chapter -5 acute and chronic toxicity...
TRANSCRIPT
78
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
2 c-EACA 2000 1-Male 190 gm 195 gm 14 days No abnormalities detected
3 c-EACA 2000 1-Male 190 gm 195 gm 14 days No abnormalities detected
4 c-EACA 2000 1-Female 200 gm 205 gm 14 days No abnormalities detected
5 c-EACA 2000 1-Female 205 gm 208 gm 14 days No abnormalities detected
6 c-EACA 2000 1-Female 195 gm 202 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
2 c-ECA 2000 1-Male 185 gm 192 gm 14 days No abnormalities detected
3 c-ECA 2000 1-Male 192 gm 196 gm 14 days No abnormalities detected
4 c-ECA 2000 1-Female 205 gm 210 gm 14 days No abnormalities detected
5 c-ECA 2000 1-Female 200 gm 206 gm 14 days No abnormalities detected
6 c-ECA 2000 1-Female 190 gm 194 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),
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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
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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.
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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.
90
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).
91
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
(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-ECA 500 mg/kg)
(Group I – Control; Group II, Group III, Group IV and Group V – Treated)
Fig. 5.3: HISTOPATHOLOGICAL STUDIES ON LIVER
94
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-ECA 500 mg/kg)
(Group I – Control; Group II, Group III, Group IV and Group V – Treated)
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
Body weighton 60th day
254.67±1.333 253.17±1.470a 251.17±1.167a 252.67±1.145a 251.17±1.108a
Body weighton 90th day
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
a-Group I & II Vs group III, IV &V. *P < 0.05; **P < 0.01 as compared to control group.
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
Food intakeon 30th day
24.5±0.7638 22.67±0.4216a 21.83±0.6009*a 22.83±0.4014a 21.17±0.3073*a
Food intakeon 60th day
24.33±0.4216 22.5±0.5627a 21.83±0.6009*a 22.5±0.6708a 21.33±0.4216*a
Food intakeon 90th day
24±1.033 22.83±0.7032a 21.83±0.3073*a 22.83±0.4773a 21.17±0.4773*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.
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
a-Group I & II Vs group III, IV &V. *P < 0.05; **P < 0.01 as compared to control group.
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
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.
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.
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.
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.
Each value denotes the mean ± S.E.M six rats in each 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
Statistical significant test for evaluation was done by ANOVA followed by Dunnett’s test.
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
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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-
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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.