Open Access Repository of Indian Theses

H.P., Mohan-Kumari

ABSTRACT

Aminoglycoside antibiotics are valuable therapeutic agents and amongst the oldest discovered antibiotics. Gentamicin is a complex of numerous deoxysteptamine antibiotics used in the treatment of several gram-negative bacterial infections. The present study has been undertaken to improve the gentamicin yields from a Micromonospora echinospora MTCC 708 using different approaches like medium optimization, immobilization, and strain improvement followed by fluidized bed and stirred tank reactor studies for the mutant strain. Based on the optimization studies, a low-cost medium was formulated. The highest gentamicin production recorded in this optimized medium on cultivation for 120 h was 445 mg l-1 (1.2 fold increase) as compared to the basal medium. Further, response surface methodology (RSM) for the optimization of medium components for the production of gentamicin was applied. The R2 value of the model was 0.9723, which indicated that the model was best fit for the present studies. The optimized medium formulated by RSM had a composition (g l-1): Starch, 9; soyabean meal, 3; K2HPO4, 0.9 and CoCl2, 0.01 produced 880 mg l-1 of gentamicin which was about 110% higher than that of the un-optimized medium. 

	Gentamicin production by immobilized cells of M. echinospora has been studied in repeated-batch process using different immobilizing carriers such as Ca-alginate, carrageenan, agar, PVA, celite, polyurethane foam and activated carbon. With Ca-alginate, gentamicin production was maximum (1096 mg l-1) and the bead stability was good, whereby alginate was selected for further experimentation. The role of alginate concentration, CaCl2 concentration and bead diameter, curing time and bead inoculum quantity were studied. Better gentamicin outputs (1549 mg l-1) were attained with alginate 4% (w/v), CaCl2 0.2M, bead diameter 2.69mm, curing time 18h and inoculum quantity 20% v/v for six repeated cycles in shake flask. Under the optimal immobilization conditions, M. echinospora could afford 1549 mg l-1 and 1938 mg l-1 of gentamicin in shake flask and fluidized bed reactor respectively. Productivity of gentamicin was increased to 1.8 fold in fluidized bed reactor (FBR) using optimized alginate immobilized beads with increased reusability. The FBR was operated for 30 days in fed batch mode of operation.  

M. echinospora was treated with chemical mutagens like EtBr and MNNG and physical mutagens such as UV was carried out to obtain a mutant with enhanced production of gentamicin. After inducing mutations screening for penicillin and kanamicin resistant mutants was done. M. echinospora EtBr-22 (Me-22) strain was obtained by mutations and its gentamicin production in shake flask reaches 1354 mg l-1 which is 1.53 fold higher than that of the parent strain. Application of different stress conditions like heat shock, feeding high ethanol and high NaCl concentrations during fermentation has found to be effective for the increased production of gentamicin. Production of gentamicin was increased to 1.26 fold in medium supplemented with 0.6% NaCl to 48h old culture. 

	Further optimization studies includes effect of production medium components, initial starch and soyabean meal concentrations, for the enhanced production of gentamicin by Micromonospora echinospora (Me-22) was studied in a lab scale stirred tank reactor. Also effect of different aeration (0.5, 1, 2, and 4 vvm) and agitation rates (100, 200, 300 and 400 rpm) in stirred tank reactor were examined. A maximum gentamicin concentration of 2.68 g l-1 was achieved in the medium having low concentrations of initial starch (7.5 g l-1) and high concentrations of initial soyabean meal (4g l-1). Both aeration and agitation significantly affected gentamicin concentration, productivity and biomass formation. The highest gentamicin concentration of 4.12 g l-1 and the highest yield of gentamicin with respect to substrate 0.967 g g​-1 were obtained at low impeller speed of 200 rpm and moderate aeration rate of 2 vvm. Under optimal culture conditions in STR, the production of gentamicin has been increased to 3 fold when compared with shake flask.




English

Monascus purpureus in relation to statin and sterol production and mutational analysis

Sharma, Phani Krishna M.V.

Synopsis


SYNOPSIS
The thesis entitled “Towards the Synthesis of Biologically Active Molecules: Zanamivir, Pinellic acid and (4R)–Dodecanolide” has been divided into four chapters.

Chapter-I: This chapter has been further divided into two sections.
Section A: This section deals with the introduction and previous synthetic approaches of Zanamivir. 
Section B: This section deals with the present work of Zanamivir. 
Chapter-II: This chapter has been divided into two sections.
Section A: This section deals with the introduction, previous synthetic approaches of Pinellic acid
Section B: This section deals with the present work of Pinellic acid. 
Chapter-III: This chapter has been divided into two sections.
Section A: This section deals with the introduction and previous synthetic approaches of (4R)-Dodecanolide.
Section B: This section deals with the present work of (4R)-Dodecanolide.
Chapter-IV: This chapter has been divided into two sections.
Section A: This sections deals with the brief review on BiCl3 and Imino Diels-Alder reactions. 
Section B: Stereoselective synthesis of octahydro-3b H-[1, 3] dioxolo [4'', 5'': 4', 5'] furo [2', 3': 5, 6] pyrano [4,3-b]- quinolines via intramolecular hetero-Diels-Alder reactions catalyzed by Bismuth (III) Chloride.

Chapter I: Towards the synthesis of Zanamivir
	Influenza viruses are the most serious viral cause of respiratory illness both in terms of morbidity and mortality, and influenza is a disease of immense economic importance. In 1918-1919 influenza is thought to have been responsible for the deaths of 20 million people. Influenza is also called the flu. It's an infection that causes fever (​http:​/​​/​kidshealth.org​/​kid​/​ill_injure​/​sick​/​fever.html​), chills, cough, body aches, headaches (​http:​/​​/​kidshealth.org​/​kid​/​ill_injure​/​sick​/​headache.html​), and sometimes earaches (​http:​/​​/​kidshealth.org​/​kid​/​ill_injure​/​sick​/​ear_infection.html​) or sinus problems (​http:​/​​/​kidshealth.org​/​kid​/​ill_injure​/​sick​/​sinuses.html​). The flu is caused by the influenza virus (​http:​/​​/​kidshealth.org​/​kid​/​talk​/​qa​/​germs.html​) A.
	



Zanamivir 1 inactivates viral influenza neuraminidase,1 an enzyme responsible for cleaving sialic acid residues on newly formed virions as they bud off from the host cell. This inhibition results in aggregation of virions on the surface of the host cell, which limits the extent of infection and speeds recovery from illness. Clinical studies have shown that neuraminidase inhibitors can decrease the median duration of influenza related symptoms by  1 day if initiated within 48 hours of the onset of symptoms of influenza. This section deals with the biological significance; clinical applications and gives brief account of the work carried out by the various research groups reported the total synthesis of Zanamivir.
Our synthetic strategy is based on the inexpensive starting material D-Sorbitol which has three hydroxyl groups having same configuration as in side chain of the target molecule and permits characterization of the intermediates with simple techniques such as NMR, IR and Mass etc.

Retrosynthetic strategy for Zanamivir
The synthesis of Zanamivir requires introduction of 2, 3 double bond and in particular a stereospecific introduction of nitrogen based substituents at C-4 and C-5 positions. The synthetic strategy that we adopted towards the total synthesis of Zanamivir started from inexpensive commercially available D-Sorbitol 7 as indicated in the following retro synthesis (Fig 2).












Fig 2
Present work
D-Sorbitol 7 was converted to chiral propargyl alcohol 17 by the following sequence of reactions in a known route.2 Thus, D-Sorbitol 7 was treated with 37% formaldehyde solution in presence of conc. HCl to get tri methylene protected compound 8 in 68% yield (Scheme 1). The selective cleavage of 1, 3 and 5, 6-O-methylene groups of compound 8 was achieved with a mixture of acetic acid, acetic anhydride and conc. H2SO4 to get the tetra acetate 9 in 52% yield. The tetra acetate 9 was deacetylated with 0.2 N sodium methoxide in chloroform to afford the tetrol 10 in 60% yield. 


























Scheme 1
The tetrol 10 was converted to diacetonide 11 in 62% yield by using 2, 2-dimethoxy propane in presence of catalytic amount of PTSA in dry acetonitrile. The selective deprotection of 1, 3 -O-isopropylidene ring of 11 was achieved by using 30% acetic acid in methanol to provide the diol 12 in 60% yield. The primary hydroxyl group of compound 12 was protected as its silyl ether by using TBDMSCl and imidazole in dry DCM at room temperature to get compound 13 in 90% yield. The secondary hydroxyl group of compound 13 was protected as its PMB ether by using PMBBr and NaH in dry THF at room temperature for 4 h to afford the compound 14 in 85% yield. Compound 14 was subjected to deprotection of silyl group by using 1 eq of TBAF (1 M in THF) at 0oC to room temperature for 2 h to give alcohol 15 in 90% yield. The six membered 1,3-dioxolane alcohol 15 was converted to corresponding six membered 1, 3-dioxolane chloride 16 in 85% yield by using TPP and NaHCO3 in dry CCl4 on refluxing for 4 h. The chloro compound 16 was converted to 17 in 76% yield by using lithium metal and catalytic amount of ferric nitrate in liquid NH3.3
















Scheme 2
The compound 17 was protected as its methoxy methyl ether 6 by using iPr2EtN, and MOMCl in dry DCM at room temperature for 20 h in 80% yield4 (Scheme 2). The ring opening of the epoxide 6a with alkyne 6 using n-BuLi (1.6 M, in hexane) and BF3.Et2O (Yamaguchi’s method) in dry THF at -78oC to gave the coupled product 5 in 70% yield.5 Controlled hydrogenation of the alkyne 5 to its cis alkene compound 18 in 90% yield, was achieved by using Pd/CaCO3 and quinoline (Lindlar’s catalyst) in EtOAc under H2 gas. 
 Oxidation of compound 18 by using Dess-Martin Periodinane in dry DCM for 30 min at room temperature afforded the compound 19 in 90% yield.6 The cyclized compound 20 was obtained by the deprotection of p-methoxy benzyl group in compound 19 by using DDQ, silica and DCM:H2O (9:1) in 70% yield.7 The cyclic compound 20 when treated with dry MeOH and catalytic amount of PPTS in dry DCM at room temperature for 6 h afforded 4 in 70% yield (Scheme 2). 


















Scheme 3
The compound 4 was converted to bromohydrin 21 (Dalton and Dutta method) as a single isomer by treating with NBS, water in DMSO at –10oC in 70% yield8 (Scheme 3). The base mediated cyclization of bromohydrin 21 using K2CO3 in MeOH at room temperature for 4 h furnished the epoxide 22 in 80% yield (Scheme 3). The epoxide 22 when opened by azide functionality using NaN3 and aq DMF at 125oC for 2 h afforded the mixture of regioisomers of azido alcohols 23 and 24 in 70% yield 9(Scheme 3). Generally nucleophile attacks from less hindered side, but in this case azide ion attacked abnormally and resulted in 70% yield of 23 and in 30% yield of 24. The secondary hydroxyl groups of azido alcohols 23 and 24 were converted as their mesylates 25 and 26 respectively in 90% yield using MeSO2Cl and TEA in dry DCM at 0oC. The stereochemistry of azide and –OMs groups were characterized by nOe studies. Mesylazide compounds 25 and 26 were treated with TPP and iPr2EtN in aq THF at reflux temperature to give aziridine 27 in 85% yield. The ring opening of the aziridine 27 was demonstrated with sodium azide and aq DMF at 125oC. After purification the compound was acetylated with acetic anhydride, Et3N and catalytic amount of DMAP in dry DCM to get 3 in 70% yield (Scheme 3). The ring opening of aziridine 27, unlike epoxide 22 as indicated by nOe studies underwent nuceleophile attack from less hindered side. Nucleophilic attack from more hindered side would have resulted in desired stereochemistry to complete the synthesis of Zanamivir effectively, efforts are still on for the total synthesis of Zanamivir. 

Chapter II: Total synthesis of Pinellic acid 
Pinellic acid10 (28) isolated from an oriental medicine, pinellae tuber, is a novel and potentially useful oral adjuvant when used in conjunction with intranasal inoculation of influenza HA vaccines 



Pinellic acid 
Fig 3

	Among the C-9-isomer of Pinellic acid, 9S-compound showed much stronger activity compared with 9R-compounds. Thus stereochemistry at the C-9 hydroxyl group is very important for adjuvant activity. Among the 9S-derivatives, the adjuvant activities of 13S-compounds are stronger than that of 13R-compounds. However, the stereochemistry of the C-12 hydroxyl group is not important for adjuvant activity.
Retrosynthetic strategy for Pinellic acid
	We chose a strategy for the synthesis of Pinellic acid 28 by disconnecting the carbon backbone at C11-C12, thus dividing the target into two key fragments 30 and 31. Both C1-C11 fragment 30 and C12-C18 fragment 31 possess a common precursor 33, which with respective alkyl halides results in corresponding compounds (Fig 4).











Fig 4
Construction of the C12-C18 fragment 
Initially propargyl alcohol 33 was treated with 1-bromo pentane 34 in liquid ammonia using lithium amide at –33oC to give compound 35 in 65% yield.11 Acetylenic alcohol 35 was reduced with LiAlH4 in refluxing dry THF to afford the trans -allylic alcohol 36 in 90% yield. The E-allyl alcohol 36 was subjected to Sharpless asymmetric epoxidation protocol. Accordingly, treatment of 36 with L (+) DET, Ti (OiPr)4 and TBHP (3.3 M in toluene) in dry DCM at -24oC afforded the epoxy alcohol 37 in 80% yield.12 The epoxy alcohol 37 was converted to corresponding epoxy iodide 38 in 80% yield using triphenylphosphine, iodine and imidazole in a mixture of dry ether and acetonitrile in 3:1 ratio at room temperature (Scheme 4).













Scheme 4
Compound 38 were converted into a secondary allylic alcohol 39 in 80% yield by refluxing with activated zinc and sodium iodide in dry methanol.13 The allylic compound 39 was converted into methoxy methyl ether 40 in 90% yield using Hunig’s base iPr2NEt and MOMCl for 2 h at room temperature. The compound 40 was subjected to ozonolysis to give the required aldehyde 31 in 80% yield (Scheme 4). 

Construction of C1-C11 fragment
This fragment was synthesized by following a known route. Propargyl alcohol 33 was treated with bromide 32 in lithium amide at liquid ammonia temperature to give compound 41 in 70% yield. Acetylenic alcohol 41 was reduced with LiAlH4 in refluxing dry THF to afford the trans-allylic alcohol 42 in 80% yield. The E-allyl alcohol 42 was subjected to Sharpless asymmetric epoxidation protocol. Accordingly, treatment of compound 42 with L (+) DET, Ti(OiPr)4 and TBHP (3.3 M in toluene) in dry DCM at -24oC afforded the epoxy alcohol 43 in 80% yield. The epoxy alcohol 43 was converted to corresponding epoxy chloride 44 in 90% yield by using TPP and NaHCO3 in dry CCl4 on refluxing for 4 h. Accordingly compound 44 was subjected to lithium amide in liquid ammonia to get the chiral propargyl alcohol 45. The secondary hydroxyl functionality of compound 45 was protected as its methoxy methyl ether using Hunig’s (iPr2EtN) base and MOMCl in dry DCM at room temperature to afford the compound 30 in 90% yield (Scheme 5).








Scheme 5


Coupling of C1-C11 and C12-C18 fragments
 Compound 31 was treated with n-BuLi, HMPA and coupled with aldehyde 31 at -78oC to get compound 29 (20:1) in 75% yield. The coupled compound 29 was treated with LAH to afford trans-allyl alcohol 46 in 80% yield. 





























Scheme 6

The secondary hydroxyl functionality of compound 46 was protected as its methoxy methyl ether using Hunig’s (iPr2EtN) base and MOMCl in dry DCM at room temperature to afford the compound 47 in 90% yield. THP deprotection of 47 was carried out by using PPTS in methanol to afford the compound 48 in 80% yield.14 The compound 48 was oxidized with DMP in DCM to afford the aldehyde 49 in 85% yield (Scheme 6). The compound 49 was further oxidized with NaClO2 and NaH2PO4 in DCM to afford the corresponding acid 50 in 79% yield.15 Finally deprotection of three MOM groups by using 6 N HCl to afforded the target molecule 28 in 70% yield (Scheme 6).

Chapter III: Total synthesis of (4R)-Dodecanolide
Lactonic functionality is present in a large number of natural products and biologically active compounds. Lactone derivatives are very common flavor components16 used in the perfume industry. They have also been reported to be sex attractant pheromones of different insects and to be plant growth regulators and they are also useful intermediates in the synthesis of natural products.



Fig 5
 	(4R)-Dodecanolide is a pheromone component, which was isolated from the pygidal glands of rove beetles Bledius mandibularies and Bledius spectabilis by Wheeler et al. 
Retrosynthetic route of (4R)-Dodecanolide
The synthetic strategy adopted for the synthesis of (4R)–Dodecanolide is outlined below (Fig 6).




Fig 6
Present work
Initially propargyl alcohol 33 was treated with 1-bromooctane 55 in liquid ammonia using lithium amide at -33oC to give compound 56 in 75% yield. 















Scheme 7
Acetylenic alcohol 56 was reduced with LiAlH4 in refluxing dry THF to afford the trans-allylic alcohol 57 in 85% yield The E- allyl alcohol 57 was subjected to the Sharpless asymmetric epoxidation protocol. Accordingly, treatment of compound 57 with D (-) DET, 0.2 eq of Ti (OiPr)4 and TBHP (3.3 M in toluene) in dry DCM at -24oC afforded the epoxy alcohol 58 in 85% yield. The epoxy alcohol 58 was converted to corresponding epoxy chloride 59 in 90% yield by using TPP and NaHCO3, in dry CCl4 on refluxing for 4 h. Accordingly, compound 59 was subjected to LiNH2 in liquid ammonia to get the chiral propargyl alcohol 60 in 76% yield (Scheme 7). The secondary hydroxyl functionality of 60 was protected as its methoxy methyl ether using Hunig’s base (iPr2EtN) and MOMCl in dry DCM at room temperature to afford 53 in 90% yield (Scheme 7). The acetylene 53 was subjected to methoxy carbonylation in presence of 1 eq of n-BuLi (1.6 M in Hexane) and 1 eq of 54 at -78oC to give the compound 61 in 90% yield.17 The ester 61 was reduced with 10% Pd/C under hydrogen atmosphere to get the saturated ester 52 in 90% yield (Scheme 7). Finally the Cyclization of 2 with PTSA in methanol afforded 51 in 85% yield (Scheme 7). 

Chapter IV: BiCl3 Catalyzed Intramolecular imino Diels-Alder reactions.
Stereoselective synthesis of octahydro-3b H-[1, 3] dioxolo [4'', 5'': 4', 5'] furo [2', 3': 5, 6] pyrano [4,3-b]- quinolines via intramolecular hetero-Diels-Alder reactions catalyzed by Bismuth (III) Chloride.
The chemistry of tetrahydroquinoline derivatives has been long area of intense for organic chemists due to presence of these scaffolds within the framework of numerous biologically interesting natural products and pharmaceutical agents. The most attractive strategy for these derivatives is the acid-promoted imino-Diels-Alder reaction between N-arylaldimines and electron rich alkenes. 
Carbohydrates have been used as chiral auxiliaries or chiral building blocks and used in asymmetric transformations due to their known absolute stereochemistry, readily availability and often their low cost.18 In continuation of our interest on intramolecular hetero-Diels-Alder reactions, we report herein BiCl3 catalyzed highly efficient and stereoselective synthesis of 2,2,6,6-tetramethyl3a,5,5a,6,11,11a,11b,12a-octahydro-3bH 1,3]dioxolo[4'',5'':4',5']furo[2',3':5,6] pyrano[4,3-b]quinolines using [4+2] cycloaddition reactions of anilines with the O-allyl derivative of the sugar derived  aldehyde19 63 from D-glucose (Scheme 8).













Scheme 8
The reaction of aniline 62a with the O-allyl derivative of the aldehyde 63 in acetonitrile in the presence of 10 mol% BiCl3 furnished a diastereomeric mixture of trans and cis products 64 and 65 in 93:7 ratio in 81% yield. The reaction was very rapid and complete in 30 minutes as indicated by TLC. The structure of compound confirmed by inter proton-coupling constants and characteristic nOe’s were used to obtain the proposed structures. These structures were further supported by minimum energy calculations.23 Under similar conditions, several aromatic amines 62a-i were treated with the O-allyl derivative 63 to illustrate the novelty of the present strategy and the results are presented in the Table 2. In all cases the reactions are highly stereoselective, leading to the formation of trans isomers as the major products and cis isomers as minor products. When a bulky group such as tert-butyl is presenting to ortho to the amine (entry 3) only one diastereomer was isolated.


























References:
1). Ryan, D. M,; Ticehurst, J,; Dempsey, M. H,; Penn, C. R. Antimicrob. Agents Chemother. 1994, 38, 2270. 
2). Hann, R. M.; Ness A. T.; Hudson, C. S. J. Am. Chem. Soc. 1944, 66, 73.
3). Yadav, J. S.; Madhavi, C.; Srinivas Rao, C. Tetrahedron Lett. 1989, 30, 5455.
4). Stork. G.; Takahashi. T. J. Am. Chem. Soc. 1977, 99, 1275.
5). Yamaguchi, M.; Hirao, I. Tetrahedron Lett. 1983, 24, 391.
6). Dess, D. B.; Martin, J. C. J. Org. Chem. 2004, 69, 2831.
7). Horita, K.; Yoshika, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu, O. Tetrahedron 1986, 42, 3021.
8). Hudlicky, T.; Tian, X.; Kinigsberger, K.; Mourya, R.; Rouden, J.; Fan, B. J. Am. Chem. Soc. 1996, 118, 10752.
9). Suzuki, M.; Kambe, M.; Tokuyama, H.: Fukuyama, T. J. Org. Chem. 2004, 69, 2831.
10). Sanazuka, T.; Shirahata, T.; Yoshida, K.; Yamamoto, D.; Harigaya, Y.; Nagai, T.; Kiyohara, H.; Yamada, H.; Omura, S. Tetrahedron Lett 2002, 43, 1265.
11). Yadav, J. S.; Geetha, V.; Raju, A. K., Gnaneshwar, D.; Chandrashekar, S. Tetrahedron Lett. 2003, 44, 2983.
12). Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5954.
13). Nicolaou, K. C.; Duggan, M. E.; Ladduwahetty, T. Tetrahedron Lett. 1984, 25, 2069.
14). Miyasita, M.; Yoshikoshi, A.; Grieco, P.A. J. Org. chem. 1977, 42 3772.
15). Mann, J.; Thomos, A. Tetrahedron Lett. 1981, 37, 2091.
16). a) Willhalm, B.; Palluy, E.; and Winter, M. Helv. Chem. Acta. 1966, 49, 66. b) Broderick. Am. Perfumer. Cosmet. 1966, 86, 43. c) Tang, C. S.; and Jennigs, W. G. J. Agr. Food. Chem. 1968, 16, 252.
17). Nakata, M.; Ohashi, Junji.; Ohsawa, K.; Nishimura, T.; Kinoshita, M.; Tatsuta, K. Bull. Chem. Soc. Jpn. 1983, 66, 3464.
18). Hultin, P. G., Earle, M. A.; Sudarshan, M. Tetrahedron 1997, 53, 14823.
19). Sharma, G. V. M.; Srinivas Reddy, I.; Goverdhan Reddy, V.; Rama Rao, A. V. Tetrahedron: Asymmetry 1999, 10, 229.




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Study and development of TIO2 - based photocatalysts for pesticide degradation

NAIK, SUSHANT SURESH

LATE QUATERNARY FLUCTUATIONS IN CARBONATE AND CARBONATE ION CONTENT IN THE NORTHERN INDIAN OCEAN

PARIMALAN, R.

PAGE  



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Synopsis


Diabetes is a disease of great concern to many all over the world and is characterized by its complications such as diabetic neuropathy, retinopathy and nephropathy. Diabetic nephropathy leading to kidney damage is one of the serious concerns and has aroused lot of research interest. During diabetic nephropathy changes in glomerular basement membrane (GBM) are known to take place making the glomeruli thicker. The structure and composition of GBM is of importance for its function as filtration barrier in the glomerulus. The glomerular filtration matrix, which is essential for normal filtration to take place is formed of heparan sulphate, type IV collagen and laminin. During diabetes thickening of GBM is known to take place due to reduction in heparan sulfate and laminin and accumulation of type IV collagen leading to abnormal filtration.  

Dietary management of diabetes is now well established to be one of the means in the management of diabetes. In this category, dietary fibers and foods rich in dietary fibers are well accepted to have beneficial functions in the management of diabetes. But there are not many scientific investigations on the role of these dietary fibers in minimizing complications of diabetes, such as diabetic nephropathy. In recent years, fermentation of dietary fibers to short chain fatty acids (SCFA), such as acetate, propionate and butyrate is being increasingly recognized to play beneficial functions in normal physiological condition and also in alleviating pathological conditions such as cancer, diabetes, etc. Our laboratory has shown that butyric acid ameliorates diabetic and diabetic nephropathy state. 
Among the foods that possess beneficial effects on diabetes, dietary fiber rich foods are one of the important classes.  Spent turmeric, a by-product of turmeric industry is one of the rich sources of dietary fibers. Hence,  one of the objectives was to study the effects of dietary fiber rich food, spent turmeric, a by-product of curcumin industry, on diabetic and diabetic nephropathy state with particular emphasis on glycoconjugate metabolism in glomerular basement membrane.   

The thesis contains four chapters. Chapter I; Introduction, II; Materials and Methods, III; Results and Discussion along with Summary and Conclusions; IV Common Bibliography. Results and Discussion of the thesis presented in Chapter II is divided into three subsections. Section 3.1 deals with studies on the role of spent turmeric on diabetic status in streptozotocin induced diabetic rats and summary and conclusions of this section are presented in 3.1.1. The section 3.2 is divided into two subsections. Section 3.2.a deals with studies on the role of Emblica officinalis on diabetic status in streptozotocin induced diabetic rats and section 3.2.b.  deals with chemical and biological studies on the polysaccharides of Emblica officinalis. Summary and conclusions of these two subsections (3.2.a and 3.2.b) are presented in section 3.2.1. The studies carried out on phenolic acids of Curcuma longa and Emblica officinalis is presented in section 3.3 and its summary and conclusions are presented in sections 3.3.1.

Chapter I: Consists of general introduction on diabetes, complications of diabetes such as diabetic nephropathy, nutritional management of diabetes and biological activity of polysaccharides are provided with proper literature citation. At the end, scope of the present study is given.

Chapter II: This chapter deals with Materials and Methods that are employed in this investigation. Colorimetric, spectroscopic, microscopic, analytical methods, isolation procedures, methods employed in structural analysis and histopathological methods are detailed.

Chapter III: In this chapter results obtained are discussed with appropriate literature citations. Section 3.1 deals with effect of spent turmeric in streptozotocin induced diabetic rats. Spent turmeric is used as a rich source of dietary fiber. Water intake, urine sugar, urine volume, fasting blood sugar were increased during diabetes in the starch fed diabetic group (SFD) and these were alleviated to different extents by feeding spent turmeric (TFD). Diet intake in the spent turmeric fed group was more and could be due to caloric dilution and palatability. Decreased body weight in the starch fed diabetic group was augmented by spent turmeric (TFD) feeding.

Activities of disaccharidases such as maltase, sucrase and lactase were increased in the intestine and decreased in the kidney during diabetes when compared to controls and these enzyme activities were alleviated in spent turmeric fed diabetic group.

Glomerular filtration rate (GFR) was tested in terms of creatinine clearance. Weight of the kidney and size of the glomeruli were increased in the starch fed diabetic group (SFD) and the feeding of spent turmeric ameliorated them.

Weights of various organs (expressed as g/100g body weight) such as liver, spleen, lungs, brain, heart and testis were increased to different extents during diabetes and was prevented by feeding spent turmeric in the diet. Effect of spent turmeric on glycoconjugate constituents in different tissues were analysed in terms of total sugar, uronic acid, amino sugar, sulfate and proteins and these were ameliorated to different extents by the feeding of spent turmeric. Not much effect was observed in the sulfate content in kidney. Protein content was decreased during diabetes and was prevented by the feeding of spent turmeric to some extent. 

Glycosaminoglycans (GAGs) were isolated from different organs and their content was decreased in the starch fed diabetic group (SFD) and was ameliorated by the feeding of spent turmeric in the diet. Constituents of GAGs such as total sugar, uronic acid, aminosugars and sulfate were decreased during diabetes and they were alleviated in the spent turmeric fed diabetic group (TFD).

Effect of spent turmeric on some of the enzymes of glycoconjugate metabolism like L-glutamine fructose-6-phosphate amino transferase (GFAT), N-acetyl--glucosamindase (NAG) and -glucuronidase in kidney were studied. The activities of GFAT, N-acetyl--glucosaminidase increased during diabetes and was ameliorated by the feeding of spent turmeric. There was no significant change in -glucuronidase activity during diabetes.

Since the major emphasis was to study changes in glomerular basement membrane during diabetic nephropathy state, an attempt was made to study in depth, the role of spent turmeric in minimizing the complications of diabetic nephropathy state with particular emphasis on glycoconjugate metabolism. Hence GAGs were isolated from the kidney and the contents of GAGs were analysed.  In the starch fed diabetic group (SFD) the content of GAGs was decreased and was ameliorated by feeding spent turmeric (TFD). Total sugar, uronic acid and amino sugar contents decreased during diabetes and spent turmeric feeding prevented the decreased contents. The decrease in sulfate content indicated decrease in sulfated GAGs and it was ameliorated by the feeding of spent turmeric diet. Further total GAGs were fractionated into heparan sulfate and chondroitin sulfate using chondroitinase ABC. Analysis of this fraction clearly indicated that heparan sulfate was the major GAG in GBM. Results indicated that during diabetes, heparan sulfate content decreased and the feeding of spent turmeric in the diet ameliorated it. This was further confirmed by agarose gel electrophoresis. The electrophoretic pattern revealed the presence of heparan sulfate as the predominant GAG and chondroitin sulfate was in minor amounts. The results clearly indicated a decrease in heparan sulfate content during diabetes and was alleviated in spent turmeric fed diabetic group.

Histopathological studies of kidney sections was carried-out by a combination of alcian blue and Periodic Acid Schiffs (PAS) staining. Alcian blue stains glycosaminoglycans, which is blue in color and was positive in control kidney sections. Trace intensity of pink color was observed due to PAS stain. PAS positive glycoproteins were elevated during diabetes and there was alleviation in spent turmeric fed diabetic group. The observed alterations due to Alcian blue-PAS stain during diabetes was alleviated by the feeding of spent turmeric during diabetes.

Immunohistochemsitry was done with primary antibody for type IV collagen and secondary antibody tagged with FITC. The results clearly indicated increased content of type IV collagen during diabetes, which was ameliorated during diabetes by the feeding of spent turmeric. The changes in type IV collagen were further confirmed by estimating the content of hydroxyproline and was estimated as a measure of type IV collagen. Our studies clearly showed that the content of type IV collagen increased during diabetes (SFD) and was modulated by the feeding of spent turmeric (TFD). This clearly depicted that spent turmeric has beneficial effect in countering diabetic nephropathy status that occurs in the glomerular basement membrane with particular emphasis on heparan sulfate and type IV collagen. Section 3.1.1 contains summary and conclusion of this section.

The other food material studied for the effect on diabetes and diabetic nephropathy status was Emblica officinalis. Many Ayurvedic preparations use Emblica officinalis in many of the ailments such as cancer, immunomodulatory disorders, atherosclerotic, including diabetes.

Section 3.2 is subdivided into “a” and “b” sub-sections. 3.2.a deals with Results and Discussion of the studies on the role of Emblica officinalis on diabetic status in streptozotocin induced diabetic rats. 

Diabetes was induced in rats using streptozotocin and the rats were grouped into starch fed control and diabetic (SFC/SFD), Emblica officinalis fed control and diabetic (EFC/EFD). The rats fed with Emblica officinalis at 1.0% level developed diarrhea with in 3 days and hence the experiment was terminated. The 0.5% Emblica officinalis fed diabetic group (SFD) showed increased diet intake and loss of body weight when compared to starch fed diabetic group (SFD). Urine sugar, urine volume, fasting blood glucose and glomerular filtration rate were increased in Emblica officinalis fed diabetic group (EFD) when compared to starch fed diabetic group (SFD) and hence Emblica officinalis did not shown beneficial effects at 0.5% concentration on diabetic and diabetic nephropathy status. At lower concentration of Emblica officinalis i.e. 0.1% and 0.025% levels also no significant alleviations in the diet intake and body weight were observed when compared to control and diabetic groups. No beneficial effects were observed in terms of urine sugar, urine volume, water intake, fasting blood glucose and glomerular filtration rate by the feeding of Emblica officinalis during diabetes when compared to starch fed diabetic group.

Sections 3.2.b deals with chemical and biological studies on the polysaccharides of Emblica officinalis. 

Since no beneficial effects were observed by Emblica officinalis at 1.0, 0.5, 0.1, and 0.025% concentrations on diabetic and diabetic nephropathy state, an attempt was made to study the polysaccharides present in Emblica officinalis, if they have any biological functions and then study chemical nature of such polysaccharides. Various polysaccharides were isolated from Emblica officinalis such as water soluble, pectic polysaccharide, hemicellulose A and hemicellulose B and were analyzed for the carbohydrate composition, antioxidant activity and wound healing activity. Pectic polysaccharide showed potential antioxidant activity and wound healing property.

The pectic polysaccharides isolated from Emblica officinalis was fractionated on DEAE-cellulose and the column was eluted with water, ammonium carbonate (0.1 to 0.5 M) and alkali (0.1,0.2 M) and checked for the antioxidant activity. The fraction eluted with 0.2 M ammonium carbonate showed good antioxidant activity. Carbohydrate composition of DEAE eluted fractions was studied. The 0.2 M ammonium carbonate eluted fraction had 37% of uronic acid and 51% was galactose and contained trace amounts of arabinose and xylose. Since, 0.2 M Ammonium Carbonate eluted Fractions (ACF) showed good antioxidant activity it was taken for detailed investigation. Homogeneity criteria and molecular weight determinations were done with cellulose acetate electrophoresis, HPLC and Sepharose CL-4B column chromatography. Purity and molecular weight were found to be 96% and 55 Kda, respectively. The purified pectic polysaccharide (ACF) was subjected to structural analysis using methylation anlysis, GC-MS, Smith degradation, periodate oxidation, formic acid release, optical rotation, FT-IR and NMR. By combination of these structural studies, the ACF having potential antioxidant activity was found to have galacturonic acid backbone in -1,4 linkages with branches through O-6. The branches had galactose, arabinose and rhamnose. Galactose was the terminal sugar. Section 3.2.1 deals with summary and conclusions of 3.2.a  and 3.2.b.

Section 3.3 deals with content of free and bound phenolics of Emblica officinalis and Curcuma longa and their antioxidant activity was studied using reducing power ability, free radical scavenging activity and DNA damage. Results clearly showed that phenolics of Emblica officinalis have better antioxidant activity when compared to phenolics of Curcuma longa.

Separation and identification of free and bound phenolics of Emblica officinalis and Curcuma longa was done using HPLC.

Section 3.3.1 deals with summary and conclusions of 3.3. 

Chapter IV: Finally the thesis concludes with a collective bibliography citation for all the chapters.



VIII








Micropropagation of Bixa orellana L. and regulation of annatto dye synthesis

DOMALAPALLY, SREENU

Chocolate, confectionery and bakery require special types of fats to impart desirable textural and other quality attributes. Though, cocoa butter is the ultimate fat used in chocolates  because of its unique physico-chemical characteristics, it has certain drawbacks like high cost, wide variation in properties depending on the origin,  requirement of careful processing conditions and softening in warm climates.  Due to this, alternatives came into existence.  In  the present study, cocoa butter extenders similar to cocoa butter and which have heat resistance property for use in tropical climates were prepared from mahua oil/kokum fat blends,  simarouba and sal fats by fractionation and blending techniques.

Plastic fats normally prepared by hydrogenation are used in bakery, and also as vanaspati for culinary purposes. These hydrogenated fats had considerable amounts of trans fatty acids, which are reported  to be the risk factors for coronary heart diseases. In the present study, trans free plastic fats were prepared from mango fat, mahua oil and palm oil by fractionation, blending and interesterification techniques. 


The word ‘fats’ refers to the lipid group, and is used to mean both fats and oils. They are generally, insoluble in water but soluble in organic solvents such as petroleum ether, diethyl ether, chloroform etc.  The lipids which are liquid at room temperature are traditionally referred to as ‘oils’ while those which are solid at room temperature are called ‘fats’. More than 95% of most of the oils and fats are composed of triacylglycerol (TAG) molecules.  TAGs, also called triglycerides (TGs) are formed  if all of the -OH groups of a glycerol molecule are esterified with fatty acid moieties.
The hydrocarbon chain length of the fatty acids ranges from 4 to 30 carbons – the most common being 12 to 24 in naturally occurring fats and oils.  The chain is typically linear and usually contains an even number of carbons in vegetable oils and fats.  However, in the presence of one or more double bonds, the chain becomes kinked. 
Oils and fats exhibit a wide range of physical properties which are influenced by the degree of unsaturation, the length of the carbon chain, the isomeric form of the fatty acids, the molecular configuration of the triglyceride and the polymorphic state of the fat (Dziezak 1989).  Fats consisting of highly saturated or long chain fatty acids will generally have a higher melting point than those possessing a high content of unsaturated or short chain fatty acids. Unsaturated fatty acids can have different isomeric forms which have different melting points.  For instance, a cis isomer oleic melts at 16C while the trans isomer elaidic acid has the melting point of 44C.  The type, quantity and distribution of fatty acids and the glycerol molecule also affect the properties of fats to a greater extent.  The number of double bonds greatly affect the fat properties as does the orientation of the isomer. For example, the melting point of a linolenic ester with three double bonds is -13C while the stearic ester without double bonds will melt at 70°C.  Clearly these wide swings in properties are of great interest to the modern food processor.
There are four basic types of triglycerides (TG) depending on the fatty acids present in them (Charley, 1982).
(i)	Saturated fatty acids are shaped like a straight line. TGs consisting of saturated fatty acids can easily align themselves in a close packing to form a compact mass.  For instance a saturated fat is one that stays hard at room temperature and is not easily oxidized. Palm kernel oil, cocoa butter, sal fat and coconut oil are good examples of the saturated fats. 
(ii)	Monounsaturated fatty acids have a single ‘kink’ in their molecular strand.  Unsaturated fatty acids on the central (sn-2) or on the terminal (sn-3) carbon atoms of the glycerol molecule interfere with the close packing of triacylglycerols. Such a disrupted packing of TAGs makes crystal formation more difficult, thereby lowering their melting point and increasing their susceptibility to oxidative deterioration. Olive and canola oils are rich in monounsaturated fatty acids. 
(iii)	Polyunsaturated fatty acids have two kinks and this makes the resulting TGs more reactive than the TGs consisting only of monounsaturated fatty acids. Safflower oil and corn oil are well known polyunsaturated oils.  Both require careful handling to prevent rancidity due to oxidation. 
(iv)	The super-unsaturated fatty acids are molecules with three kinks. These are found mostly in fish tissue and in the seeds of black currant, evening primrose and flax plants. These must be carefully protected to preserve their nutritional qualities. 
1.1 Functions of fats
	Fat constitutes one of the three major constituents of foods on line with carbohydrates and proteins. It is an important ingredient in many food products, contributing to their functional, nutritional and organoleptic properties.  They interact with other ingredients to develop characteristic mouthfeel and aroma of a food. Fats carry flavor, they impart richness to foods and give the impression that a food is moist. Fats and oils play an important role in human diet; some of their important functions are listed below (Boothby, 1983; Griffiths, 1985; Sreenivasan, 1978).
i)	They are essential nutrients in the human diet and are the most concentrated sources of energy. They supply about 9 Kcal/g as compared to about 4 Kcal/g from proteins and carbohydrates.
ii)	They serve as carriers for fat soluble vitamins like A, D, E and K and  also for the provitamin A, carotene.
iii)	They furnish the essential fatty acids without which the organism cannot thrive (e.g., linoleic, linolenic and arachidonic acids).
iv)	Besides their direct nutritional value, they have the virtue of making other foods more appetizing and palatable.
v)	They are indispensable in practical cooking and baking since many foods cannot be made fit to eat without fats. 
vi)	They also act as a heat transfer medium, allowing even heat transfer in deep and shallow fat frying.
vii)	Satiety values of food - fats give a feeling of satisfaction as they stick to our ribs. The length of time food stays in the stomach is responsible for giving a feeling of satiety.
viii)	Fat gives marbling effect to the texture of food. Though fats are hidden in most of the foods they are often essential to obtain foods having a desirable texture and eating qualities. 
Fats and oils play a vital role in the formulation and preparation of a variety of foods. Icings, mayonnaise, margarine, chocolate, confectionery coatings, imitation dairy coffee whiteners, toppings, cheeses and dips possess desirable qualities that are attributed to the fats they contain. Chocolate, confectionery and imitation dairy products require special type of fats for their formulation. These fats are replacements or substitutes for cocoa butter and butter fat in confectionery and imitation dairy products, respectively (Paulicka, 1976). They are hard and brittle at and below room temperature and melt completely or nearly completely at body temperature. They are known commonly as hard butters.
More than 50% of our normal fat intake is in the form of ‘invisible’ fat i.e as unseparated oils and fats in foods such as grains, nuts, dairy products, eggs, meat, etc. In such natural food products, which include vegetable oils and fats, the lipid content and composition are more or less fixed, with minor variations depending on the season. Therefore, the nutritional and functional properties are also fixed and are not suitable for use in many application to impart desirable quality attributes.  
Specialty fats are tailored fat systems whose nutritional and functional properties have been manipulated in order to deliver specific consumer needs. These modified fats provide special functional utility in baking, confectionery, and cooking applications.  They have to satisfy a host of physical functionality and health/nutritional requirements. There are various types of speciality fats such as:
i)	Cocoa butter alternatives
ii)	Margarines and shortenings for bakery applications
iii)	Filling fats
iv)	Speciality fats in ice-cream production
v)	Oils and fats in deep frying
Oils & fats in infant nutrition



STEROSELECTIVE TOTAL SYNTHESI OF FUNGAL METABOLITE HERBARUMIN-I, PLANT NATURAL PRODUCT LEIOCARPIN-A AND DEVELOPMENT OF SYNTHETIC METHODOLOGIES

ARORA, NEELIMA

PRIORITIZATION OF MALARIA ENDEMIC ZONES IN ARUNACHAL PRADESH THROUGH INFORMATION TECHNOLOGY TOOLS

Y., VIJAYA BHASKAR

 i
A  B  S  T  R  A  C  T 
 
 
Dharwar craton of southern Peninsular India has well preserved volcano-
sedimentary assemblages presented in different greenstone belts which range in age from 
3.4 – 2.7 Ga.  The western and eastern sectors of Dharwar craton have been divided by 
Closepet Granite.  The western Dharwar craton (WDC) is dominated by komatiite-
tholeiite sequences emplaced by plume magmatism along with sedimentary rocks formed 
at different tectonic settings whereas the eastern Dharwar craton (EDC) has 
predominance of tholeiite-bimodal sequences with limited occurrence of sedimentary 
rocks.  Most of the greenstone belts present in EDC are of Neoarchaean in age containing 
a great compositional diversity of subduction related volcanic rocks such as boninites, 
basalt-andesite-dacite-rhyolite (BADR) and adakites. 
 Gadwal greenstone belt of eastern Dharwar Craton is a linear belt running 
discontinuously from Narayanpet in the North to Veldurti in the South. It disappears 
below the Proterozoic Cuddapah Basin and reappears near Veldurti in the south.  The belt 
is ~85 km long and ~1 - 15 km wide.  Apart from different events of volcanic activity 
within this greenstone belt, the emplacement of kimberlites in Siddampalli area of 
Gadwal belt has been dated at 1094 + 8 Ma, which appears to be coeval with the 
emplacement of other kimberlite pipes in the Eastern Dharwar Craton (EDC). The EDC 
has got certain unique characteristics that are not found in Western Dharwar Craton 
(WDC).  The seismic tomographic data have shown relatively a lower crustal thickness in 
the EDC (~35 km) in comparision to the WDC (~55 km), which differs with the gravity 
and magnetic data of Ramadass et al. (2006; average crustal thickness of WDC= 39 km; 
EDC= 37 km).  The geological setting and lithologies of the greenstone belts in both the 
 ii
above sectors of Dharwar Craton in the light of their comparative crustal thickness, 
appear to reflect  the geodynamic setting of these two parts of Dharwar Craton. 
 Gadwal belt is made up of metabasalts along with felsic, intermediate volcanic 
rocks, thin band of pyroxenites and minor banded iron formations. This belt has been 
intruded by granitoids of different phases which have not been studied and dated as yet.   
Moyen et al. (2003) have identified four types of granitoids like 1) TTG, 2) Sanukitoid, 
3) Biotite granite and 4) Closepet granite (high-Mg, high-HFSE type) in Eastern Dharwar 
Craton.  The rocks of this belt have been subjected to three phases of deformation (DF1 – 
DF3) and metamorphosed to amphibolite facies along the margins and lower greenschist 
facies in the interior parts (Matin, 2001). 
 Geochemical studies on metavolcanic rocks of the Gadwal greenstone belt 
(GGB), Eastern Dharwar Craton, have documented several rock types that are indicative 
of subduction zone tectonics reflecting on the crustal growth processes in the Dharwar 
craton. The dominance of komatiites in the western Dharwar Craton and the arc volcanics 
in the Eastern Dharwar Craton is an indication of dominance of plume magmatism in the 
WDC and intraoceanic subduction zone processes in EDC which together played a 
significant role in the growth and evolution of continental crust in the Dharwar craton.  
Boninites of GGB are high calcic type with high MgO (20-23 wt %) and the 
characteristic MREE depleted U-shaped REE patterns whereas generally the basalts have 
flat REE patterns with no Eu anomalies, with a few exhibiting transitional characteristics 
having variably enriched LILE and LREE patterns.  Nb-enriched basalts exhibit slightly 
fractionated REE patterns with high Nb (8-26 ppm) content compared to the arc basalts.  
Andesite-Dacite-(Rhyodacite) Rhyolite of GGB are medium to high-K calc-alkaline 
 iii
rocks, enriched in LILE and LREE, exhibiting highly fractionated REE patterns and 
elevated total REE contents with progressing fractionation, indicative of low degree 
partial melting and fractional crystallization of a metasomatized mantle wedge. Adakites 
of GGB are Sr depleted with highly fractionated REE patterns and no Eu anomaly 
compared to rhyolites.   
Magmatism within plate and along destructive plate margins is the widely 
preferred hypothesis for the generation of new continental crust (Hawkesworth and 
Kemp, 2006 and references there in).  Most estimates suggest that the average crust is 
andesitic in composition but andesite is not the most abundant rock type erupted along 
the continental margin arcs as modeled by Taylor and McLennan (1985). Subsequent 
studies showed that andesite model does not explain the growth of continental crust 
(Rudnick and Fountain, 1995).  Most arcs are basaltic, intracrustal melting is required to 
generate an andesitic crust from these basalts (Rudnick and Gao, 2004).  Many recent 
models for Archaean continental evolution indicate a peak of crustal growth during the 
Neoarchaean, and parts of Archaean crust have been considered to have evolved through 
terrane accretion including amalgamation of oceanic plateaus and island arcs at 
convergent margins (Polat et al., 1998; Kerrich and Polat, 2006; Polat et al., 2006; 
Manikyamba and Kerrich, 2006).   
 Most of the products of such magmatic and tectonic processes are apparently well 
preserved in the Neoarchaean greenstone belts of Superior Province, Canada (Schreiber-
Hemlo, White River-Dayohessarah (Polat et al., 1998; Polat and Kerrich, 1999), Wawa 
(Polat and Kerrich, 2001), Birch-Uchi (Hollings and Kerrich, 2000), Abitibi greenstone 
belts (Kerrich et al., 1998).  These greenstone belts are characterized by two types of 
 iv
volcanic associations.   While tholeiite-komatiite suites are interpreted to have erupted 
from mantle plumes, tholeiitic to calc-alkaline suites have been suggested to have been 
erupted in island arc settings (Campbell, 1992; Wyman et al., 1999; Manikyamba et al., 
2004a; Kerrich and Polat, 2006). Late Archaean greenstone belts contain a great 
compositional diversity of subduction related volcanic rocks such as basalt-andesite-
dacite-rhyolite (BADR), adakites, low and high TiO2 basalts (Wang et al., 2004; 
Samsonov et al., 2005; Niu et al., 2006). In addition to bimodal tholeiite-calc-alkaline 
suites made up of basalts to rhyolites; there are adakites, Mg-andesites, Nb-enriched 
basalts and boninites, which have been reported from a few Archaean greenstone belts of 
Superior Province, Quebec, Canada, Greenland, Baltic Shield and Western Australia 
(Kerrich and Polat, 2006).  Individual suites of boninites and adakites were also identified 
from other greenstone belts of the world (Smithies, 2002; Polat and Munker, 2004; 
Smithies et al., 2005).  Kerrich and Polat (2006) have made a compilation on the 
Archaean hot subduction volcanic rocks and their Phanerozoic analogs reported from 
different cratons of the world, in which they discuss their geodynamic processes and 
demonstrate the prevalent operation of plate tectonics involving a thin oceanic crust and 
shallow angle subduction during Archaean. The geochemical characteristics of these 
volcanic rocks are consistent with diverse petrological processes operative in Archaean 
subduction zones including slab melting, slab dehydration, mantle wedge metasomatism 
and crustal recycling that have played a significant role in the growth and evolution of 
continental crust in the Dharwar Craton. 
The occurrence of boninites along with arc basalts, Nb-enriched basalts-
(tholeiitic) basalt- (calc-alkaline) andesite-dacite-rhyolites and adakite association in 
 v
Gadwal greenstone belt indicate intraoceanic subduction zone processes with a clear cut 
evidence of partial melting of a metasomatized mantle wedge (boninites), melting of 
subducting slab (adakites) and residue of adakite-wedge hybridization (Nb-enriched 
basalts) which all have played a significant role in the growth of continental crust in the 
Dharwar Craton during the Neoarchaean. 
 
OBJECTIVES 
¾ Fieldwork in the study area of Gadwal belt to understand the lithotectonic 
configuration, associated rock types, structural disposition and metamorphic 
pattern, and to collect samples representative of various lithologies for laboratory 
studies.  
¾  Detailed petrographic studies of the collected samples representing various 
lithologies to understand their mineralogy and textures. 
¾ Microprobe studies on selected samples for the estimation of mineral 
composition, P-T and depth of melting conditions.  
¾ Geochemical studies for the estimation of major, Trace, REE and PGE to 
understand the chemical composition of these metavolcanic rocks. 
¾ To evaluate the process (es) involved in the genesis of these metavolcanics and 
the development of Gadwal Greenstone Belt and  
¾ To develop a conceptual model for the genesis of these metavolcanic rocks and 
evolution of Gadwal Greenstone Belt. 
HYPOTHESIS TO BE TESTED 
 The convergent plate margin model for the generation of volcanic rocks 
i.e. Boninites, NEB and Adakites associated with Basalt-Andesite-Dacite-
 vi
(Rhyodacite) Rhyolite, by the melting of a metasomatized mantle wedge, 
subducting slab and their interaction during Archaean has been identified  
and studied from different greenstone belts of the world by various 
workers. Identification of these rock types from Neoarchaean Indian 
greenstone belts of Dharwar Craton suggest similar geotectonic 
environment for the generation of continental crust in the Dharwar Craton. 
APPROACH AND ORGANIZATION OF THE THESIS 
(1) Geological maps, Toposheets, Quadrangle maps and district resource maps, 
published by the Geological Survey of India (SR) and Survey of India, were 
utilized for carrying out the fieldwork.  
(2) Detalied petrography at NGRI and selective EPMA studies at GSI (SR), were 
carried out. 
(3) Major, Trace, REE studies of representative samples, and PGE studies of 
selected samples were carried out at NGRI. 
(4) Compilation, classification, processing and interpretation of data have been 
carried out for arriving at a conceptual genetic model.  
Chapter I describes the geology of the Dharwar Craton with an overview of inherent 
greenstone belts, spatial distribution of metavolcanic rocks and the lithotectonic 
distinction between WDC and EDC.  
Chapter II deals with the geology of the Gadwal Greenstone Belt (GGB) with 
associated lithologies, structure, metamorphism and location map of the samples 
collected for petrographic and geochemical studies.  
 vii
Chapter III elaborates the sampling and analytical procedures applied to carryout the 
geochemical and mineralogical studies.  
Chapter IV describes petrography and major, trace, REE and PGE geochemistry; 
mineral chemistry (EPMA) of Boninites from GGB. 
Chapter V explains in detail the petrography, major, trace, and REE geochemistry; 
mineral chemistry (EPMA) of associated Arc basalts/ Meta basalts from GGB. 
Chapter VI describes in detail the petrography and major, trace and REE 
geochemistry of meta-andesites from GGB. 
Chapter VII deals with the petrography, major, trace and REE geochemistry of 
metadacites from GGB. 
Chapter VIII describes the petrography, major, trace and REE geochemistry of 
metarhyolites from GGB. 
Chapter IX elaborates the petrography, major, trace, and REE geochemistry; mineral 
chemistry (EPMA) of adakites from GGB. 
Chapter X presents the discussion and applicability of various tectonic models to 
arrive at a suitable model for the genesis of these metavolcanics and conclusions 
drawn from the present study towards the evolution of the Gadwal greenstone belt.   
 


STUDIES ON BIOREMEDIATION AND SIMULTANEOUS BIOHYDROGEN GENERATION FROM INDUSTRIAL WASTEWATER BY ANAEROBIC PROCESS

DAS, PRANAB

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, beta- and gamma-crystallins, are the prototype and founding members of the betagamma-crystallin superfamily. betagamma-Crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of betagamma-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in betaB2-crystallin and other betagamma-crystallins from fish and mammalian genomes, it was argued that lens betagamma-crystallins should not bind calcium. In order to probe whether beta-crystallins can bind calcium, we selected one basic (betaB2) and one acidic (betaA3) beta-crystallin for calcium-binding studies. Using calcium-binding assays such as (45)Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both betaB2- and betaA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, (15)N-(1)H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to betaB2- and betaA3-crystallins, we suggest that all beta-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.

It has been established that many heterologously produced proteins in E. coli accumulate as insoluble inclusion bodies. Methods for protein recovery from inclusion bodies involve solubilization using chemical denaturants such as urea and guanidine hydrochloride, followed by removal of denaturant from the solution to allow the protein to refold. In this work, we applied on-column refolding and purification to the second crystallin domain D2 of Yersinia crystallin isolated from inclusion bodies. We also purified the protein from the soluble fraction (without using any denaturant) to compare the biophysical properties and conformation, although the yield was poor. On-column refolding method allows rapid removal of denaturant and refolding at high protein concentration, which is a limitation in traditionally used methods of dialysis or dilution. We were also able to develop methods to remove the co-eluting nucleic acids during chromatography from the protein preparation. Using this protocol, we were able to rapidly refold and purify the crystallin domain using a two-step process with high yield. We used biophysical techniques to compare the conformation and calcium-binding properties of the protein isolated from the soluble fraction and inclusion bodies.
Betagamma-crystallin is a superfamily with diverse members from vertebrate lens to microbes. However, not many members have been identified and studied. Here, we report the identification of a putative exported protein from Yersinia pestis as a member of the betagamma-crystallin superfamily. Even though calcium has been known to play an important role in the physiology and virulence of the Yersinia genus, calcium-binding proteins have not yet been identified. We have studied the calcium-binding properties of two of the three crystallin domains present in this putative exported protein designated "Yersinia crystallin." These two domains (D1 and D2) have unique AA and BB types of arrangement of their Greek key motifs unlike the domains of other members of the betagamma-crystallin superfamily, which are either AB or BA types. These domains bind two calcium ions with low and high affinity-binding sites. We showed their calcium-binding properties using various probes for calcium and the effect of calcium on their secondary and tertiary structures. Although both domains bind calcium, D1 underwent drastic changes in secondary and tertiary structure and hydrodynamic volume upon calcium binding. Domain D1, which is intrinsically unstructured in the apo form, requires calcium for the typical betagamma-crystallin fold. Calcium exerted an extrinsic stabilization effect on domain D1 but not on D2, which is also largely unstructured. We suggest that this protein might be involved in calcium-dependent processes, such as stress response or physiology in the Yersinia genus, similar to its microbial relatives and mammalian lens crystallins.
BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-exchange with gel filtration chromatography. In the case of betaB2-crystallin too, different approaches have been used to obtain the purified protein, majority of which use a combination of ion-exchange and gel filtration chromatography. We present a new approach to purify betaB2-crystallin using hydrophobic interaction chromatography. In this method, the protein is bound to the hydrophobic matrix in the presence of high concentration of a non-chaotropic salt and eluted by decreasing the salt concentration. The method that we have used for the purification of this globular protein has definite advantages over the earlier methods in its simplicity and efficiency. The most noted advantage of this procedure is the rapid purification with a relatively purified product and a comparatively high yield (>20 mg/L of culture). Over all, the present protocol provides a rapid, efficient and simplified procedure for the preparation of betaB2-crystallin in large yield, sufficient for structural and functional studies
Chapter 1: Introduction
	This chapter introduces the crystallin proteins of eye lens with focus on the -crystallin superfamily. -Crystallin and taxon-specific crystallins are the other major components of eye lens of vertebrates. The founding members - and -crystallins have been described which were classified separately in the early days. The basis of double crystallin-type Greek key motifs and their types in -crystallin proteins have been described. -Crystallin members isolated from bacteria, amphibians and yeast have been described. The calcium-binding properties and three-dimensional structures determined for some of these proteins have been mentioned. Effect of calcium in tissues or microorganisms where these proteins are expressed and their possible roles are also described. Observations about this superfamily of proteins leading to a hypothesis behind this work are described in the last section of this chapter.

Chapter 2: Experimental Procedures
	All the experimental procedures used in this work are described in this chapter excluding the cloning, overexpression and purification procedures of individual proteins, which have been mentioned in their respective chapters. A set of common biochemical, spectroscopic and calorimetric techniques applied to this study are described.

Chapter 3: Calcium-binding properties of lens -crystallins
	Calcium plays an important role in the pathophysiology of cataractogenesis. It is known that free calcium rises several fold in the cataractous lens compared to normal lens. This free calcium could activate calpains, calcium-dependent proteases and transglutaminases leading to precipitation of proteins, and covalent crosslinking in the lens thereby increasing the light scattering resulting in blurred vision. In spite of many studies, calcium-binding targets in lens are not known. Since crystallins are the major proteins in lens, they could act as calcium-binding agents. -Crystallin is known to function as molecular chaperone whereas -crystallins’ function are not yet known. I hypothesized that -crystallins could be calcium-binding proteins. -Crystallins are known to bind calcium whereas concrete evidences of such property of -crystallins are lacking. It was shown that L-crystallin fraction from bovine eye lens binds calcium with micromolar affinity. Since L fractions consists of several subunits of -crystallins, calcium-binding ability of individual subunits needs to be investigated. In this chapter, I have addressed the issue of calcium-binding to individual subunits namely bovine B2- and A3-crystallins as representative members of -crystallins. I have used recombinantly produced protein for a homogenous protein preparation using hydrophobic interaction chromatography. Different calcium-binding assays such as Stains-all, terbium binding and calcium-45 overlay were used to establish calcium-binding to these proteins. Calcium-binding affinity for B2-crystallin was determined using isothermal titration calorimetry (ITC) which revealed four calcium-binding sites with low to moderate affinity. However, this technique was not suitable for determining very low binding affinity of A3-crystallin for calcium instead terbium, a calcium mimic was used. Effect of calcium-binding on the conformation of B2- and A3-crystallin was probed using fluorescence and CD spectroscopy, which indicated no significant change in the apo to holo transition of these -crystallins. Holo form of B2-crystallin does not have significantly different chemical or thermal stability compared to apo form. Since all -crystallins share high sequence identity, these studies clearly prove that all -crystallins are calcium-binding proteins with low to moderate affinity for the cation. These studies fill a missing gap in our understanding of the calcium induced cataractogenesis and the functional role played by -crystallins in general.

Chapter 4: Calcium-binding properties of two single -crystallin domains from Yersinia pestis
	Studies on -crystallins in the previous chapter indicated that -crystallins bind calcium. However, they lack any of the known calcium-binding motifs of EF-hand, annexin, C2 domain or gelsolin type. It is important to identify the calcium-binding motif of -crystallins because few more proteins of this superfamily are now known to bind calcium such as -crystallins, Proteins S and Spherulin 3a. I hypothesize that -crystallin superfamily is a novel calcium-binding superfamily and calcium binds at the Greek key motif. In order to establish this, I identified many new putative protein sequences containing this motif mostly from microbes described in the following chapters. From the genome sequence of Yersinia pestis, I identified a pair of paralogous proteins containing the characteristic Greek key motifs. I identified four Greek key motifs organized as two -crystallin domain in one of the proteins which I named as “Yersinia crystallin”. The nucleic acid sequence corresponding to these two domains were cloned into an expression vector and overexpressed in E. coli as single domain for the production of recombinant proteins. These isolated domains were named as D1 and D2. D1 was isolated from the soluble fraction whereas D2 was refolded and purified to homogeneity from inclusion bodies using on-column refolding technique for various biochemical and biophysical assays for calcium-binding. Sequence analysis of these domains revealed the unique nature of these proteins due to the presence of one type of Greek key motif combination AA and BB in D1 and D2, respectively. These proteins tested positive for calcium-binding using the conventional methods of Stains-all, terbium binding and calcium-45 overlay assays. I then looked at the conformational changes upon calcium-binding using florescence and CD spectroscopy. Surprisingly, these domains are intrinsically unstructured proteins (IUP) in apo form. Calcium addition changes the random coil content to -sheet conformation in D1, whereas D2 retains its predominantly unstructured nature even in holo form. Intrinsically unstructured nature of the proteins was further confirmed by analytical gel filtration. As expected for an IUP, apo D1 elutes at a volume higher than expected of a monomer whereas holo D1 behaves as a globular monomer. Even though apo D1 elutes at a higher volume, it was a monomer as seen by glutraldehyde crosslinking. Thus, these studies challenge the conventional notion that -crystallin domains are compact rigid globular proteins. These studies also indicate that minor sequence variations can affect the globular nature of these proteins. The only prevalent nature of these proteins is the calcium-binding property. It has been known for a long time that calcium regulates the virulence of Yersinia species which are pathogenic to humans and animals. In spite of several studies, presence of a calcium sensor has not been identified in Yersinia species. I suggest that yersinia crystallin is a potent candidate for such calcium sensing function because of the conformational transitions from unstructured form to beta-sheet upon binding calcium. However, further in vivo studies are required for deciphering the function of this protein in Yersinia species.

Chapter 5: Calcium-binding and stability of an archaeal -crystallin domain from Methanosarcina acetivorans
	In the earlier chapter 4, the detailed studies on pathogenic bacterial -crystallin domains have been described. In continuation of above studies, I have for the first time identified a -crystallin domain in the genome of Methanosarcina acetivorans, a methanogenic archaebacteria. I have named it as “M-crystallin”. Using a number of methods, I established that M-crystallin is a calcium-binding protein. Unlike Yersinia crystallin, calcium-binding does not cause significant changes in the secondary or tertiary structure as probed by CD and fluorescence spectroscopy. Each molecule binds two calcium ions with micromolar affinity as determined by isothermal titration calorimetry (ITC). However, calcium-binding significantly enhances the stability of the domain against chemical and thermal denaturation unlike its lens homologues. Its solution structure was solved both in apo and in holo form. This single domain calcium-binding protein shows high structural similarities to mammalian lens -crystallins from the lens. It is clear from these studies on the ancient homologue of mammalian -crystallins that even though both share high sequence similarity, their calcium-binding properties and effects are significantly different.  Since archaebacteria are thought to be the oldest of all living organisms on this planet, this domain could be a direct ancestor of lens -crystallin domains. Extrinsic stabilization of this protein by calcium could help this methanogen counter stress, which is usually found in extreme environmental conditions.

Chapter 6: Calcium-binding to paralogous -crystallin domains from Caulobacter crescentus
	Our studies on the diverse -crystallin domains from mammals, bacteria and archaea described in previous chapters clearly established that calcium-binding is a prevalent property of the proteins of this superfamily. To further validate our hypothesis that -crystallin domains bind calcium, I selected a pair of paralogous proteins from the genome of an eubacterium, Caulobacter crescentus.  Both proteins contain a single -crystallin domain in the C-terminal end and an unusually long N-terminal extension. These proteins named as “caulollin1 and caullolin2” were over expressed in E. coli. Full length caullolin2 was not expressed instead its domain part was selected for overexpression and purified. Each of these proteins binds two calcium ions with micromolar affinity. CD and fluorescence spectroscopy indicate that caulollin2 is partly unstructured in apo form and gains -sheet conformation upon binding calcium.  Analytical gel filtration also confirms this observation. Caullollin1 on the other hand shows no significant conformational changes upon binding calcium. Based on these results, it can be decidedly concluded that -crystallin is a novel calcium-binding superfamily.

Chapter 7: Conclusions and future prospectus: -Crystallins is a novel calcium-binding superfamily
Chapter 7 concludes the thesis work by emphasizing on novel findings of this work. This work identified and characterized several new and unique domains from organisms spanning extremes of the spectrum of life. Low affinity calcium-binding to mammalian -crystallins fills a vital missing gap in our understanding of calcium homeostasis, disturbances of which results in cataractogenesis. Of general importance is the calcium-binding motif in these proteins, which appears to have very few members. Identification of more members is boasted by the various genome sequencing projects, using which I identified several new and diverse members and characterized their calcium-binding properties. These include the -crystallin domains from a pathogenic bacterium, Yersinia pestis and the only known domain from an archaebacteria, Methanosarcina acetivorans. I have also for the first time characterized a pair of paralogous -crystallin domains from Caulobacter crescentus. Calcium plays an important role in structural stabilization and conformation of many of these domains. Based on this and the regulation exerted by calcium on the phenotype exhibited by some of these organisms, the possible physiological role of these proteins looks important. Although the exact role can further be ascertained by in vivo studies, the most important finding of these studies is that the function of -crystallin proteins irrespective of the source should be seen in the context of calcium. These results can be summarized as the discovery of a novel calcium-binding superfamily with crystallin type Greek key motif as the calcium-binding motif.
In addition, this work has demonstrated that various domains exhibit different conformational and thermodynamic properties with respect to calcium-binding, which are likely due to the diversity of their sequences. Therefore, more structural studies need to be directed on the diverse members of -crystallin domains. This would add further knowledge to our understanding of the calcium related functions of these diverse proteins.






Morphotectonic Evolution and Petrochemistry of Central Indian Ocean Floor

Spices are known to add taste, flavor, aroma, color to food. These spices also have been the many traditional medicines in India and elsewhere to provide treatment to various diseases. Elettaria cardamomum is one of the spices used in traditional medicines for several diseases. It is used in treating various gastrointestinal, cardiovascular and neuronal disorders. The inhibitory activity of cardamom extract was studied on human platelets has also been reported. This review aims to highlight the ethno botany, pharmacognostic and pharmacological uses of Elettaria cardamomum. This review will also highlight the patenting trends and the new compositions developed using the actives from Elettaria cardamomum

Dhulap, Sivakami

Phyto-pharmacology of Elettaria cardamomum

KUMAR, SREE S.

Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
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Silica based materials are inert and possess high surface area and hence these 
materials are highly suitable to disperse active components or to incorporate large 
number of acidic sites, basic sites and functional groups. Among the silica based 
materials, SBA-15 type materials are relatively new and possess uniform pores in 
meso region which are capable for accommodate variety of organic molecules for 
converting them into useful products. The other advantage associated with SBA-15 
mesoporous silica material is its high robustness, thermal stability and easy way to 
modify in order to incorporate various functional groups. The modifications of SBA-
15 materials pave a way to conduct variety of organic reactions. Thus the work 
presented in this thesis is carefully planed to yield different types of reactions like 
alkylation, condensation, oxidation, lactonization etc. Most of these reactions on 
modified silica are reported for the first time. The conclusion part highlights briefly 
the modifications done and the characteristic facture for getting responsible high 
activity.  
Al-PHTS and Al-SBA-15 are prepared successfully by doping method, which 
are an eco-friendly and hydrothermally robust solid acid catalysts. The higher activity 
of Al-PHTS over Al-SBA-15 for isopropylation of m-cresol has been attributed to the 
presence of more number of medium strength acid sites formed by the plugging of 
silica and higher AlCl3 heterogenization in the former catalyst as observed from 27Al 
MAS NMR. The plugging of silica and immobilization of AlCl3, which resulted in 
Al-PHTS, is found to be a better catalyst and also the ratio of Td/Oh aluminum is 
much higher on Al-PHTS samples (1.69) compared to that in Al-SBA-15 (1.09). Thus 
the results clearly indicate that Al-PHTS sample has higher acidity than Al-SBA-15. 
Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
            232
 Mesoporous SBA-15 silica has been functionalized with propylsulfonic acid 
groups post-synthetically using 3-mercapropropyl-trimethoxysilane as sulfur source. 
Sulfonic acid groups anchored to SBA-15 silica pore surfaces are thermally stable up 
to 653 K, hydrothermally robust in boiling water and resistant to leaching in organic 
and aqueous solvents under mild conditions. The resultant mesoporous material 
(SBA-15-SO3H) exhibited hexagonal mesoscopic ordering, which possess 1.3 meq 
H+/g SiO2 exchange capacity, 96 Å mesopore size diameter, and 660 m2/g surface 
area.   
0
10
20
30
40
50
60
70
80
90
100
(m
ol
e 
%
) 
H2SO4 H-ZSM-5 Amberlite-
120
HY(Si/Al=11) SBA-15-
SO3H
Conversion  Styrene
Selectivity 4-phenyl-1, 3- Dioxane
Selectivity Others
 
Figure 9.1 Prince Condensation of styrene over different acid catalysts using 
dichloromethane as solvent 
Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
            233
 The Prins condensation results on SBA-15-SO3H solid acid catalyst with 
dichloromethane solvent seem to be much better for the synthesis of 4-phenyl-1,3-
dioxane, compared to the other acid catalysts like H2SO4,  H-ZSM-5, Amberlite-120 
and HY(Si/Al=11). The conversion and selectivity of all above catalysts are shown 
from Figure 9.1, which clearly shows that the selectivity was not maintained 100% 
selectivity except in the case of SBA-15-SO3H catalyst. Both the conversion of 
styrene and the selectivity of 4-phenyl-1,3-dioxane is are nearly 100% is due to 
presence of more number of acid sites available on SBA-15-SO3H catalyst surface 
and the uniform mesoporous net work.  
 Co-SBA-15 catalysts are synthesized by direct synthesis and post synthetic 
impregnation methods with two different loadings of cobalt viz., 1.2 and 2wt%. The 
encapsulation of cobalt in SBA-15 structure is successfully achieved and the structure 
of SBA-15 remained intact even after the incorporation of cobalt as observed from 
the low-angle XRD. The encapsulation of cobalt in SBA-15 was found to be highly 
advantageous in yielding significant amounts of the desired products viz., 
cyclohexanone and cyclohexanol in the oxidation of cyclohexane in liquid phase 
without using any solvent under moderate pressure of O2 at 433 K. Highly stable 
cobalt species in the SBA-15 framework as observed from TPR peaks are responsible 
for the catalysts prepared by direct synthesis method to show high turn over numbers 
towards the oxidation of cyclohexane to cyclohexanone. 2%CoSYN catalyst showed 
a maximum of 9.4% conversion of cyclohexane with the selectivity of 78% towards 
the formation of cyclohexanone.   
Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
            234
Trifluoromethane sulfonicacid (triflic acid) groups are anchored to the surface 
of mesoporous SBA-15 silica, and the material is found to be an efficient reusable 
catalyst for the condensation of styrene with aqueous formaldehyde and phenol with 
acetone respectively to get desired product with high conversion and high selectivity. 
In conclusion, triflic acid-anchored SBA-15 (SBA-15-O(Tf)3) have been found to be 
very effective in the synthesis of p,p-bisphenol-A and 4-phenyl-1,3-dioxane with very 
high selectivity at relatively low reaction temperatures.  
The results on SBA-15-O(Tf)3 solid acid catalyst shows all most equilibrium 
level conversion for phenol acetone condensation reaction compare to some of acid 
catalysts, which are shown in Figure 9.2. And also SBA-15-O(Tf)3 catalyst was much 
better for the synthesis of 4-phenyl-1,3-dioxane to wards conversion and selectivity 
under solvent free conditions reported so far. The conversion and selectivity of some 
acid catalysts for Prins condensation reaction are shown in Figure 9.3. 
0
10
20
30
40
50
60
70
80
90
 (m
ol
e 
%
)
Amberlite-120 HZSM-5 HY H-beta  SBA-15-
SO3H
SBA-15-
O(Tf)3
Phenol conversion p,p- bisphenol-A Selectivity o,p- bisphenol-A Selectivity Others
 
Figure 9.2 Condensation of phenol over different acid catalysts at 353 K  
Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
            235
Catalysts of this type represent an important class of superacid catalysts. New, 
simplified and high-yielding routes to these compounds open up a wide variety of 
opportunities. The relatively low volatility and high boiling point translate to safer 
handling of these materials. Supported versions of these acids enable reactions in 
non-polar reaction media. The silica supported superacids described in this thesis 
have been found to show high catalytic activity with low leaching in a number of 
reactions of industrial importance. Further more, SBA-15-O(Tf)3 solid acid catalyst is 
in expensive, non-toxic and reusable which makes the process convenient, 
economical and environmentally benign. 
0
10
20
30
40
50
60
70
80
90
100
(m
ol
e 
%
)
F3C-
SO3H
H2SO4 H-ZSM-5 Amberlit
e-120
HY H-beta SBA-15-
SO3H
SBA-15-
O(Tf)3
Conversion Styrene Selectivity 4-phenyl 1, 3-dioxane Selectivity Others
 
Figure 9.3 Solvent-free Prins condensation styrene over different acid 
catalysts at RT 
Functionalization of cobalt and ferrocene on the surface mesoporous SBA-15 
was successfully achieved. The structure of SBA-15 remained intact even after the 
Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
            236
functionalization of cobalt and ferrocene as observed from the low-angle XRD. 
Co(III)-SBA-15 catalysts showed significantly high turn over numbers and good yield 
towards the formation of cyclohexanone from the oxidation of cyclohexane. Co(III)-
SBA-15 yielded a maximum of 39.3% cyclohexanone from cyclohexane oxidation at 
room temperature. In the case of Ferro-SBA-15 yields only 29.6% cyclohexanone 
conversion from cyclohexane oxidation at room temperature. The hybrid material 
exhibits high catalytic activity for cyclohexane oxidation in presence of oxygen and it 
is recyclable and reusable without any apparent reduced activity.  
Syntheses of lactones from diols by oxidative lactonization over copper-based 
catalysts in vapor phase are studied. Copper silica-supported catalysts exhibits good 
conversion and selectivity to words formation of lactones. A variety of diols with 3 to 
10 carbon atoms can be converted in to the corresponding lactones in high yields 
(Table-X.X). The surface areas of the all copper silica catalysts are higher compared 
to the bulk CuO probably due the presence of copper in more dispersed form as 
compared to that in bulk CuO. The XRD analysis of the all calcined catalysts shows 
the presence of CuO phase only, where as their reduced forms showed the presence of 
only Cu0 only. The copper crystallite size plays an important role in determining the 
activity. 
The oxidative lactonization reaction of diols is studied on copper supported on 
different supports like Al2O3, MgO, HT, SiO2, carbon etc… but only Cu-SiO2 system 
exhibits more conversion and selectivity towards desired product. The result on 
copper silica (20%Cu-SiO2) catalyst shows 99% convention and 99.9% selectivity 
towards the formation of p-dioxanone at 553 K for more than 10 hours and all other 
Chapter – 9                                                                                                   Conclusions                              
 
                                                                         
            237
loadings showed were decrease with time. Such a high conversion and selectivity to 
nearly 100% is reported for the first time on copper silica system. In addition, copper 
silica solid catalyst is inexpensive, non-toxic and reusable which makes the process 
convenient, economical and environmentally benign. Therefore, these materials have 
tremendous potential for use in reactions of industrial importance for the production 
of lactones.  


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