Upload
lykien
View
235
Download
1
Embed Size (px)
Citation preview
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].
406 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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.
407 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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.
408 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
• 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.
409 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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
410 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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
Values are expressed as Mean ± S.E.M
*- significantly different from normal control (p < 0.05)
# - 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).
411 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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
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) .
412 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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##
Values are expressed as Mean ± S.E.M
***- significantly different from normal control (p < 0.001)
## - significantly different from diseases control (p < 0.01)
# ##- significantly different from diseases control (p < 0.001)
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).
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].
413 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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.
414 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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.
415 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
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.
416 Full Text Available On www.ijupbs.com