synthesize it. This chapter deals with the total synthesis of natural (+) Membrenone C and its 7- epimer utilizing desymmetrization approach.
	Restrosynthetically, (+) Membrenone C 1 can be obtained via tetrone 2.  The construction of tetrone was based on a disconnection between the C4-C5 & C11-C12 bonds via double aldol.  The sequence of five contiguous stereogenic centres C6 to C10 in 3 was envisaged from the chiral bicyclic lactone 5 which was generated from bicyclic ketone 6.
Restrosynthetic strategy of (+) membrenone C :




	













SCHEME 1



The synthesis of (+) Membrenone C starts with the common bicyclic ketone 6. The bicyclic ketone 6 was prepared by employing a (3+4) cycloaddition reaction of oxyallyl cation and furan as reported by Hoffmann and co-workers.  The acid catalysed dibromination of 3-pentanone 7 afforded the dibromo compound 8.  The dibromo compound 8 when treated  with furan 9 in the presence of Zn-Cu couple underwent (3+4) cycloaddition reaction to afford 2,4-dimethyl-8-oxabicyclo[3.2.1]oct-6-ene-3-ones 6, 10 and 11 in the ratio 8:1:1. These bicyclic ketones on selective reduction with DIBAL-H gave the corresponding alcohols (Scheme 2). 


SCHEME 2


	The required major alcohol 12 was separated from the other isomers using column chromatography and the structure was confirmed by spectral studies.

	The hydroxyl group of compound 12 was protected as its 4-methoxybenzyl (PMB) ether 13 using NaH and 4-methoxybenzyl bromide.    Asymmetric hydroboration of olefin 13 using (+) - Diisopinocampheylborane (IPC2BH) proceeded smoothly to give the alcohol 14 with high enantiomeric purity.  The alcohol 14 was converted into the lactone 16 by a two step sequence. PCC oxidation of alcohol 14 followed by Baeyer-Villiger oxidation of the resulting ketone 15 afforded lactone 16.  Alkylation of lactone 16 using LHMDS and methyl iodide gave 5. Reductive opening of bicyclic lactone 5 with LAH afforded the triol 17 (Scheme 3).











SCHEME 3

Triol  17 was selectively protected as its di silyl ether 18 using TBDMSCl and imidazole. Acetyl protection of alcohol 18 using acetic anhydride ,TEA and DMAP afforded 19. Deprotection of PMB ether of 19 in presence of DDQ in aq. DCM afforded compound 20 which was oxidised with Dess-Martin periodinane to give ketone 21 (Scheme 4).








SCHEME  4
	Next attention was directed towards one pot deprotection of 21 and diastereoselective 1,3 syn selective reduction . Reaction of 21 with DIBAL-H in dry ether at -100º C afforded the undesired product 23 as the major isomer with high diastereoselectivity (Scheme 5).



SCHEME 5
The stereochemistry of 23 was assigned by the total synthesis of final product. The diol of major isomer 23 was protected as di tert-butylsilylene 24 using di tert-butyl trifluoromethane sulfonate and 2,6 lutidine. Treatment of silylether 24 with 2 eq. of PPTS in methanolic DCM produced 25, which was subjected to PCC (8 eq.) oxidation to give stable dialdehyde 26 (Scheme 6).







SCHEME 6
	Next the two directional chain extending double aldol was achieved  by treating 26 with Ti (IV) enolate of diethyl ketone  in DCM at -78º C to afford the double aldol product 27. Since the stereo centres produced in formation 27 are not present in final product, we proceeded with diastereomeric mixture. Double Swern oxidation of diol 27 using oxalyl chloride, DMSO and triethylamine readily provided tetraone 28. Di tert-butyl silylene group of tetraone 28 was removed by treatment with HF-pyridine buffered with excess pyridine giving a mixture of diols and hemiacetals. Rapid acid catalyzed cyclisation dehydration was achieved by treatment with TFA giving a single product 29 (Scheme 7).



	






SCHEME 7

After comparision of optical rotation and 1H NMR data of synthetic 29 and that of natural 1, we realized that the synthetic compound was the 7-epimer of (+) Membrenone C. It was reasoned that 1,3 syn selectivity was not observed in case of DIBAL-H reduction of compound 21 (Scheme 5). Probably because DIBAL-H was not chelating with the oxygen when the hydroxyl  was protected as acetate in 21. Deprotection of acetate of 21 under various experiments led to eliminated or cleaved products.(Scheme 8)

















SCHEME 8
Since the acetate deprotection failed, protecting groups strategey was changed. The two primary hydroxyl of triol 17 were  protected as di benzylether 30, which was further subjected to silylation using TBSOTf and 2,6 lutidine to afford 31. Deprotection of PMB ether 31 using DDQ in aq. DCM afforded 32, which was subjected to Dess Martin oxidation to give ketone 33. Treatment of 33 with 2 eq.. of PTSA in methanolic DCM afforded hydroxyl ketone 34 (Scheme 9).












SCHEME  9
Diastereoselective reduction of 34 using DIBAL-H in ether at -100º C afforded syn diol 35 as the major isomer with high diastereoselectivity (Scheme 10)




SCHEME 10
	Di tert butyl silylene protection of the diol 35 using di tert butyl trifluoromethane sulfonate and 2,6 lutidine afforded compound 36. Debenzylation of 36 under catalytic (Pd/C) hydrogenolysis afforded diol 37, which was subjected to PCC oxidation to give dialdehyde 3 (Scheme 11)








SCHEME 11
	Next, the double aldol was achieved by treating 3 with the Ti (IV) enolate of diethyl ketone 4 in DCM at -78º C to afford 38. Double Swern oxidation of double aldol product 38, using oxalylchloride, DMSO and triethylamine afforded tetraone 2. Di tert butyl silylene group of tetraone 2 was removed by treatment with HF-pyridine buffered with excess pyridine to afford mixture of diols and hemiacetals. Rapid cyclisation/dehydration of the mixture using TFA afforded the  solid product 1 (Scheme 12)













SCHEME 12

	The spectral and optical properties for 1 were in agreement with those reported earlier and we have prepared for the first time (+)7-epi-Membrenone C 29.

DIRECT SYNTHESIS OF (+)7-epi-MEMBRENONE C :

	Synthesis (+)7-epi Membrenone C 29 was achieved directly from the dibenzyl ether 30, without using oxidation/reduction strategy. Removal of PMB group of 30 using TFA/anisole in DCM afforded diol 39, which was protected as di tert butyl silylene to give 40. Treatment of 40 with catalytic amount of Pd/C under H2 atmosphere gave the known diol 25 (Scheme 13). Further treatment of 25 to 29 following same steps as depicted in Scheme 6 & 7.
















SCHEME 13















CHAPTER II  :

STUDIES DIRECTED TOWARDS THE TOTAL SYNTHESIS OF SIPHONARIN B 

	In 1984, Faulkner, Ireland and co-workers first reported isolation of unusual γ-pyrone polypropionate, containing a characteristic spiroacetal ring system, siphonarin B 41 from the marine molluscs , Siphonaria zelandica and S. atra, collected on the coast of New South wales, Australia, Siphonarin B was found to possess anti-microbial activity.


	





SIPHONARIN B

The architecturally complex structure, biological activity and scarcity of Siphonarin B make it an attractive target for total synthesis. As part of our studies towards the total synthesis of Siphonarin B, we chose to adopt a highly stereoconvergent strategy, disconnecting the carbon back bone at C2-C3 via Kishi-Nozaki coupling, reveals two sub units 44 and 45.  Further disconnection of  acyclic precursor 44 at C8-C9 via aldol coupling leads to subunits 46 and 47.  The synthesis of 46 and 47 are envisaged from common precursor triol 51, which was generated from the known bicyclic ketone 6.



  RETROSYNTHETICANALYSIS OF SIPHONARIN B :


























SCHEME  14


Synthesis of Intermediate triol  51 :

	Asymmetric hydroboration of known olefin 13 using (-)-Diisopinocampheylborane (Ipc2BH) proceeded smoothly to give the alcohol 52 with high enantiomeric purity. The alcohol was converted into the ketone 53 by PCC oxidation. Baeyer-Villiger oxidation of the resulting ketone 53 gave the lactone 54 which on treatment with  methyl iodide and
















SCHEME 15

lithiumhexamethyl disilazide (LHMDS) at -78º C furnished compound 55.  Reductive opening of bicyclic lactone 55 with LAH afforded the triol 51 (Scheme 15 ), which was employed as a common precursor for the synthesis of  both the key fragments.



Synthesis of C3-C8 fragment :

	The synthesis of C3-C8 fragment 46 was started from triol intermediate which has five contiguous stereogenic centers. The two primary hydroxyl groups of triol 51 were selectively protected as their tert-butyldimethyl silyl ether 56 (Scheme 16 ).  Inversion of the hydroxyl group configuration at C-5 center of compound 56 was achieved by oxidation reduction strategy.  Oxidation of compound 56 using Dess-Martin periodinane followed by reduction using DIBAL-H afforded the required isomer 58 as the major product.  Compound 58 was then converted into the triol 48 by desilylation with TBAF (Scheme 16).













SCHEME 16

The triol 48 was subjected to 2,2 dimethoxy propane and CSA (cat.) to give the acetonide product 59.  The hydroxyl group of compound 59 was protected as the tert-butyldimethyl silyl group followed by the deprotection of PMB ether using DDQ afforded the compound 61.  Oxidation of 61 using Dess-Martin periodinane followed by desilylation with TBAF to give the hydroxy ketone 63 (Scheme 17).



SCHEME 17


Now we focused on decarbonylation of β-keto aldehyde 64 through retero-Aldol pathway.  Under various oxidation conditions we failed to get the β-keto aldehyde.  Further attempts to complete the synthesis of C3-C8 fragment and total syntheis  of Siphonarin B are still under progress.

Synthesis of C9-C21 Fragment :
	The triol 51 was subjected to 2,2 dimethoxypropane and CSA (Cat.) to give the acetonide product 65.  The alcohol 65 was protected as benzyl ether using NaH and BnBr followed by deacetonization with aq.2N HCl to afford the diol 67.The primary alcohol of 67 was selectively protected as its benzoate ester 68 (Scheme 18 ).


	




SCHEME 18
The secondary hydroxyl group of compound 68 was protected as methoxy methyl  ether using MOMCl and DIPEA to afford 69.  Hydrolysis of benzoate ester 69 using aq 3N KOH followed by the oxidation with IBX-DMSO afforded aldehyde 49.




	








	SCHEME 19
Addition of the titanium enolate of 3-pentanone to propanal provided diastereomeric mixture 72  which was oxidized with Dess-Martin periodinane to yield diketone 50.
	




SCHEME 20
Our subsequent task was the coupling of fragments 49 and 50 to construct the γ-pyrone moiety.  The diketone 50 was converted to a dianion with 2.5 eq.. of LDA and the dianion was reacted with the aldehyde 49 to afford the diastereomeric mixture of aldol products 73.  Rapid cyclization / dehydration of the mixture 73 (without  purification) was achieved by treatment with CSA (cat) to give the diastereomeric mixture of γ-dihydropyrone 74 which is not easily separable by column chromatography (Scheme 21 ).









SCHEME 21

Next attention was directed to bring the unsaturation between C16 and C15 of  γ-dihydropyrone.  Inorder to obtain that, γ-dihydropyrone 74 was subjected to various types of reactions like dehydrogenation, halogenation-dehydrohalogenation, selenylation-selenoxide elimination etc, under different experimental conditions as shown in the scheme 22.   But to our extreme surprise, none of the approaches led to the desired product.



                                                          	


SCHEME 22
Since the desired product could not be obtained from 74, the diastereomeric aldol mixture 73 was immediately subjected to careful oxidation with DMP to give the triketone 76.  The resulting β-triketone 76 without purification was directly cyclised by TPP-CCl4 system to form the required γ-pyrone 75 (Scheme 23).







SCHEME 23
	To our relief and extreme satisfaction this approach afforded the much desired product. So we successfully completed the synthesis of C9-C21 fragment. Efforts are in progress to couple the C3-C8 and C9-C21 fragments  and  total synthesis of Siphonarin B. 

















CHAPTER III

This Chapter is divided into two sections .Section ‘A’ deals with the synthesis of Tetrahydrochromanoquinolines using LPDE catalysed intramolecular aza-Diels-Alder reaction.

Aza-Diels-Alder rection is one of the most powerful synthetic routes for construction nitrogen containing six-membred heterocycles. The tetrahydrochromanoquinoline derivatives are exhibit a vast range of biological activities including psychotropic, anti-allergic, anti-inflammatory and estrogenic behaviour.

The recent surge in the use of LPDE medium as a mild Lewis acid prompted us to disclose a simple and efficient procedure for the synthesis of tetrahydrochromanoquinolines. 5M LiClO4 in diethyl ether was found to be effective Lewis acid in promoting intramolecular imino-Diels-Alder reaction of aldimines derived from aromatic amines and O-prenyl derivatives of salicylaldehydes to produce the corresponding tetrahydrochromanoquinoline derivatives (Scheme 24).








SCHEME 24


































SECTION ‘B’ deals with the synthesis of 4-chlorotetrahydropyrans under microwave irradiation. 

The tetrahydropyran ring is part of the back-bone of various important carbohydrates and natural products such as avermectins, aplysiatoxin, oscillatoxins, latrunculins, talaromycins and acutiphycins.

The acid catalyzed condensation of olefins with carbonyl compounds known as Prins reaction, is an important carbon-carbon bond forming reaction. In this section a mild and efficient procedure for the synthesis of 4-chloropyrans through the Prins type cyclization reaction of homoallyl alcohols with aldehydes in the presence of BiCl3 is described (Scheme 24).
	
In recent years microwave assisted organic reactions is an area of growing interest because of distinct advantages like rapid reaction, easy work up and solvent free conditions. The general Scheme to synthesize 4-chlorotetrahydropyrans is outlined below and results are listed in Table 2.




                               1                                       2                                             3
	
SCHEME  25



































































PAGE  



I





Site suitability analysis for solid waste disposal in Bangalore using GIS

Deka, Rekha

Neurospora crassa homologue of Neuronal Calcium Sensor-I has a role in growth, calcium stress tolerance, and ultraviolet survival.

S, Kamatchi

Abstract
Modulating Conformation and Organization of Protein Assemblies Through Interfacial Rheology
This thesis describes a perspective on the role of interfacial rheology on the conformations of different classes of proteins at air/buffer, water/solvent and solid/solution interfaces and possible folding/misfolding events that lead to both organized and disorganized assemblies of proteins near the interface. Further, the role of interfacial viscosity in the adsorption and adhesion of the proteins to solid substrates has been analyzed. The influence of pH, electrolytes and viscogens on the rheological properties for different classes of proteins and poly aminoacid has been analyzed.




MODULATING CONFORMATION AND ORGANIZATION OF PROTEIN ASSEMBLIES THROUGH INTERFACIAL RHEOLOGY

N, Suganthy











Abstract

	The thesis entitled “Multipotent action of Rhizophora mucronata (Red mangrove) as neuroprotective drug against Alzheimer’s disease – Evidence from preclinical studies” deals with the study of neuroprotective effect of Rhizophora mucronata against Alzheimer’s disease (AD) through its multipotent action in  in vitro (PC12 cells) and in vivo model systems (mice). The thesis is typically divided in six chapters.

	Chapter I: In vitro screening of Rhizophora mucronata for its multipotent neuroprotective effect - Antioxidant, Cholinesterase inhibitory, Metal chelating and Anti-amyloidogenic property.
	Chapter II: Nutritional evaluation of Rhizophora mucronata leaves.
	Chapter III: Safety and Toxicity evaluation of Rhizophora mucronata methanolic leaf extract: in vitro and in vivo study.
	Chapter IV: Neuroprotective effects of Rhizophora mucronata against beta-amyloid-induced cell death in PC12 cells via its antioxidant mechanism.
	Chapter V: Anti-amnestic and neuroprotective effect of methanolic leaf extract of Rhizophora mucronata against A (25-35) induced toxicity in Swiss Albino mice.
	Chapter VI: Bioassay-directed isolation of active compounds with neuroprotective effect from Rhizophora mucronata leaf extract.
	
	Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized clinically by insidious onset of memory and cognitive impairment, emergence of psychiatric symptoms and behavioral disorder, and impairment of activities of daily life. AD one of the fourth leading causative of death, affects about 26.6 million people worldwide, which might quadruple to 106.8 million by 2050 if no therapy intervenes. Multiple causative factors such as - amyloid (A) aggregation,-protein aggregation, oxidative stress and cholinergic hypothesis plays major role in the pathogenesis of AD, among which, Aβ peptide plays a pivotal role. Currently AChE inhibitors such as tacrine, donepezil, rivastigmine and galanthamine are used for the treatment of mild to moderate AD as approved by Food and Drug Administration. However these drugs possess severe gastrointestinal side effects and are effective only for mild to moderate forms of AD. Diverse pathogenic factors and safety limitation of currently used synthetic drugs necessitates exploration of multipotent drug from natural source to hit more than one target implicated in AD. 
	Rhizophora mucronata commonly termed as Asiatic mangrove have been valued for its timber and ethanomedicinal properties. In traditional medicine, leaves of R. mucronata have been used for the treatment of elephantitis, hematoma, hepatitis, ulcers, inflammation and febrifuge.  In addition the leaves have been used as alternative source of tea and as animal feed. The leaves have also been reported for its pharmacological properties such as anti-diabetic, anti-inflammatory, anti-viral activity and anti-diarrheal effect. Despite of its wide pharmacological properties, the application of R. mucronata in the treatment of neurological disorder is still at its infancy. Mangroves in general are halophyte which possess rich source of antioxidant system to overcome the oxidative stress induced by salt stress environment. Since A peptides leads to neuronal death and cognitive deficits through oxidative stress, it was attempted to screen the mangrove R. mucronata, which has rich antioxidant and anti-inflammatory property, for its neuroprotective effect. We have attempted to evaluate the neuroprotective activity of Rhizophora mucronata against AD through its multipotent action.
	The chapter I “In vitro screening of Rhizophora mucronata for its multipotent neuroprotective effect - Antioxidant, Cholinesterase inhibitory, Metal chelating and Anti-amyloidogenic property” deals with screening different mangroves for its potent antioxidant, cholinesterase inhibitory activity, metal chelating and antiaggregation property. Nine mangroves (Avicennia marina, Rhizophora mucronata, Rhizophora lamarkii Rhizophora apiculata, Rhizophora annamalayana, Ceriops decandra, Sweda maritima, Pruscaria celligrica and Lumnitzera racemosa) collected from Pichavaram, Vellar estuary, Tamil Nadu, India were used for the present study. Screening for cholinesterase and antioxidant activity of mangroves showed that of the nine mangroves screened, leaf extract of R. mucronata exhibited potent antioxidant and dual cholinesterase inhibitory activity. Further, successive solvent extracts from the leaves of R. mucronata were assessed for its multipotent neuroprotective effect (antioxidant, cholinesterase inhibitory and metal chelating activity).  Of the various solvents, methanolic leaf extract of R. mucronata (MERM) displayed remarked inhibition against both AChE and BuChE exhibiting dual cholinergic effect.  In vitro antioxidant assays showed that MERM showed excellent reducing power and free radical scavenging activity. Assessment of antiamyloidogenic property illustrates that MERM inhibits Aβ fibrillogenesis and also destabilizes the preformed fibrils. To the best of our knowledge, this is the ﬁrst study describing anticholinesterase and antioxidant activity using ferrous ion-chelating, nitric oxide, hydroxyl radical scavenging and ferric-reducing antioxidant power (FRAP) assays of the afore-mentioned mangrove species. Since MERM acts as multifunctional agent exhibiting cholinesterase inhibition, antioxidant, metal chelating property, antiaggregation and deaggregation of Aβ fibrils, it can act as a potent drug to combat the multiple targets of AD. The multipotent activity of MERM could be attributed to the presence of high amount of total phenol and ﬂavonoid contents.
 	The chapter 2 “Nutritional evaluation of Rhizophora mucronata leaves” deals with nutritional analysis of R. mucronata, its proximate composition, amino acid profiles and physicochemical properties in comparison with green leafy vegetables consumed in India.  Proximate composition analysis showed that R. mucronata act as a good source of dietary fiber, lipid and protein content suitable for human consumption. The study also detected the presence of seventeen amino acids with higher essential amino acid content which were comparable to FAO/WHO requirement. Presence of higher levels of Linoleic acid, alpha linolenic acid and low levels of saturated fatty acid signifies the leaves as nutritionally balanced diet. Leaves also act as rich source of ascorbic acid, thiamine, riboflavin, tocopherol, Na+, Fe2+ and Ca2+ which could help in overcoming micronutrient malnutrition at a negligible cost. Physicochemical properties of leaves had a high positive correlation with their total amount of fiber and protein content. Overall, the leaves of R. mucronata could be used as a potential health food in human diets and may be of use to the food industry as a source of ingredients with high nutritional value.
	Safe use as plant-based medicine, beside historical and experienced applications on humans and animals, requires toxicity evaluation of these medicinal herbs.  Hence Chapter III “Safety and Toxicity evaluation of Rhizophora mucronata methanolic leaf extract: in vitro and in vivo study” deals with assessment of toxic effect of MERM under in vitro (mutagenic, genotoxic, cytotoxic) and in vivo conditions (Acute and Sub-acute toxicity). Results of Salmonella/microsome assay shows that MERM produced neither frame shift mutation in TA98 and TA1538 nor base pair substitution in TA100 and TA1535.  Lack of mutagenic effect of MERM at concentrations up to 4000 µg/plate in all the tester strains provides reassurance on the safety of the extracts widely used in traditional medicine. In addition MERM exhibited antimutagenic effect against direct acting mutagens (Sodium azide) in both TA98 and TA 100 tester strains. Investigation of cytotoxic effect by trypan blue exclusion assay and MTT assay showed that MERM had no cytotoxic effect on PBMC up to 24 hrs. Evaluation of genotoxicity (comet assay) revealed that MERM (2000 µg/ml) showed no significant difference in comet tail moment and tail scores which concludes that MERM has no genotoxic effect. In vitro hemolytic activity showed that MERM showed no significant (p  0.05) hemolytic effect even at the maximum dose (2000 µg/ml), which indicates that leaf extract preserves the integrity of cell membranes. In acute toxicity studies oral administration of MERM (250-2000 mg/kg) in Wistar rats produced neither mortality nor any noticeable changes in behavior. Hematological and biochemical parameters showed no difference, except for a significant increase in ALT and AST at the highest dose. Histopathological findings revealed hepatotoxicity and neurotoxicity at highest dose of extract. In sub-acute toxicity studies administration of MERM (1000 mg/kg) for 28 consecutive days neither altered the body weight gain nor behavioral parameters. No significant change was observed in the hematological and biochemical parameters analyzed. Histopathological examination showed normal architecture suggesting no morphological disturbances. Collectively, these data demonstrate that consumption of MERM for various medicinal purposes is safe.
	Chapter IV “Neuroprotective effect of Rhizophora mucronata against beta-amyloid-induced cell death in PC12 cells via its antioxidant mechanism” deals with various mechanism by which MERM protects against A induced toxicity in PC12 cells. PC12 cells used for the current study are widely used for in vitro research on AD as these cells contain many membrane-bound and cytosolic molecules associated with neurons, and are electrically excitable and neurosecretory. In addition PC12 cells were found to be more sensitive to Aβ insult hence it has been used extensively to study Aβ induced cellular toxicity. A (25-35) treatment in PC12 cells (2  105 cells/ml) for 24 hrs at 20 M concentration reduced cell viability, altered the morphology,  induced ROS/RNS production, decreased mitochondrial membrane potential, deregulated intracellular calcium levels and caused macromolecular damage. MERM pretreatment prior to A (25-35) exposure significantly elevated cell viability, reduced A (25-35) induced apoptosis, decreased ROS/RNS production, stabilized mitochondrial membrane potential, attenuated caspase 3 activation, maintained intracellular calcium homeostasis and  inhibited protein and lipid oxidation.  MERM also restores the antioxidant status of the cell by maintaining the antioxidant enzymes (SOD, CAT). MERM maintains the intracellular Glutathione level by regulating glutathione metabolizing enzyme (GR, GPx and GST). MERM also inhibits both acetyl and butyryl cholinesterase activity. Our findings suggest that MERM, a multiple-target drug holds potential for neuroprotection and therefore, may be promising for the treatment of AD. This effect of MERM could be due to the active compounds present in it, which may increase the capacity of endogenous antioxidant defense and may modulate the cellular redox state.
	Animal models play a critical role in understanding the pathology and screening of therapeutics for AD.  Although no available model can meet all the full pathologic spectrum of AD disease, the injection of Aβ into the brain was shown to impair learning and memory, and elicit a degree of AD type neurodegeneration. Chapter V “Anti-amnesic and neuroprotective effect of methanolic leaf extract of Rhizophora mucronata against A (25-35) induced toxicity in Swiss Albino mice” deals with possible mechanism by which MERM combats A (25-35) induced toxicity under in vivo condition. Mice were fed orally with 200 and 400 mg/kg bw for 21 days followed by single intracerebroventricular (i.c.v.) injection of A (25-35) (10 g/mouse). Behavioral changes in the mice were evaluated using water maze, Y-maze and passive avoidance tests. The consumption of MERM significantly ameliorated the cognitive deficits caused by i.c.v. injection of A (25-35) by restoring spatial memory, improving short term memory (STM) and long term memory (LTM). MERM enhances cholinergic effect by inhibiting both AChE and BuChE, attenuating lipid peroxidation and protein oxidation, reducing ROS and RNS level by activating antioxidative defense system (SOD, CAT) and  restoring glutathione metabolizing enzyme systems (GPX, GR and GST). MERM inhibits BACE1 activity thereby reducing the formation of neuronal tangles as observed in histopathological analysis. MERM inhibits neuronal apoptosis by suppressing both caspase-3 and MAO-B activity. Taken together, the results conclude that exogenous administration of MERM provides neuroprotective effect against toxic effect of Aβ peptide in regions related to cognitive functions providing new insight for future therapeutic approaches of AD.
	Chapter VI “Bioassay-directed isolation of active compounds with neuroprotective effect from Rhizophora mucronata leaf extract” deals with isolation and identification of bioactive compound responsible for the neuroprotective effect. MERM was fractionated using silica column chromatography (60-120 mesh) with solvents from nonpolar to polar (benzene to water) in various ratios and the eluted fractions were freeze dried. The fractions were subjected to bioactive guided fractionation using in vitro antioxidant and cholinesterase inhibitory assays. Among the various fractions Ethyl acetate: Methanol (1:1) fraction (Fr 14) showed the highest antioxidant and cholinesterase inhibitory activity. TLC analysis of the active fraction using Vanillin-Sulphuric acid showed the presence of four pink and two yellow colored bands. Preliminary phytochemical analysis showed the presence of flavonoids, terpenoids and glycosides. TLC chromatogram sprayed with p-anisaldehyde sulphuric acid and 1% Alcoholic aluminium chloride showed purple colored bands in visible light and fluorescent yellow colored bands under UV 366 nm, confirming the presence of terpenoids and flavonoids. The active fraction 14 was further fractionated by HPLC using methanol : water by binary gradient method. Among the eight fractions eluted, fraction 3 showed the highest AChE and BuChE inhibitory activity with IC 50 value of 32.25  0.001 and 40  0.002 g/ml respectively. Enzyme kinetic studies showed that the active fraction exhibited competitive and mixed type of inhibition against AChE and BuChE. 
TLC chromatogram analysis of active fraction sprayed with alcoholic aluminum chloride showed single fluorescence band with Rf value of 0.6 which confirmed the presence of flavonoids. Structural elucidation using FTIR, UV scan, C13 and H1 NMR spectrum illustrated that the content present  in bioactive fraction 3 was (+) - catechin (C15H14O6) a flavones-3-ol with molecular weight of 290.27. The identity was further confirmed by comparison with authentic sample (Sigma Aldrich, Bangalore) using LC-MS. 
	To conclude, the present work represents a comprehensive study of multipotent neuroprotective role of Rhizophora mucronata and its constituent (+)-Catechin against AD. Overall results of in vitro and in vivo studies validated the hypothesis that exogenous administration of MERM exerts neuroprotective actions against toxic effects of Aβ peptides in regions related to cognitive functions providing new insight for future therapeutic approaches of AD. Results of safety evaluation illustrates that MERM used for the present study is devoid of toxic effect both under in vitro and in vivo conditions which assures the safe usage of traditional drug. Despite of its neuroprotective effect, the rich nutritive value indicates that leaves can be used as nutraceuticals.  Moreover this is the first report regarding the neuroprotective role of mangrove, hence the present work offers new insight for exploration of drugs from marine natural products for the treatment of AD. 























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Multipotent action of Rhizophora mucronata (red mangrove) as neuroprotective drug against Alzheimer's disease ? Evidence from preclinical studies

PARTE, Seema C

Abstract/Synopsis 
The research work in the present Thesis emphasizes the development of green catalytic routes for the 
synthesis of fine and commercially important chemicals using eco–friendly heterogeneous catalysts via 
hydroformylation, condensation and isomerization reactions. Chapters from second to fourth of this thesis are 
related to the development of multi-functional catalyst to reduce the multi–steps processes into a single pot for 
the synthesis of higher carbon chain length aldehydes/alcohols starting from low carbon number alkene as a 
reactant. For example, 2–ethylhexanal and 2–ethylhexanol, which are the commercially important 
intermediates for the synthesis of dioctylphthalate (DOP), are synthesized industrially from propylene in three 
steps process. The existing commercial processes for the manufacturing of C8 aldol derivatives have drawbacks 
like, i) being a multi–step process, ii) use of hazardous liquid base KOH or NaOH in stoichiometric amount for 
aldol condensation reaction, iii) involving post synthesis work–up in the separation of spent KOH or NaOH from 
product mixture, iv) lower selectivity of product, corrosion of reactors and storage vessels. Therefore, the main 
focus was on the replacement of liquid bases, which are being used in stoichiometric amount, by the eco–
friendly heterogeneous catalyst to carry out all three steps into a single pot by altering the reaction conditions. 
The multi-functional [HF/HT] catalyst system was synthesized by the impregnation (Chapter 2) and 
intercalation (Chapter 3 and 4) of rhodium complex [HF] onto/into the interlayer space of hydrotalcite [HT] and 
its multi–functional potential was evaluated for the single pot selective synthesis of C8 aldol derivatives 
(aldehydes/alcohols) from propylene. The Mg/Al ratio of [HT], amount of [HF] complex and [HT], and reaction 
temperature showed pronounced effect on the selectivity of C8 aldol derivatives. Aldol condensation 
temperature T2 played a significant role in the formation of 2–ethylhexanol in a single pot. As Mg/Al molar ratio 
and amount of [HT] increased, the selectivity of 2–ethylhexanal also increased due to the enhancement in the 
basicity of the catalyst. From kinetic experiments, it was observed that the rate of formation of 2–ethylhexanal 
is dependent on the rate of aldol condensation which is catalyzed by hydrotalcite present in the [HF/HT] 
catalyst.  
Other commercial examples are the synthesis of perfumery chemicals such as, jasminaldehyde by the 
condensation of heptanal with benzaldehyde; trans–anethole and trans–isoeugenol by double bond 
isomerization of methyl chavicol and eugenol, respectively, using liquid KOH or NaOH in stoichiometric 
amounts. With increased environmental awareness, replacement of stoichiometric technologies by atom 
efficient greener catalytic routes with low         E factor (kg of byproducts generated per kg of desired product) 
and substitution of toxic and/or hazardous solvents/reagents with cleaner alternatives is today’s demand. 
Therefore, it is desirable to find solid base catalysts which could substitute liquid bases.  
The hydrotalcite samples of varied Mg/Al molar ratio were used as catalysts for synthesis of 2-
methypentenal by aldol condensation of propanal (Chapter 5) and jasminaldehyde in solvent free condensation 
of 1–heptanal with benzaldehyde (Chapter 6). 97% conversion of propanal with 99% selectivity of 2–
methylpentenal was achieved using activated hydrotalcite of Mg/Al molar ratio 3.5. From the kinetic data for 
aldol condensation of propanal using activated hydrotalcite (Mg/Al molar ratio 3.5), the initial rate of reaction 
was observed to increase on increasing the amount of catalyst. The activation energy for propanal condensation 
was calculated by Arrhenius plot and found to be 58 kJ/mol. The catalyst was recycled upto six cycles without 
loss in its activity. The selectivity of jasminaldehyde was observed to increase on increasing the M(II)/Al molar 
ratio of as–synthesized as well as activated hydrotalcite. The maximum selectivity of jasminaldehyde (86%) 
with 98% conversion of 1–heptanal was observed using as–synthesized Mg–Al hydrotalcite of Mg/Al molar ratio 
of 3.5 as a catalyst. The effect of activation of as–synthesized Mg–Al hydrotalcite samples of varied Mg/Al molar 
ratio on catalytic activity was studied and correlated with their basicity as determined from the model test 
reaction. The rate of reaction was calculated as 11.6 x 10–4 mol/(gcat min) at optimum reaction conditions and 
catalyst was reused several times for the synthesis of jasminaldehyde..  
The regioselective synthesis of trans–anethole from methyl chavicol via double bond isomerization of 
methyl chavicol using RuCl2(PPh3)3 and RuCl3(AsPh3)2.CH3OH complexes with detailed kinetics has been studied 
in Chapter 7. The incorporation of ruthenium in the hydrotalcite matrix was also carried out for double bond 
isomerization of perfumery related chemicals such as, methyl chavicol, eugenol, safrole, allylbenzene, 
dimethoxy allylbenzene, 3–carene. 98% conversion of methyl chavicol with 88% selectivity of trans–anethole 
was observed in 1 h reaction time. The effect of reaction temperature showed 41% conversion of methyl 
chavicol and 74% selectivity of trans–anethole at 100 °C which increased to 98% with 88% selectivity of trans–
anethole on increasing temperature to 210 °C using Ru–Mg–Al as a catalyst. The activity of Ru–Mg–Al was 
compared with the various ruthenium impregnated catalysts such as, Ru–HT, Ru–MgO, Ru–CaO, Ru–SiO2 and 
Ru–alumina. The Ru–Mg–Al catalyst was recycled upto fourth cycle without loss of its activity. 
 
 
 
 


DYNAMICS OF FOLLICLE GROWTH & PUTATIVE STEM-LIKE CELLS IN ADULT MAMMALIAN OVARY

K, Anbarasu

Abstract

Characterization of Aspergillus carbonarius mutant in relation to carotenoid production and its application

Fungi are a unique group of organisms, different from all others in their behavior and cellular organization. They have an enormous range of activities – as pathogens of crop plants or humans, as decomposer organisms, as experimental “model organisms” for investigating genetics and cell biology and as producers of many important metabolites. Thousands of secondary metabolites have been described from fungi. For example, the penicillins (from Penicillium chrysogenum), the cephalosporins (from Cephalosporium or Acremonium species), and griseofulvin (from P. griseofulvum) are antibiotics produced commercially from fungi. Some secondary metabolites are plant hormones, like gibberellins produced by the parasitic fungus Gibberella fujikuroi. Secondary metabolites are marketed as pharmaceuticals, including ciclosporin A which suppresses organ rejection in transplant surgery. Further examples include the mycotoxins, such as the aflatoxins produced by Aspergillus flavus and A. parasiticus and the ergot alkaloids produced by Claviceps purpurea and related species. 

Many fungi have the potential to synthesize pigments as secondary metabolites. Most fungal pigments are melanins and are associated with sporulation structures.  These compounds help to protect cells from damage caused by reactive oxygen species such as hydrogen peroxide and superoxide. They are generally dark brown pigments formed by oxidative polymerization of phenolic compounds and are deposited in the cell wall. Species of Aspergillus glaucus, reported to produce pigments like erytroglaucin (red), auroglaucin (orange) and flavoglaucin (yellow). Species of Aspergillus tawarii and Aspergillus sydowii are well known for the production of Xanthomegnin (yellow) and viomellein (Yellow) pigments. Some filamentous fungi such as Phycomyces blakesleeanus, Mucor circinelloides, and Blakeslea trispora possess the ability to synthesise carotenoid pigments. Many microorganisms were exploited for the production of carotenoids such as Blakeslea trispora for β-carotene, Rhodosporidium for γ-carotene, Mycobacterium lacticola & Phaffia rhodozyma for astaxanthin, Brevibacterium for canthaxanthin and Monascus purpureus and Streptomyces coelicolor for polyketides,. Canthaxanthin has been used as food and feed additive for eggyolk, fish and crustacean farms, in cosmetics and as an orally administered pigmenting agent for human skin in pharmaceutical applications. It plays important roles in animal displays of maturity and in the protection of tissues against oxidizing free radicals.

In this laboratory a mutant Aspergillus carbonarius isolated for polygalacturonase over production in shake flasks secreted copious quantities of the enzyme when the growth medium acidified to low pH. During growth under these conditions, the mutant fungus showed features different from that of the wild type. Importantly, it synthesized a yellow pigment, partially saturated canthaxanthin (PSC) which found pharmaceutical applications. This thesis describes studies on the mutant A. carbonarius.

The characterization of the mutant fungus and the possible mechanism involved in its ability to grow in shake flasks at extreme acidic conditions are described. Though the mutant was morphologically distinct from the wild type due to yellow pigmented mycelia, RAPD analysis confirmed its identity as the variant of the wild type A. carbonarius. The pigment (partially saturated canthaxanthin) production and deposition in the mycelia membrane caused a permeability barrier to H+ ions for intracellular pH homeostasis. Thus the mutant was able to grow in extremely acidic conditions and secrete the enzyme under such condition. Probable synthetic pathway of the pigment suggested that the carotenoid originated from the mevalonate pathway. Some other intermediates, not phytoene, were apparently involved in the pigment synthesis. Occurrence of other cyclases, instead of lycopene cyclase, involved in the cyclization reactions to cause pigment synthesis. Results also suggested the possible occurrence of mutation in the isoprenoid pathway for higher production of carotenoid as well as ergosterol. Increased ergosterol concentration with decreased PSC concentration during growth in presence of diphenylamine and vice versa, when grown in the medium containing nicotine, revealed a flow of carbon either for sterol biosynthesis or for carotenoids. 

	For studies on the absorption and bioavailability of A. carbonarius carotenoid, PSC was fed to Wistar rats. These results showing increased ascorbic acid and decreased lipid peroxides in liver suggested the importance of PSC as a dietary compound. The reduction in total cholesterol in the rats fed with PSC was apparently due to decreased serum LDL cholesterol. Dietary PSC in rats showed elevated levels of catalase and glutathione transferase apparently activating endogenous antioxidant defense mechanism. Dietary intake of PSC even at 0.25%, did not result in any treatment related adverse effects in Wistar rats. Thus its possible use in food and therapeutical applications appear feasibility. Upon oral administration, the occurrence of maximum concentration of PSC in the plasma of rats within short period of 3 – 6 h, suggested its consumption to exert health benefits. 

Based on the results above, potential food applications of the pigment, as nutraceutical, were explored.  Preparation of spray dried product with antioxidant property and shelf life are explained. The spray drying of PSC resulted in a free flowing, dry yellow to light brown coloured powder and showed greater storage stability and improved antioxidant property.




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