CHAPTER 4 IN VIVO TOXICITY EVALUATION OF LAUHA...

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CHAPTER 4 –IN VIVO TOXICITY EVALUATION OF LAUHA BHASMA

4.1 INTRODUCTION

The usage of Ayurvedic herbo-metallic preparations has been in vogue over several

centuries in clinical practice [1, 2]. There is a global perception that Ayurvedic medicines

are toxic, based on several reports that indicate the presence of heavy metals like mercury

and lead above the permissible limits in these preparations [2–4]. The toxic effects of

metals in case of improper bhasma preparations have also been portrayed in the literature

[5]. Iron is also known for its toxic nature, apart from its essential functions in the biological

systems. Iron can produce free radicals, which ultimately result in alteration of metabolic

pathways and alteration in the biological systems [6]. Due to these toxicity concerns,

several questions have been raised about the safety of these preparations. Some of them are

Do Ayurvedic purification and calcination procedures convert iron into medicine?

Whether the prepared Lauha bhasma is safe or toxic?

To address these questions, attempts have been made here to study the in vivo toxicity

profile of Lauha bhasma in Wistar rats. Acute oral toxicity (single dose) and sub acute

toxicity studies (repeated dose) were carried out by oral administration of Lauha bhasma.

All the experiments were carried as per the WHO guidelines.

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4.2 MATERIALS AND METHODS

4.2.1 Study design

Wistar rats of both sexes in the age group of 4-6 weeks (Acute oral toxicity) and 6- 8

weeks (Sub acute oral toxicity) were used for the study. The rats were housed in

polypropylene cages and kept in a 12 h light/dark cycle .The room temperature was

maintained at 21±2°C and they were fed with standard rat feed pellet and deionized water

ad libitum throughout the entire study. The animals were acclimatized for 5 days before

the initiation of the study. The procedures used were reviewed and approved by the

Institutional Animal ethics committee (120/SASTRA/IAEC/RPP, SASTRA University).

4.2.2 Acute oral toxicity study (14-days single dose toxicity study)

A total of 12 rats were divided into two groups (I & II) with equal number of male and

female. The body weight of male ranged from 105 - 125 g and female from 85 - 100 g.

Group I rats were treated as normal control where they received honey with distilled water

in the ratio of 2:3 through oral route. Group II animals were administered with single dose

of Lauha bhasma (2000 mg/ kg) as a suspension in honey with distilled water in the ratio

of 2:3 through oral route. Body weight was measured on Day 0, Day 7 and Day 14. The

fecal samples were collected for 14 days to determine the elimination profile of iron. On

the 14th day, animals were sacrificed and major organs including liver, spleen, intestine,

brain and kidney were collected for bio-distribution studies.

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4.2.3 Bio-distribution studies and Fecal analysis

The organs and fecal samples were subjected to microwave digestion using ultra-pure nitric

acid (Merck) and 50 % Hydrogen peroxide(Merck) in the ratio of (4:1) using microwave

digestor (Anton-Paar, Graz, Austria). The digested samples were then quantitatively

estimated for the iron content using atomic absorption spectroscopy (AAnalyst 400, Perkin

Elmer, USA).

4.2.4 Sub acute oral toxicity study (28-days repeated dose toxicity study and 14 days

recovery study)

A total of 60 rats (n=10) were divided into six groups (Group I to Group VI) with equal

number of male and female. The therapeutic dose of Lauha bhasma for humans is 250mgs

[7]. This dose was converted to animal dose [8] as follows:

Human equivalent dose (mg kg⁄ ) =Rat dose (mg kg⁄ ) × Rat Km (6)

Human Km (37)

Human equivalent dose (mg/kg) = Rat dose (mg/kg) x Rat Km (6) / Human Km (37)

where, Km is the dose conversion factor based on body surface area.

The human equivalent dose was found to be 25 mg/kg. By fixing 25mg/kg as normal dose,

the medium and high doses were calculated as 100 mg/kg and 400 mg/kg respectively.

Group I was treated as normal control and received honey with distilled water in the ratio

of 2:3 for 28 days. Lauha bhasma was administered to Group II to Group IV animals at

different doses and was given as a suspension in honey and distilled water (2:3) for 28

days.

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Group II received the therapeutic dose (25mg/kg body weight) of Lauha bhasma, Group

III received the medium dose (100 mg/kg) and Group IV received the high dose (400

mg/kg). Group V and Group VI were treated as satellite groups in which group V was

treated as satellite control and received honey with distilled water in the ratio of 2:3. Group

VI received the high dose (400 mg/kg body weight). Weekly body weight was measured.

On the 28th day animals from Group I to Group IV were sacrificed. Blood was collected by

retro orbitary puncture for biochemical and hematological analysis. Major organs were

collected for histopathological studies. Group V and Group VI were treated with standard

rat feed pellet and deionized water ad libitum for the next 14 days. On the 42nd day the

satellite group animals were sacrificed as mentioned above.

4.2.5 Biochemical and Hematological analysis

Biochemical analysis was performed using Auto Analyzer (A15 Biosystems, Spain) and

the kits were procured from Bio Systems. Hematological parameters were analyzed using

Mythic 22 Hematology Analyzer (Orphee SA, Switzerland)

4.2.6 Histopathology

Tissues were collected and preserved in 10 % buffered formalin. All tissues required for

histopathological evaluation were embedded in paraffin wax, sectioned at a thickness of

approximately 3-5 microns, stained with hematoxylin-eosin and examined by light

microscopy.

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4.2.7 Statistical analysis:

All the values were expressed as Mean±SD. A level of p< 0.05 was considered statistically

significant and accordingly data were interpreted. Two way anova was performed to find

the significant difference between the control and Lauha bhasma treated groups in

bodyweight and iron excretion pattern. Bonferroni post test was done in iron excretion

pattern followed by two way anova. One way anova was performed to find the significant

difference between the control and Lauha bhasma treated groups in biochemical and

hematological parameters.

4.3 RESULTS AND DISCUSSION

4.3.1 Effects on body weight

Oral administration of Lauha bhasma did not cause any mortality or adverse effects

throughout the study period. No significant difference was observed in body weight

between the control and the Lauha bhasma treated groups in both the acute oral toxicity

study and sub acute oral toxicity study.

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Figure 4.5 Weekly body weight in control and different doses of Lauha bhasma treated

male rats in sub acute oral toxicity study. The values are expressed as Mean± SD for 5

animals.

Figure 4.1 Body weight of control and Lauha bhasma treated animals in acute oral

toxicity study. The values are expressed as Mean ± SD for 3 animals.







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Figure 4.6 Weekly body weight in control and different doses of Lauha bhasma treated

female rats in sub acute oral toxicity study. The values are expressed as Mean± SD for 5

animals.

4.3.2 Bio-distribution studies

In acute oral toxicity studies there was no deposition of iron in major organs including

liver, kidney, brain intestine and spleen when compared to control group in both male and

female rats. From Figure 4.4 it was apparent that there was no significant difference

between the control and Lauha bhasma treated groups in the concentration of iron in the

organs. This also correlated reasonably very well with the iron excretion pattern (Figure

4.5) as high amount of iron excretion was seen on day 1 in Lauha bhasma treated group.

Machado et al. [9] reported that lower deposition of iron was observed in liver when

administered as iron peptide complex (30mg/kg and 60 mg/kg body weight). However

when administered as iron sulfate at same doses, excess deposition of iron was observed in

liver and spleen [9]. Papanastu et al. [10] reported that under normal circumstances, the

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concentration of iron was found to be higher in spleen when compared to liver. After

administration with high dose of iron, the concentration was found to be higher in liver due

to excess deposition when compared to spleen [10].

Figure 4.7 Bio distribution studies of control and Lauha bhasma treated animals in acute

oral toxicity study. The values are expressed as Mean± SD for 3 animals in each group of

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both male and female rats.

Figure 4.8 Fecal sample analysis of control and Lauha bhasma treated animals in acute

oral toxicity study. The values are expressed as Mean± SD for 3 animals in each group of

both male and female rats (* p<0.05).

However in the present study there were no deposition of iron in major organs including

liver, kidney, brain, intestine and spleen in Lauha bhasma treated groups when compared

to control group which may be attributed to the addition of triphala.

4.3.3 Effect on Biochemical & Hematological parameters:

The Lauha bhasma treated groups of three different doses did not exhibit any significant

changes in the biochemical parameters including glucose, cholesterol, protein, urea,

creatinine, SGOT, SGPT when compared to the control group (Figure 4.6- Figure 4.8)

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Figure 4.9 Biochemical parameters of control and different doses of Lauha bhasma

treated groups, (A) Glucose, (B) Protein. The values are expressed as Mean± SD for 5

animals.

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treated groups, (C) Cholesterol,(D) Creatinine. The values are expressed as Mean± SD for

5 animals.

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treated groups, (E) ALT, (F) AST. The values are expressed as Mean± SD for 5 animals.

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No significant changes were observed in major hematological parameters including RBC,

hemoglobin and hematocrit in Lauha bhasma treated groups when compared to control

group. Lymphocytes count was also found to be normal between the groups.

Figure 4.12 Hematological parameters of control and different doses of Lauha bhasma

treated groups (A) RBC, (B) Hemoglobin. The values are expressed as Mean± SD for 5

animals.

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Figure 4.13 Hematological parameters of control and different doses of Lauha bhasma

treated groups (C) Hematocrit. The values are expressed as Mean± SD for 5 animals.

Sarkar et al. [11] performed investigations on toxicity of Lauha bhasma in experimental

animals, followed by recovery studies. Lauha bhasma was administered to experimental

animals (Albino rats) at a dosage of five times the therapeutic effective dose. The drug

was administered for 60 days and 45-day recovery period was observed. Hyperglycemia

was observed in Lauha bhasma treated group along with elevated levels of serum liver

enzymes and the levels were restored to normalcy in the recovery period [11].

In another study iron administered as iron dextran injection in mice resulted in iron

overload and caused liver injury that resulted in significant elevation of serum enzymes

including ALT, AST, ALP and bilirubin. Further an increase in liver iron content was also

observed. However when the iron overloaded mice was treated orally with Terminalia

chebula extract there was a decline in the level of serum enzymes and the liver iron content

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also got lowered. Terminalia chebula scavenges free radicals and inhibits the free radical

formation by chelating excess iron with its active components [12]. Hence it is evident that

iron should not be administered alone.

It has been reported that iron supplementation when administered continuously to Sprague-

Dawley rats resulted in increased serum cholesterol and triglycerides level. This is due to

the decreased antioxidant levels which resulted in increased lipid peroxidation in liver and

leads to leakage of hepatic enzymes in the circulation [13].

However, in the present study, Lauha bhasma even at high doses did not cause any adverse

effects as evidenced from the biochemical and hematological parameters.

4.3.4 Histopathological Studies

Liver and spleen play a major role in iron metabolism[7,15]. Normal cyto architecture of

liver and spleen were observed in the high dose treated group. Liver exhibited normal

hepatocytes and sinusoids. No signs of iron deposition in the form of hemosiderin as brown

patches, were observed in liver and spleen in the high dose treated group (Figure 4.11)

Sarkar et al. [11] reported liver toxicity in Albino rats after administering Lauha bhasma

in high dose. Histopathological findings of liver showed congestion of central vein,

sinusoidal dilation with mild to moderate fatty changes [11].

Meltem et al. [15] studied the effect of chronic iron toxicity in Sprague-Dawley rat liver

by administering iron-sorbitol for two days per week for a period of eight weeks. Iron

overload in hepatocytes was observed by appearance of characteristic yellowish brown

depositions when stained with hematoxylin-eosin. The iron overload in liver was further

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confirmed by Perls prussain blue stain, Massons trichrome stain and periodic acid Schiff

reagent (PAS) [15].

Figure 4.14 Representative histopathology images of liver: [A] - Control; [B] - High dose

of 400 mg/kg of Lauha bhasma. Hepatocytes (H) and sinusoids (S) are shown.

Representative histopathology images of spleen: [C] - Control; [D] - High dose of 400

mg/kg of Lauha bhasma. Peri-arteriolar lymphoid sheath (PALS) and central splenic

arteriole (A) are shown. Representative histopathology images of kidney: [E] - Control;

[F]- High dose of 400 mg/kg of Lauha bhasma. Glomerulus (G) and tubules (T) are shown.

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In another study, attempts were made to study the role of iron supplementation in treatment

of anemia as well as when administered during normal conditions. Iron was administered

to rabbits in fumarate form for a period of three weeks. Signs of anemia characterized by

decrease in animal size and activeness, decolorized organs (pale) were observed in starved

group. After iron supplementation, the starved animals showed moderate recovery from

the anemia during the first week. During the third week, the cyto architecture of liver and

kidney were restored along with increased iron deposition with time. In case of well-fed

groups, excess iron deposition with disruption of liver and kidney cyto architecture was

observed. Liver sections of such animals showed dark stained iron accumulation with

ruptured hepatic cell membrane. Iron deposition in the form of granules scattered in the

cytoplasm of kidney was noticed, which increased in volume and number by time. The

renal tubules appeared irregular, dilated with flattened epithelial lining and shrinkage of

the glomerolus leading to dilated urinary space [16].

It has been reported in a 28-day study that the iron deposition in liver and spleen was

reduced when supplemented as iron-peptide complex rather than iron sulphate [9].

Aggressive behavior was observed in ferrous sulfate treated group [9].

Feeding rats with iron rich diets led to the accumulation of iron in periportal region of the

liver, mostly in the cytoplasmic region of the hepatocytes. In spleen, splenic atrophy was

observed by loss of cells in white pulp region and deposition of iron as hemosiderin in

sinusoidal macrophages[17]. Iron administration either as diet or intravenous or

intraperitoneal administration led to deposition of iron as hemosiderin in hepatocytes and

kuppfer cells surrounding the entire hepatic lobule of liver of Wistar male rats.

Hemosiderin laden macrophages were observed in the spleen in iron overloaded conditions

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[10]. However, the results in the present study showed that the rats administered even with

high dose of Lauha Bhasma did not show any signs of toxicity in liver, spleen and kidney.

Representative histopathology images of thymus, oesophagus and pancreas are shown in

Figure 4.12

Figure 4.15 Representative histopathology images of thymus: [A] – Control; [B] - High

dose of 400 mg/kg of Lauha bhasma. Cellular cortex (C) is shown. Representative

histopathology images of eosophagus: [C] – Control; [D] - High dose of 400 mg/kg of

Lauha bhasma. (SS) Stratified squamous epithelium (SS), stratum corneum (SC) and

submucosa (SM) are shown. Representative histopathology images of pancreas: [E] –

Control; [F] - High dose of 400 mg/kg of Lauha bhasma. Exocrine pancreas (EX) and

endocrine pancreas (EN) are shown.

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In an earlier study, porta caval shunt was surgically created in male Sprague-Dawley rats

to study iron accumulation in pancreas [18].The rats were fed with carbonyl iron diet for

17 weeks. Histopathological section revealed chronic pancreatitis characterized by acinar

atrophy, proliferation of intercalated ducts, mononuclear cell infiltration and fibrosis.

Oxidative stress was also confirmed with the elevated levels of glutathione disulfide [18] .

However, the results in the present study showed that the rats administered even with high

dose of Lauha Bhasma did not show any signs of toxicity in pancreas.

Representative histopathology images of intestine, brain and ovary are shown in Figure

4.13.

Mohammed et al. [19] studied the effect of changes in brain neuro transmitters of rats due

to administration of iron fortified diet. The levels of serotonin and dopamine were found

to be reduced significantly. Increase in lipid peroxidation in brain as a result of excess iron

deposition led to oxidative stress that ultimately resulted in neuro degenerative disorders.

Histopathology studies revealed the conditions of meningeal haemorrhage, congestion and

edema due to excessive iron overload. Further degenerated neurons, satellitosis and

neuronophagia were also observed in cerebal cortex [19].

Sobotka et al. [20] investigated toxicity and neuro behavioral changes in rodents fed with

different concentrations of dietary iron. Dose dependent toxicity and behavioral changes

were observed [20]. Another study revealed behavioral impairments combined with brain

iron accumulation in rats injected with ferrous sulphate for five days [21].

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Figure 4.16 Representative histopathology images of intestine: [A] – Control; [B] - High

dose of 400 mg/kg of Lauha bhasma. Intestinal crypts (C) are shown. Representative

histopathology images of brain: [C] – Control; [D] - High dose of 400 mg/kg of Lauha

bhasma. Neurons (N), eosinophilic cytoplasmic processes (C) and glial cells (G) are

shown. Representative histopathology images of ovary: [E] – Control; [F] - High dose of

400 mg/kg of Lauha bhasma. Ovarian stroma (OS) and ovarian follicle (OF) are shown.

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In vitro studies were carried out using PC 12 cells incubated with ferrocene and dopamine.

The addition of Fe2+ (in the form of ferrocene) increased the oxidation of monoamines.

The oxidation products were bound covalently to sulfhydryl groups of serotonin binding

proteins leading to apoptosis in PC-12 cells [22].

The effect of ferrous fumarate at low dose on dextran sulfate sodium (DSS) induced colitis

in rats has been reported [23]. The signs of intestinal inflammation and plasma redox status

were observed in DSS-administered groups with respect to control groups. A significant

increase in colitis score was observed in DSS-administered groups. Colitis score in DSS-

administered groups was further increased due to administration of ferrous fumarate. Rats

that received ferrous fumarate only showed normal cyto –architecture with no excess iron

deposition. No changes were observed in plasma redox status in DSS induced colitis rats

when compared to control groups [23].

In another study, the effect of oral iron supplementation at two different doses was

investigated in DSS induced colitis rats. Iron with doses of 0.3 % and 3 % showed higher

histological scores of colitis accompanied by heavier rectal bleeding and shortening of the

colon. The levels of antioxidant vitamins were also decreased with increased lipid

peroxidation [24]. The free radical generating capacity and lipid peroxidation of the

intestine in rats were assessed through administration of iron enriched diets for six months

[25]. Though the chronic administration of iron did not increase crypt cell proliferation,

increased free radical generation in colon and lipid peroxidation in caecum were observed

[25]. However, the results in the present study showed that the rats administered even with

high dose of Lauha Bhasma did not show any signs of toxicity in intestine and brain.

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Histopathology of other organs were also carried out and normal cyto articheture was

observed in high dose Lauha bhasma treated group, when compared to control group.

Representative histopathology images of thyroid, lungs and heart are shown in Figure 4.14

Figure 4.17 Representative histopathology images of thyroid: [A] – Control; [B] - High

dose of 400 mg/kg of Lauha bhasma. Cuboidal epithelium (CE) and homogenous colloid

(C) are shown. Representative histopathology images of lungs: [C] – Control; [D] - High

dose of 400 mg/kg of Lauha bhasma. Alveolar wall (Arrow mark) and alveolar lumen (AL)

are shown. Representative histopathology images of heart: [E] – Control; [F] - High dose

of 400 mg/kg of Lauha bhasma. Cardiac myocytes (CM) and cytoplasm (C) are shown.

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Representative histopathology images of adrenal gland, pituitary gland and bone

marrow are shown in Figure 4.15

Figure 4.18 Representative histopathology images of adrenal gland: [A] – Control; [B] -

High dose of 400 mg/kg of Lauha bhasma. Cortex (C) and medulla (M) are shown.

Representative histopathology images of pituitary gland: [C] – Control; [D] - High dose of

400 mg/kg of Lauha bhasma. Pars distalis, (D) pars intermedia (I) andpars nervosa (N) are

shown. Representative histopathology images of bone marrow: [E] – Control; [F] - High

dose of 400 mg/kg of Lauha bhasma. Erythroid and myeloid precursors (P) and

megakaryocytes (M) are shown.

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4.4 CONCLUSION

Iron, despite its essential role in biological system can cause adverse toxic effects ranging

from male sterility to tumorogenesis. Lauha bhasma even when administered at a high dose

was not toxic to the vital organs of the rats. Histopathological studies showed that the cyto

architecture of rats treated with high dose of Lauha bhasma was comparable to normal

control. This was further supported by the biochemical and hematological parameters

which showed that Lauha bhasma did not cause any significant changes in the levels of

those parameters.

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