56708443-Antibiotic (1)
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Transcript of 56708443-Antibiotic (1)
Contents
Introduction Classification Isolation Structure Determination Stereochemistry Biological Activity
2Dr. Harish & Purvi Kakrani
INTRODUCTION…
A chemical compound acting against life is called an ‘Antibiotic’,obtained either from natural source or by a synthetic method.
The term ‘Antibiotic’ has been derived from the word “antibiosis” which according to biological concept means survival of fittest means a process in which one organism may destroy another to preserve itself.
3Dr. Harish & Purvi Kakrani
The word ‘Antibiotic’ was first introduced by vuillemin in 1889 and defined by waksman and latter modified by benedict &langlykke.
4Dr. Harish & Purvi Kakrani
DEFINITION OF ANTIBIOTIC:
“It is a chemical substance produced by or derived from living cell which is capable ,in small concentration ,of inhibiting the life process or even distroying the microorganism.”
5Dr. Harish & Purvi Kakrani
Conditions for chemical compound
being an antibiotic.1)Originally ‘Antibiotic’ must have been a product
of metabolism although it might have been synthesized.
2) If ‘Antibiotic’ is a synthetic product then it should be a structural analogue of naturally occurred ‘Antibiotic’.
3) The ‘Antibiotic’ must be effective at low concentration.
4)The ‘Antibiotic’ must be antagonise the growth and/or survival of microorganism.
6Dr. Harish & Purvi Kakrani
In order for a particular ‘Antibiotic’ to act as a therapeutic agent,it has to satisfy the following conditions:
1) It must be effective against a pathogen.2) It must not cause significant toxic effect.3) It should be stored for a long time period
without loss of its activity.4) Its stability must be high so that it can be
isolated and processed into suitable forms of dosage which are readily absorbed in to human body.
7Dr. Harish & Purvi Kakrani
8
IMPORTANCE OF ANTIBIOTIC:
• Clinically effective in protozoal infection and fungal infection.
• Carcinostatic activity• Antitumour activity• Antibacterial activity• Antiviral activity• Important tool for study of biochemical
cellular mechanism
Dr. Harish & Purvi Kakrani
Three ‘ANTIBIOTICS’ should be discussed here…
PENICILLIN STREPTOMYCIN GRISEOFULVIN
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CLASSIFICATION...
First classification(the broad based classification):
a) Broad spectrum Antibiotics:
PENICILLINS
Tetracycline
Chloramphenicol
b) Relative broad spectrum Antibiotics:
Ampicillin
Cephalosporin10Dr. Harish & Purvi Kakrani
c) Narrow spectrum Antibiotic:
STREPTOMYCIN(gm -ve)
GRESEOFULVIN(antifungal)
Amphotericin Second classification(type of bacteria
can antibiotic destroy):
a) Effective against Gm +ve bacteria:
PENICILLIN
Erythromycin
Bacitracin
11Dr. Harish & Purvi Kakrani
b) Effective against Gm –ve bacteria:
STREPTOMYCIN
Neomycin
Gentamicin
c) Effective against both Gm +ve & Gm –ve:
Ampicillin
Cephalosporin
Tetracycline
Chloramphenicol
12Dr. Harish & Purvi Kakrani
Third classification (chemical
structure):
a) PENICILLIN and related Antibiotics:
PENICILLIN
SEMISYNTHETIC PENICILLIN
b) Aminoglycoside antibiotics:
STREPTOMYCIN
Neomycin
c) Macrolide Antobiotics:
Erythromycin 13Dr. Harish & Purvi Kakrani
d) Tetracycline Antibiotics:
Tetracycline
Oxytetracycline
Chlorotetracycline
e) Peptide Antibiotics:
Bacitracin
Gramicidin
f) Chloramphenicol and its synthetic analogues
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g)Antifungal Antibiotics:
GRISEOFULVIN
Amphotericin
h) Unclassified Antibiotics:
Cycloserine
Fusidic acid
Vancomycin
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‘Penicillin’ is the first antibiotic discovered by alexander flemming .
It was first extracted from P.notatum then P.chrysogenum but now by biofermentation process.
It is also known as beta lactam antibiotics.
The basic structure of Penicillin consists of thiazolidine ring(A) fused with beta lactam ring(B).both these unit form the 6-amino penicillinamic acid.
19Dr. Harish & Purvi Kakrani
CLASSIFICATION… Acid resistant Penicillin:
Penicillin V
Phenethicillin Penicillinase resistant Penicillin:
Methicillin
Oxacillin Extended spectrum Penicillin:
Ampicillin
Amoxicillin
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Reversed spectrum Penicillin:
Temocillin β-lactamase inhibitors:
Clauvanic acid
Sulbactam
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ISOLATION…
Various culture method known which are used to produce Penicillin.
The strains of Penicillin species are grown in a nutrient medium which is obtained from sugary materials and proteinous substance.
P .chrysogenum (NRRL 1951,Stanford 25099,X-1612,176) are widely used strains for the commercial production of Penicillin.
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Typical nutrient medium for penicillin
Component %
Corn steep liqour solid
3.5
Lactose 3.5
Glucose 1
CaCO3 1
KH2PO4 0.4
Edible oil 0.25
Penicillin precursor
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Penicillin produced on larger scale by 3 methods:
Liquid surface culture method Bran method Submerged method
In all these methods submerged method is widely used.
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Liquid surface method:The molases containing pH 7-8 is used as medium for microorganisms.After few days the growth of microorganism starts and after 6-7 days,concentration of Penicillin become 0.3-0.4 mg/ml.This method mainly used for lab.studies.
Bran method:The use of moist bran is considered to be substrate for mould growth and resultant Penicillin may be extracted in a liquid or Penicillin containing bran.
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Submerged culture method:This is widely used method for commercial production of Penicillin.In this method P.chrysogenum is used for the culture.Here culture and culture medium are taken in large tanks.The medium is agitated by a stream of sterile air at 24 c for 2-3 days.After that fermentation vessel is sealed and positive air pressure is maintained.under this condition mould grows throughout the bulk of liquid.
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After fermentation the contents of
vessel should be rapidly cooled and mycellium filtered on sterile rotary filter.
Adjust the pH of fitrate depends upon the nature of solvent extraction that carried out with amyl or butyl alcohol now pet.ether is added and shaken with dil.Na2CO3,adjust the pH 6-7 and rapidly freeze evaporated to yield Na-salt.
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STRUCTURE DETERMINATION… From analytical data the molecular
formula of Penicillin is C9H11N2O4SR. As Penicillin forms mono salts it
should be revealed that it contains carboxyl group.
From the chemical test it has been proved that penicillin does not contain free amino group.
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When penicillin are hydrolysed by hot dilute inorganic acids,they degraded to the equimolar amount of D-penicillimine and penilloaldehyde.
C9H11N2O4SR+2H2O C5H11NO2S+C3H4NO2R+CO2
31Dr. Harish & Purvi Kakrani
Structure of D-Penicillamine:• Molecular formula of Penicillamine is
C5H11NO2S.• Penicillamine gives colour reaction
with sodium nitroprusside this reveals that it contains thiol group so it is probably substituted cysteine.
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• When Penicilline is treated with
diazomethane, it is converted into its methyl ester.the later compound when treated with aqueous solution of mercuric chloride gives methyl ester of Penicillamine.this reaction reveals that carboxyl group in Penicillamine is carboxyl group in penicillin itself.
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• Like cysteine D-Penicillamine also react with acetone to yield isopropylidene derivative.the later compound does not contain a free amino or thio group and reconverted into Penicillamine on hydrolysis.
• Further the oxidation of Penicillamine with bromine water gives sulphonic acid.
So, D-Penicillamine is β ,β-dimethylcysteine,which is confirmed by its synthesis.
34Dr. Harish & Purvi Kakrani
Structure of Penilloaldehyde:• Molecular formula of Penilloaldehyde
found to be C3H4NO2R.• When hydrolysed vigorously,all
Penilloaldehyde yields a substituted acetic acid and amino acetaldehyde.this reaction reveals that Penilloaldehyde are acyl derivatives of amino acetaldehyde.
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• The latter structure of Penilloaldehyde
has been confirmed by its synthesis from corresponding acid chloride and aminoacetal.
RCONHCH2CHO+H2ORCOOH+
NH2CH2CHO RCOCl+NH2CH2CH(OC2H5)2→
RCONHCH2CHO.
37Dr. Harish & Purvi Kakrani
Mode of linking of Penicillamine and Penilloaldehyde:
• When Penicillin is hydrolysed with dilute alkali it yields Penicilloic acid which is dicarboxylic acid and readily eliminates a CO2 to yield monocarboxylic acid,Penilloic acid.this suggests that in Penilloic acid one of the carboxyl group is in β-position with respect to electrone attracting group.
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• When Penilloic acid hydrolysed with aqueous mercuric chloride,yields Penicillamine and Penilloaldehyde.this type of hydrolysis is characteristic of compound having a thiozolidine ring.
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• if (і) is the structure of Penilloic acid
then the structure of Penicilloic acid represented as (ii)
• (iii) and (iv)are also possible structures of Penicillin.
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Desulphurisation of benzyl penicillin with raney nickel to yield desthiobenzylpenicillin which on hydrolysis by acid gives desthiobenzylpenicilloic acid it proves existance of β-lactam ring in Penicillin.
42Dr. Harish & Purvi Kakrani
The infrared spectral studies of oxazolone and β-lactam structure,and correlation between various band and functional group is worked out.
This could be understood by considering the methyl ester and Na-salt of benzylpenicillin.
43Dr. Harish & Purvi Kakrani
Ir maxima of methyl ester & Na-salt benzyl penicillinIr maxima of methyl ester
Ir maxima of Na-salt
assignment
3333 3333 NH- group
1770 1770 β-lactam
1748 1613 >C=O
1684 1681 2ֹamide
1506 1515 2 ֹamide
44Dr. Harish & Purvi Kakrani
The ir spectra of number of β-lactam & fused thiazolidine β-lactams are recorded.
The β-lactam do not exhibit band at 1770 cm ֿwhile the other exhibit band at 1770 cm ֿ,this explain the 5th band so it would be concluded that (iv) is the correct structure for penicillin.
45Dr. Harish & Purvi Kakrani
STEREOCHEMISTRY…
Penicillin contains a 4 membered lactam ring which is fused through the nitrogen and tetrahedral carbon to a second heterocyclic ring.
All the natural Penicillin are strongly dextrorotatory .
It has been denoted that there are 8 stereoisomers of benzyl penicillin,achieved by changing the orientation of three carbon in structure namely 4,10,14.
47Dr. Harish & Purvi Kakrani
The incorporation of an ionized or polar group at the α- position of the side chain benzyl carbon atom of benzylpenicillin imparts clinical activity against Gm –ve bacilli.
If we were to find the stereochemistry of cyclisation which forms the β-lactam ring in Penicillin,we would have to synthesized cysteine with chiral tritium labelling at C-3 by cis-addition of H atom to an olefin of well defined stereochemistry.
49Dr. Harish & Purvi Kakrani
BIOLOGICAL ACTIVITY…
Penicillin inhibits bacterial cell wall formation.the cell wall of bacteria is essential for their normal growth and development.
Peptidoglycan is a component of the cell wall and provides rigid mechanical stability by virtue of its highly crossed linked lattice work structure.
51Dr. Harish & Purvi Kakrani
Penicillin inhibits the enzyme-transpeptidase that brings about cross linking between 5th glycine of the already existing peptidoglycan in the cell wall and 4th amino acid of newly formed peptidoglycan.
In addition, to the peptidoglycan synthesis,Penicillin also inhibits penicillin binding proteins at the cell wall causing lysis of the bacterial cell through autolysing enzymes.
52Dr. Harish & Purvi Kakrani
Penicillin is bactericidal in action.In its presence the bacterial cell wall become soft and finally undergoes lysis.
It is more effective on actively multiplying organisms.
53Dr. Harish & Purvi Kakrani
Ineffectiveness of Penicillin towards Gm –ve organisms leading to search for newer antibiotics.
Streptomycin was developed as a result of a well planned scientific research by waksman and coworkers in 1944.
58Dr. Harish & Purvi Kakrani
It was first isolated from Streptomyces griesus by schatz & waksman in 1944.
It is active against Gm –ve, Gm +ve organism including acid fast bacteria.
Streptomycin and other related antibiotics like kanamycin,Tobramycin,Framycetin etc. are termed as aminoglycoside antibiotics
59Dr. Harish & Purvi Kakrani
chemically aminoglycoside antibiotics,consists of two or more amino sugars joined in glycosidic linkage to a hexose nucleus.
This hexose is either Streptidine(in streptomycin)or 2-Deoxy streptamine(in other aminoglycoside antibiotics).
60Dr. Harish & Purvi Kakrani
The number of the amino sugars attached are also different.
kanamycin ,Gentamicin,Tobramycin consists two amino sugars,neomycin consists three aminosugar.
61Dr. Harish & Purvi Kakrani
ISOLATION… Initially,Streptomycin was prepared by
surface culture technique but now a days submerged culture technique is widely used.
The yield of Streptomycin depends greatly on medium used.the addition of corn steep liquor, meat extract, soyabean meal enhances the yield.
The medium composed of glucose ,1% peptone, 0.3% meat extract, Nacl 0.5% in water.
62Dr. Harish & Purvi Kakrani
The culture solution is kept in large
vats. The growth of micro organism begins at 24-28 ֹC and maximum yield achieved after 3-5 days.
After seperating mycellium and other wastes,Streptomycin is extracted from filtrate, either by adsorption on charcoal or on base exchangable resin.
Sreptomycin is eluted from column by means of dil.aqueous or alcoholic mineral acid,then purify it by passing
63Dr. Harish & Purvi Kakrani
through ion exchangable resin. The pure form of Streptomycin has
been isolated as sulphate or crystalline double salt of calcium chloride.
To get sterile drug crystaline Streptomycin is dissolved to yield 25% solution which is free from impurity by passing through seitz filter and then freeze dried then transferred aseptically to small vials.
64Dr. Harish & Purvi Kakrani
STRUCTURE DETERMINATION…
From essential analytical data molecular formula of Streptomycin has been found to be C21H39N7O12.
As Streptomycin form tri hydrochloride, this shows that its three N atoms must be strongly basic in nature.
C21H39N7O12 +3Hcl→C21H39N7O12.3Hcl
66Dr. Harish & Purvi Kakrani
When Streptomycin is hydrolysed in
strongly acid solution,it yields one molecule of Streptidine and one molecule of Streptobiosamine.(folkers et al)
Streptomycin+H2O→Streptidine+
Streptobiosamine. On the other hand alkaline hydrolysis
gives maltol which is believed to be derived from one of the fragment of Streptobiosamine.
67Dr. Harish & Purvi Kakrani
Structure of Streptidine:• From analytical data molecular
formula is found to be C8H18N6O4.• When it is oxidised with pottasium
permenganate,it yields two molecules of guanidine it reveals that Streptidine has two guanidino groups.
68Dr. Harish & Purvi Kakrani
• When Streptidine is subjected to
alkaline hydrolysis,it yields Streptamine and ammonia.(brink et al)
Streptidine+4H2O→Streptamine+ 4NH3+2CO2
69Dr. Harish & Purvi Kakrani
70
•As Streptamine is obtained by alkaline hydrolysis of Streptidine,the following structure may be assigned to Streptidine.this structure is confirmed by its synthesis from Streptamine(wolfrom et al)
Dr. Harish & Purvi Kakrani
• Streptidine is not opticaly active,therefore it was assigned mesoconfiguration with two guanidino group in a cis position.
71Dr. Harish & Purvi Kakrani
Structure of Streptobiosamine:• From analytical study the molecular
formula found to be C13H23NO9.• When Streptomycin is subjected to
methanolysis then hydrolysed finally acetylated,the penta acetate of N-Methyl L-glucosamine which on hydrolysis yields N-Methyl L-glucosamine.
72Dr. Harish & Purvi Kakrani
methyl streptobiosamine dimethyl acetal.
• When methyl Streptobiosamine dimethyl acetal is subjected to drastic alkaline hydrolysis yields methyl acetal,indicating the presence of methyl amino group.
73
• When Streptomycin is reacted with methanolic hydrochloric acid it gives Streptidine di hydrochloride and
Dr. Harish & Purvi Kakrani
• On the other hand less drastic
degradation of the Methyl Streptamine by means of acetic anhydride and hydrochloric acid yields N-Methyl glucosamine and Streptose so it would be concluded that Streptobiosamine may be glycoside of N-Methylglucosamine and Streptose.
74Dr. Harish & Purvi Kakrani
• When N-Methylglucosamine is treated with phenylhydrazine,it yields a phenylhydrazone.the later can be converted in to phenylosotriazole which has same m.p and an equal but opposite specific rotation as of D-glucose phenylosotriazole.
75Dr. Harish & Purvi Kakrani
• When Streptobiosamine is oxidised with bromine,it yields a product which on hydrolysis yields Streptosonic acid monolactone.the lactone is converted into amide which consumes two moles of periodic acid ,indicating the presence of three hydroxyl group.so the structure of Streptosonic Acid Diamide,Streptosonic Acid Monolactone and Streptose may be written as follows:
76Dr. Harish & Purvi Kakrani
Streptamine N-Methylglucosamine
-
Streptose
Streptidine Streptobiosamine
Streptomycin
80Dr. Harish & Purvi Kakrani
Point of linkage between Streptidine
and Streptobiosamine:• In 1948-49 merck group of chemist
established the point of linkage between Streptidine and Streptobiosamine by carried out hydrolysis of benzolyated streptomycin very carefully without disturbing the ester linkage to yield optically active heptabenzoylstreptidine.
81Dr. Harish & Purvi Kakrani
• The oxidation product clearly established the structure of dibenzoyl derivative it reveals that –OH group is adjacent to one of the carbon attached to the guanidino group of streptidine.
82Dr. Harish & Purvi Kakrani
• C1 of Streptose unit is involved in linkage between Streptidine and Streptobiosamine.
• But later compound do not have substituents on C1 it means that substituent must be present for maltol production.
• Hence,C1 of Streptose is linked glycosidically to the C4 of the Streptidine to yield following structure of Streptomycin.
83Dr. Harish & Purvi Kakrani
STEREOCHEMISTRY… Streptomycin has two important
structural features namely aminosugar portion and centrally placed hexose ring.
If Amino sugar portion at C-6 or C-2 position,serve as major target sites for bacterial inactivating enzyme.
If modification at C-1 carbon of centrally placed hexose ring,helps to retain the antibacterial potency.
86Dr. Harish & Purvi Kakrani
Streptidine is not optically active.therefore,it was assigned mesoconfiguration with two guanidino groups in a cis position.
88Dr. Harish & Purvi Kakrani
BIOLOGICAL ACTIVITY… It is bactericidal in therapeutic dose
and bacteriostatic in lower dose.it interferes with protein synthesis.
The initial event is penetration of drug into the cell.
The 30s subunit of cell ribosomes is primarily affected which in turn causes inhibition of protein synthesis.it also causes elongation of the cell without permiting final division.
91Dr. Harish & Purvi Kakrani
Ribosomal protein synthesis is
inhibited by aminoglycoside by three ways:
1)They interfere with the initiation complex of peptide formation.
2)They induce misreading of the code on the mRNA template.
3)They cause a break up of polysomes into non-functional monosomes.
92Dr. Harish & Purvi Kakrani
Before producing inhibition of protein synthesis ,it is necessary for aminoglycoside to enter into cytosol of the bacteria by diffusion through aqueous channel or energy dependent electrone transport.
93Dr. Harish & Purvi Kakrani
Non functional proteins produced due to effect of Streptomycin,may be incorporated into the cell membrane leading to further stimulation of aminoglycoside transport and leakage of small and larger molecule.this is the reason of bactericidal action of Streptomycin.
94Dr. Harish & Purvi Kakrani
Griseofulvin is an antifungal agent produced by the cultures of P.griseofulvum other penicillium species including P.notatum,P.paltatum.
Grisoefulvin is the drug of choice for widespread or itractable dermatophyte infection.
99Dr. Harish & Purvi Kakrani
Griseofulvin contain neither sulphur nor nitrogen,but contains chlorine.chlorine is supplied as pottasium chloride throughout its production,otherwise the product is dechlorogriseofulvin which is identical to griseofulvin.
100Dr. Harish & Purvi Kakrani
ISOLATION… The principle in Griseofulvin
fermentation is to encourage early growth and then,by nutrient limitation,to maintain a cell population just within the aeration potential of the fermentor.
Griseofulvin remains intracellular,at harvest it is the cell that are significant and the filtrate is no of use.the cell can be easily removed by fitration.
101Dr. Harish & Purvi Kakrani
The first step in the recovery of Griseofulvin is to extract the cell with acetone.
The resulting extract is subjected to concentration,thus a crude product is obtained.
102Dr. Harish & Purvi Kakrani
This crude product is washed with further organic solvent to remove acetone soluble impurities.
The washed Griseofulvin is redissolved in acetone and solution is mixed carefully with water so it would be precipitated.the large volume of acetone used are recovered for further use.
103Dr. Harish & Purvi Kakrani
STRUCTURE DETERMINATION… There is no particular details about
structure determination for Griseofulvin.
From essential analytical data molecular formula of Griseofulvin is C17H17ClO6.
By the usual chemical test it has been shown that Griseofulvin do not have free amino or thiol group.
104Dr. Harish & Purvi Kakrani
Initial inspection of structure of Griseofulvin shows the alternative oxygenation pattern and also methyl group which identifies the start of the polyketide chain.
105Dr. Harish & Purvi Kakrani
From IR spectra studies of Griseofulvin the principal peaks at wave number 1220,1611,1210,1583,1135,800(KBr disc).
From Mass spectral studies of Griseofulvin principal peaks acc. to m/z 138,352,215,310,214,69,321,354.
106Dr. Harish & Purvi Kakrani
BIOLOGICAL ACTIVITY… A prominent morphological
menisfastation of the action Griseofulvin is the production of multinucleate cells as the drug inhibit fungal mitosis.the explanation for this phenomenon appears to come from the studies of effects on mammalian cells of higher concentration of the antibiotic.
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Griseofulvin causes disruption of mitotic spindle by interacting with polymerized microtubules,thus action similar to colchicine and vinca alkaloids.
109Dr. Harish & Purvi Kakrani
Griseofulvin‘s binding site on microtubular protein is distinct.there is evidence that it binds to a microtubule associated protein in addition to its binding to tubulin.
Here,by destruction of microtubules responsible for the synthesis of various substance.
110Dr. Harish & Purvi Kakrani
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Kar Ashutosh,Pharmacognosy & Pharmabiotechnology 2nd revised edition;New age international publisher.
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Agarwal O P,Organic Chemistry of Natural Products Vol.2,28th edition;Goel publication merut. Page:98-122.
111Dr. Harish & Purvi Kakrani
Comprehensive Medicinal Product,vol 4. Page:100-02.
Peter K,Volhardt C,Organic Chemistry: Structure & Function, 3rd edition;W H Freeman & Company,New york Page:898,1102.
Indian Pharmacopoiea’96 vol-1,Published by controller of publication,Delhi.Page:353.
British Pharmacopoiea’93 vol.1 International edition,Page:316,635.
112Dr. Harish & Purvi Kakrani
Devik Paul,A Biosynthetic Approach to Medicinal Natural Product,2nd edition;Willey publication Page:78,437-444,478-480.
Patel A H,Industrial Microbiology,Macmillan India ltd, Page:112-22.
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Kar Ashutosh,Medicinal Chemistry;2nd edition, New age publication Page:455-61.
113Dr. Harish & Purvi Kakrani
Manfred Wollf,Berger’s Medicinal Chemistry &Drug discovery vol 4:Therapeutic agent;5th edition Page:285-93.
Goyal R K, Element of Pharmacology 14th edition 2004-05;B S Shah Prakashan, Page:486-88,500-02,530.
Rang &Dale,Pharmacology,5th edition;elsevier publication,page:639-47,666-69.
114Dr. Harish & Purvi Kakrani
Clarke’s Isolation &Identification of Drug in Pharmaceutical Body Fluid and Postmorterm Material,2nd edition;The Pharmaceutical press.Page:645.
Foye Willium,Williams A David,Principal of Medicinal Chemistry 4th edition;B I Waverly Pvt ltd,New Delhi.Page:1190-93,1222.
Tripathi K D,Essentials of Medical Pharmacology,5th edition;Jaypee Page:653-57,681-82.
115Dr. Harish & Purvi Kakrani
Bothra K G, Kadam S S,:Principles of Medicinal Chemistry vol 2 15th edition; Nirali Prakashan Page:69-72,84.
Munson Paul,Maller,Breese: Principles of Pharmacology;Basic Concept &Clinical application, Hall publication Page:1319-22,1351-57,1409.
Gilman,limbird;Goodman &Gilman’s Pharmacological Basis of Therapeutics,10th edition; International edition Page:1189-93,1219-24,1305-06.
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