Peptides in anemia

Department of Pharmaceutical Sciences
Birla Institute of Technology
Mesra, Ranchi – 835 215.
2012
A Thesis
Submitted in partial fulfillment of the requirements for
the award of the Degree of
MASTER OF PHARMACY
IN
PHARMACEUTICAL CHEMISTRY
By
Shroff Prashantkumar Balubhai
(MPH/1014/2010)
Under the guidance of
Dr. S. SAMANTA
(Professor)

 INTRODUCTION
 LITERATURE REVIEW
 OBJECTIVE OF THE WORK
 PLAN OF WORK
 EXPERIMENTAL WORK
 RESULT AND DISCUSSION
 SUMMARY AND CONCLUSION
 FUTURE SCOPE
 REFERENCES
19-May-12 2BIT, MESRA.

NUTRACEUTICAL1,2,3
•The term “Nutraceutical” was coined from “Nutrition”
& “Pharmaceutical” in 1989 by Stephen DeFelice, MD
Founder and Chairman of the Foundation for Innovation
in Medicine (FIM).
1
•Nutraceuticals may range from isolated nutrients, herbal products, dietary supplements
and diets to genetically engineered ''designer'' foods and processed products such as cereals,
soups and beverages. Doubtlessly, many of these products possess pertinent physiological
functions and valuable biological activities.
DEFINITION :
•Food, or parts of food, that provide medical or health benefits, including the prevention and
treatment of disease.
•The term also has been defined as a product isolated from food and generally sold in
medicinal forms not usually associated with food and demonstrated to have physiological
benefit or provide protection against chronic disease.

• It is important to note that this definition pertains to all categories of foods. It ranges
from dietary supplements such as folic acid used for the prevention of spine bifida, to
chicken soup taken to lessen the discomfort of the common cold. It includes a
bioengineered designer vegetable food rich in antioxidant ingredients to a stimulant
functional food or pharmafood.
• With the passage of the Dietary Supplement Health and Education Act of 1994 in
US, the definition of nutraceuticals has been expanded to include vitamins, minerals,
herbs and other botanicals, amino acids and any dietary substance for use by humans
to supplement the diet by increasing total dietary intake and subsequently increased the
use of nutraceuticals dramatically.
 Nutraceuticals can be grouped into the following three broad categories:
• Nutrients- The most commonly known nutrients are antioxidant, water and fat-soluble
vitamins.
• Herbals- Numerous nutraceuticals are present in medicinal herbs as key components.
E.g. Aloe Vera, Ephedra, Garlic etc.
• Dietary supplements- For e.g. Ketogenic diets, comprised of foods high in fat and low
in protein and carbohydrate content, have been reported to improve seizure control.

• Iron, as a therapeutic agent, was first documented about 2735, B.C., when it
was declared by the Chinese Emperor, Shen Nung, as a cure for “Anemia”.4
Iron is an essential in human body for5:
• the formation of hemoglobin and certain enzymes, many proteins and
enzymes that maintain good health
• transporting oxygen in the blood to all parts of the body
• many metabolic reactions and the regulation of cell growth and differentiation
• immune activity
• proper functioning of the liver
• protection against the actions of free radicals.
Iron(Fe) is an absolute requirement for most forms of
life, including humans and most bacterial species,
because plants and animals all use iron; hence, iron
can be found in a wide variety of food sources.

Fig 1.1: Iron transport through the enterocytes6
Nonetheless, intestinal iron absorption and cellular iron transport are poorly
understood. Iron deficiency anemia is, in part, a result of diet.
 The availability of iron from many foods is very low. No more than 5% of
vegetable iron is absorbed and while the absorption of iron from meat, poultry
and fish may be somewhat higher, significant worldwide consumption of
animal proteins is limited to the more affluent.

•Consumption of certain foods, such as coffee or tea, phosphates, phytates and
bran will generally reduce iron absorption. Besides a diet, an individual’s iron
status is also related to age, sex, lifestyle, lactations etc.
•The use of oral contraceptives, aspirin, antacids, antiinflammatories,
anticoagulants and steroids may all increase the risk of iron deficiency.
•Diseases of the gastrointestinal tract, including cancer, hemorrhoids will result
in a greater risk of iron deficiency due to increased iron requirements and/or an
increased inability to efficiently absorb iron.
•Illness associated with a fever may also reduce iron utilization, even if the
iron is absorbed.

• Iron deficiency is the most common micronutrient deficiency in the world and has
far-reaching and serious adverse effects on health. Nearly one fourth of the world’s
population is currently anemic.
• As per WHO, Anaemia(Anemia) is a condition in which the number of red blood
cells or their oxygen-carrying capacity is insufficient to meet physiologic needs,
which vary by age, sex, altitude, smoking, and pregnancy status.7
• Anemia detection is often used as a screening test for iron deficiency. Anemia is a
late sign of deficient iron stores.
Figure 1.2: Anemic blood cells60

Anemia has four basic causes. One or more of these causes must be operating to
produce anemia8:
•Hemorrhage -- bleeding
•Hemolysis -- excessive destruction of red blood cells
•Underproduction of red blood cells
•Not enough normal hemoglobin
There are many forms of anemia, some of them are common. They include, for
example:8
Iron deficiency anemia: ( iron deficiency results in anemia)
Aplastic anemia: (due to failure of the bone marrow to produce blood cells)
Fanconi anemia: (A genetic disease of the bone marrow elements, mainly in
children)
Pernicious anemia: (A blood disorder caused by inadequate vitamin B12 in the
blood.)
Sickle cell disease: (A genetic blood disorder caused by the presence of an
abnormal form of hemoglobin.)

Figure 1.3: Symptoms of Anemia61

The World Health Organization considers Iron Deficiency is the number one
nutritional disorder in the world. As many as 80% of the world's population may
be iron deficient, while 30% may have iron deficiency anemia.
In iron deficiency anemia, the red cells appear abnormal and are unusually small
(microcytic) and pale (hypochromic). The pallor of the red cells reflects their low
hemoglobin content.
The prevalence of iron deficiency anemia is the highest in children and women of
childbearing age (particularly pregnant women).
The treatment of iron deficiency anemia, whether it be in children or adults, is
with iron and iron-containing foods.

How anemia is detected?7
Anemia is most commonly detected by measuring Hemoglobin or by determining
Hematocrit (the volume of red blood cell in the specific amount of blood).
WHO proposes the following cut-off hemoglobin(Hb) values:
WHO lists the following ranges for normal hematocrit(Hct) values:
Children under 5 year of age Hb < 110g/L
Non-pregnant women Hb < 120g/L
Pregnant women Hb < 110g/L
Men Hb < 130g/L
Children under 5 year of age Hct 38-44%
Women Hct 37-43%
Men Hct 40-50%

Oxidative stress in Anemia9
Figure 1.4: A hypothetical mechanism of anemia and autoantibody production
against erythrocytes due to reduced activity of the antioxidant system in blood.9

Fenton reaction in Erythrocytes10
The erythrocytes represent an important component of the antioxidant capacity
of blood, comprising in particular intracellular enzymes, e.g. superoxide
dismutase and catalase, but also the glutathione system.
Iron deficiency anemia enhances red blood cell oxidative stress and reduces
the erythrocyte levels of catalase, superoxide dismutase and glutathione.10

GLUTATHIONE REDUCTASE11
Glutathione reductase, also known as GSR or GR, is an enzyme that
reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an
important cellular antioxidant. GSR is used as an indicator of oxidative stress
in red blood cell in anemia.
Reaction mechanism of human glutathione reductase:
NADPH reduces FAD present in GSR to produce a transient FADH- anion. This
anion then quickly breaks a disulfide bond (Cys58 - Cys63) and leads to Cys63's
nucleophilically attacking the nearest sulfide unit in the GSSG molecule, which
creates a mixed disulfide bond (GS-Cys58) and a GS- anion. His467 of GSR then
protonates the GS- anion to form the first GSH. Next, Cys63 nucleophilically
attacks the sulfide of Cys58, releasing a GS- anion, which, in turn, picks up a
solvent proton and is released from the enzyme, thereby creating the second
GSH. So, for every GSSG and NADPH, two reduced GSH molecules are gained,
which can again act as antioxidants scavenging reactive oxygen species in
the cell. 9,10

Iron - Amino acid chelates12:
In order to enhance iron bioavailability and still avoid interaction with food
ingredients, chelating iron with amino acids has been employed.
A nutritionally viable iron amino acid chelate must have a stability constant which
would result if the iron were chelated or complexed to the food ligands found in the
stomach and intestines.
If the chelate dissociates in the gut, it has no more value than ionized iron from a
soluble salt. The stability constant should also be high enough to allow chelate to cross
the intestinal cell membrane into the cytoplasmic ligands are capable of removing the
iron from the absorbed amino acid chelate by complexing with the absorbed iron.17
For an iron amino acid chelate to be absorbed into the mucosal tissue, it must be a low
molecular weight chelate less than 1500 daltons if it is to be absorbed in humans. Or
we can prepare iron chelated peptides with amino acids which can be absorbed from
gastrointestinal track into the mucosal cell as a chelate than a soluble salt of iron in
treatment of iron deficiency anemia.4

Bioactive Antioxidant Peptides13,14:
• Opportunities have arisen to formulate food products which deliver specific
health benefits, in addition to their basic nutritional value. In this respect, bovine
milk and colostrum are considered the most important source of natural bioactive
components.
• All amino acids are susceptible to oxidation, although their susceptibilities vary
greatly. Organisms have evolved complex antioxidant defenses to minimize
oxidative damage to proteins and other macromolecules.
• The antioxidative activities of peptides generated from the digestion of various
proteins have been reported. Several amino acids, such as Tyr, Met, Asp, Pro,
His, Lys, and Trp are generally accepted to be antioxidative and exhibit higher
antioxidative activities when incorporated into peptides. However, neither the
structure-activity relationship nor the antioxidant mechanism of peptides is fully
understood.

IRON AND ANEMIA
•William Dameshek in 1950
Has shown that Ferrous ion has become established as the material of choice and
ferrous sulphate is highly acceptable; however oral preparations are good, but
injectable iron is better due to severe reactions in GIT with oral iron.15
•Dainel et al. in 1955
Have shown that iron stores are exhausted before anemia occurs and patients shows
response to oral iron but if they are unable to take iron because of gastrointestinal
symptoms, parenteral administration of iron has advantages.16
•Hugh in 1958
Has shown that local factors ( reducing agents, GI acidity, presence of phosphate)
and General factors (Diet etc…) are influencing absorption of iron, has also shown
supplementation with inorganic iron is a more effective way of increasing iron
retention.17

•Chan et al. in 1959
Have shown that Anemia usually responds to Oral iron therapy in any ferrous salt
like sulphate, fumarate, succinate or gluconate.18
•Stephen H. Robinson in 1969
Has shown bilirubin formation has been increased in rats with iron deficiency
anemia which can originate from hemolysis of circulating red blood cell, iron
deficiency disorders heme metabolism in bone marrow which can be overcome with
iron therapy.19
•Gordeuk et al. in 1986
Have shown that Carbonyl iron is safe, effective, well tolerated in treatment of iron
deficiency anemia and has less toxicity in children than with ferrous sulfate.20

OXIDATIVE STRESS IN IRON DEFICIENCY ANEMIA
•Ramachandran in 1985
Has shown that lipid peroxidation plays a major role in the reported decrease in
red cell life-span in iron deficiency.21
•Jansson et al. in 1985
Have found the higher content of superoxide dismutase in iron deficient RBC
than control, which suggests an increased formation of SOD compensatory to
an increased oxidative stress.22
•Mehmat et al. in 2002
Have shown that Oral iron treatment improved the IDA and recovered
antioxidant defense system by increasing SOD and GSH-Px activity.23

•Farshad et al. in 2008
Have shown that the activities of erythrocyte cytoprotective enzyme decrease and
lipid peroxidation increases in women with IDA which may lead to other
degenerative disorders.24
•Coghetto et al. in 2009
Have shown that the patients with IDA are subjected to chronic oxidative stress.25
•Jong-Ha Yoo et al. in 2009
Have shown that blood reactive oxygen species was lower and total antioxidant
activity was higher after treatment which supports the higher oxidative stress
hypothesis in IDA.26
•Krishnamurthy et al. in 2010
Have shown that Oral iron is safe and efficacious in children with IDA.27

ANTIOXIDANT NUTRACEUTICAL PEPTIDES
•Rodney et al. in 1996
Have shown that Cysteine and Methionine residues function in the catalytic cycle of
several enzymes and constitute an important antioxidant defense mechanism.28
•Chan et al. in 1998
Have shown that in the antioxidant peptide (LLPHH) derived from proteolytic digest of a
Soybean protein, HH plays a major role in antioxidant activity.29
•Kunio S. and Jiun-Rong C. in 2002
Have shown that peptides having potent antioxidant activity were from the hydrolysate
of Wheat gluten with amino sequences Leu-Gln-Pro-Gly-Gln-Gln-Gly and Ala-Gln-Ile-
Pro-Gln-Gln.30
•Saito et al. in 2003
Have constructed combinatorial libraries with histidine, tryptophan, alanin and glycin,
based on antioxidative peptide isolated from a soybean protein hydrolysate and
concluded their antioxidant activity against the peroxidation of linoleic acid, the reducing
activity, the radical scavenging activity and the peroxynitrite scavenging activity.31

•Zhao et al. in 2004
Have developed peptide antioxidants that target the inner mitochondrial membrane
which potentially reduced intracellular reactive oxygen species and can be beneficial
in the treatment of ageing and diseases associated with oxidative stress.32
•E Aida et al. in 2004
Have shown that the ability of Whey protein hydrolysate fractions to delay lipid
oxidation was found to be related to the prevelence of histidine and hydrophobic
amino acids.33
•Hannu K. and Anne P. in 2006
Have shown that naturally formed bioactive peptides have been found in fermented
dairy products such as yoghurt, sour milk and cheese and the peptide with amino acid
sequence Ala-Arg-His-Pro-His-Pro-His-Leu-Ser-Phe-Met shows antioxidant
activity.34
•Anne Pihlanto in 2006
Has shown that milk-derived antioxidant peptides are composed of 5-11 amino acids
including hydrophobic amino acids, proline, histidine, tyrosine or tryptophan in
sequence.35

•Wakabayasi et al. in 2006
Have reviewed on an iron binding glycoprotein present in milk which is considered to
be an important host defence molecule and has a diverse range of physiological
functions such as antimicrobial/antiviral activities, immunomodulatory activity,
anticancer, antioxidant, anti-infective and anti-inflammatory activity.36
•Chen et al. in 2006
Have investigated peanut protein hydrolysate for its antioxidant activities, including its
ability the autooxidation of linoleic acid, the scavenging effect on the 1,1-diphenyl-2-
dipicrylhydrazyl (DPPH) free radical, the reducing power and the inhibition of liver
lipid oxidation.37
•Kati et al. in 2008
Have shown that bioactive peptides are found in milk, egg, meat, fish as well as in
many plants and amongst them peptides with a sequence Pro-His-His showed the
greatest antioxidant activity.38
•Dan et al in 2008
Have shown that brain permeable iron chelators may present therapeutic benefits and
the NAP(Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) peptide showed inhibition of lipid
peroxidation and hydroxyl radical formation.39

•Minelli et al. in 2007
Have shown that neuroprotective activity of cyclo(His-Pro) deals with increase
antioxidant protection and its oral administration improves a synergistic mechanism
the glycaemic control in diabetes.40
•Mine Y. and Katayama S. in 2008
Have shown that phosphopeptides from hen egg yolk act as inhibitor of lipid
peroxidation and radical scavengers.41
•Wu-Yang et al. in 2010
Have shown that the oxygen radical-scavanging effects of egg white protein
ovotransferrin might be constitutive amino acid tryptophan and the bond between
tryptophan and arginine.42
•Bahareh H. Sarmadi. and Amin Ismail in 2010
Have reviewed the antioxidative activity of bioactive peptides can be attributed to
their radical scavanging, inhibition of lipid peroxidation and metal iron chelation
properties of peptide which may be affected by amino acid sequence.43
•Anusha G. P. and Li-Chan C. Y. in June,2011
Have reviewed the food derived peptides which are being considered as potential
sources to control various oxidative processes in the human body as well as in food.44

IRON ABSORPTION AND IRON-AMINOACID CHELATE
•Adelia et al. in 2000
Have shown that in whole maize meal, iron from ferrous bisglycinate is better
absorbed than iron from ferrous sulfate or ferric triglycinate.45
•H. Dewayne Asmead in 2001
Has reviewed on the absorption and metabolism of iron aminoacid chelate and
has shown that intestinal absorption of iron from iron amino acid chelate
compared to the inorganic salts and iron amino acid chelate is both a safe and
effective source of iron for treatment of iron deficiency.46
•Marcos et al. in 2002
Have shown that Fe+3-peptide complex is a potential compound for use as an
iron source in treatment of patients with IDA.47

•Bertille et al. in 2005
Have shown that when bound to Fe, caseinphosphopeptides derived from
milk proteins resist luminal digestion, enhance Fe solubility and could
improve its bioavailability.48
•Yeung et al. in 2006
Have shown that casein phosphopeptides enhances the iron availability from
foods with low availability.49
•Konstantina et al. in 2007
Have shown that milk proteins produce a range of bioactive peptides and
among those are peptides that may enhance iron absorption.50

Iron-deficiency anemia is a common anemia (low red blood
cell level) caused by insufficient dietary intake and absorption of iron,
and/or iron loss from intestinal bleeding, parasitic infection,
menstruation, etc. Red blood cells contain iron and are not formed when
iron is deficient.
The mechanism by which iron enters the mucosal cells lining
the upper gastrointestinal tract is unknown. Most cells in the rest of the
body are believed to acquire iron from plasma transferrin (an iron-
peptide chelate), via specific transferrin receptors and receptor-mediated
endocytosis.
No more than 5% of vegetable iron is absorbed and while the
absorption of iron from meat, poultry and fish may be somewhat higher,
significant worldwide consumption of animal proteins is limited to the
more affluent.

A literature survey on antioxidant activity of some nutraceutical peptides,
iron absorption and anemia shows that food like whole milk, soybean, egg
have some bio active peptides which are useful to treat oxidative stress
during anemia and increase absorption of iron in human body.
The objective of our work is
 To design some small synthetic antioxidant peptides which will be useful
in treatment of anemia by lowering red blood cell oxidative stress and by
increasing iron absorption in body.
 To synthesize the peptides using more economical liquid phase method
(CPE method) and characterize by M.P, TLC, FT-IR, NMR and Mass
spectral analysis.
 To evaluate the synthesized compounds for antioxidant activity followed
by prediction of their biological activity spectra for antianemic activity
using the chemistry software server PASS for prediction of biological
activity spectra (http://195.178.207.233/PASS/).

1. Designing of some nutraceutical based linear and cyclic peptide molecules
based on rational structure based drug design.
2. Docking of modeled peptides with the receptor (Glutathione reductase) by
using software- Glide (version: 5.0).
3. Checking the ADME profile of best docked compounds with the help of
software- QikProp (version: 3.1).
4. Synthesis of selected peptides by liquid phase peptide synthesis method.
5. Synthesis of iron peptide chelates.
6. Characterization of the synthesized compounds:
• Physicochemical characterization (M.P, Rf value)
• Spectral analysis (FT-IR, NMR and Mass)
7. To evaluate of antioxidant activity of synthesized peptides followed by
prediction of biological activity spectra.

Experimental work
Designing of new molecule
and docking studies
Prediction of ADME
properties of compound
Prediction of biological
activity
Synthetic studies

MOLECULAR MODELING AND DOCKING STUDIES
•Docking is the process of fitting of the ligand into the receptor.
•It does not only give an idea about how the ligand is going to bind with the
receptors but also about the extent to which conformational changes can be
brought in the receptor structure.
Softwares used :-
Maestro – Glide 8.3
Chemdraw ultra 10.0
Chem3D ultra 10.0
Qikprop version3.1
PDB file used :- 1GRE (Substrate crystal structure of human glutathione
reductase enzyme complexed with FAD as a ligand)

Ligand preparation
Ligand structures were prepared by
using software chemdraw ultra10.0 and
chem3D ultra and saved in .mol file
Structures were imported in Maestro
programme.

Energy minimization was
carried out corresponding
low energy 3D conformer of
ligands.
Ligands with least energy were
selected for docking.

Protein preparation
The X-ray crystal structure of glutathione
reductase enzyme (pdb code: 1GRE) was
selected from Protein Data Bank
(www.rcsb.org/pdb) into Maestro.
Workflow →Protein Preparation Wizard →
Preprocess
Preparation was done by deleting substrate cofactor
& water molecule
Energy minimization of receptor was done.
[Application → Macromodel → Minimization →
mini → Start]

Ligand-Receptor Docking
Active site of the protein was defined by generating the
Grid
[Application → Glide → Receptor grid generation]
Application → Glide → Ligand docking.
After standard precision (SP) docking, Extra
precision (XP) docking was done.

PREDICTION OF ADME PROPERTIES :
ADME properties of designed compounds were found out by using software Qikprop
version 3.151
SYNTHETIC STUDIES :
Synthesis of peptides
Amino acids can be considered as having two main functionalities to manipulate, i.e.
the amino and carboxyl terminals. Functional groupings are also present in the side
chain of many of the principal amino acids. These functionalities must be protected so
that they do not interfere with the formation of the peptide bond.52
With respect to peptide bond formation there are found main steps,
1.) Protection 3.) Coupling
2.) Activation 4.) Selective deprotection

NH2
OH
O
R
2
NH
OH
O
R
2
P
2
NH
Cl
O
R
2
P
2
NH2
OH
O
R
1
NH2
O
O
R
1
P
1
Amine
protection
Activation of
carboxylic group
Carboxyl
protection
NH
O
O
NH
P
2
R
2
R
1
O
P
1
NH2
O
O
NH
R
2
R
1
O
P
1
Selective deprotection
of amino group
NH
O
OH
R
2
P
3
N
O
O
NH
R
2
R
1
O
P
1NH
R
3
H
O
P
3
Coupling
General Method For Synthesis Of Linear
Peptides52,53
R1, R2, R3 are alkyl chain of amino
acids. P1, P2, P3 are protecting
groups to respective amino acid.
(Pthaloyl protection)

C
C
C
CC
H
HC
HC
H
C
O
O
O H2N CH C
CH3
OH
O
Alanine
Phthalic anhydride
C
NCH
O
HO C
C
C
C
H
C
CH
CH
C
H
O
O
phthaloyl alanine
H3C
C
N CH
O
O
C
C
C
C
C
H
HC
HC
H
C
O
O
CH3
P
O
OEt
OEt
Activation
PCl5
Et-OH
STEP:1
STEP:2
Anhydride moiety (A)
STEP:3
Protection
heating on sand bath
Coupling
Triethylamine is added to maintain
pH 7
temp: below 5ºC
NH2
CH
H2
C
C
CHHN
N
O
OH
Histidine
C
N CH
O
C
C
C
CC
H
HC
HC
H
C
O
O
CH3
NH
HC
H2C
C
C
OH
CH
N
H
C
HN
O
A +
Phthaloyl Ala-His
till it melts
Scheme of synthesis of linear tripeptide (Ala-His-Cys)

Phthaloyal Ala-His
PCl5
Et-OH
Anhydride moiety (C)
STEP:5
C Cysteine Phthaloyal Ala-His-Cys
(D)Triethylamine is added to maintain
pH 7
Temp. : below 5ºC
Coupling
STEP:6
D
Deprotection
NH2NH2/ HCl
C NH2
C
H
H2C
C
O
NH2
OC
H
NH
C
H2
C
C
CHHN
HC
N
O
C
NH
C
HH2C
SH O
OH
Ala-His-Cys
STEP:4

Scheme of synthesis of cyclic peptide52,53
N
O
O
CH2CONHCH2CONHCH2COOC2H5 NH2CH2CONHCH2CONHCH2CONHNH2
Phthaloyl Tripeptide Ethyl Ester
NH2CH2CONHCH2CONHCH2CON3
Tripeptide azide
H2C
HN
C
O C
H2
N
H
C
O
CH2
NH
O
C

Preparation of phthaloyl alanine:54
• A mixture of purified phthalic anhydried (2.2gm, 0.015mol) and alanine
(1.25gm, 0.015mol) was heated on sand bath, within 25 minutes the mixture
melted.
• Heating was continued for another 15 min. The reaction mixture was cooled,
the residue was dissolved in ethanol and filtered off, water was added to the
clear filtrate till turbid and then the solution was cooled at 0ºC for 24 hours.
• Fine crystals of phthaloyl alanine separated were filtered and dried and their
melting point and Rf value were found out for their physical characterization.
Protection of α-Amino Group

Preparation of Ala-His-Cys :
Step-1: (Condensation of Phthaloyl Ala-His)
The CPE reagent was added to phthaloyl Alanine (1.095gm, 0.005mol)
and stirred to a clear solution. Then (0.078gm, 0.005 mol) of Histidine
was added to the mixture and stirred to a clear solution. To this mixture,
triethylamine was added to maintaining the pH 7 and the reaction
temperature kept below 5ºC. This mixture was then left for 6 hrs at 0oC,
the product was filtered out and washed with solvent ether and dried.
Peptide synthesis:54

Step-2: (Condensation of second amino acid)
The CPE reagent was added to phthaloyl ala-his (0.356gm, 0.001mol) and stirred to
a clear solution. Then (0.121gm, 0.001 mol) of cysteine was added to the mixture
and stirred to a clear solution. To this mixture, triethylamine was added to
maintaining the pH 7 and the reaction temperature kept below 5ºC.This mixture
was then left for 6 hrs at 0oC, the product was filtered out and washed with solvent
ether and dried.
Step-3: Deprotection
Phthaloyl ala-his-cys (0.32, 0.0007mol.) was dissolved in ethanol (25 ml).
Hydrazine hydrate (0.1 ml, 70 %) was added to it and the mixture was heated on a
steam bath for 2 hrs. The reaction mixture was cooled, acidified with conc. HCl
and again heated on water bath at 50oC for 1 hr. Phthaloyl-hydrazide was removed
by filtering it out. The filtrate was neutralized with triethylamine to liberate free
peptide which is held back in solution. Removal of triethylamine was carried out
under vaccum distillation followed by crystallization from aqueous ethanol gave
the tripeptide as colourless crystal.

General scheme for synthesis of Iron-Peptide chelates55:
The chelate-containing solution was evaporated in a rotary evaporator,
product is dried and stored at 25-270C.
Insoluble form of Fe+3-Peptide complex was obtained by adjusting the pH
to 3.5 with 0.1N HCl.
Addition of FeCl3.6H2O was stopped when free Fe+3 is detected in the
mixture after reaction with potassium thiocyanate(KSCN).
2.5gm of FeCl3.6H2O was slowly added with constant mixing to a solution
containing the peptide (10%w/v) at 270C.
pH 7.8 maintained with 0.1N NaOH.
Fe+3 and thiocynate show intense red color.

Determination of DPPH radical scavenging activity:
The standard (Ascorbic acid) and samples (synthesized peptides) were prepared in
different concentrations 50, 100, 150, 200, 250µg/ml with methanol and then 2ml of
sample was added to 2ml of 0.15mM DPPH in 100ml methanol. The mixture was
allowed to stand at room temperature in the dark for 30 min. The absorbance of the
mixture was measured at 517nm using a spectrophotometer (Shimadzu, Japan). The
control was conducted in the same manner where methanol was used instead of sample.
DPPH radical scavenging activity was calculated as per following equation:
RSA (%) = (Acontrol −Asample)/Acontrol ×100, where Asample is the absorbance of sample
and Acontrol is the absorbance of the control. The IC50 value was defined as an effective
concentration of peptide that is required to scavenge 50% of radical activity. All the
compounds were compared with standard (Ascorbic acid) for antioxidant activity.
Antioxidant activity56,57,58:

The PASS internet tools were used for prediction of biological activity of
synthesized compounds. The input MDL Mol files (*.mol) file of compounds were
drawn with the help of Chemdraw Ultra 10.0 and Chem3D ultra 10.0 softwares. This
software gave Pa and Pi ratio (active and inactive ratio) in Pa > 30%, Pa > 50% and
Pa > 70% levels.
Pa (probability "to be active") estimates the chance that the studied
compound is belonging to the sub-class of active compounds (resembles the
structures of molecules, which are the most typical in a sub-set of "actives" in PASS
training set).
Pi (probability "to be inactive") estimates the chance that the studied
compound is belonging to the sub-class of inactive compounds (resembles the
structures of molecules, which are the most typical in a sub-set of "inactives" in
PASS training set).
The PASS tools available at “http://195.178.207.233/PASS/ pre-dict.php”
were used in April 2012.
PREDICTION OF BIOLOGICAL ACTIVITY SPECTRA59:

We have designed some linear and cyclic small peptides containing
different amino acid combinations of Alanine, Glycine, Asparagine,
Glutamic acid, Histidine, Cysteine, Proline and Glutathione. The designed
compounds were docked into crystal structure of Glutathione reductase
(pdb: 1GRE) which is the most accurate structure available. The interaction
energy between designed molecule and receptors were calculated and the
results are presented in the Table-4.1. The score represented in terms of
Gibbs free energy (∆G).

NAME OF LIGAND CHEMICAL NAME
DOCKING SCORE
IN SP MODE
DOCKING SCORE
IN XP MODE
FAD Flavine Adanine Dinucleotide -13.86 -14.03
SSPS1 Ala-Asn-Gsh -12.58 -13.20
SSPS2 Ala-Asn-Glu-Asn -11.48 -11.74
SSPS3 Ala-Tyr-Gln-Glu -9.04 -8.97
SSPS4 Ala-Trp-Gsh -10.23 -9.98
SSPS5 Ala-Gsh-Pro-His -8.63 -9.08
SSPS6 Ala-His-Gsh -8.53 -7.55
SSPS7 Ala-Pro-Gsh -11.67 -12.32
SSPS8 Ala-His-Cys -11.96 -12.62
SSPS9 C(Ala-His-Cys) -10.69 -11.79
SSPS10 C(Ala-Asn-Gsh) -8.01 -7.98
SSPS11 C(Ala-Asn-Glu-Asn) -3.05 -5.59
Table 4.1: Docking scores of designed linear and cyclic peptides in 1GRE (Glutathione
Reductase) receptor grid:

NAME OF LIGAND CHEMICAL NAME
DOCKING SCORE
IN SP MODE
DOCKING SCORE
IN XP MODE
SSPS12 C(Ala-Pro-Gsh) -7.89 -8.12
SSPS13 C(Ala-His-His-Gsh) -5.82 -6.03
SSPS14 Gly-Asn-Gsh -11.02 -11.59
SSPS15 Gly-His-Gsh -10.84 -9.95
SSPS16 Gly-Trp-Gsh -11.45 -11.78
SSPS17 Gly-Ser-Val-Cys-Ser -8.59 -9.87
SSPS18 Gly-Gsh-Pro-His -7.93 -8.79
SSPS19 Gly-Asn-Glu-Asn -7.86 -8.86
SSPS20 Gly-His-Pro-Glu -7.54 -8.83
SSPS 21 Gly-Pro-His-His -11.90 -12.04
SSPS22 C(Gly-Pro-His-His) -6.33 -7.02
SSPS23 C(Gly-Trp-Gsh) -4.71 -5.33
SSPS24 C(Gly-Asn-Gsh) -5.32 -5.04

Figure 4.1: Crystal structure of Glutathione reductase (1GRE) in ribbon form

XP Glide-predicted pose of molecule SSPS1. Active site amino acid residues and inhibitor are
represented as ball and sticks. While the inhibitor SSPS1 is colored with the atoms as carbon: green,
hydrogen: cyan, nitrogen: blue, and oxygen: red. Hydrogen bond interactions are represented by
yellow dotted lines.
SSPS1 is forming six hydrogen bonds with Histidine 52, Histidine 129, Alanine 115, Serine 51,
Asparagine 294, Glutamic acid 50 and Aspartic acid 331. The distance of hydrogen bonding is
1.91A0, 2.297A0, 2.184A0, 1.978A0, 2.154A0, 2.067A0 and 2.082A0 respectively.(Fig. 4.2)
Figure 4.2: Docking of SSPS1 with 1GRE

Figure 4.3: Docking of SSPS9 with 1GRE
XP Glide-predicted pose of molecule SSPS9. Active site amino acid residues and inhibitor are
represented as sticks. While the inhibitor SSPS9 is colored with the atoms as carbon: green,
hydrogen: cyan, nitrogen: blue, and oxygen: red. Hydrogen bond interactions are represented by
yellow dotted lines.
SSPS9 is forming three hydrogen bonds with Glycine 37, Threonine 156 and Serine 51. The
distance of hydrogen bonding is 1.507A0, 1.734A0 and 2.275A0 respectively.(Fig. 4.3)

ADME PROPERTIES PREDICTION:
Compound
Molecular
weight
QPlogPo/w QPlogS
Human oral
absorption
%Oral
absorption
SSPS1 492.50 -2.115 -0.576 2 33.66
SSPS2 446.41 -3.028 +0.261 1 26.03
SSPS3 509.50 -3.440 -0.280 1 16.79
SSPS4 564.6 -3.110 -0.099 1 22.79
SSPS5 612.6 -2.870 -0.039 2 24.21
SSPS6 515.54 -1.942 -1.536 1 26.06
SSPS7 475.52 -1.814 -1.095 1 35.05
SSPS8 372.40 -1.984 -1.708 1 24.95
SSPS9 354.38 -3.712 +0.299 2 67.48
SSPS10 474.48 -2.302 -0.962 2 40.88
SSPS11 428.39 -1.841 -1.574 2 45.66
Table 4.2: Predicted ADME properties of designed molecules:

Compound
Molecular
weight
QPlogPo/w QPlogS
Human oral
absorption
%Oral
absorption
SSPS12 457.50 -2.535 -1.520 2 45.39
SSPS13 634.66 -1.767 -1.587 2 54.32
SSPS14 478.78 -1.530 0.712 1 37.52
SSPS15 501.51 -0.532 -0.292 2 47.10
SSPS16 550.58 -1.801 -0.449 2 49.98
SSPS17 451.49 -1.690 +0.791 1 50.05
SSPS18 598.60 -0.420 -0.428 2 47.06
SSPS19 432.38 -2.604 +1.304 2 44.90
SSPS20 438.43 -1.059 +0.064 2 35.32
SSPS21 446.46 +0.018 -0.969 2 55.23
SSPS22 428.44 -3.120 -0.456 2 65.54
SSPS23 532.56 -2.436 -0.845 2 56.54
SSPS24 460.46 -2.541 -0.554 2 52.63

In table 4.2-
QP log Po/w: Predicted octanol /water partition coefficient; Range, -2.0 to 6.5
QP logS: Predicted aqueous solubility, log S. S in moles/liter is the concentration of the solute in a
saturated solution that is in equilibrium with the crystalline solid ,log S; Range, -6.5 to 0.5
Human Oral Absorption: Qualitative; 1→Low, 2→Medium, 3→High
Percent Human Oral absorption: 0 to 100% scale; [>80%→ High, <20%→ Poor]
From results, obtained by Qikprop software, we can see that cyclization of peptide improves its oral
absorption. All cyclic peptides i.e SSPS9 to SSPS13 and SSPS22, SSPS23, SSPS24 shows more
predicted % oral absorption than linear peptides i.e SSPS1 to SSPS 8 and SSPS14 and SSPS21. For
e.g. SSPS8 i.e. Ala-His-Cys is a linear tripeptide. Its predicted oral absorption is 24.95% when its
cyclic form i.e SSPS9 has predicted oral absorption 67.48%.

SYNTHETIC STUDIES:
1. SSPS1 (Ala-Asn-Gsh)
Structure:
H2C
HC
NHC
CH2H2C
CHHN
C OH
O
O C
HN CH2
C OH
OO
C
HN
HC
H2C
C
O
NH2
O
C
NH2
CHH3C
O
SH
Chemical formula : C17H28N6O9S
Molecular weight : 492.50
Melting point : 2880C
Rf value (Butanol:Acetic acid:Water) : 0.56
IR spectral data:
3404.47cm-1 (N-H str of amide), 2980cm-1 (C-H str of alkane), 2679.21cm-1 (O-H str of –COOH),
1739.85cm-1 (C=O str of –COOH), 1616.40cm-1 (C=O str of amide), 1485.24cm-1 (C-H def), 1178.55cm-1
(C-N str)

2. SSPS2 (Ala-Asn-Glu-Asn)
Structure:
IR spectral data:
3420cm-1 (N-H str of 20 amide), 2891.39cm-1 (C-H str of alkane), 2617.49cm-1 (O-H str of –
COOH), 1925.02cm-1 (C=O str of –COOH),1680.05cm-1 (C=O str of amide), 1475.59cm-1 (C-H
def ), 1178.55cm-1 (C-N str)
C
H2N C
H
CH3
O
H
N
C
H
H2
C
C O
H2N
O
C
H
N
C
HH2C
C O
H2N
O
OHC
NH
C
H
H2
C
C
H2
C
O
HO
O
Chemical formula : C16H26N6O9

3. SSPS7 (Ala-Pro-Gsh)
Structure:
IR spectral data:
3412.19cm-1 (N-H str of amide), 2800.73 cm-1(C-H str of alkane), 2677.29 cm-1(O-H str of –
COOH), 1928.88cm-1 (C=O str of –COOH),1606.76cm-1 (C=C str), 1737.93cm-1 (C=O str of –
COOH), 1479.45cm-1 (C-H def ), 1176.62cm-1 (C-N str)
C
NH2
H
C
H3C
O
N
C
H
H2
C CH2
CH2
O
OC
HN
CH2
CHO
O
HCH2C
HS
NH
C
H2C CH2
CH NH
C
OH
O
O

4. SSPS8 (Ala-His-Cys)
Structure:
IR spectral data:
3361.48cm-1 (N-H str of amide), 2891.39cm-1 (C-H str of alkane), 2492.11 cm-1 (O-H str of –
COOH), 1739.85 cm-1 (C=O str of –COOH), 1685.84 cm-1 (C=O str of amide), 1475.59 cm-1 (C-H
def), 1176.62cm-1 (C-N str), 2492.11cm-1 (S-H str)
C NH2
HC
H2C
C
O
NH2
OC
H
N
H2
C
C
CHHN
HC
N
O
C
NH
CH
H2C
SH
O
OH

5. SSPS9 [C (Ala-His-Cys)]
Structure:
IR spectral data:
3254.02cm-1 (N-H str of amine) ,2943.47cm-1 (C-H str of alkane), 2629.06cm-1 (O-H str of –
COOH), 1722.49cm-1 (C=O str of –COOH) , 1577.82cm-1 (C-H def ), 1174.69cm-1 (C-N str),
2494.04cm-1 (S-H str)
C NHH
C
H2C
C
O
NH2
O
C
HN
CH
H2
C
C
H
C
HN
HC N
O
C
H
N
HC
CH2
SH
O

6. SSPS14 (Gly-Asn-Gsh)
Structure:
IR spectral data:
3390.97cm-1 (N-H str of amide), 2891.39cm-1 (C-H str of alkane), 2492.11cm-1 (O-H str of –
COOH), 1739.85cm-1 (C=O str of –COOH), 1475.59cm-1 (C-H def ), 1176.62 cm-1(C-N str),
2492.11cm-1 (S-H str)
O
C
HN
C
H2
C
OH
O
CH
CH2
HS
N
H
C
C
H2
H2
C
CH
H
N
C
HO O
O
C
NH2
CH2O
CHN
CH
H2C
C
O
NH2
O

7. SSPS16 (Gly-Trp-Gsh)
Structure:
IR spectral data:
3448.84cm-1 (N-H str of amide), 2891.39cm-1 (C-H str of alkane), 2675.36cm-1 (O-H str of –
COOH), 1737.92cm-1 (C=O str of –COOH), 1477.52cm-1 (C-H def), 1176.62cm-1 (C-N str),
2494.04(S-H str)
O
C
HN
CH2
C
OH
O
HC
H2C
SH
H
N
C
CH2
H2C
C
H
HN
C
OH
O
O
C
NH2
H2C
O
C
H
N
CH
H2
C
C
CHHN
CHC
HC
H
C CH
C
O

8. SSPS21 (Gly-Pro-His-His)
Structure:
IR spectral data:
3360cm-1 (N-H str of amide), 2748.65 cm-1(C-H str of alkane), 2492.11 cm-1(O-H str of –COOH),
1925.02 cm-1 (C=O str of amide), 1477.52 cm-1 (C-H def), 1176.62 cm-1 (C-N str)
C
NH
HCH2C
C
CH
H
N
HC
N O
HO
C
NH
CH CH2
C
HC
H
N
CH
NO
C
N HC
CH2
C
H2
H2C
O
C
H2N
H2C
O
Chemical formula : C19H28N8O5

9. Fe+3- C (Ala-His-Cys) chelate:
Structure:
C N
H
C
H2C
C
O
NH2
O
C
N
CH
H2
C
C
H
C
HN
HC N
O
C
N
HC
CH2
SH
O
Fe
Chemical formula : C13H15FeN6O4S

•Antioxidant assays:
Antioxidant assays were performed using DPPH and percentage scavenging is shown in
the table 4.3 below.
Table 4.3: Comparison of IC50 values of synthesized peptides in DPPH radical
scavenging assay:
Sr. No. Compounds
PERCENTAGE SCAVENGING % IC50
(µg/ml)50 µg/ml 100 µg/ml
1. Ascorbic acid 89.91 91.92 24.30
2. SSPS1 88.26 94.26 27.25
3. SSPS2 66.42 82.16 60.87
4. SSPS7 71.64 82.58 55.72
5. SSPS8 72.87 83.76 50.93
6. SSPS9 63.74 81.10 65.61
7. SSPS14 79.45 88.25 42.98
8. SSPS16 67.30 84.14 57.06
9. SSPS21 70.14 86.18 53.49

Figure 4.4: Comparison of IC50 values of synthesized peptides in DPPH radical
scavenging assay
Among the eight synthesized peptides, SSPS1 is showing the highest radical scavenging
activity (IC50= 27.25 µg/ml) than other compounds as compared to Ascorbic acid (IC50=
24.30 µg/ml) as a standard. Also the percentage scavenging activity of all peptides was
increasing consistently with increasing concentration.

PREDICTION OF BIOLOGICALACTIVITY:
Table 4.4: Prediction of possible biological active spectra of compound for antianemic
activity (Possible activities at Pa > 30%)
Compound Pa Pi
SSPS1 0.826 0.012
SSPS2 0.846 0.004
SSPS7 0.687 0.013
SSPS8 0.564 0.046
SSPS9 0.763 0.024
SSPS14 0.858 0.004
SSPS16 0.765 0.009
SSPS21 0.753 0.007
Fe+3-(SSPS9) chelate 0.831 0.009
Ferrous bisglycinate 0.580 0.039
Ferrous fumarate 0.559 0.048
Ferrous gluconate 0.327 0.238
Ferrous sulphate 0.547 0.054
*Standard drugs available in market are shown as Bold.

In table 4.4,
 Pa= possibility to be active
 Pi = possibility to be inactive
From result shown in table 4.4, we can see that almost all the synthesized compounds
have shown better “Pa” scores than the available drugs for anemia as standard
compounds. If further animal experiment studies will be carried out for antianemic
activity or for iron absorption then these compounds may show good activity.

The present work aims to study the status of research going on in the field of
nutraceutical and anemia. Peptides help to improve intestinal absorption of iron
and their antioxidative property helps to prevent the lysis of erythrocytes.
Keeping these things in mind, we have designed and synthesized nutraceutical
based linear and cyclic antioxidant peptides and iron chelates.
The purpose of the present work was to do the CADD studies by docking a
number of linear and cyclic peptide ligands to glutathione reductase enzyme and
to evaluate the binding free energy between a target protein and a ligand. On the
basis of the scoring results some linear and cyclic peptides are synthesised. The
biological activity of the synthesised compounds was then studied by the use of
PASS software.

 Our designed molecules show good docking scores comparable with FAD. The
docking score of FAD is -13.86, while that of SSPS1, SSPS8 and SSPS21 are -12.58, -
11.96 and -11.90 respectively.
The ADME properties of designed compound were checked by using software
QikProp 3.0 which shows that cyclic peptide has better oral absorption. SSPS8 i.e Ala-His-
Cys is a linear tripeptide, its predicted oral absorption is 24.95% when its cyclic form i.e
SSPS9 has predicted oral absorption 67.48%.
Based on these studies and feasibility for the synthesis, some new linear and cyclic
peptides were synthesized in the laboratory by the liquid phase synthesis method.
 Chlorophosphate ester was used as condensing agent.
 Phthalic anhydride was used for functional group protection.
 Hydrazine hydrate was used as cleavage agent for protecting groups.

The synthesised compounds were characterised by determination of melting point, Rf
values and spectral data using FTIR and NMR.
The synthesized compounds were characterized using FTIR instrument has
shown characteristic peaks of the functional groups i.e 3500-3300 cm-1 (N-H str of amide),
2950-2850 cm-1 (C-H str of alkane), 3300-2500 cm-1 (O-H str of –COOH), 1715cm-1 (C=O
str of –COOH), 1700-1670 cm-1 (C=O str of amide), 1500-1430 cm-1 (C-H def), 1200-
1100 cm-1 (C-N str).
 Antioxidant assays were performed for all the synthesized compounds and they show
good free radical scavenging activity as compared to the standard. SSPS1 compound has
shown almost same activity (IC50=27.25µg/ml) like the standard Ascorbic acid
(IC50=24.30µg/ml).
 Predicted values of antianemic activity were investigated by using the chemistry
software server PASS for prediction of biological activity spectra
(http://195.178.207.233/PASS/). The compounds were predicted to have much better
antianemic activity as compared to the standard compounds.

This study has a scope for the development of nutraceutical which have peptide
and iron combination and can be useful in prevention or treatment of anemia in future.
This work can be further extended to check bioavailability of iron if given in amino acid
or peptide chelate form.
Also, industrial or semi-industrial scale processing techniques are available for
fractionation and isolation of such components from foods like milk. These components
present an excellent source for different applications in health-promoting foods. Much as
a result of this development, the nutraceutical industry has achieved a leading role in the
development of functional foods and has already commercialised many milk protein and
peptide-based products which can be consumed as part of a regular healthy diet. It can be
envisaged that in the near future more similar products may be launched on worldwide
markets. They can be targeted to infants, elderly and immune-compromised people as
well as to maintain good health status and prevent diet-related chronic diseases.

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