acute and 28 days repeated oral toxicity study of the

www.wjpr.net Vol 6, Issue 5, 2017.

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Sridevi et al. World Journal of Pharmaceutical Research

ACUTE AND 28 DAYS REPEATED ORAL TOXICITY STUDY OF THE

SIDDHA DRUG VEERA AYA CHENDHURAM (VAC)

1J. Sridevi* and

2P. Shanmuga Priya

1Siddha Consultant, Srihari Siddha Clinic, Salem.

2Lecturer, Nanjunoolum Maruthuva Neethi Noolum, National

Institute of Siddha, Chennai.

ABSTRACT

According to the text kannusamiyam parambarai vaithiyam, the drug

Veera aya chendhuram(VAC) is used in the treatment of Anemia.

Anemia is a global health problem affecting both developing and

developed countries with major health consequences for human health

as well as social and economic development. It is generally attributed

that 50% of anemia is due to iron defeciency. Hence it is an need for an

hour to evaluate safety and efficacy of the drug VAC which is

indicated for anemia. Acute and 28 days repeated oral toxicity study

was performed in wistar albino rats according to OECD guidelines. Acute toxicity single dose

of 5mg, 50mg, 300mg and 2000mg was administered to rats and monitored for 14 days. 28

days repeated oral toxicity was carried out in two group of 10 animals (5 male and 5 female).

VAC was administred in the dose of 4.68mg, 23.4mg, 46. 8mg to the rats for the period of 28

days. Detailed hematological, biochemical, histopathological evaluation of different organs

were performed in all animals. The acute toxicity study results concludes that the LD 50 cut

off value of VAC may be above 2000 mg/kg body weight. The histopathological report

shows that the drug is considered safe in low and mid doses.

KEYWORDS: Veera aya chendhuram, Anemia, Siddha drug, toxicity study.

INTRODUCTION

Siddha system of medicine is an integrated part of Indian system, which is very potent &

unique system in existence. It is a complete holistic medical system that has been practiced in

Southern parts of India for two thousands of years and above. It is the treasure house of secret

science, embodying the results of the ardent pursuit there of by the ancient Siddhars. Siddhars

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 6, Issue 5, 1390-1401. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. J. Sridevi

Siddha Consultant, Srihari

Siddha Clinic, Salem.

Article Received on

17 March 2017,

Revised on 07 April 2017,

Accepted on 27 April 2017

DOI: 10.20959/wjpr20175-8455


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were the people who achieved Siddhi which means perfection. They were the greatest

spiritual scientist on those days and also philosophers, healers and men with super natural

powers. The contribution of Siddhars to Siddha literature with its boundless therapeutics and

wonderful pharmaceutical preparation of medicine is acclaimed pre- eminent even in this

20th century and worthy of its remarkable results.

Siddhars realized that if the body could may be strong and perfect, they could get rid of death

& diseases. They have fully investigated and studied the cause and effect of the diseases and

they imparted their knowledge for the upliftment of the human life. They defined health as a

perfect state of physical, physiological, social and spiritual well being of an individual.

Siddha medicine revitalises and rejuvenates dysfunctional organs that cause the disease and

to maintain the ratio of vadham, pitham and kabam. The equilibrium of humour is considered

as health and its disturbance or imbalance leads to a diseased state.

This treatment deals not only as a curative but also as a preservative, taking care of the

external body with its internal being - soul. The system not only deals with medicine but also

with spirituality, righteous way of living, rejuvenation and it's main aim is attainment of

perfection.

This system of medicine uses a fascinating combination of herbs, minerals, metals and animal

products to promote good health and longevity. Metallic preparations have been used to treat

chronic diseases since time immemorial. At present scenario, the toxicity manifestation and

related health problems produced in human and environment by heavy metals like mercury,

lead, arsenic etc are considerded to be a great threat to the society. Siddha proponents believe

that the toxicity of these materials which are used in the Siddha formulations is reduced

through purification process such as suddhi muraigal. These methods not only deals with a

purification of heavy metals and minerals, but also increases the effectiveness of the raw

drugs.

Many research workers have conducted a number of pharmacological and toxicological

experiments which revealed that the toxicity of the crude drug is quite different from that of

the finished Siddha formulation. In the broadest sense toxicology is the science of poisons

and the harmful or noxious effects of these substance have on living things, and is responsible

for predicting the toxic or harmful nature of a substance by designing experiments that will


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supply data necessary to asses the toxicity of the substances. These data help clinicians to

make predictions about the hazardous nature of the material tested and their potential impart

on the environment and on human population.

Hence I have chosen metallic based preparation Veera Aya Chendhuram which includes

Veeram, Ayam, Gandhagam, Lingam, Pooneeru, Vediuppu for the evaluation of its toxicity

profile. As per the text it is indicated for anemia, jaundice, dropsy. Anemia is a global health

problem affecting both developing and developed countries with major health consequences

for human health as well as social and economic development[1]

It occurs in all stages of life

cycle. Although the prevalence of anemia is estimated at 9% in countries with higher

development,the countries with low development the prevalence is 43%.[2]

It is a mild health

problem, when prevalence of anemia is <20%, a moderate public health problem,when the

prevalence of anemia is between 20 and 40%, a severe health problem when the prevalence

of anemia is >40%.[3]

Many herbs and minerals have been used in the treatment of anemia.[4]

Till now there is no toxicity evaluation carried out for the drug VAC. So there is need for an

hour to evaluate toxicity studies to find safe dose for human trial.[5]

MATERIALS AND METHODS

STANDARD OPERATING PROCEDURE OF VEERA AYA CHENDHURAM

Ayapodi(Iron) - 1 palam(35gm).

Gandhagam (sulphur) - 1 palam(35gm).

Lingam(Red sulphide of mercury) - 1 palam(35gm).

Veeram(Hydragyrum perchloride) - 1 palam(35gm)

Vediuppu(Potassium nitrate) - 1 palam(35 gm)

Pooneeru(Fullers earth) - 1 varagan(4.2gm)

Drugs were identified and authenticated from dept. of Pharmacognosy in Siddha Central

Research Institute, Chennai and botanist in National Institute of Siddha, Chennai.

After purifying all the drugs, the Ayam and Gandhagam is placed in kalvam and rubbed with

lemon juice till it reaches waxy consistency (mezhugu) for about 4 samam(12 hours) and

made it into a single pellet and dried under the sunlight. Then it is placed in the mudplate and

closed it with similar plate. The margins are covered with 7 layers of clay cloth and it is

dried. Then it is subjected to pudam with 15 cow- dung cakes. The finished product which is

red in colour. It is kept in kalvam along with other drugs and rubbed with lemon juice for 2-3


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samam(6-9hours). Then it is made into a single pellet and pudam is done as mentioned above.

The whole process is repeated for 3 times. Finally dark brown colour chendhuram is obtained

which is stored in a air tight container.[6]

ANIMALS

Test animals were obtained from King Institute Of Preventive Medicine, Guindy and kept at

animal house, National Institute of Siddha, Chennai. The animals had free access to RO water

and standard pellet diet (Sai Meera foods pvt. Ltd, Bangalore). The animals was kept in a

acclimatization for a period of 7 days. The principles of laboratory animal care were

followed.

The toxicity studies were evaluated after getting permission from the Institutional Animal

Ethical Committee.(IAEC Approved No.NIS/IAEC/1/2013/10).

ACUTE ORAL TOXICITY STUDY OECD 423 GUIDELINES

HOUSING AND FEEDING CONDITIONS

The animals were housed in polypropylene cages provided with bedding of husk. The

temperature in the experimental animal room was 22ºC ±3ºC. Although the relative humidity

was at least 30% and preferably not exceed 70% other than during room cleaning the aim was

50-60%. Lighting was artificial, the sequence being 12 hours light, 12 hours dark.

IDENTIFICATION OF ANIMAL

By cage number and individual marking on the fur of each animals with picric acid.

ROUTE OF ADMINISTRATION

Oral route was selected, because it is the normal route of clinical administration.

TEST SUBSTANCE AND VEHICLE

The Veera Aya Chendhuram is dark brown in colour, without taste and odour. In order to

obtain and ensure the uniformity in drug distribution; the drug is dissolved in honey and

distilled water.

ADMINISTRATION OF DOSES

Veera Aya Chendhuram was suspended in honey and distilled water . It was administered to

the test groups in a single oral dose by oral gavage. The control groups were received equal

volume of the vehicle. Animals were fasted overnight to food, but not to water prior to drug


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administration. The animals were weighed before giving the drug. Test drug at four different

dose (5, 50, 300mg, 2000mg /kg.b.w) was administered stepwise. After that food was

withheld for 3-4 hours. Then it was observed for every half an hour till 4 hours untill 24

hours. Finally, the number of survivors was noted. After 24 hours these animals were then

maintained for further 14 days and observations made daily. The toxicological effect was

assessed on the basis of mortality and behavioural observations.

ACUTE ORAL TOXICITY STUDY (OECD guidelines – 423)

Species and strain : Wistar Albino rat

Sex : Female

Age/Weight : 6 weeks/150-200 gm

Test guideline : OECD guidelines - 423

Groups/treatment : Grouped by randomization

Duration of exposure to the

“Veera Aya Chendhuram” : Single dose

Study duration : 14 days

Number of animals : 6 females/group,

Route of administration : Oral

Groups No of Rat

Group I Vehicle control (Honey) 6 female

Group II Test drug – 5 mg/kg b. wt 6 female

Group III Test drug – 50 mg/kg b. wt 6 female

Group IV Test drug – 300 mg/kg b. wt 6 female

Group V Test drug – 2000 mg/kg b. wt 6 female

Acute oral toxicity of the formulations were evaluated in rats following OECD guideline -

423. Animals were divided into five groups, each group containing 3 females weighing 150 -

200g with age of 6 weeks. One group as control and the other four groups were treated with

test drug at four different doses (5mg, 50mg, 300mg, 2000mg /kg.b.wt) by oral gavages.

Behaviour: The animals were observed closely for behaviour in the first four hours which

includes abnormal gait, aggressiveness, exophthalmos, ptosis, akinesia, catalepsy,

convulsion, excitation, head twitches, lacrimation, loss of corneal reflex, loss of traction,

piloerection reactivity of touch, salivation, scratching, sedation, stereotypes (chewing),

stereotypes(head movements), stereotypes (sniffing), tremor and writhes, diarrhoea, lethargy,

sleep and coma.


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Body weight

Body weights were recorded at day 1, 2, 7 and 14 of the study.

Mortality

Animals were observed for mortality throughout the entire period.

Gross necropsy

At the end of 14th

day animals were sacrificed by excessive anesthesia for gross necropsy. It

includes examination of the external surface of the body, all orifices, and organs like brain,

lungs, heart, spleen, liver, stomach, kidneys, adrenals and sex organs of all animals.

28 DAYS REPEATED ORAL TOXICITY STUDY OF VEERA AYA CHENDHURAM

OECD-407

Species and strain : Wistar albino rats

Sex : Male and Female

Age/Weight : 6 weeks/150-200mg

Test guideline : OECD guidelines – 407

Groups/treatment : Grouped by randomization

Duration : 28 days

Number of animals : 10/group (5/sex)

Route of administration : Oral

Groups No of Rats

Group I Vehicle control (Honey) 10 (5male,5 female)

Group II test drug - low dose X (4.68 mg) 10 (5male,5 female)

Group III test drug - Mid dose 5X (23.4mg) 10 (5male,5female)

Group IV test drug - High dose 10X( 46.8mg) 10(5male,5female)

The study will be carried out as per OECD Guideline 407 (Repeated Dose 28-Day Oral

Toxicity in Rodents). The animals will be divided in four groups each group consist of 10

animals (5 males and 5 females). One group will serve as control and the other three groups

for test drug at three different dose levels (low, mid and high) for 28 days.

DOSE SELECTION

Repeated oral toxicity study was carried out at different dose levels x (4.68mg), 5x (23.4mg),

10x (46.8mg).


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PREPARATION AND ADMINISTRATION OF DOSE

Veera Aya chendhuram was suspended in honey and distilled water. It was administered to

groups II, III, IV at dose levels of X(4.68mg), 5X (23.4mg), 10X(46.8mg). The control

animals were administered vehicle only. Administration was given orally by using an oral

gavage once in daily for 28 days.

OBSERVATIONS

BODY WEIGHT

During the study, Body weight of the animals was evaluated weekly once.

FOOD AND WATER INTAKE

Water and food consumption were calculated daily.

MORTALITY

Animals were observed for mortality daily.

LABORATORY INVESTIGATIONS

Collection of blood: Blood was collected in all overnight (12 hours) fasted rats through retro

orbital plexus and it was processed for below mentioned investigations.

Laboratory test

Complete Haemogram

Renal function test

Liver function test

NECROPSY

By the end of 28 days, the animals were sacrificed by excessive anesthesia. Animals were

subjected to gross necropsy. Vital organs collected from the animals were subjected to

histopathology.

HISTOPATHOLOGY

Animals were subjected to histopathological investigations. Organs like heart, lungs, kidney,

liver, spleen, stomach, brain were collected from all animals and preserved in 10% buffered

neutral formalin, sliced 5 or 6µm sections and it will be stained with hematoxylin and eosin,

examined for histopathological changes.


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RESULTS

STATISTICAL ANALYSIS

Findings such as clinical sings of intoxication, body weight changes, food consumption,

hematology and biochemical parameters were subjected to one-way ANOVA followed by

Dunnet “t” test using a computer software programme-Graphad INSTAT-V3.1.

DISCUSSION

In Acute toxicity study period the trial drug shows no abnormal toxicity signs in the dose

level 2000 mg/kg body weight. There were no reduction in body weight of animals and no

mortality were observed in the study period. It concludes that LD 50 cut-off of Veera aya

chendhuram may be above 2000mg/kg body weight CATEGORY-5(GHC) as per the

guideline OECD- 423.

In 28 days Repeated oral toxicity study as per the OECD guideline-407 VAC was

administered in low dose(4.68), mid dose(23.4), high dose(46.8). Animals were observed

throughout the period. After 28 days animals were sacrificed and blood samples were

collected, investigated and the results revealed that there were slight increase in

haematological parameters(Hb, PCV,MCV,MCHC,MCH) and no changes in biochemical

parameters compared to control group. It may be due to the iron present in the drug. Finally

all the reports were statistically calculated. There was no significant change in body weight,

water and food intake, hematological and biochemical parameters.

The histopathological study on the organs such as brain, heart, lungs, kidney, spleen, liver

and Stomach was normal in control, low dose, and mid dose groups. In high dose group the

liver shows congestion and bile duct hyperplasia, focal mononuclear cell infiltration, focal

necrosis of hepatocytes. The stomach shows focal hyperplasia of mucosal epithelial cells. The

lung shows congestion. It reveals that the drug is considered safe in low and mid doses.

Acute oral toxicity study of Veera aya chendhuram

Table 1: Dose finding experiment and its behavioral Signs of Toxicity

No Dose mg/kg 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1. 5 + - - + - + - - - - - - - - - - - - - -

2 50 + - - - + + - - - - - - - - - - - - - -

3 300 + - - + + + - + - - - - - - - - - - - -

4 2000 + - - + + + - + - - - - - - - - + - - -


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1. Alertness 2. Aggressiveness 3. Pile erection 4. Grooming 5. Gripping 6. Touch Response

7. Decreased Motor Activity 8. Tremors 9. Convulsions 10. Muscle Spasm 11. Catatonia 12.

Muscle relaxant 13. Hypnosis 14. Analgesia 15.Lacrimation 16. Exophthalmos 17. Diarrhoea

18. Writhing 19. Respiration 20. Mortality (+ Present, - Absent).

28 Days Repeated oral toxic study of Veera aya chendhuram

Table 2: Body weight of wistar albino rats group exposed to Veera aya chendhuram

NS- Not Significant, **(p > 0.01),*(p >0.05), n = 10 values are mean ± S.D (One way

ANOVA followed by Dunnett’s test).

Table 3: Water intake (ml/day) of Wistar albino rats group exposed to Veera aya

chendhuram

DOSE DAYS

1 7 14 21 28

CONTROL 60.5 ± 8.95 60±6.23 58.5±6.23 59±8.096 60.5±4.97

LOW DOSE 55.1±3.41 55.5±3.62 55.8±4.26 56.4±4.29 56.8±4.13

MID DOSE 57±4.34 57.4±4.11 58.1±2.84 58.3±2.21 57.3±3.35

HIGH DOSE 58±2.44 58.2±2.14 58.9±2.13 58.2±2.82 58.9±2.96

P value (p)* NS NS NS NS NS

N.S- Not Significant, **(p > 0.01), *(p >0.05), n = 10 values are mean ± S.D (One way


Table 4: Food intake (gms/day) of Wistar albino rats group exposed to Veera aya

chendhuram

DOSE DAYS

1 7 14 21 28

CONTROL 37±5.37 38.5±4.11 39.5±4.37 38.5±3.37 37±4.21

LOW DOSE 34.7±2.98 35.4±2.24 35.1±2.28 35.2±3.32 35.9±2.51

MID DOSE 36.4±3.65 37.1±3.60 37.2±3.25 39±4.98 39.1±4.53

HIGH DOSE 40.2±3.45 40.3±3.88 39.9±3.78 40.3±3.40 40.1±3.84




DOSE DAYS

1 7 14 21 28

CONTROL 163.6±33.683 169.4 ± 29.315 172.8 ± 28.870 173.8 ± 29.73 174.8 ± 28.310

LOW DOSE 150.1 ± 21.105 155.9 ± 20.88 161.5 ± 21.706 161 ± 20.77 166.7 ± 14.88

MID DOSE 158.6± 13.57 174.2 ± 20.70 175.7 ± 29.01 179.1 ± 33.37 186.8 ± 32.11

HIGH DOSE 178.5± 28.75 187.7 ± 33.87 192.8 ± 32.48 199 ± 30.93 208 ± 28.94



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Table 5: Haematological parameters of Wistar albino rats group exposed to Veera aya

chendhuram

Category Control Low dose Mid dose High dose P value (p)*

Haemoglobin(g/dl) 13.7±0.88 13.41±0.96 13.81±1.53 15.91±0.96* N.S

Total WBC (cells/cu.mm) 10450±574.45 8950±880.340 9483.33±491.59 9300±878.63 N.S

Neutrophils(%) 48.25±7.93 44.66±11.75 48.16±5.98 53.33±8.68 N.S

lymphocyte (%) 48.5±8.54 53.83±11.23 48.33±5.88 42.66±8.84 N.S

Monocyte (%) 0±0 0±0 0±0 0±0 N.S

Eosinohil(%) 3.25±2.06 1.5±0.836 3.16±1.16 4±1.54 N.S

Platelets cells/ul 2.07±0.16 2.33±0.16 2.13±0.48 2.36±0.30 N.S

Total RBC (cells/cu.mm) 4.03±0.28 4.54±0.31 4.52±0.10* 5.36±0.30* N.S

PCV% 40.25±2.75 41.83±2.78 42±1.78* 48.83±2.56* N.S

MCH pg 26.72±0.095 29.21±0.21 29.48±0.34* 29.61±0.26* N.S

MCHC g/dl 30.6±0.14 32.03±0.31 32.96±0.52* 33±0.44* N.S

MCV Fl 90.2±0.37 92.11±0.38 92.03±0.89* 92.63±0.70* N.S



Table 6: Biochemical Parameters of of Wistar albino rats group exposed to Veera aya

chendhuram

Biochemical

Parameters Control Low Dose Mid Dose High Dose P Value (p)*

GLUCOSE

(R) (mg/dl) 105.75±9.21 105.16±8.44 97.33±7.60 96.16±9.80 N.S

T.CHOLOSTEROL

(mg/dl) 130.5±47.17 99.16±5.70 103.33±9.20 106.33±9.68 N.S

HDL(mg/dl) 29±3.74 25.83±3.76 28±2.36* 30.16±2.92* N.S

LDL(mg/dl) 40±12.75 41.5±4.92 43.16±8.44 49.5±9.81 N.S

VLDL(mg/dl) 29±3.74 30.166.17 31.166.91 28.335.39 N.S

TRIGLY(mg/dl) 161.75±37.45 137.83±15.09 154±31.40 120.66±9.33* N.S

NS- Not Significant, **(p > 0.01), * (p >0.05), n = 10 values are mean ± S.D (One way


Table 7: Renal function test of of Wistar albino rats group exposed to Veera aya

chendhuram


UREA (mg/dl) 24.17±3.86 21.833±3.06 20.66±2.73 18.5±2.88 N.S

CREATININE(mg/dl) 0.90±0.031 0.816±0.045 0.803±0.75 0.83±0.069 N.S

URIC ACID(mg/dl) 4.37±0.35 5.11±0.43 5.9±1.25* 4.48±0.43 N.S

NS- Not Significant, **(p > 0.01), * (p >0.05) , n = 10 values are mean ± S.D (One way



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Table 8: Liver Function Test of of Wistar albino rats group exposed to Veera aya

chendhuram


T.BILIRUBIN(mg/dl) 0.6±0.081 0.68±0.116 0.75±0.104 0.7±0.14 N.S

D.BILIRUBIN(mg/dl) 0.27±0.05 0.33±0.516 0.33±0.081 0.3±0.063 N.S

I. BILIRUBIN(mg/dl) 0.32±0.09 0.35±0.104 0.41±0.752 0.4±0.141 N.S

SGOT(U/dl) 17.5±1.29 18.66±2.33 21±4.14 18.83±4.95 N.S

SGPT(U/dl) 22±3.55 23.166±4.020 24.16±6.17 21.5±4.88 N.S

ALP(U/dl) 196.25±8.77 151±16.17 148.16±24.07* 154.33±14.65* N.S

T.PROTEIN(mg/dl) 6.32±0.38 7.48±0.34 7.016±0.23 6.53±0.46 N.S

ALBUMIN(mg/dl) 3.55±0.23 3.55±0.32 3.51±0.19 3.35±0.266 N.S

GLOBULIN(mg/dl) 2.77±0.15 3.76±0.28 3.5±0.25 3.18±0.240 N.S

NS- Not Significant, **(p > 0.01), * (p >0.05), n = 10 values are mean ± S.D (One way


Histopathology report of Veera aya chendhuram

ORGANS LOWDOSE MID DOSE HIGH DOSE

KIDNEY

LIVER

STOMACH

LUNGS

SPLEEN

Fig.1


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CONCLUSION

The Acute and 28 days Repeated oral toxicity study of Veera aya chendhuram shows no

mortality and the histopathology report reveals that the drug is considered safe in the mid and

low dose.

Finally the mild increase in haematological paramaters (Hb, PCV, MCH, MCHC, MCV)

reveals that test drug may have the ability in the management of anaemia in the therapeutic

dose as mentioned in the literature.

ACKOWLEDGEMENT

I am grateful to my college National Institute Of Siddha, Chennai and my parents.

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low income and middle income countries, The Lancet, 2011; 378, no.9809: 2123-2135.

3. WHO Iron deficiency Anemia, Assessment, Prevention and control. A guide for program

managers world health organization, Geneva, Switzerland, 2001.

4. A.Kumar, A.K.Garaj, A Clinical study on panduroga, iron deficiency anemia, with

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acute and 28 days repeated oral toxicity study of the

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