chapter -5 acute and chronic toxicity...

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CHAPTER - 5

ACUTE AND CHRONIC TOXICITY STUDIES

Chapter No Contents Page No.

Acute Toxicity Study on Combined Extract of Cissus

Quadrangularis and Aegle Marmelos

5.1. Introduction 79

5.2. Materials and Methods 80

5.3. Experimental Procedure 80

5.4. Results and Discussion 81

5.5. Conclusions 81

Chronic Toxicity Studies on Combined Extract of Cissus

Quadrangularis and Aegle Marmelos

5.6. Introduction 86

5.7. Materials and Methods 87

5.8. Experimental Design 87

5.9. Results andDiscussion 90

5.10. Conclusions 103

79

ACUTE STUDIES ON COMBINED EXTRACT OF CISSUS QUADRANGULARIS

AND AEGLE MARMELOS

5.1. Introduction

Toxicity is defined as the substance which is able to harm or damage the exposed

organism. These toxicities are possible to effect on a whole organism like an animal, bacteria

or plant. It includes the effect on substructure of organism akin a cell (cytotoxicity) and organ

(organotoxicity) e.g. liver (hepatotoxicity).

Acute toxicity is described as the undesirable changes or signs of toxicity occur

immediately with in 24 hr or a short period of denudation to substances as toxic effect occurs

within 14 days as per guidelines of OECD.

The need of acute oral toxicity screening is to get the information on the biological

mechanism & systemic effect of chemical substances. Usually long term studies starts with

dose finding method under acute conditions. Additionally, the acute systemic toxicity

generates information by the chemical substance which is used in risk management in the

production, handling, hazard identification and use of chemical substances. Currently, the

precise or approximate value of LD50 calculated is based on the toxicological classification

of chemicals. The animals were observed closely after administration of substances during

first 24 hr and up to 2 weeks and changes in behaviors & appearance of animals were

detemined. The most of the clinical signs were evaluated by characterizing acute systemic

toxicity and describe its amelioration1.

5.1.1. Principle

The method was followed by means of OECD 423 (Acute Toxic Class

Method). The acute toxic class process is a step wise procedure with three rats of a

single sex per step. Depending on the mortality of the rats and the standard two to

three steps may be essential to allow conclusion on the acute toxicity of the test

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material. This method results in the use of rats while allowing for satisfactory data

based systematic conclusion. The process is used to defined doses (2000, 300, 50 and

5 mg/kg) the reports allow a material to be ranked and classified base upon the

Globally Harmonized System (GHS) for the categorization of chemicals which

produce acute toxicity.

5. 2. MATERIALS AND METHODS

5.2.1. Materials

Combined ethyl acetate extract of stem bark of Cissus quadrangularis and

fruit pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of

Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA), Vehicle -1% tween

80, Oral needle.

5.2.2. Experimental Animals

Albino Wistar rats (150-220gm) were used for the present study. These animals were

maintained in Santhiram College of Pharmacy, Nandyal, Andhra Pradesh, India. According to

CPCSEA guidelines Albino Wistar rats were kept in a proper ventilated room at 23°C and

12:12 hr light & dark cycle in polypropylene cages. Institutional Animal Ethical Committee

(IAEC) clearance was done with reference no 1519/PO/a/11/CPCSEA).

5.3. Experimental Procedure

The two different extracts were selected for acute oral toxicity study. The

initial dose level of combined ethyl acetate extract of stem bark of Cissus

quadrangularis and fruit pulp of Aegle marmelos(c-EACA) and combined ethanol

extract of stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos(c-

ECA) were 2000 mg/kg, p.o. for different groups.

The crude extracts have LD50 higher than 2000 mg/kg; p.o. Single dose of

extract was given 0.2 ml/100 gm body weight to rats which are kept fasted for

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overnight.

Food was withdrawn for remaining 3-4 hr after given of c-EACA and c-ECA

for determining signs of toxicity. The weights of the rats were notified before and

after given. The signs of changes in skin and fur, mucous membranes, eyes,

circulatory, respiratory, autonomic and central nervous system, motor activity and

behavior pattern were noted. The toxicity signs of fits, excessive salivation, tremors,

diarrhea, lethargy, sleep and coma, as well as the appearance of toxicity and signs of

toxicity were too noted2.

5.4. RESULTS AND DISCUSSION

Individual mortality data and necropsy were observed. Acute oral toxicity studies

revealed that combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit

pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark ofCissus

quadrangularis and fruit pulp of Aegle marmelos (c-ECA) is non-toxic nature, after the

administration of c-EACA and c-ECA, the both group rats were observed immediately

observed for 4 hrs for autonomic and central nervous system, motor activity and behavior

pattern for any changes or lethality for the next 14 days. Absence of lethality or toxic

reactions such as tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma were

found at the dose of 2000 mg/kg till the last part of the study period.

5.5. CONCLUSIONS

As per guidelines of Organisation for Economic Cooperation and Development for

acute oral toxicity, a lethal dose of c-EACA and c-ECA were calculated to be more than 2000

mg/kg body weight and both the extracts determined to be harmless up to 2000 mg/kg body

weight.

Therefore, 1/8th dose of 250 mg/kg body weight and 1/4th dose of 500 mg/kg body

weight were choosed for remaining pharmacological studies.

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Table 5.1: Individual Mortality data of c-EACA and c-ECA in acute oral toxicity study

Maximumdose level

sex No of animals diedduring day ofdosing (hr)

No of animals died during periodafter dosing (days)

Deaths

½ 1 2 3 4 1 2 3 4 5 6 7 8-14

c-EACA

2000mg/kg

M/3 0 0 0 0 0 0 0 0 0 0 0 0 0 0/3

F/3 0 0 0 0 0 0 0 0 0 0 0 0 0 0/3

c-ECA

2000mg/kg

M/3 0 0 0 0 0 0 0 0 0 0 0 0 0 0/3

F/3 0 0 0 0 0 0 0 0 0 0 0 0 0 0/3

83

Table 5.2: Individual necropsy findings in acute oral toxicity study of c-EACA

S. No TreatmentDose

(mg/kg)

Animal

Number

and Sex

Weight of RatsDuration of

studyMacroscopic observationsBefore

Test

After

Test

1 c-EACA 2000 1-Male 180 gm 185 gm 14 days No abnormalities detected



4 c-EACA 2000 1-Female 200 gm 205 gm 14 days No abnormalities detected



84

Table 5.3: Individual necropsy findings in acute oral toxicity study of c-ECA

S. No TreatmentDose

(mg/kg)

Animal

Number

and Sex

Weight of RatsDuration of

studyMacroscopic observationsBefore

Test

After

Test

1 c-ECA 2000 1-Male 180 gm 185 gm 14 days No abnormalities detected



4 c-ECA 2000 1-Female 205 gm 210 gm 14 days No abnormalities detected



85

Fig. 5.1: Flow chart for acute toxic class method (OECD guidelines 423) starting dose of

86

CHRONIC TOXICITY STUDIES ON COMBINED EXTRACT OF

CISSUS QUADRANGULARIS AND AEGLE MARMELOS

5.6. Introduction

Toxicology is the study of poisons and any material that produces a dangerous cause once

administered, either by mistake or intend, to a human being. The purpose of toxicology is to pave

the way to protect not only the humans and also the environment from the deadly effects of

toxicants besides to assist the growth of more selective toxicants like anticancer and other clinical

drugs and pesticides. Poisonous substance is a quantitative theory, nearly any material being

unsafe at some doses; the same material is safe at some minimum dose. Vinyl chloride is an

example. It is an effective hepatotoxicant at higher doses, at lower doses, Vinyl chloride is said to

be harmful and actually lacking effect at lower doses3, 4.

Clinical drugs are very beneficial at some doses, but it may be fatal at a high dose. For

example, Aspirin (acetyl salicylic acid) is a quite harmless drug at suggested doses and is used by

many people globally. Chronic use can leads to harmful effects on the gastric mucosa and it is

found to be lethal at a dose of about 0.2 to 0.5 g/kg. Around 15% of accidental deaths were

reported from poisoning in kids is caused by salicylates, mainly aspirin5.

The measurement of toxicity is a difficult task. Toxicity can be acute or chronic, and can

differ from one organ to another as well as with age, heredity, sex, diet, physiological state or the

strength status of the organism. The simplest measure of toxicity, the LD50 (the dose necessary

to kill 50% of a population under stated conditions) is very much dependent on the degree to

which the above variables are controlled. LD50 values may vary depending upon the route of

administration.Thus the toxicity obtained from intraperitoneal (IP), intravenous (IV),

87

intramuscular (IM), or subcutaneous (SC) injection and oral route of a known compound may

also vary. Toxicity may differ as tenfold with the route of administration.

A Word Health Organization study revealed that about 70–80% of the world’s

populations depends on synthetic medicines, largely of herbal resource, in their major healthcare6,

7. Although medicinal plants may cause numerous biological actions in humans, commonly very

little is well-known for their toxicity and the same applies for Combined ethyl acetate extract of

stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos (c-EACA) and combined

ethanol extract of stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA).

5.7. MATERIALS AND METHODS

5.7.1 Materials

Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit

pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of Cissus

quadrangularis and fruit pulp of Aegle marmelos (c-ECA), Vehicle -1% tween 80, Oral

needle.

5.7.2. Experimental Animals

Wister albino rats weighing between 150-220 gm each were used. They were maintained

in Santhiram College of Pharmacy, Nandyal, Andhra Pradesh, India. The animals were kept in a

well-ventilated room with at 12:12 hr light, dark cycle in polypropylene cages.Institutional

Animal Ethical Committee (IAEC) clearance was done with reference no

1519/PO/a/11/CPCSEA).

5.8. EXPERIMENTAL DESIGN

Animals were separated into 5 groups. Each group is comprised of six rats.

Group I

88

Vehicle 1% Tween 80 (5 ml/kg, p.o) once daily for 90 days.

Group II & III

Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit pulp of Aegle

marmelos (c-EACA) 250 & 500 mg/kg, p.o

Group IV & V

Combined ethanol extract of stem bark of Cissus quadrangularis and fruit pulp of Aegle

marmelos (c-ECA) 250 & 500 mg/kg, p.o once daily for 90 days respectively.

Toxic manifestations and death rate were observed daily. At the end of 7th, 30th ,60th &

90thday, all groups of animals feed intake were recorded. Weights of the animals were recorded

on initial day, 30th, 60th& 90th day. From each group animals were sacrificed at the 90thday,

various haematological and serum biochemical tests were performed after the last dose8.

5.8.1. Collection of blood and serum samples

After termination of the study, by cervical decapitation from cardiac puncture matching

blood samples were collected from rats which are anaesthetized by using diethyl ether and stored

in heparinised bottles for haematological analyses and to make the blood samples clot, it is stored

in clean non-heparinised bottles. For biochemical analysis, by using centrifugation process the

serum was separated from the clot and it is stored in clean bottles. Biochemical analyses were

done in serum which is obtained by centrifugation of total blood devoid of anticoagulants, at 2500

rpm for 15 min.

89

5.8.2. Estimation of haematological parameters

Estimation of RBC count, Hemoglobin, WBC count, clotting time, neutrophils,

eosinophils, lympocyte, monocyte and haemoglobin9, 10 were estimated by labor-intensive

techniques recognized in the laboratory.

5.8.3. Estimation of biochemical parameters

Blood Glucose, Serum Gluconate Oxaloacetate Transaminase (SGOT), Serum Glutamic

Pyruvate Transaminase (SGPT), Serum Bilirubin, Serum Alkaline Phosphatase (ALP), Blood

Urea, Blood Cholesterol, Creatinine and Serum protein11, 12, 13 were measured by commercially

available diagnostic kits (Span Diagnostics, India).

5.8.4. Histopathological studies

All animals were autopsied after euthanasia and from each animal sections of stomach,

liver and kidney were collected. For histological processing, the sections were dipped in 10%

formalin. The Section should be of 5 µm thicknesses were obtained on a rotary microtome and it

was stained by hematoxylin-eosin (HE) 14. Microscopic analyses were assessed for

histopathological changes such as congestion, edema, hemorrhage and necrosis.

5.8.5. Statistical analysis

The datas were recorded as mean ± standard error mean (S.E.M).The Significance of

variations between the groups were calculated using one way and multiple way analysis of

variance (ANOVA). The test followed by Dunnet’s test and the pvalues less than 0.05 were noted

as significance.

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5.9. RESULTS AND DISCUSSION

5.9.1. Effect of chronic oral administration of c-EACA and c-ECA extracts on the body

weight and death rate

The chronic oral administration of c-EACA and c-ECA extracts produced no apparent

variation in the common behaviour of the rats and, compared to the control group. Prior to

sacrifice, the animals were survived throughout the study time. The gain in bodyweight changes

was significantly reduced during the study period in the c-EACA 500 and c-ECA 500 mg/kg

group (P < 0.05, Table 7.1). In addition, throughout the 90 days treatment no lethality was

determined for any dose up to the highest of 500 mg/kg. Both the control and treated rats

appeared equally in good physical state till the end and all through the 90 days.

5.9.2. Effect of chronic oral administration of c-EACA and c-ECA extracts on the Feed

intake

A considerable reduction in food intake was determined in the c-ECA 500 mg/kg

treatment group (at day 7, 30, 60 and 90, P < 0.05), c-EACA 500 mg/kg (at day 30, 60 and 90, P

< 0.05). But c-EACA 500 and c-ECA 500 mg/kg treatment groups not showed significant

changes in feed intake (Table 7.2).

5.9.3. Effect of chronic oral administration of c-EACA and c-ECA extracts on the

haematological parameters

The neutrophil, lymphocyte and monocyte in the c-EACA and c-ECA extracts treated

rats do not vary much (P > 0.05) as of the control group and all the values remained in ordinary

limits all through the trial period. But remaining haematological parameters, Clotting time,

hemoglobin concentration, white and red blood cells in the c-EACA and c-ECA extracts treated

rats significantly (P > 0.05 &P > 0.01) increased whereas count of eosinophils considerably (P >

0.05) decreased as compared to control group (Table 7.3).

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5.9.4. Effect of chronic oral administration of c-EACA and c-ECA extracts on the

biochemical parameters

As shown in Table 7.4, no significant c-EACA and c-ECA treatment associated changes

in the quantities of plasma analytes such as SGOT, SGPT, bilirubin, and ALP, urea and serum

protein were estimated at the termination of the study. Whereas glucose, total cholesterol and

creatinine parameters decreased significantly. All the values remained in ordinary limits at the

termination of the study.

5.9.5. Effect of chronic oral administration of c-EACA and c-ECA extracts on the organs

weight

Total organ wet weights of rats treated with oral administration of c-EACA and c-ECA

extracts are shown in Table 7.5. There was no major change in the wet weights of the heart in the

treated rats compared to that of the control group. However, there is a considerable increase in

kidneys (left & right), spleen and liver were noted in the c-EAC and c-ECA extracts treated

groups compared to the control group. There is no dose dependent factor.

5.9.6. Histopathological observation

In the present study, there were no histopathological results that could be recognized to

the c-EACA and c-ECA treatment groups compare to control group. No variations in the organ

morphology for all the different dose groups. No histological changes were observed in stomach,

liver and kidney.

92

Group I Group II

(1% Tween 80 (5ml/kg, p.o) (c-EACA 250 mg/kg)

Group III Group IV

(c-EACA 500 mg/kg) (c-ECA 250 mg/kg)

Group V

(c-ECA500 mg/kg)

(Group I – Control; Group II, Group III, Group IV and Group V – Treated)

Fig. 5.2: HISTOPATHOLOGICAL STUDIES ON STOMACH

93

Group I Group II


Group III Group IV


Group V

(c-ECA 500 mg/kg)


Fig. 5.3: HISTOPATHOLOGICAL STUDIES ON LIVER

94

Group I Group II


Group III Group IV


Group V

(c-ECA 500 mg/kg)


Fig. 5.4: HISTOPATHOLOGICAL STUDIES ON KIDNEY

95

Table 5.4: Effect of chronic oral administration of c-EACA and c-ECA extracts on the body weight

Week oftreatment

(g)

Treatment

Group I

(1% Tween 80

5 ml/kg, p.o)

Group II

(c-EACA

250 mg/kg)

Group III

(c-EACA

500 mg/kg)

Group IV

(c-ECA

250 mg/kg)

Group V

(c-ECA

500 mg/kg)

Initial Bodyweight

187.5±1.544 190.33±1.892a 189.83±1.833a 188.17±1.579a 191±1.844a

Body weighton 30th day

216.17±0.833 209.5±1.335a 207.67±1.174a 207.67±1.229a 206.67±1.358a


254.67±1.333 253.17±1.470a 251.17±1.167a 252.67±1.145a 251.17±1.108a


290.17±0.654a 285.5±0.957a 275±1.125*a 282.83±0.945a 272.67±0.988*a

a-Group I & II Vs group III, IV &V. *P < 0.05; **P < 0.01 as compared to control group.

Each value denotes the mean ± S.E.M six rats in each group.

Statistical significant test for evaluation was done by ANOVA followed by Dunnett’s test.

96

Initial 30th day 60th day 90th day0

100

200

300IIIIIIIVV

Bod

y w

eigh

t in

gms

Fig.5.5: Effect of chronic oral administration of c-EACA and c-ECA extracts on the body weight


97

Table 5.5: Effect of chronic oral administration of c-EACA and c-ECA extracts on food intake

Week oftreatment

(g/day/rat)

Treatment

Group I

(1% Tween 80

5 ml/kg, p.o)

Group II

(c-EACA

250 mg/kg)

Group III

(c-EACA

500 mg/kg)

Group IV

(c-ECA

250 mg/kg)

Group V

(c-ECA

500 mg/kg)

Food intakeon 7th day

23.67±0.5578 23.17±0.4773a 22.17±0.6009a 22.5±0.4282a 20.83±0.3073*a


24.5±0.7638 22.67±0.4216a 21.83±0.6009*a 22.83±0.4014a 21.17±0.3073*a


24.33±0.4216 22.5±0.5627a 21.83±0.6009*a 22.5±0.6708a 21.33±0.4216*a


24±1.033 22.83±0.7032a 21.83±0.3073*a 22.83±0.4773a 21.17±0.4773*a




98

7th day 30th day 60th day 90th day0

10

20

30IIIIIIIVV

Fee

d i

nta

ke(g

/day

/rat

)

Fig.5.6: Effect of chronic oral administration of c-EACA and c-ECA extracts on food intake


99

Table 5.6: Effect of chronic oral administration of c-EACA and c-ECA extracts on hematological parameters

HematologicalParameters

TreatmentGroup I

(1% Tween 805 ml/kg, p.o)

Group II(c-EACA

250 mg/kg)

Group III(c-EACA

500 mg/kg)

Group IV(c-ECA

250 mg/kg)

Group V(c-ECA

500 mg/kg)Neutrophil(%) 24.83±0.3073 26.17±0.4773a 26.5±0.4282a 25.5±0.4282a 27.17±0.3073*a

Eosinophil(%) 0.97±0.0421 0.95±0.0223a 0.87±0.0210*a 0.97±0.0211a 0.85±0.0223*a

Lymphocyte(%) 69.5±0.4282 68.33±0.2108a 68.67±0.5578a 69.33±0.3333a 68.33±0.3333a

Monocyte(%) 2.83±0.0309 2.82±0.0333a 2.79±0.0135a 2.91±0.0413a 2.78±0.0261a

Clotting time (seconds) 80.17±0.9804 82.83±0.7491a 84±0.8563*a 82.83±1.046a 85.33±0.6146*a

Haemoglobin(g/dL) 11.67±0.1585 12.48±0.0945*a 12.8±0.0365**a 12.35±0.1088*a 13.18±0.0909**a

RBC cells (×106

RBC/mm3)6.62±0.0792 6.73±0.0557a 6.93±0.0628*a 6.68±0.06a 6.92±0.0792*a

WBC cells(×103WBC/ mm3)

5.67±0.0614 5.85±0.07188a 6.27±0.0882**a 6.03±0.0803*a 6.50±0.07303**a




100

Table 5.7: Effect of chronic oral administration of c-EACA and c-ECA extracts on biochemical parameters

Biochemicalparameters

Treatment

Group I

(1% Tween 80

5 ml/kg, p.o)

Group II

(c-EACA

250 mg/kg)

Group III

(c-EACA

500 mg/kg)

Group IV

(c-ECA

250 mg/kg)

Group V

(c-ECA500mg/kg)

Glucose mg/dl 108.5±3.191 96.5±1.118*a 96.17±0.7032*a 85±1.238**a 79.83±0.8724**a

Bilirubin mg/dl 0.32±0.0167 0.32±0.0167a 0.33±0.0211a 0.34±0.028a 0.31±0.0117a

SGOT Unit/L 59.5±1.204 62.5±1.025a 64.17±1.078a 63.33±1.022a 65.67±1.308a

SGPT Unit/L 35.67±1.054 38.33±1.174a 42.17±0.8333a 37.17±0.9458a 40.17±0.8724a

ALP Unit/L 42.17±0.9458a 41.83±1.195a 43.67±0.7601a 42.83±1.078a 44.83±0.7032a

Total Cholesterol

mg/100ml

154.5±2.643a 146.33±1.606*a 139±1.065**a 141±1.528**a 132.5±1.176**a

Urea mg/dl 45.17±0.7923 46±0.9661a 46.17±0.8724a 47.33±0.6667a 47.5±0.5627a

Creatinine mg/dl 0.56±0.0104 0.51±0.0101*a 0.48±0.0124**a 0.49±0.0079**a 0.45±0.0562**a

Protein mg/dl 7±0.1414 6.72±0.0703a 6.77±0.0614a 6.7±0.0683a 7.05±0.05a

a-Group I & II Vs Group III, IV &V. *P < 0.05; **P < 0.01 as compared to control group.



101

Table 5.8: Effect of chronic oral administration of c-EACA and c-ECA extracts on organ weight (g) in rats

a-Group I & II Vs Group III, IV &V. *P < 0.05; **P < 0.01 as compared to control group.


Group Treatment

Kidney

Heart Spleen Liver

Left Right

I 1% Tween 80 (5 ml/kg, p.o) 0.59±0.0084 0.56±0.0086 1.0±0.0287a 0.84±0.0120 7.47±0.0477

II c-EACA 250 mg/kg 0.57±0.0057a 0.55±0.0115a 1.09±0.0333a 0.87±0.0112a 7.82±0.0439a

III c-EACA 500 mg/kg 0.63±0.0081**a 0.59±0.0138a 0.99±0.1738a 0.88±0.0114*a 8.06±0.0561**a

IV c-ECA 250 mg/kg 0.59±0.0088a 0.55±0.014a 1.09±0.0249a 0.88±0.007*a 7.75±0.0428**a

V c-ECA 500 mg/kg 0.64±0.0062**a 0.64±0.0067**a 1.15±0.0215a 0.88±0.0075**a 8.16±0.0417**a

102


103

5.10 CONCLUSION

Herbalism, a natural therapy has become generally admired in most important healthcare systems,

mainly in rising nations including India. Herbal sources from medicinal plants are recognized to be

harmless exclusive of any compromising health effect, thus extensively used as self-medication15.

Though, there is a lack of evidential scientific researches on the toxicity and undesirable effect of these

remedies, More investigations are truly needed and the same applies for Combined ethyl acetate extract of

stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos (c-EACA) and combined ethanol

extract of stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA). Because

protection should be the superseding criterion in the choice of medicinal plants to apply in healthcare

systems16. Everyone should, counting to the utilization of past citations on combined ethyl acetate extract

of stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos (c-EACA) and combined ethanol

extract of stem bark of Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA) also contains

proper toxicological estimation of this plant to optimize its harmless use as a medicine. But previous to

such valuation can be completely warranted in humans, the preclinical estimation of the safety of the

EACA and c-ECA is required.

The present study indicates that the Combined ethyl acetate extract of stem bark of Cissus

quadrangularis and fruit pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of

Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA), used in the conventional medicine for

numerous therapeutic conditions in India, was comparatively non-toxic when given to rats as a chronic

oral administration for 90 days.

In the 90 days chronic toxicity study, the c-EACA and c-ECA extracts at the dose of 250 & 500

mg/kg, slightly decreased body weight but did not appear to affect the behaviour of the rats and produced

no considerable changes in their food intake and consumption of food signifying regular metabolism in

the animals and telling that, at the oral doses administered, c-EACA and c-ECA extracts did not delay the

104

growth of rats. At the same time a reduction in feed intake and body weight by administration of the c-

EACA and c-ECA extracts demonstrated that the plants have anti-obesity activity.

After 90 days treatment, no treatment associated changes in the haematological parameters (i.e.

hemoglobin concentration, clotting time, neutrophils, eosinophils, lymphocytes, monocytes, red and white

blood cells) between control and treated groups representing that the c-EACA and c-ECA extracts was

not lethal to the circulating red cells, nor interfered with their production. It does not affect the

hematopoiesis and leucopoiesis although the haematopoietic system is one of the main responsive targets

for poisonous compounds17 and an vital index of physiological and pathological grade in man and

animals18.

In addition, most of the hepatological and renal parameters (i.e. Creatinine, Bilirubin, SGOT,

SGPT, ALP, Urea, Uric acid, Protein) were also unchanged by the doses of c-EACA and c-ECA 250 &

500 mg/kg. The lack of considerable alterations in the amount of ALP, Creatinine, Bilirubin, SGOT and

SGPT, good indicators of liver and kidney functions, respectively19. The SGOT and SGPT are familiar

enzymes used as biomarkers predicting potential toxicity20. Commonly, when there is a damage in the

parenchymal liver cells there will be an elevations in both of these transaminases21. The transaminases

were not considerably greater than before at the doses of c-EACA and c-ECA 250 & 500 mg/kg. It

suggests that chronic ingestion of c-EACA and c-ECA did not modify the hepatocytes and kidneys of the

rats and additionally the regular metabolism of the animals.

The importance of this outcome may be related to the biological value of the c-EACA and c-ECA

extracts. It may be concluded that the c-EACA and c-ECA extracts did not stimulate any considerable

harm to these organs. A decline in plasma glucose and total cholesterol by administration of the c-EACA

and c-ECA extracts demonstrated that the plants have hypoglycemic activity and hypolipidemic activity.

In general, this work provides helpful preliminary information on the toxicity report of c-EACA and c-

105

ECA extracts that should be valuable for the development of future pre-clinical and clinical studies of this

plant medicine.

In conclusion, this research work demonstrates that at doses consumed in the traditional

medicine, the c-EACA and c-ECA extracts considered as relatively harmless, as it did not produce either

any lethality or differences of in the common behavior in chronic toxicity studies in rodents and in high

doses may have a hypoglycemic, hypolipidemic and anti-obesity effect. Additionally, no histological

changes were observed in stomach, liver and kidney.

chapter -5 acute and chronic toxicity...

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