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Comparative anti anaemic activity of Murraya koenigii (Linn.) Spreng leaves and its combination with Emblica officinalis in aluminium chloride induced anaemia using rodents.

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  • East West Group of Institutions Bangalore

Abstract

ABSTRACT−Murrraya koenigii (curry leaves) and its combination with Emblica officinalis (amla) were evaluated for their antianaemic activity in aluminium chloride induced anaemic animals. Rats were divided into 7 groups of 6 each. Group 1 was given normal saline and served as control and all other groups were given 0.5 mg/kg b.w of AlCl3 for 30 days to induce anaemia. Group 2 served as positive control and Group 3 was treated with synthetic iron (40mg/kg) and served as standard .Group 4 and 5 were treated with different doses (200 and 400 mg/kg b.w.) of aqueous extracts of curry leaves respectively. Whereas Group 6 and 7 were treated with combination of aqueous extract of curry leaves and amla fruit (200 and 400 mg/kg b.w.) respectively. All the treatments were given orally and continued upto 30 days. On 0 and 31 day blood samples were collected by retroorbital puncture and haematological parameters such as haemoglobin (Hb) concentration, RBC count, Mean Haemoglobin Volume (MHV), serum iron, ferritin, and antioxidant parameters such as lipid peroxidation (LPO), superoxide dismutase (SOD) and catalase (CAT) were estimated. Results showed that both curry leaves and its combination with amla showed significant anti anaemic and antioxidant activity but compared to curry leaf extract alone, its combination with amla showed better activity. This may be due to synergistic action of herbs when used in combination and this combination may be an alternative to synthetic iron therapy in anaemia. Keywords: Aluminum chloride, anaemia, haematological parameters, antioxidant.
International Journal of Advanced Research in
Medical Science
Volume: 1 Issue: 1 12-May-2014,ISSN_NO: xxxx-xxx
Comparative anti anaemic activity of Murraya
koenigii (Linn.) Spreng leaves and its
combination with Emblica officinalis in
aluminium chloride induced anaemia using
rodents.
Purnima A1, Vamsee Veena A2, Renuka S3 and Rajendra R4
Professor, Dept. of Pharmacology, KLE University’s College of Pharmacy, Bangalore, India,1
Asst .Professor, Dept. of Pharmacology, KLE University’s College of Pharmacy, Bangalore,
India2
Research Scholar, Dept. of Pharmacology, KLE University’s College of Pharmacy, Bangalore,
India3
Green Chem Herbal Extracts and Formulations, Bangalore, India4.
ABSTRACTMurrraya koenigii (curry leaves) and its combination with Emblica officinalis (amla)
were evaluated for their antianaemic activity in aluminium chloride induced anaemic animals. Rats were
divided into 7 groups of 6 each. Group 1 was given normal saline and served as control and all other
groups were given 0.5 mg/kg b.w of AlCl3 for 30 days to induce anaemia. Group 2 served as positive
control and Group 3 was treated with synthetic iron (40mg/kg) and served as standard .Group 4 and 5
were treated with different doses (200 and 400 mg/kg b.w.) of aqueous extracts of curry leaves
respectively. Whereas Group 6 and 7 were treated with combination of aqueous extract of curry leaves
and amla fruit (200 and 400 mg/kg b.w.) respectively. All the treatments were given orally and continued
upto 30 days. On 0 and 31 day blood samples were collected by retroorbital puncture and haematological
parameters such as haemoglobin (Hb) concentration, RBC count, Mean Haemoglobin Volume (MHV),
serum iron, ferritin, and antioxidant parameters such as lipid peroxidation (LPO), superoxide dismutase
(SOD) and catalase (CAT) were estimated. Results showed that both curry leaves and its combination
with amla showed significant anti anaemic and antioxidant activity but compared to curry leaf extract
alone, its combination with amla showed better activity. This may be due to synergistic action of herbs
when used in combination and this combination may be an alternative to synthetic iron therapy in
anaemia.
Keywords: Aluminum chloride, anaemia, haematological parameters, antioxidant.
1. INTRODUCTION
Anaemia is defined as reduction of haemoglobin concentration, RBC count, or packed cell
volume to below normal levels. As a result the oxygen carrying capacity of blood is reduced.
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Anaemia is a common disease that affects people of all ages but elder population, young women
of child bearing age and infants are at greater risk. There are many types of anaemia and in all
types there is decrease in circulating RBC [1,2].
Aluminium is ubiquitous element present in every food product. When it is accumulated in
humans it can cause bone diseases, CNS disorders, hepatotoxicity and anaemia. Aluminium
leads to impaired erythropoiesis and can produce peroxidative damages in erythrocyte membrane
leading to haemolysis. Iron is an essential nutrient and critical for haemoglobin synthesis [3].
Ferritin is major cellular iron storage protein, and by sequestering iron, it protects cells from iron
induced oxidative damage. Several lines of evidence suggest that aluminium interferes in
fundamental ways with iron and ferritin metabolism [4].
Murrraya koenigii is commonly called ‘curry patta’, belongs to family Rutaceae, is traditionally
used in India as spice for its characteristic flavour and aroma. The aromatic leaves are considered
as tonic, antihelminthic, analgesic, digestive and appetizer. The leaves are used for treatment of
piles, inflammation, itching, fresh cuts, dysentery, vomiting and dropsy. M.koenigii leaves
contains a range of active pharmacological agents including carbazole alkaloids, flavonoids,
furanocoumarins, terpenoids and tannins. It has been reported that curry leaves are rich source of
magnesium, zinc, iron and copper [5].
Emblica officinalis commonly called amla, belongs to Euphorbiaceae. Amla fruits are acrid,
cooling, refrigerant, astringent, diuretic and laxative. The fermented liquor prepared from fruits
is used in jaundice, dyspepsia and cough. E.officinalis fruits contain vitamin C, tannins,
flavonoids, ellagic acid, gallic acid, quercetin and other constituents which are responsible for its
immunomodulatory and anticancer activity [6].
In this context, we have under taken anti anaemic and antioxidant activity of curry leaves and its
combination with amla in aluminium chloride induced anaemia using rats.
2. MATERIALS AND METHODS
2.1 Animals
Healthy male Wistar albino rats (200-230g) were procured from Venkateshwara enterprises,
Bangalore and housed in polypropylene cages at KLE University’s College of Pharmacy,
Bangalore , animal house facilities and maintained under standard conditions (12 h light and dark
cycle, 22±2oC ,and 55±5% relative humidity) in accordance with CPSCEA guidelines. All
animals were given standard rat pellet diet and water ad libitum. The study protocol was
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Volume: 1 Issue: 1 12-May-2014,ISSN_NO: xxxx-xxx
approved by IAEC, KLE University’s College of Pharmacy, Bangalore (Ref No:
IAEC/07/PA/2011-12).
2.2 Plant extracts
Murraya koenigii leaf aqueous extract and its combination with Emblica officinalis fruit
aqueous extract were gift samples from Green Chem Herbal Extracts and Formulations,
Bangalore.
2.3 Chemicals
All chemicals used were of analytical grade, obtained from commercial source and synthetic iron
was a marketed product of Himalaya Drug House Ltd., Bangalore.
2.4 Experimental protocol
Animals were divided into 7 groups of six each. All animals, except normal group received 0.5
mg/ kg b.w of AlCl3 for 30 days by oral route. Rats received single oral dose of selected
treatment every day 15 minutes before administration of AlCl3 and treatments were as follows:
Group 1: Normal (Normal saline)
Group 2: Positive control (AlCl3alone)
Group3: Synthetic iron (40 mg/kg) + AlCl3
Group 4: Curry leaves (200 mg/kg) + AlCl3
Group 5: Curry leaves (400 mg/kg) + AlCl3
Group 6: Curry leaves + amla (200 mg/kg) + AlCl3
Group 7: Curry leaves + amla (400 mg/kg) + AlCl3
Aluminium chloride and extracts were dissolved in distilled water before administration.
Synthetic iron was in the form of syrup and all were given p.o. using oral feeding needle.
2.5 Biochemical estimations
Blood samples were collected on day 0 and 31 through retroorbital puncture under mild ether
anaesthesia. Fresh blood was immediately collected into heparinised tubes for routine
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haemotological parameters such as RBC count, Hb concentration and MCV[7,8]. A second
blood fraction was collected without anticoagulant and centrifuged at 5000 rpmfor 10 minutes
and used for determining iron content and ferritin in serum. Serum analysis of iron was done by
Atomic absorption spectrometer (Shimadzu AA 6300) and serum ferritin level estimation was
done by immuno chemiluminescence commercial kit. Haemolysate was prepared from
heparinized blood and used for determination of SOD [9], CAT [10] and LPO in terms of
TBARS [11 ].
2.6 Statistical analysis
Statistical analysis was done by One-way ANOVA followed by Dunnet multiple comparision
test. All values of p<0.05 were considered statistically significant.
3. RESULTS:
Oral AlCl3 treatment resulted in significant increase in MCV (p<0.001) and decrease (p<0.001)
in total erythrocytes count, blood hemoglobin (Hb), serum iron and ferritin levels compared to
normal group (Table1).
Both the extract treated groups at different doses showed a significant decrease (p<0.001) in
MCV and increase (p<0.001) in RBCs, haemoglobin content, serum iron and ferritin levels
compared to positive control group and these are not significantly differ from synthetic iron
treatment.(Table1). AlCl3 treatment increased lipid peroxidation (p<0.001) and decreased SOD
and CAT enzyme levels significantly (p<0.001) compared to normal group. Administration of
extracts significantly reversed lipid peroxidation and normalized SOD and CAT levels and this
did not significantly differ from synthetic iron treatment (Figure1,2,3).
Table 1: Haematological parameters in control rats and different treatment groups
S.N.
GROUPS
RBCs x 106
Hb(g/dl)
MCV(fl)
Serum iron µg/ml
Serum ferritin
ng/ml
1.
NC
7.28 ± 0.160
15.33 ± 0.331
47.48 ± 0.44
2.56± 0.143
186±0.00
2.
PC
4.82 ± 0.295 a***
7.86 ± 0.166 a***
61.32 ± 0.396 a***
1.17±0.04 a***
150±0.0 a***
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All values are mean ±SEM (n=6).*p<0.01 **p<0.05, ***p<0.0001. a= when PC was compared with NC,
b= when groups compared with PC,c= when groups compared with STD.NC= Normal Control, PC=
Positive Control (AlCl3 Treated), STD= standard (Synthetic Iron Treated), CL200= Curry leaves extract
treated (200mg/kg),CL400= Curry leaves extract treated (400mg/kg), CLA400= Curry Leaves + Amla
extract Treated (400mg/kg) and CLA400= Curry Leaves + Amla extract Treated (400mg/kg).
Fig 1: LPO measured as TBARS in RBC’s of control and different treatment groups
NC
PC
STD
CL 200
CL 400
CLA 200
CLA 400
0
2
4
6
8NC
PC
STD
CL 200
CL 400
CLA 200
CLA 400
a***
b*** b***,c*
b***,c**b***,c**
b***,c**
TBARS(nmoles/ml of blood)
3.
STD
8.02 ±0.106b***
16.02 ± 0.14b***
49.93 ± 0.136b***
3.42±0.08b***
180±0.00b***
4.
CL 200
6.83 ± 0.348b***,c**
12.76 ± 0.23b***,c*
46.27 ± 0.628b***
2.274±0.015(b,c)**
158±0.01(b,c)***
5.
CL 400
7.53 ±0.20b***
15.38 ±0.35b***
48.95 ± 0.295b***
2.665±2.57b***,c*
165±0.00 (b,c)***
6.
CLA 200
7.66 ± 0.145b***
14.36 ± 0.211b***
46.82 ± 0.192b***
2.882±0.03 b***,c*
171.0±0.01b***, c**
7.
CLA 400
8.03 ± 0.088b***
16.64 ± 0.224b***
48.25 ± 0.169b***
3.301±0.104b***
180.5±0.01b***
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All values are mean ±SEM (n=6).*p<0.01 **p<0.05, ***p<0.0001. a= when PC was compared with NC,
b= when groups compared with PC,c= when groups compared with STD. NC= Normal Control, PC=
Positive Control (AlCl3 Treated), STD= standard (Synthetic Iron Treated), CL200= Curry leaves extract
treated (200mg/kg),CL400= Curry leaves extract treated (400mg/kg), CLA400= Curry Leaves + Amla
extract Treated (400mg/kg) and CLA400= Curry Leaves + Amla extract Treated (400mg/kg).
Fig 2: SOD activity in RBC of control and different treatment groups
NC
PC
STD
CL 200
CL 400
CLA 200
CLA 400
0
20
40
60 NC
PC
STD
CL 200
CL 400
CLA 200
CLA 400
a***
b*** b*** b*** b*** b***
SOD(U/mg protein)
All values are mean ±SEM (n=6).*p<0.01 **p<0.05, ***p<0.0001. a= when PC was compared with NC,
b= when groups compared with PC,c= when groups compared with STD. NC= Normal Control, PC=
Positive Control (AlCl3 Treated), STD= standard (Synthetic Iron Treated), CL200= Curry leaves extract
treated (200mg/kg),CL400= Curry leaves extract treated (400mg/kg), CLA400= Curry Leaves + Amla
extract Treated (400mg/kg) and CLA400= Curry Leaves + Amla extract Treated (400mg/kg).
Fig 3: .CAT activity in RBC of control and different treatment groups
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NC
PC
STD
CL 200
CL 400
CLA 200
CLA 400
0
1
2
3
4
5NC
PC
STD
CL 200
CL 400
CLA 200
CLA 400
a*** b** b** b*** b*** b***
CAT(U/mg protein
All values as mean ±SEM (n=6).*p<0.01 **p<0.05, ***p<0.0001. a= when PC was compared with NC,
b= when groups compared with PC,c= when groups compared with STD. NC= Normal Control, PC=
Positive Control (AlCl3 Treated), STD= standard (Synthetic Iron Treated), CL200= Curry leaves extract
treated (200mg/kg),CL400= Curry leaves extract treated (400mg/kg), CLA400= Curry Leaves + Amla
extract Treated (400mg/kg) and CLA400= Curry Leaves + Amla extract Treated (400mg/kg).
4. DISCUSSION
The present results showed haematological modifications and decreased serum iron levels
associated with orally administered aluminum chloride in rats and the reversible effect of curry
leaves and its combination with amla on these modifications.
There are reports on the reduction in erythrocyte count, haemoglobin levels and hematocrit
values on oral administration of aluminum chloride in rats [12,13]. The present work supports
this statement as evidenced by results and our treatment with curry leaves and its combination
with amla reversed the above mentioned effects with AlCl3.
The ferritin levels measured usually have a direct correlation with the total amount of iron stored
in the body [14]. Ferritin levels indicate stores of iron and in this work there is a substantial
increase in ferritin levels more than synthetic iron by the samples tested which is an added
advantage in anaemic conditions.
Since curry leaves are a rich source of iron [5] and amla [6] contains ascorbic acid which
facilitates iron absorption, the synergistic effect seen with the combination in this work is
convincing.
The UNICEF report states, the possibility that certain individuals may at some time develop iron
overload is a risk-benefit question. Direct absorption of synthetic iron can upset the
gastrointestinal tract. Most commonly, the side effects viz., nausea or, more rarely, diarrhoea and
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constipation are untoward effects to who consumes direct iron tablets, which may be greatly
reduced by organic supplement of curry leaves with amla especially in the condition of
pregnancy. The present findings support the treatment in the conditions like insufficient dietary
intake and absorption of iron, and/or iron loss from intestinal bleeding, parasitic infection,
menstruation, etc. with curry leaves or its combination with amla.
Moreover the most significant cause of iron-deficiency anemia is parasitic worms: hookworms,
whipworms, and roundworms. The worms cause intestinal bleeding which isn't always
noticeable in faeces and is especially damaging to growing children and develops anaemia of
chronic disease (ACD) is also referred to as anemia of inflammatory response. This can nullify
by herbal supplements powerful antioxidant and anti-inflammatory activity.
AlCl3 caused damage was corrected by oral treatment with these herbs as they normalised the
levels of enzymatic components of antioxidants such as LPO, SOD and CAT in RBC’s of the
treated groups. The protective effect of curry leaves and its combination with amla against AlCl3
induced anaemia and increased levels of oxidative stress can be attributed to their antioxidant
property.
Thus, the present work reveals the fact that curry leaves or its combination with amla will protect
from the hazards of AlCl3 in rodents and can be thought as a supplement to synthetic iron
preparations. Since combination of curry leaves with amla enhanced the absorption of iron due to
its the ascorbic content which by itself is an antioxidant agent, this might be an added advantage
to use this combination in place of synthetic iron.
5. CONCLUSION
In conclusion, our findings support the fact that AlCl3 is capable of causing marked alteration in
some haematological parameters, inducing anemia and inhibiting the antioxidant components in
the RBC, curry leaves and its combination with amla will minimize these hazards. The increase
in iron and ferritin levels observed with curry leaves as well as curry leaves with amla is
comparable to synthetic iron and can be thought as an alternative to marketed synthetic iron
preparations. Further, of the two herbs tested, combination proved to be a better alternative than
curry leaves alone in alleviating the symptoms of anaemia produced by AlCl3.
6. REFERENCES
[1]. S.Sathyayea, Y.Bagula, S.Gupta, H.Kaura, R.Redkara. Hepatoprotective effects of aqueous
leaf extract and crude isolates of Murraya koenigii against in vitro ethanol-induced hepato-
toxicity model. Exp Toxicol Path. 2011; 63(6): 587-91.
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[2]. A.Muller, H.Jacobsen, E.Healy, S.Mcmickan et al. Hazard classification of chemicals
inducing haemolyticanaemia: An EU. Regul Toxicol Pharmacol. 2006; 45(3): 229-41.
[3]. MI.Yousef. Aluminium-induced changes in hemato-biochemical parameters, lipid
peroxidation and enzyme activities of male rabbits: protective role of ascorbic acid. Toxicol.
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[4]. J.Han, MA.Dunn. Effect of dietary aluminium on tissue non haeme iron and ferritin levels in
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[14].C.Itoa, M.Itoigawa, K Nakao, T. Muratac, M.Tsuboic, N. Kanedac, H.Furukawa. Induction
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BIOGRAPHY
Author: Dr.Purnima Ashok, M.Pharm, Ph.D.
E-mail: purnimaal1@yahoo.com.
Present designation: Professor & Head, Dept. of Pharmacology.
Also worked as principal from August 2006 to March 2009 at KLE’s University’s College of Pharmacy,
Bangalore. Have around 57 total publications of which 29 are national and 28 are international. Has
undertaken 25 R & D projects from different industries.
Awards and achievements:
Dr.P.S.Lalitha award for UG student (guide).
Best teacher award for the year 2011 from KLE University, Belgaum.
Presented a poster at Bangkok, Thailand-2006.
Co-chairman for Indian Pharmaceutical Congress, Varanasi - 2007.
Loc member - 46th annual conference of Indian Pharmacological Society,2013, Bangalore.
PATENT - Extraction of phytochemicals from Trichilia connaroides.
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Aluminum toxicity is well documented but the mechanism of action is poorly understood. In renal failure patients with aluminum overload, disturbances in iron metabolism leading to anemia are apparent. Few animal models, however, have been used to study the effects of dietary aluminum on iron metabolism. The purpose of this study was to determine if dietary aluminum exposure alters tissue iron and ferritin concentrations in the chick, as has been found in cultured human cells exposed to aluminum. Groups of day-old chicks were fed purified diets containing one of two levels of iron (control or high iron), and one of three levels of aluminum chloride in a 2 x 3 factorial design. Diets were consumed ad libitum for 1 week, then pair-feeding was initiated for 2 more weeks. A seventh group consumed a low iron diet ad libitum for comparative purposes. After the 3-week feeding period, samples of kidney, liver, and intestinal mucosa were analyzed for nonheme iron and ferritin concentrations by a colorimetric assay and SDS-PAGE, respectively. Results showed that dietary aluminum intake reduced iron stores in liver and intestine, but had no effect on nonheme iron levels in the kidney. Ferritin levels were reduced by aluminum intake in all tissues studied. The decreases in tissue ferritin levels were proportionately more than the decreases in tissue nonheme iron levels. This resulted in increased nonheme iron to ferritin ratios that amounted to as much as 140 and 525% in kidney and intestine, respectively. These findings are consistent with the interpretation that, in the growing chick, dietary aluminum can inhibit iron absorption, disrupt the regulation of tissue ferritin levels by iron, and potentially alter the compartmentalization and protective sequestration of iron within cells.
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For a long time, aluminium (Al) has been considered an indifferent element from a toxicological point of view. In recent years, however, Al has been implicated in the pathogenesis of several clinical disorders, such as dialysis dementia, the fulminant neurological disorder that can develop in patients on renal dialysis. Therefore, the present experiment was carried out to determine the effectiveness of l-ascorbic acid (AA) in alleviating the toxicity of aluminium chloride (AlCl3) on certain hemato-biochemical parameters, lipid peroxidation and enzyme activities of male New Zealand white rabbits. Six rabbits per group were assigned to 1 of 4 treatment groups: 0mg AA and 0mg AlCl3/kg body weight (BW) (control); 40 mg AA/kg BW; 34 mg AlCl3/kg BW (1/25 LD50); 34 mg AlCl3 plus 40 mg AA/kg BW. Rabbits were orally administered their respective doses every other day for 16 weeks. Evaluations were made for lipid peroxidation, enzyme activities and hemato-biochemical parameters. Results obtained showed that AlCl3 significantly (P<0.05) induced free radicals and decreased the activity of glutathione S-transferase (GST) and the levels of sulfhydryl groups (SH groups) in rabbit plasma, liver, brain, testes and kidney. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AlP), acid phosphatase (AcP), and phosphorylase activities were significantly decreased in liver and testes due to AlCl3 administration. While, plasma, liver, testes and brain lactate dehydrogenase (LDH) activities were significantly increased. Contrariwise, the activity of acetylcholinesterase (AChE) was significantly decreased in brain and plasma. Aluminium treatment caused a significant decrease in plasma total lipids (TL), blood haemoglobin (Hb), total erythrocytic count (TEC) and packed cell volume (PCV), and increased total leukocyte count (TLC) and the concentrations of glucose, urea, creatinine, bilirubin and cholesterol. Ascorbic acid alone significantly decreased the levels of free radicals, TL, cholesterol, glucose and creatinine, and increased the activity of GST, SH groups, Hb, TEC and PCV. While, the rest of the tested parameters were not affected. Also, the present study showed that ascorbic acid can be effective in the protection of aluminium-induced toxicity.