international standard serial number (issn): 2319-8141 ...ijupbs.com/uploads/33. rpa1300104.pdf ·...

International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(3): May-June 2013

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES

Pharmaceutical Sciences Research Article……!!!

Received: 01-05-2013; Accepted: 02-06-2013

EVALUATION OF BENEFICIAL EFFECTS OF MEDICAGO SATIVA (ALFALFA) ON

HEMATOLOGICAL PARAMETERS IN IRON-OVERLOAD CONDITIONS

Rinal Patel1*, Dr. Pravin Tirgar

2

1Student, 4

th Sem., M. Pharm (Pharmacology) School of Pharmacy, RK University, Rajkot,

Gujarat, India. 2

M. Pharm (Pharmacology), Ph. D, Associate Professor, Department of Pharmacology School

of Pharmacy, RK University, Rajkot, Gujarat, India.

KEYWORDS:

Iron, Medicago sativa,

desferoxamine,

Hematological

parameters, bleeding

time, clotting time.

For Correspondence:

Patel Rinal Sanjaybhai*

Address:

School of Pharmacy, RK

University,

Kasturbadham-Tramba,

Rajkot-Bhavnagar

Highway,

Rajkot-360020.

Mb. No: 9428755721.

ABSTRACT

Iron plays an important role in biology, forming complexes with

molecular oxygen in hemoglobin and myoglobin and used at the active

site of many important redox enzymes dealing with cellular respiration

and oxidation. In medicine, iron overload indicates accumulation of iron

in body from any cause. The most important causes are thalassemia,

hereditary haemochromatosis (HHC) and transfusional iron overload,

which can result from repeated blood transfusion. The emergence of new

orally effective iron chelators gives hope to both patients as well as

physicians caring for thalassemia patients. Herbs with higher phenol and

flavanoids content have been noted to possess antioxidant and blood

purifier. Aim of present study is to investigate in-vivo beneficial effects

of Medicago sativa on hematological parameters and bleeding and

clotting time in iron overload conditions. Beneficial effects on

hematological parameters by methanolic (250 mg/kg) and water (500

mg/kg) extracts of Medicago sativa along with standard drug

desferoxamine were assessed against iron-dextran induced iron overload

models in Wistar rats which results in condition of chronic iron overload

found in thalassemia patients. At the end of 15 and 30 days of in-vivo

trial, blood were collected by retro orbital and all hematological

parameters (RBC, WBC, platelet counts and erythrocyte indices, bleeding

and clotting time) were estimated. There were significant increase in

hematological parameters and decreased in bleeding and clotting time

were observed compared to iron overloaded rats. Data of our study

confirmed that the methanol and water extracts of Medicago sativa have

beneficial effects on hematological parameters in iron overload

conditions. Thus M. sativa is very beneficial in management of iron

overload disorders like thalassemia, haemochromatosis like conditions.

And due to effects on bleeding and clotting time possess beneficial

activity in thrombocytopenia and hemophilia.

405 Full Text Available On www.ijupbs.com

International Standard Serial Number (ISSN): 2319-8141

1. INTRODUCTION:

Iron is an absolute requirement for most forms of life, including humans and most bacterial

species, because plants and animals all use iron. Iron is essential to life because of its unusual

flexibility to serve as both an electron donor and acceptor. Iron can also be potentially toxic[1].

Most well-nourished people have 4 to 5 grams of iron in their bodies. Of this, about 2.5 g is

contained in the hemoglobin needed to carry oxygen through the blood, and most of the rest is

contained in ferritin complexes that are present in all cells, but most common in bone marrow,

liver, and spleen. The liver's stores of ferritin are the primary physiologic source of reserve iron

in the body [2].

The human body needs iron for oxygen transport. That oxygen is required for the production

and survival of all cells in our bodies. Human bodies tightly regulate iron absorption and

recycling. Iron is such an essential element of human life, in fact, that humans have no

physiologic regulatory mechanism for excreting iron [3].

In medicine, iron overload disorders are diseases caused by the accumulation of iron in the

body. Iron toxicity results when the amount of circulating iron exceeds the amount of

transferrin available to bind it. The type of acute toxicity from iron ingestion causes severe

mucosal damage in gastrointestinal tract, among other problems. Iron overload is one of the

major causes of morbidity in all patients with severe forms of thalassemia, regardless of

whether they are regularly transfused. A variety of other iron overload diseases are present.

These are thalassemia, sideroblastic anemia, abnormal red cell production (dyserythropoiesis),

iron overload secondary to IV therapy, chronic liver disease secondary to alcohol, porphyria

cutanea tarda [3].

Excess iron in vital organs, even in mild cases of iron overload, increases the risk for liver

disease (cirrhosis, cancer), heart attack or heart failure, diabetes mellitus [4-5] osteoarthritis,

osteoporosis, metabolic syndrome [6], hypothyroidism [7], hypoparathyroidism [8],

hypogonadism [8-9], impaired growth [10], numerous symptoms and in some cases premature

death. Iron mismanagement resulting in overload can accelerate such neurodegenerative

diseases as Alzheimer’s, early-onset Parkinson’s, Huntington’s, epilepsy and multiple sclerosis

[11-12]. Iron overload is major problem found in thalassemia major patients. Thalassemia is a

hereditary anemia resulting from defects in hemoglobin production [13]. β-Thalassemia, which

is caused by a decrease in the production of β-globin chains, affects multiple organs and is

associated with considerable morbidity and mortality [14]. Accordingly, lifelong care is

required [15], and financial expenditures for proper treatment are substantial [16].


http://www.ijupbs.com/


Thalassemia is among the most frequent genetic disorders worldwide; frequency of the globin

variants in the world’s population is 4.83 %, including 1.67 % of the population who are

heterozygous for a-thalassemia and b-thalassemia. In addition, 1.92 % carries sickle

hemoglobin, 0.95 percent carry hemoglobin E, and 0.29 % carry hemoglobin C. Consequently,

the worldwide birth rate of people who are homozygous or heterozygous for symptomatic

globin disorders, including a-thalassemia and b- thalassemia, is not less than 2.4 per 1000

births, of which 1.96 have sickle cell disease and 0.44 have thalassemias [17]. In untransfused

patients with severe ß-thalassemia, abnormally regulated iron absorption results in increases in

body iron burden that may, depending on the severity of erythroid expansion, vary between 2

and 5 grams per year[18,19]. Regular transfusions may double this rate of iron accumulation.

Synthetic agents like desferrioxamine and deferiprone used for the treatment of iron overload in

thalassemia are accompanied by serious side effects and certain limitations including need for

Parenteral administration, arthralgia, nausea, gastrointestinal symptoms, fluctuating liver

enzyme levels, leucopenia, agranulocytosis and zinc deficiency and obviously the heavy cost. In

addition, they are not suitable for use during pregnancy [20-22]. Compared to synthetic drugs,

herbal preparations are frequently less toxic with fewer side effects. Therefore the search for

more effective and safer treatment of thalassemia and other iron overload conditions are new

area of research.

Thus objective of present study is to evaluate new herbal drug having protective effects on

hematological parameters in iron overload condition and with least adverse effects.

The plant Medicago sativa Linn. (Alfalfa) belongs to family Fabaceae is locally known as

‘buffalo herb of Lucern’, in gujarati its known as ‘Rajko’ or ‘Gadab’ [23].

Evidence have also found that Medicago sativa have iron chelation activity and also presence of

phenol and flavanoid content and antioxidant activity [24]. M. sativa contains other chemical

constituents like cystine, phytosterol, saponin, coumarins, amino acids, Vit. A, K, C, E, Zn,

Selenium, minerals.

Medicago sativa is taken for a wide range of conditions, including allergies, morning sickness,

arthritis, digestion, gout, anemia, rheumatism, blood clotting agent, blood purifier, tooth decay,

bone strengthener and urinary problems [25]. Medicago sativa is useful for breaking down

toxins in the blood system [26].

Objective of my work is find out beneficial effect of various extracts of Medicago sativa on

hematological parameters, bleeding and clotting time in iron-dextran induce iron overload in

rats.




Materials and methods:

Collection and Authentification

The arial part of Medicago sativa was collected from local area of Rajkot region, Rajkot,

Gujarat. The crude drug was authentified by Prof. Vishal Muliya, botanical department, Christ

College, Rajkot, Gujarat, India. Herbarium authentified sample of M. sativa was prepared

according to standard procedure (Herbarium No. SOP/COG/1/2013).

Preparations of Various Extracts

The crude drugs was collected, dried and pulverized to fine powder. Powder was macerated first

with cold water and then with methanol. The extracts were filtered and concentrated under

reduced pressure and adjusted with known volume. Resulting extracts were transferred into

evaporating disc and left overnight in a stream of air to produced dry residue, which was further

used for evaluation process [27].

Selection of Animals

Either sex Wistar rats of weighing 200-220 g were used for the study. The animals were

collected from Animal House, Department of Pharmacology, School of Pharmacy, RK

University Rajkot, India. The animals were placed at random and allocated to treatment groups

in polypropylene cages with paddy husk as bedding. Animals were housed at a temperature of

24±2˚C and relative humidity of 30 – 70 %. A light and dark cycle was follow. All animals

were fed on standard balance diet and provided with water ad labium.

All the experimental procedures and protocols used in study was

reviewed and approved by the Institutional Animal Ethical Committee (IAEC) and care of

laboratory animals were taken as per the guidelines of Committee for the purpose of control and

supervision of experiments on animals (CPCSEA), Government of India (Protocol No.

RKCP/COL/RP/13/39).

Induction of Iron overload

Either sex of Wistar rats of initially weighing 200-220 gm were used for present study. The rats

were given six Intraperitoneal injections of iron-dextran (12.5 mg/l00 g body wt.) evenly

distributed over a 30 days of period which results in condition of chronic iron overload[28,29].

Control rats were injected with an equal volume of dextrose at the same time. The experimental

animals were divided into five groups, (n=6).

• Group 1(n=6):Normal control received dextrose solution (NC)

• Group 2(n=6): Disease control treated with Iron Dextran (DC) (12.5mg/l00g body wt.)

Intraperitoneal injections for 30 days.




• Group 3(n=6): Disease control treated with Desferoxamine(DCD) (40 mg/kg, p.o.,/ day)

• Group 4(n=6):Disease control treated with aqueous extract of M. sativa (DCWM)

(500mg/kg/day p.o. per day)

• Group 5(n=6):Disease control treated with methanolic extract of M. sativa

(DCMM)(250mg/kg p.o. per day )

Blood samples were collected on 15 and 30 days under fasting conditions and hematological

parameters like, Hb, RBC, WBC, platelet counts, erythrocyte indices, bleeding and clotting

time were measured in iron overloaded rats.

Biochemical Parameters

At the end of 15 days and 30 days of treatment period, blood samples were collected from rats

under fasting conditions, from retro orbital plexuses using light ether anesthesia, in clean dry

centrifuge tubes. Blood samples were subjected for estimation of –

Hematological Parameters

a. Hemoglobin content

b. Total RBC, MCV, MCH, MCHC

c. Total WBC count

d. Differential WBC count Neutrophil, Lymphocyte, Eosinophil, Basophile counts

e. Platelet counts

f. Erythrocyte indices

f. Bleeding tine and clotting time

The time elapsed between the moment blood escapes from vessel and the cessation of its flow is

defined as the bleeding time the usual bleeding time is 1-3 minutes and clotting time is 4-10

minutes. Bleeding time is prolonged during thrombocytopenia.

During coagulation sol form of the blood is changed to gel from. The time elapsed between the

moment of escape of blood outside the vessel and the observation of physical change is taken as

clotting time. Collected blood samples were allowed to clot for 30 min at room temperature and

serum was separated by cooling centrifugation at 5000 rpm for 20 min and stored at –20˚ C

until the analysis was carried out using three part cell counter.

Statistical Analysis:

To checking the significance of data, following statistical tests were performed:

ANOVA: to see the variability within all the groups.

Tuckey’s test: for the same purpose mentioned in above test.

INSTAT software: to derive all the statistical terms like Standard Error of Mean (SEM),

ANOVA, P – value, Degree of freedom, Standard deviation, etc.




Results: Hematological Parameters

Beneficial effects of M. sativa on hemoglobin and RBC parameters on iron overloaded rats

Iron overloaded group rats exhibited significant decrease in Hb count and RBC count as

compared to normal control group rats suggesting toxic effect of iron excess on Hb and RBC

synthesis. 15 days treatment with desferoxamine, water extract and methanol extract in iron

over loaded rats produced significant increase in Hb levels and RBC counts. Over all, there was

improvement in Hb level and RBC count after treatment with M. sativa extracts after 15 and 30

days in iron overload rats (Table 1).

TABLE 1: ERYTHROPOETIC EFFECTS OF VARIOUS EXTRACTS OF ALFALFA

ON DAY 15 AND 30 ON IRON OVERLOAD INDUCED RATS.

Hematological

Parameters

NC

(n=6)

DC

(n=6)

DCD

(n=6)

DCWM

(n=6)

DCMM

(n=6)

15

days

30

days

15

days

30

days

15

days

30 days 15 days 30 days 15

days

30 days

Hb gm/dl 14.9 ±

0.35

14.3 ±

0.90

11.7 ±

0.52**

11.7 ±

0.51**

14.5 ±

0.32#

14.4 ±

0.73 #

14.9

±0.44##

14.8 ±

0.38 ##

14.5±

0.30 #

14.6 ±

0.34#

RBC 106/µl 8.44

± 0.30

8.18 ±

0.21

6.91 ±

0.29*

6.57 ±

0.41*

8.33 ±

0.41 #

7.78 ±

0.40#

8.48 ±

0.44#

8.07 ±

0.28 #

8.00 ±

0.46#

7.97 ±

0.32

Values are expressed as Mean ± S.E.M

*- significantly different from normal control (p < 0.05)

**- significantly different from normal control (p < 0.01)

# - significantly different from diseases control (p < 0.05)

# #- significantly different from diseases control (p < 0.01)

NC: Normal control received dextrose solution

DC: Disease control treated with iron dextran (12.5mg/l00g body wt.)

DCD: Disease control treated with desferoxamine (40 mg/kg, p.o., per day)

DCWM: Disease control treated with water extract of M. sativa (500 mg/kg, p.o., per day)

DCMM: Disease control treated with methanol extract of M. sativa (250 mg/kg, p.o., per day).

Beneficial effects of M. sativa on defense system (WBC count) and platelet count on iron

overloaded rats

Accumulation of iron in body leads to suppression of bone marrow resulting in reduction of

total and differential leucocytes counts. This was observed in iron overloaded rats of disease

control group as compared to placebo group. After 15 and 30 days treatment with

desferoxamine, water extract and methanol extract of alfalfa in iron over loaded rats, total WBC

count was significant increased (Table 2). The increase in leukocyte count indicates that alfalfa




may have stimulating effect on bone marrow and also on synthesis of all types of leucocytes.

Similarly, treatment with methanol extract and water extract of M. sativa significantly increased

platelet counts in iron over loaded group rats as compared to diseases control group rats. These

data indicate beneficial effect of M. sativa in platelet deficiency disorders (Table 2).

TABLE 2: EFFECT OF M. SATIVA ON PARAMETERS RELATED TO PLATELET

AND NATURAL DEFENSE MECHANISM OF THE BODY.

Hematologic

al

Parameter

NC

(n=6)

DC

(n=6)

DCD

(n=6)

DCWM

(n=6)

DCMM

(n=6)

15

days

30

days

15

days

30

days

15

days

30

days

15

days

30 days 15 days 30

days

Platelet

(103/µl)

1305

±

184.9

1281

±

78.43

852.8

±

44.1*

708.6

±

103.6*

1202

±186.

4 ##

1165

± 101

##

1083

±27.77

#

1155.8

± 59.3

##

1016.1

± 78.29

#

1049.6

±

107.1#

Total WBC counts

WBC

(103/µl)

10.20

±

0.10

10.07

±

0.17

6.77

±

0.32*

6.83

±

0.67*

9.95

±

0.7#

10.07

±

1.26#

9.70

±

1.22#

10.02

±

0.47#

9.55

±

0.92#

9.68

±

0.33#

Differential WBC counts

Neutrophil

(103/ µl)

2.94±

0.38

2.97

±

0.40

1.68

±

0.17*

1.58

±

0.20*

2.65

±

0.18#

2.67

±

0.28 #

2.58

±

0.28

#

2.50

±

0.23 #

2.30

±

0.33

#

2.35

±

0.32

#

Lymphocyte

s (103/ µl)

1.50

±

0.11

1.51

±

0.14

0.97

±

0.07 *

0.94

±

0.10*

1.49

±

0.14#

1.51

±

0.16#

1.48

±

0.14#

1.50

±

0.12#

1.32

±

0.17#

1.43

±

0.17#

Eosinophil

(103/ µl)

0.13

±

0.02

0.13

±

0.02

0.06

±

0.01*

0.05

±

0.01*

0.10

±

0.02#

0.09

±

0.01#

0.09

±

0.01#

0.10

±

0.01#

0.10

±

0.01#

0.11

±

0.02#

Basophil

(103/ µl)

0.12

±

0.01

0.11

±

0.01

0.06

±

0.01*

0.06

±

0.01*

0.08

±

0.01#

0.08

±

0.01#

0.07

±

0.01

0.08

±

0.01#

0.07

±

0.01

0.07

±

0.01













Beneficial effects of Medicago sativa on different hematological parameters on iron

overload rats

Iron overloaded rats showed significant reduction in HCT (%), MCV(fL), MCH(pg),

MCHC(g/dl), RDW-CV(%), PDW(fL), MPV (fL), and P-LCR(%) counts as compared to

normal control rats which indicates toxic effects of iron on blood (Table 3). After 15 and 30

days treatment with water and methanol extracts of M. sativa showed increase in these

parameters which indicates beneficial effects of M. sativa on various blood disorders.

TABLE 3: BENEFICIAL EFFECTS OF M. SATIVA ON DIFFERENT

HEMATOLOGICAL PARAMETERS ON IRON OVERLOADED RATS.

Hematologi-

cal

Parameters

NC

(n=6)

DC

(n=6)

DCD

(n=6)

DCWM

(n=6)

DCMM

(n=6)

15

days

30

days

15

days

30 days 15

days

30

days

15

days

30

days

15

days

30

days

HCT (%) 41.9 ±

1.36

44.4±

2.68

34.8 ±

1.96*

36.1 ±

1.47*

40.4 ±

1.36#

40.4±

3.59#

41.4 ±

2.79#

43.0 ±

2.50#

40.0 ±

2.74#

42.9 ±

2.10#

MCV(fL) 61.4 ±

5.70

62.3 ±

6.4

43.5 ±

2.4*

39.8±

4.07*

57.6 ±

2.4

60.2 ±

6.76 #

58.6 ±

4.41

60.8 ±

5.38#

54.9 ±

6.4

57.2 ±

3.70

MCH (pg) 22.1 ±

1.19

22.2 ±

1.26

15.4 ±

1.72*

15.5 ±

1.75*

19.7 ±

1.07#

19.4 ±

0.76

19.3 ±

1.57

19.7 ±

1.84#

16.5 ±

1.67

18.3 ±

1.10

MCHC

(g/dl)

32.6 ±

1.90

32.9 ±

1.59

25.2 ±

2.03*

24.6 ±

3.41*

29.1 ±

0.71

29.3 ±

1.65

28.4 ±

1.57

30.9 ±

2.37#

27.4 ±

1.89

29.7 ±

1.66

SRDW-CV

(%)

19.9 ±

0.59

20.1 ±

0.94

16.1 ±

0.37*

16.9 ±

1.46*

17.5 ±

0.50

17.9 ±

0.80

17.9 ±

0.33

19.5 ±

1.20#

17.2 ±

0.60

17.7 ±

0.57

PDW (fL) 8.28 ±

0.47

8.83 ±

0.34

5.93 ±

0.64*

5.98 ±

0.69*

6.82 ±

0.48

7.60 ±

0.90#

7.43 ±

0.45

8.23 ±

0.72#

#

7.00 ±

0.53

7.88 ±

0.80#

MPV (fL) 12.43

± 0.06

12.1 ±

0.36

10.6 ±

0.43

**

7.82 ±

0.62*

11.3 ±

0.32#

#

10.5 ±

0.86#

11.3 ±

0.48#

#

10.4 ±

0.85#

11.0 ±

0.38#

#

10.3 ±

0.61

P-LRC (%) 11.17

± 0.37

10.8 ±

0.39

8.83 ±

0.62*

8.62 ±

0.50*

9.00 ±

0.46

9.85 ±

0.78

9.28 ±

0.63

9.98 ±

1.35

8.73 ±

0.62

9.93 ±

1.15



**-significantly different from normal control (p< 0.01)







DCMM: Disease control treated with methanol extract of M. sativa (250 mg/kg, p.o., per day) .




Beneficial effects of M. sativa on bleeding time and clotting time on iron overloaded rats

Platelets play an important role in clotting and bleeding. Disease control group rats which

received Iron dextran showed significant increase in bleeding and clotting time as a result of

reduction in platelet counts compared to normal healthy group rats. Treatment with methanol

and water extract of M. sativa produced significant reduction in bleeding and clotting time in

disease suffering rats. Increases in bleeding and clotting time in rats were significantly

prevented by treatment with methanol and water extract of M. sativa. Thus M. sativa seems to

help in reducing bleeding and clotting time, near to normal (Table 4).

TABLE 4: BENEFICIAL EFFECT OF METHANOL AND WATER EXTRACT OF M.

SATIVA ON BLEEDING AND CLOTTING TIMES ON IRON OVERLOADED RATS.

Blood

Parameters

NC DC DCD DCWM DCMM

Bleeding

time (sec)

88.3

±

3.3

145.7

±

6.1***

99.4

±

8.8##

104.7

±

10.8##

107

±

5.5##

Clotting

time(sec)

110.0

±

3.4

188.3

±

0.11***

120.3

±

10.1###

127.7

±

10.3###

135.5

±

5.2##


***- significantly different from normal control (p < 0.001)

## - significantly different from diseases control (p < 0.01)

# ##- significantly different from diseases control (p < 0.001)






DISCUSSION:

Iron overload conditions are major problem arise in the iron overload disorders like

thalassemia, haemosiderosis, neuroferritinopathy, anemia of chronic disease, and iron overload

neuronal disorders [30]. Patient with chronic iron overload conditions required regular

transfusion of blood because of abnormalities are found in hematological parameters in order to

improve quality of life [31]. Physiology of human body indicates that, there is no excretory

system for elimination of iron from body so, in iron overload conditions excess iron is

deposited as hemosiderin and ferritin in the liver, spleen, endocrine organs and myocardium

[32].




The accumulation of iron damage the organ results in poor liver, kidney and cardiac function,

thus because of tissue damage complications such as heart failure, endocrine abnormalities like

diabetes, hypothyroidism, liver failure and ultimately early death and imbalance in

hematological parameters [4,5].

Water and methanol extracts of M. sativa were subjected to in-vivo testing on rats and their iron

chelating activities were compared with that of desferoxamine. Iron overload was induced in

rats by intra peritoneal injections of iron-dextran (12.5 mg/l00 g body wt.) evenly distributed

over 30 days period, which resulted in condition of chronic iron overload that resembled with

other iron overload disorders [28,29].

Iron overload is disorder of blood, thus blood parameters were estimated after 15 and 30 days of

treatment period of study. For that blood samples were collected by retro orbital method and

hematological parameters were estimated.

While, there were significant decrease in hemoglobin content and RBC count in iron

overloaded rats compared to normal control rats. These parameters were significantly increased

in water extracts group and methanol extract group, compared to disease control group at the

end of 15 days and 30 days treatment so, M. sativa improves synthesis of Hb and RBC in iron

overloaded rats.

There were significant increase in total and differential WBC counts in water and methanol

extracts groups compared to diseases control group. These data indicate that M. sativa improves

defense mechanism of body in iron overloaded patients.

There was significant increase in platelet counts in water and methanol extract groups as

compared to diseases control group. Thus, this effect on platelet counts decrease bleeding and

clotting time in water and methanol extract treated groups as compared to disease control group.

Thus, data indicate beneficial effect of M. sativa in thrombocytopenia and hemophilia.

REFERENCES:

1. Andrews N, Disorders of iron metabolism. (2000), New Engl J Med. Related

correspondence, published in NEJM, 342(17): 1293-1294.

2. Schrier SL, Regulation of iron balance.( 2005), Up-to-Date (Subscription required)

Accessed December.

3. Schrier SL, Bacon BR, Iron overload syndromes other than hereditary hematochromatosis.

Accessed December 2005.

4. Grundy RG, Woods RA, Savage MO, et al. (1994), Relationship of endocrinopathy to iron

chelation status in young patients with thalassaemia major. Arch. Dis. Child., 71:128.




5. Kwan EYW, Lee ACW, Li AMC, et al. (1995), Across-section al study of growth, puberty

and endocrine function in patients with thalassaemia major in Hong Kong. J. Paediatr Child

Health, 31:83.

6. Gubatti V, Borgna-pignatti C, (1994), Clinical manifestation and therapy of transfusional

haemosiderosis. Baifferer Clin. Haematol., 7: 919-940.

7. Landau H, Matoth I, Landau-Cordova Z, et al. (1993), Cross-sectional and longitudinal

study of the pituitary-thyroidaxis in patient with thalassaemia major. Clin. Endocrinol, 38:

55.

8. McIntosh N, (1976), Endocrinopathy in thalassaemia major Arch Dis. Child, 51:195.

9. Maurer HS, Lloyd-Still JD, Ingrisano C, et al. (1988), A prospective evaluation of iron

chelation therapy in children with severe –thalassaemia : Asix-yearstudy. Am J Dis. Child.,

142:287.

10. DeSanctis V, Katz M, Vullo C, (1994), Effect of different treatment regimes on linear

growth and final height in b-thalassaemia major. Clin. Endocrinol, 40:91.

11. Kontoghiorghes GJ, Weinberg ED, (1995), Iron: mammalian defense systems, mechanisms

of disease, and chelation therapy approaches. Blood Review, 9: 33- 45.

12. Wangastiniotis M, Modell B, (1998), Global epidemiology of hemoglobin disorders. Ann.

N. Y. Acad. Sci., 850: 251-259.

13. Higgs DR, Thein SL, Woods WG, (2001a), The molecular pathology of the thalassemias.

In: Weatherall, D.J. and B. Clegg. eds. The thalassaemia syndromes. 4th ed. Oxford,

England: Blackwell Science, 133-191.

14. Cunningham MJ, Macklin EA, Neufeld EJ, (2004), Complications of b- thalassemia major

in North America. Blood, 104: 34-39.

15. Old JM, Olivieri NF, Thein SN, Et al. (2001), Diagnosis and management of thalassaemia.

In: Weatherall, D.J. and B. Clegg. eds. The thalassaemia syndromes. 4th ed. Oxford,

England: Blackwell Science, 630-685.

16. Karnon J, Zeuner D, Brown J, et al. (1999), Lifetime treatment costs of β-thalassaemia

major. Clin. Lab. Haematol, 21: 377-385.

17. Angastiniotis M, Modell B, (1998), Global epidemiology of hemoglobin disorders. Ann. N.

Y. Acad. Sci., 850: 251-259.

18. Pippard MJ, Callender ST, Warner GT, et al. (1997), Iron absorption and loading in beta-

thalassaemia intermedia, Lancet 2: 819–21.

19. Pootrakul P, Kitcharoen K, Yansukon P, (1998), The effect of erythroid hyperplasia on iron

balance. Blood, 71:1124–1129.




20. Hebbel RP, Leung A, Mohandas N, (1990), Oxidation-induced changes in microheological

properties of the red cell membrane. Blood, 76:1015-1022.

21. Grinberg LN, Rachmilewitz EA, Kitrossky N, et al. (1995), Hydroxyl radical generation in

ß-thalassemia red blood cells. Free Rad. Biol. Med., 18: 611-615.

22. Kukongviriyapan V, Somparn N, Senggunprai L, et al. (2008), Endothelial Dysfunction

and Oxidant Status in Pediatric Patients with Hemoglobin E-beta Thalassemia. Pediatr.

Cardiol, 29: 130-135.

23. Prajapati ND, Dr Purohit SS, Sharma AK, et al. (2007), A handbook of Medicinal plants: A

complete source book., 336.

24. Rana MG, Katbamna RV, Padhya AA, et al. (2010), In vitro antioxidant and free radical

scavenging studies of alcoholic extract of Medicago sativa l. Rom. J. Biol. Plant boi, 55(1):

15–22.

25. Mikaili P, Shayegh J, (2011), Medicago sativa: a historical ethnopharmacology and

etymological study of the alfalfa. Research opinions in animal & veterinary sciences, roavs ,

1(9): 614-618.

26. Xie Z, (2008 ), Antioxidant activity of peptides isolated from alfalfa leaf protein

hydrolysate. Food Chemistry , 111: 370-376.

27. Geissman A, (1955), Modern Methods of Plant Analysis,Heidelberg, Berlin, Springer

Verlag, 3: 471.

28. Addison GM, Beamish MR, Hales CN, et al (1972), An Immunoradiometric Assay for

Ferritin in the Serum of Normal Subjects and Patients with Iron Deficiency and Iron

Overload. J Clin Pathol., 25: 326-329.

29. British journal of hematology (1990), Studies of an oral iron chelator, 75: 274-277.

30. Anuradha Mudipall A, Srirama K, Nadadur SS, (2008), Iron transport & homeostasis

mechanisms: Their role in health & disease. Indian J Med Res., 128: 533-544.

31. Gutteridge JMC, Rowley DA, Halliwell B, (1981), Superoxide-dependent formation of

hydroxyl radicals in the presence of iron salts. Biochem. J., 199: 263 -265.

32. Fleming RE, Bacon BR, (2005) Orchestration of iron homeostasis. New England Journal of

Medicine, 352(17): 1741-1744.



international standard serial number (issn): 2319-8141 ...ijupbs.com/uploads/33. rpa1300104.pdf ·...

Documents