Turkish Journal of Zoology

TITLE

Elimination of excessive iron by Emblica officinalis in the iron overloaded rabbits

Amna SHAHID* and Azizullah

Department of Zoology, Government College University, Katchery Road, Lahore,

Pakistan.

*Corresponding Author
Email address: a_shahid13@[Link]

1
 Abstract

The elimination of excessive iron by Emblica officinalis (Amla) in the

artificially iron overloaded rabbits has been studied in the present work. The iron

overloading in higher amounts becomes toxic, as it can affect the liver, heart and

pancreas and irritate the stomach and gut, causing constipation or diarrhea. In this

experiment, 28 large male rabbits (Oryctolagus cuniculus) with body weights of 1.5

-2.0 kg were used. They were divided into four groups: control, iron overloaded

control, E. officinalis treated and iron overloaded treated with E. officinalis group.

The data shows a significant increase in the iron concentration in serum of rabbits

after daily administration of 0.3 ml/lb of Jectosol-Plus injections each for four

weeks (P < 0.01). The treatment of iron overloaded rabbits with the herb, E.

officinalis @ 250 mg/kg of body weight daily for 4 weeks led to an effective

elimination, as it showed significant decrease on the elevated serum iron levels of

iron overloaded rabbits (P < 0.05). The results indicated that E. officinalis can be

used for the elimination of excessive iron from the body of thalassemic patients in

which iron accumulates in their bodies by repeated blood transfusions.

Key words: Emblica officinalis, serum iron, artificially iron overloaded rabbits.

2
Introduction

Iron is an essential constituent of all the living organisms as it is involved in

the formation of some vital biological compounds in the body such as

haemoglobin, myoglobin, cytochromes etc which are involved in carrying out

important functions in the body (Aisen, 2001). The total content of iron in the adult

body averages about 3-5 g, of which 60-70% is present in the blood as hemoglobin.

Muscle tissue contains about 3% of iron as myoglobin and rest is stored in the liver

as ferritn, in other tissues as haemosiderin, and as the blood transport protein

transferrin.

The iron concentration in the blood serum and various organs is strictly

regulated through controlling the absorption of dietary iron in the gastrointestinal

tract. It, thus seldom increases beyond the average value in healthy individuals.

However in certain abnormal situations, iron accumulates in the body and its

excessive amount becomes toxic and damages the body.

Iron overload (hemochromatosis) can be classified as primary or secondary.

Primary hemochromatosis is a recessive autosomal genetic disorder that alters a

protein involved in the regulation of iron absorption. Most of these patients are

homozygous or heterozygous for this mutation and have iron absorption two or

three times higher than that of the unaffected population (Andrews, 1999).

Any other nongenetic cause of iron accumulation within the organs is known

3
as secondary hemochromatosis. The causes of secondary hemochromatosis

include cirrhosis, myelodysplastic syndrome, anemias related to ineffective

erythropoiesis (e.g. thalassemia) and exogenous increase by ingestion, parenteral

infusion, or multiple transfusions (Pietrangelo, 2003).

There is no specific mechanism to remove excess iron from the body. The

body iron levels are mainly regulated from the absorption of iron from the gut.

Fecal, urinogenital and integumental losses account for 4 mg/day of iron loss.

Menstruation, blood donation, and pregnancy also can cause significant iron loss.

Variations in iron status and requirements are influenced by individual genetic

makeup as well as by differences in sex, age, dietary habits, erythropoietic activity

and menstrual losses. The deficiency of iron may cause anemia and due to

defective synthesis of hemoglobin or hemolytic phenomenon, hyperferremia and

thalassemia result.

Thalassemia is a group of inherited blood disorders that varies widely in

severity involving decreased and abnormal production of protein hemoglobin. A

physician, Thomas B. Cooly, first recognized this condition in 1995. Hemoglobin

contains two different kinds of protein chains named as alpha and beta chains.

Any deficiency in these chains causes abnormalities in the formation, size, and

shape of red blood cells. There are two types of thalassemia: alpha-thalassemia

and beta-thalassemia. Alpha-thalassemia is the result of changes in the genes for

the alpha globin component of hemoglobin. Alpha thalassemias result from

mutations in these genes. Beta-thalassemia is the best-known type of thalassemia

4
and is also called Cooley's anemia. It is caused by a change in the gene for the beta

globin component of hemoglobin.

A thalassemic patient receives 20 units of blood per annum. Each unit of

blood (500 ml) contains 200-250 mg of iron. With regular transfusions, there is a

progressive accumulation of iron in the liver, heart, pancreas, bones and other

organs. This excess iron needs to be eliminated or else it causes early death in the

thalassemic children (Prati, 2000).

There is no physiological mechanism for eliminating excess iron from the

body, so patients have to undergo chelation therapy. Chelation therapy involves a

medication with drugs that can bind to the excess iron and can thus detoxify it and

remove the excess iron from the body. People may take chelators as an oral

supplement or an intravenous infusion.

Parenteral administration of chelators such as desferrioxamine B that help

patients excrete excess stores of iron can eliminate iron overload caused by

multiple blood transfusions. It also shows some side effects such as growth

retardation, hearing loss, impaired vision and bone deformities (Brill et al., 1991).

To produce a therapeutic effect parenteral administration of the drug in prolonged

infusions is needed which is very cumbersome. There is a dire need to search for

some oral and suitable iron chelator which should be cheap and without side

effects.

In some recent studies, products of plants like Terminalia belerica and

Terminalia chebula have been shown to reduce iron level in the blood sera of

5
artificially iron overloaded rabbits (Khurram, 2005; Shehzadi and Azizullah, 2009).

In the present study, it was planned to study the role of the herb “E.

officinalis” in elimination of excessive iron, which is essentially needed in certain

therapeutic situations like thalassemic patients when they have to be given

repeated blood transfusion to make good their deficiency of good hemoglobin.

Emblica officinalis (Amla) is a prestigious herb, which is extensively used in

making ayurvedic medicines because of its miraculous actions. E. officinalis is

recommended as a dietary supplement alone or in the triphala formula. Triphala is

a Ayurvedic herbal preparation consisting of equal parts of the fruits of Emblica

officinalis (Amla), Terminalia chebula (Haritaki) and Terminalia belerica (Bibhitaki).

E. officinalis is also known as the “fruit of heaven” or “nectar fruit”. It is so

called because it is rich in many desirable properties. Studies have indicated the

efficacy of E. officinalis fruit to protect against elevated cholesterol levels and the

resultant arterial damage (Mathur et al., 1996). Flavonoids from E. officinalis and

Mangifera indica effectively reduced lipid levels in serum and tissues of rats (Anila

and Vijayalakshmi, 2002). E. officinalis contains higher Vitamin C (ascorbic acid)

content increases the antioxidant activity (Scartezzini et al., 2006).

In another study, the extract of leaves of the E. officinalis has been used for

antiinflammatory and antipyretic treatments in rats (Asmawi et al., 1993). The

extract of E. officinalis possesses antisecretory, antiulcer and cytoprotective

properties. Oral administration of E. officinalis extract at doses of 250 mg/kg and

500 mg/kg significantly inhibited the development of gastric lesions in the rats (Al-

6
Rehaily et al., 2002). Dietary supplementation with extract of E. officinalis to swiss

albino mice in vivo significantly reduced the cytotoxic effects of a known

carcinogen, 3, 4- benzopyrene and sodium arsenite (Nandi et al., 1997; Biswas et al.,

1999). Due to the strong antioxidant property of Emblica officinalis, it relieved the

immunosuppressive effects of chromium on lymphocyte proliferation (Sai Ram et

al., 2002). E. officinalis is a potent radio - protector. The fruit pulp of E. officinalis

significantly reduced the bioeffects of radiation in adult swiss albino mice (Hari

Kumar et al., 2004).

The ethanol and aqueous extracts of Triphala and its individual components

(Emblica officinalis, Terminalia chebula and Terminalia belerica) have inhibitory

activites against certain bacterial isolates obtained from HIV infected patients

(Srikumar et al., 2007). It is suitable for use in antiaging, sunscreen and general

purpose skin care products (Chaudhuri, 2002). A herbal eye drop preparation

(Ophthacare) containing different herbs including E. officinalis has a useful role in

variety of infective, inflammatory and degenerative ophthalmic disorders (Biwas et

al., 2001). Recent research showed the antibacterial potential of aqueous extract of

E. officinalis and Coriandrum sativum against Gram negative urinary pathogens

(Saeed and Tariq, 2007).

In the present studies, the herb E. officinalis has been used to determine

whether it could be used as an alternative source to remove excessive iron in blood

serum of the thalassemic patients using an animal model like rabbits overloaded

with iron artificially.

7
Materials and methods

In the present study, 28 rabbits (Oryctolagus cuniculus) were used. They were

purchased from rural areas of Lahore, Pakistan. They were all males of the age

between 5.5 - 6 months, with a body weight of 1.5 - 2.0 kg. As present work

required repeated oral administration of medicinal herb E. officinalis (Amla),

intramuscular injections and serial bleedings, large rabbits were selected to work

on. Rabbits were kept in clean and spacious well-aerated rooms, the floor of the

room was kept as dry as possible, because wet warm environment provides a

favorable atmosphere for the development of coocidiosis in rabbits (……………….

All the rabbits were fed on green fodder and grams. They were divided into

4 groups with 7 rabbits each. Group 1was not given any iron injection or the herb.

Group 2 (iron overloaded control group) was given Jectosol - Plus iron injection @

0.3 ml / lb by intramuscular injections daily for 4 weeks. Group 3 (E. officinalis

treated group) was given a daily dose of E. officinalis @ 250 mg / kg daily for four

weeks. Group 4 (iron overloaded rabbits treated with E. officinalis) was iron

overloaded with Jectosol-Plus iron injection @ 0.3 ml/lb for 4 weeks and then given

daily dose of E. officinalis @ 250 mg/kg for a further four weeks. The rabbits were

iron overloaded with Jectosol-Plus @ 0.3 ml/lb body weight containing 15 mg

elemental iron. The injections were given intramuscularly, at intervals of 24 hours

for 4 - weeks. Administration of iron greater than 20 mg / kg is generally toxic and

60 mg/kg is a lethal dose. (18). The entire fruits of E. officinalis were powered and

dissolved in water. The suspensions were made and the solutions were

administered orally with the help of syringes to the rabbits.

8
 Before drawing blood, the ear of the rabbit was rubbed with xylene to make

the veins prominent. The needle of the syringe (5ml) was inserted into the vein. The

blood was sucked up in the syringe slowly. It was made sure that the needle rested

in the lumen of the vein while sucking the blood in the syringe. The blood (2-3 ml)

from the syringe was transferred to the sterile tube or eppendorf. The blood was

allowed to clot by leaving it undisturbed for 1-2 hours at 37°C without using

anticoagulants. When the clot had firmly formed and started to retract, the sample

was put in the refrigerator for over night at 4°C so that clot retraction may become

complete under condition unfavorable for the growth of bacteria. If the clot failed

to retract it was gently detached from the walls of the container by means of thin

glass rod. The serum was removed by a micropipette and transferred to another

eppendorf tube, which was then rapidly centrifuged at 4000 rpm for 5 minutes

(Mikro 22, Hettich Zentrifugen, Germany) so as to get rid of suspended red cells.

After centrifugation, serum was separated carefully for serological studies.

All the blood samples were collected in the morning (8:00 - 10:00 AM) before meal

to avoid any fluctuation in the various contents of blood which may result form the

absorption of digested food in the intestine. Blood sera are assayed for iron levels

by Colorimetric Method (Fer color, Wiener Lab. 2000 Rosario-Argentina). Iron

concentration was calculated according to following formula.

C = 100 × ΔA sample ug/dl

ΔA standard

ΔA sample = absorbance / optical density (OD) of sample.

ΔA standard = absorbance / optical density (OD) of standard.

9
Results were expressed as mean ± SER and statistical analysis was performed

using student’s t - test. P < 0.05 implied significance.

Results

The present work showed that the administration of extraneous iron

increased the iron concentration in the serum. This increase was highly significant

(P < 0.01) (table and figure 1). The increase in iron concentration in the serum was

>100 % after four weeks of iron overloading (with daily injection of Jectosol-Plus

@ 0.3 ml/lb). Whereas in the control rabbits, the iron concentration actually

decreased from 211.20 ± 10.62 to 166.22 ± 10.82 i.e. 81.72 or 21.29 % at our

experimental place.

E. officinalis treatment caused a decrease in serum iron concentration of

both the groups i.e. the control rabbits as well as the iron overloaded rabbits. In the

rabbits which were not iron overloaded, the iron concentration in the serum went

down from 227.20 ± 10.62 to 117.81 ± 1.50 i.e. it is decreased by 92.85 %. Whereas

in the iron overloaded rabbits the iron concentration decreased from 467.72 ±

10.25 to 148.65 ± 9.01 which is 202 % and is highly significant (P < 0.05) and

considerable.

On the other hand the serum iron concentration in the iron overloaded

rabbits without E. officinalis treatment decrease only by about 10.44 % (from

449.63 ± 27.74 to 407.11 ± 20.56).

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E. officinalis treatment led to a significant and considerable decrease in the iron

level of blood serum in all groups as shown in Table 2. Due to the treatment of E.

officinalis the level of iron in serum was brought to even below the normal level

from 233.50 ± 19.60 to 117.81 ± 1.50 (P < 0.01). Over all results show that the iron

concentration in serum of E. officinalis treated rabbits decreased, so this herb is an

effective iron eliminator.

The iron concentration in serum of iron overloaded rabbits on treatment with E.

officinalis for 4 weeks decreased significantly to 148.65 ± 9.01(P < 0.05) as

compared to 407.11 ± 20.56µg/dl in ion overloaded untreated group.

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Discussion
Present work shows that the administration of extraneous iron to the

rabbits by Jectosol Plus injections caused an excessive iron overloading in the

body .The increased accumulation of iron has been already reported through

artificial iron overloading in rats (19) and dogs (20). The excessive iron gets

deposited in the liver, heart, and endocrine glands and other organs. The iron

accumulation eventually causes abnormal heart rhythms or heart failure. In

addition, permanent liver scarring, diabetes, and delayed growth and delayed

sexual maturation can also occur. To minimize iron deposits in the body, people

affected by the disease must undergo chelation (iron-removing) therapy for up to

12 hours a day with intravenous (given through the skin directly into a vein) or

subcutaneous (under the skin) doses of the iron-binding agent desferrioxamine.

The side effects of desferrioxamine are mild nausea vomiting, and skin rashes as

reported at higher doses. (23). Deferiprone, CP20, L1 is an oral iron chelator of the

hydroxypyridinone family which has been studied since mid-1980’s. It is currently

being licensed in Europe and is available in India. A commonly used dose of L1 is

75 mg/kg/day divided into three doses. There is some controversy about whether

or not L1 is as effective as DF. The most common side effects of L1 are

neutropenia and arthropathy. One author suggests that L1 may be associated with

a higher incidence of hepatic fibrosis in individuals with Hepatitis C at advanced

iron overloading. (24). Another oral chelator, a compound known as ICL670, is

headed for phase III trials with thalessemia and sickle cell anemia patients. It

reduced iron by as much as 2.2 mg/g of liver in a randomized, open-label study

12
conducted in Italy with 71 people suffering from thalessemia, a rare inherited form

of anemia. (25).

A oral iron chelator deferasirox (Exjade), pill that can be dissolved in water

and taken orally once a day. The introduction of a chelating option for many

patients has had a profound effect upon their quality of life.

The present investigations have revealed that the herb E. officinalis given to

the iron overloaded rabbits led to the elimination of excessive iron from the body of

the rabbits. The greatest elimination took place in E. officinalis treated rabbits to

even below the normal level as compared with control rabbits. The iron overloaded

rabbits were treated with E. officinalis also shows elimination of iron significantly.

Previous work showed that excessive iron in iron overloaded rabbits was

eliminated by Terminalia chebula led to an effective decrease Another study

conducted in rats showed that the tannoid principles of the fruits of the plant E.

officinalis comprising of emblicanin A (37%).emblicanin B (33%), punigluconin and

pedunculagin can inhibit acute iron overload (30 mg/kg) (26).

The present study indicates that E. officinalis is a wonderful herb. It can be

very useful for the elimination of iron in thalassemic patients as compared to other

drugs like deferoxamine. E. officinalis is very cheap, required in small amounts

and can also be administered orally and comfortably to the thalassemic patients

and thus it can have an edge over the other drugs being used for this purpose so

far.

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 ACKNOWLEGEMENTS

This study was supported by the Research Funds of Zoology Department,

Government College University Lahore, Pakistan. We are thankful to Mr. Wadood

and Mr. Muhammad Ali (Senior Laboratory Attendant) for their assistance during

the work.

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17
 Table 1. Serum iron concentration (µg/dl) in control and iron overloaded rabbits
during 4 weeks of treatment (n = 7).

Weeks

Groups 0 week 1st week 2nd week 3rd week 4th

week

Control 211.20±10.62 199.01±18.62 194.85±24.68 187.05±8.42

166.22±10.82

Iron overloaded 209.20±10.62 312.04±17.40 341.80±20.84 365.83±24.51

449.63±27.74**

** Values highly significant at P < 0.01 when compared with control rabbits.
Mean ± S.E.M = mean and standard error of mean
n = number of rabbits in each group

Table 2. Serum iron concentration (µg/dl) in control, iron overloaded (untreated)

rabbits compared with Emblica officinalis treated rabbit in each group (n = 7).

Weeks

Groups 0 week 1st week 2nd week 3rd week 4th

week

18
 Control 211.20±10.62 199.01±18.62 201.85±24.68 187.05±8.42
166.22±10.82

Iron overloaded 449.63±27.74 442.34±15.20 439.53±10.51 416.24±9.01

407.11±20.56
then left untreated

Emblica officinalis 227.20±10.62 176.50±19.60 140.72±3.09 127.25±1.25

117.81±1.50**
treated

Iron overloaded + 467.72±10.25 369.62±19.78 288.98±13.16 213.16±4.35

148.65±9.01*
Emblica officinalis
treated

** Values highly significant at P < 0.01when compared with normal rabbits

* Values significant at P < 0.05 when compared with iron overloaded rabbits.
Mean± S.E.M= mean and standard error of mean
n= number of rabbits in each group

19
 500
Serum iron concentration
450 Control
400
350 Iron overloaded then left
300 untreated
250
200 Emblica officinalis treated
150
100 Iron overloaded +
50 officinalis
Emblica
µg/dl

0 treated
0 1 2 3 4

Week

Figure 1. Graph showing serum iron concentration (µg/dl) in control, Iron

overloaded then left untreated, Emblica officinalis treated and Iron overloaded +
Emblica officinalis treated rabbits during 4 weeks of treatment (n = 7).

20