Acute and sub-acute toxicological evaluation of aqueous and ethanol fractions of Annona squamosa root a traditional medicinal herb

 

C. Ronald Darwin1, C. Vijaya2, K. Sujith1, M. Sadhavi1, Haneesha Pothuri1

1Department of Pharmacology, K.K College of Pharmacy Chennai-122,

 2Department of Pharmacology, Ultra College of Pharmacy Madurai.

*Corresponding Author E-mail: ronaldpharma@gmail.com

 

ABSTRACT:

The study was designed to elucidate acute and sub acute toxicity of aqueous and ethanolic extract of Annona squamosa root in mice and rats respectively. In acute study aqueous and ethanolic extract of Annona squamosa root were administered to mice at a dose level starting from 5, 50,300 and 2000 mg/kg and in sub-acute toxicity study aqueous extract was administered at a dose of 200 mg/kg per day for 28 days. Acute toxicity data revealed that aqueous extract did not show any toxic related clinical signs, while ethanolic extract showed toxic clinical signs at dose of 300 and 2000 mg/kg. Sub-acute toxicity study with aqueous extract at dose of 200mg/kg showed no significant change in body weight, vital organs weight, biochemical and hematological parameters. Histopathological valuation has not shown any abnormalities in aqueous treated group when compared to the control group. Aqueous extract treated animal has not shown any signs of toxicity in acute and sub acute toxicity studies in rats, while ethanolic extract of Annona Squamosa  roots was found to be toxic in acute toxicity studies.

 

KEYWORDS: Annona Squamosa, Acute toxicity studies, Sub-acute toxicity study, ethanolic extract, Biochemical parameters

 


 

INTRODUCTION:

Annona squamosa L. is commonly known as custard apple and is a native of West Indies and is cultivated throughout India, mainly for its edible fruit. Annona squamosa a common plant with many folklore claims and is reported to have medicinal properties which include antifertility and anti-tumour activities in mice and rats 1,2. Annona squamosa roots are used for mental depression and spinal cord disorders 3.There was no scientific reports on toxicity data of Annona squamosa roots. So the study has been conducted for the evaluation of acute and subacute toxicity study on   Annona squamosa roots.

 

MATERIALS AND METHODS:

Plant material and extraction:

Roots of Annona squamosa were collected from the regions of Porur, Chennai, India and were identified and authenticated by Prof. P. Jeyaraman, Botanist - Plant Anatomy Research Centre, Chennai, India. A voucher specimen (Rt-Sp-03/11) has been preserved at the department for future reference.

 

Fresh roots were washed well with water. Air-dried roots (500 g) were extracted separately with water and ethanol for 7 days and filtered on muslin cloth, the filtrate were centrifuged at 10,000rpm for 10min in normal centrifuge  at room temperature and the extract was concentrated to half of its volume by slight warming. The resulting dark brown extract was concentrated on rotary evaporator under reduced pressure. The resulting crude drug (8g for aqueous and 13g for alcoholic) was used for further study.

 

Experimental Animals:

Adult female Albino mice weighing between 25 to 30 g were used for acute toxicity studies and for sub acute toxicity studies both male and female rats weighing between 150-200gm were used. Animals were housed in a well ventilated room with 12 h cycle of day and night light conditions and temperature maintained at around 32C.  The animals were fed with a standard laboratory diet and tap water ad libitum. All experiments were carried out in accordance with guide for the care and use of laboratory animals. After one week of habituation, animals were subjected to the experiments. Females were caged separately from the males to prevent mating. All tests were carried out between 9.00 am and 2.00 pm. All efforts were made to minimize animal suffering.

 

Acute toxicity studies:

Acute toxicity studies were carried out according to the OECD (Organization of Economic Co-operation and Development) guidelines 4234 .Healthy female mice, weighing 25–30 g, were selected and oral administration of the single doses of Annona squamosa extracts (aqueous and alcoholic) were done aseptically by suspending in Sodium carboxymethyl cellulose. An oral (p.o) dose of 5 mg/kg, 50 mg/kg, 300 mg/kg and 2000 mg/kg was administered step by step according to the guidelines. The general behaviors of the mice were continuously monitored for 1 h after dosing, periodically during the first 24 h (with special attention given during the first 4 h 5, and then daily thereafter, for a total of 14 days. Changes in the normal psychomotor activity and external morphology of mice and their body weights were monitored periodically before dosing and the time at which signs of toxicity or mortality were recorded.

 

Sub-acute toxicity:

Sub-acute toxicity studies were carried out according to OECD TG4076and rats were divided into 3 groups of 10 animals (5 male and 5 female). The extracts of Annona squamosa  was administered to the first group of rats at the dose of 200 mg/kg/day for 28 days, whereas an equal volume of vehicle was given to the control group. In order to access reversibility, the extracts of Annona squamosa at the dose of 200 mg/kg was given once daily to the third group of rats for 28 days, and kept for another 14 days post-treatment. The toxic symptoms such as signs of toxicity, mortality and body weight changes were monitored daily. Rats were anesthetized with ether at the end of the treatment period. All rats were sacrificed after the blood collection.

 

Blood analyses:

The blood sample was carefully collected for blood chemistry and enzyme analysis. Heparinized blood samples were used for determining total red blood cell count, total white blood cell count, platelet count haemoglobin, hematocrit, lymphocytes and neutrophils7. Non heparinzed samples were used for the estimation of biochemical parameters like creatinine8, urea9, triglycerides, total cholesterol10, total protein11, albumin12,AST, ALT13,14, ALP15 and total bilirubin16,17.

 

Histopathological examinations:

The internal organs like liver, kidney, heart, lungs, brain and spleen were isolated and weighed to determine relative organs weights and observed for gross lesions. All tissues were preserved in 10% buffered formaldehyde solution for histological examination 18,19.

 

Statistical analysis:

Statistical analysis was carried out by one way ANNOVA and the results were expressed as mean ± SEM. p values less than 0.05 were considered as significant.

 

RESULTS:

Acute toxicity studies:

Animals treated with aqueous extract of Annona squamosa at the dose level starting from 5 mg to 2000mg/ kg has survived the scheduled sacrifice, and there were no test-material-related toxic clinical signs. But one animal among the three animals treated with alcoholic extract at the dose level of 300mg/kg has not survived more than 48 hours and all the animals treated with 2000 mg/kg has not survived more than 12 hours (Table: I). Aqueous extract treated animals has not shown any signs of toxicity provided ethanolic extract treated animals show abnormal symptoms like increased sniffing, seizures, straubs tail and loss of grip strength, righting reflex and spontaneous rearing. (Table: II)


 

Table: I. Effect on Annona squamosa in mice after oral administration of single dose

Treatment

Mice/Sex

Death/ Treated

Mortality latency (hr)

Aqueous

Ethanolic

Aqueous

Ethanolic

0 mg/kg(Vehicle)

Female

0/3

0/3

-

-

5mg/kg

Female

0/3

0/3

-

-

50mg/kg

Female

0/3

0/3

-

-

300mg/kg

Female

0/3

1/3

-

<48

2000mg/kg

Female

0/3

3/3

-

<12

 

Table: II. Symptoms observed for the first four hours for the acute treatment of 2000mg/kg

Parameters observed

I  hr

II hr

III hr

IV hr

Aq.

Eth.

Aq.

Eth.

Aq.

Eth.

Aq.

Eth.

Piloerection

-

-

-

-

-

-

-

-

Edema

-

-

-

-

-

-

-

-

Urine stains

-

-

-

-

-

-

-

-

Alopecia

-

-

-

-

-

-

-

-

Loss of righting reflex

-

-

-

-

-

-

-

 

Circling

-

-

-

-

-

-

-

-

Nasal sniffing

-

-

-

+

-

+

-

+

Lacrimation

-

-

-

-

-

-

-

-

Seizures

-

-

-

-

-

+

-

+

Righting reflex (Touch)

+

+

+

-

+

-

+

-

Grip strength

+

+

+

-

+

-

+

-

Eye closure at touch

+

+

+

+

+

+

+

+

Rearing

+

+

+

-

+

-

+

-

Straub tail

-

-

-

-

-

+

-

+

Aq. –Aqueous           Eth.-Ethanolic ;       (-)            Indicates absence of symptom      (+)      Indicates presence of symptoms

Table: III. Influence of Annona Squamosa on change in body weight (200mg/kg)

Treatment

7th day

14th  day

21st day

28th day

% increase

Control

175.92±1.25

176.66±1.25

176.66±1.25

177.00±1.41*

1.04

200 mg/kg

156.16±0.29

157.16±0.79

157.83±0.74

158.16±0.70*

1.07

200mg/kg (Repeat)

155.17±1.20

155.65±1.02

156.98±0.96

157.31±1.09*

1.08

Data are expressed as mean ± SEM;’        *P<0.05vs value on 7th day

 

Table IV. Relative Organ Weight of male and female rats treated with aqueous extract of Annona squamosa

Dose

Relative Organ Weight of male and female  rats treated with aqueous extract of Annona squamosa

Liver

Kidney

Brain

Lungs

Heart

Spleen

Control

2.8±0.1

0.66±0.02

0.38±0.22

0.29±0.01

0.29±0.01

0.15±0.01

200mg/kg

2.9±0.1

0.66±0.02

0.40±0.01

0.31±0.02

0.30±0.01

0.16±0.01

200mg/kg (Repeat)

3.0±0.1*

0.67±0.03*

0.43±0.01*

0.32±0.01*

0.31±0.01*

0.17±0.01*

Data are expressed as mean ± SEM;          *P<0.05vs control

 

 

Fig: I Representative photomicrographs of H and E stained sections of rat organs over a time course after Annona squamosa administration. Control VS treated at the dose level of 200mg/kg (p.o). Fig Ia and Ib shows the normal architect of brain, Fig IIa and IIb shows the normal architect of liver, Fig IIIa and IIIb shows the normal architect of lungs, Fig IVa and IV b shows the normal architect of heart, Fig Va and Vb shows the normal architect of kidney, Fig VI a and VI b shows the normal architect of spleen


Sub acute toxicity studies:

Annona squamosa extracts at dose of 200mg/kg given orally for 28 days did not produce any mortality changes.

 

Change in body weight:

Body weight changes were monitored at weekly intervals till 28days. Body weights of rats in sub-acute toxicity studies were summarized in (Table: III). Body weights of rats at the dose of 200 mg/kg have not show any significant change over those in the untreated control group. Percentage increase in all the groups including the satellite group is found to be uniform. The extract does not show any reduction in the body weight as an evidence for absence of toxicity.

 

Organ weights:

Relative organ weight of rats treated with Annona squamosa did not show any evidence of drug-related toxicity. There were no significant changes of weight of extract treated and those of control groups. The satellite group which was kept for further 14 days was also uniform to other groups in organ weight. (Table IV)

 

Pathology—Macroscopic and Microscopic:

The histological examination of the various organs was performed in both control and treated groups. Organ weight revealed that at repeated dose administration of extract at dose of 2000mg/kg did not produce any organ swelling, atrophy and hypertrophy. All the sampling tissue sections were within the normal limits and degenerative or infiltrative lesion was not observed in the extract treated group. The results obtained revealed that there were no significant changes in the histology of the internal organs of the aqueous extract treated rats as compared to the control rats (Fig-1).

 

 

Hematological and serum biochemical parameters:

The haematological analysis shown in the (Table V), shows no significant change of blood parameters analyzed such as total red blood cell, hematocrit, hemoglobin, total white blood cell, lymphocytes, neutrophils and platelets in the treatment groups compared to the control group.

 

The biochemical analysis of creatinine, urea, triglycerides, total cholesterol, total protein, albumin, AST, ALT, ALP and total bilirubin (Table VI), shows no significant difference in any of the biochemical parameters examined in either of the control or treated groups. All the above said haematological and serum biochemical parameters are within normal limits.

 

DISCUSSION:

The results of the acute toxicity study reveal that ethanolic extract treated animal shows toxic symptoms such as change in behavior and mortality at dose of 300mg/kg and 20000mg/kg respectively. But acute toxicity study of aqueous treated animal group did not show any toxic symptoms in behavior or mortality up to dose level of 2000mg/kg. Hence, we conducted sub acute toxicity study of aqueous extract and it appeared that aqueous extract of Annona squamosa  roots at dose level of 200mg/kg did not produce any marked toxic changes in both male and female rats, as evidenced by absence of toxic symptoms, no change in water and food ingestion was noticed. Generally, the reduction in body weight gain and internal organ weights is a simple and sensitive index of toxicity after exposure to toxic substance 20. In the present study, aqueous extract of Annona squamosa  roots does not produce any statistically significant difference among three groups in body weight gain and internal organ weights.

 

 


 

Table: V. Effect of Annona squamosa Linn, on the Haematological Parameter

Haematological parameter

Control

Annona squamosa extract

200 mg

200mg (repeat)

Total R.B.C. count (×106 mm3).

07.27 ± 0.49

07.07 ± 1.86

7.01 ± 1.05

Total W.B.C. Count (×103 mm3).

07.88 ±1.97

07.54 ± 1.45

7.23 ± 1.88

Haemoglobin (Hb) (g/dl)

11.42 ±1.24

11.41 ± 1.07

11.42 ± 0.72

Hematocrit (%).

37.77±1.96

38.74±0.99

38.14±2.56

Platelets (×103 mm3).

703±123.4

701±166.74

708±135.67

Lymphocytes(%).

83.43±3.43

76.56±2.45

81±4.55

Neutrophils (%).

16.58±2.33

17.54±4.31

17.09±2.98

Data are expressed as mean ± SEM

 

Table: VI. Effect of Annona squamosa on the Biochemical Parameters

Biochemical parameter

Control

Annona squamosa extract

200 mg

200mg (repeat)

Creatinine (mg/dl)

0.72 ± 0.04

0.63±0.02

0.73±0.01

Urea (g/dl)

47.17 ± 1.92

40.75±1.65

40.17±1.75

Triglycerides (mg/dl)

48.83 ± 4.12

53.25±4.14

54.33±11.83

Total Cholesterol (mg/dl)

68.33 ± 2.03

76.00±2.66

79.25±3.95

Total protein (mg/dl)

8.53 ± 0.24

8.31±0.23

8.47±0.24

Albumin (g/dl)

4.52 ± 0.15

4.17±0.03

4.27±0.19

AST (IU/L)

219.0 ± 17.6

204±19.4

189.6±28.8

ALT (IU/L)

66.00 ± 4.19

66.5±6.36

62.67±7.61

ALP (IU/L)

289.3 ± 28.0

250.4±45.32

267.54±32.76

T. Bilirubin (mg/dl)

0.35 ± 0.15

0.37±0.13

0.35±0.19

Data are expressed as mean ± SEM


All animals survived until the scheduled euthanasia and no gross pathological alteration was found in the internal organs. Histopathological valuation did not find any abnormalities in aqueous treated group compared to the control group. The comparable biochemical results in the control groups and the extract treated groups were consistent with morphological analysis.

 

In summary, the ethanolic extract of Annona squamosa  roots was found to be toxic in acute toxicity studies , while aqueous extract treated animal was found to be  non toxic in oral acute  and sub acute toxicity studies in rats.

 

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Received on 18.06.2011          Modified on 26.06.2011

Accepted on 07.07.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1475-1479