Preliminary Toxicity Evaluation of Bidens pilosa Extracts Using Experimental Mice

S. Parimalakrishnan¹*, V.D. Sundar², I. Ulaganathan², S. Nandhakumar² and Akalanka Dey¹

¹Annamalai University, Annamalai Nagar-608002, TamilNadu, India

²GIET School of Pharmacy, Rajahmundry, AP, India

ABSTRACT:

The present study was carried out to evaluate acute and sub-acute toxicity in mice (Swiss albino mice). In acute toxicity study, the test material of methanolic extract of B. pilosa (MEBP) (Asteraceae) was administered orally at dose levels 3, 3.5, 4, 4.5, 5 and 5.5 g/kg, b.wt are observed. The mortality signs behavior using Finley’s probit analysis LD₅₀ dose was calculated as 4.5 g/kg b.wt. In the sub-acute toxicity study, methanolic and petroleum ether extracts of B. pilosa (MEBP and PEBP) were administered at dose levels of 200 and 400 mg/kg b.wt, p.o., once a day for 28 days. All the animals were sacrificed on 29^th day and blood was collected and various hematological parameters such as total count of RBC, WBC, DLC and Hb content were monitored and serum protein levels evaluated. Histopathological evidence of the viscera showed congestion and focal necrosis of the liver and renal tubules. It is apparent from the results that the MEBP is safe; however the depression in respiration mechanism that lead to the death of the mice needs to be investigated further.

KEYWORDS: Acute, Sub-acute toxicity, hematological parameters

INTRODUCTION:

Safety pharmacology is a step ahead than toxicological evaluation because ethics plays an important role in animal studies and human clinical trials. Therefore safety pharmacology is implemented animal screening stages. Traditional or folk healing herbal medicines are used approximately by 60% of the world population both in the developing and developed countries. The herbal recipes are prepared most often from a combination of two or more plant products which may contain active constituents with multiple physiological and biochemical activities and could be used in the treatment of a various diseases¹. They are administered in most disease conditions over a period of time without proper dosage monitoring and consideration of toxic effects that might result from such prolonged usage of drugs.

Bidens pilosa (Family - Asteraceae) is found in waste lands throughout India. It was been already investigated because of its wide range of medicinal applications. Its leaves are used as treatment for rheumatism, sore eyes, abdominal troubles, ulcers swollen glands and toothaches², cancer, cough³. It is commonly used for inflammatory, diuretic, jaundice, conjunctivitis, abscesses, mycosis and urinary infections and stimulates childbirth⁴. Emollient, astringent, fever, malaria, leucorrhea, diabetes, sore throat, tonsillitis and vaginal infections also treated. Apart from this plant is applied externally for fungal infections, ulcers, diaper rash, insect bites, and hemorrhoids⁵.

The scope of the present study was to evaluate the safety profile of entire plant extract of Bidens pilosa by carrying out the acute and sub acute toxicity

study in experimental mice for 28 consecutive days. The sub acute toxicity evaluation is required to establish the potential adverse effects of this valuable herbal medicine used for this study.

MATERIALS AND METHODS:

Plant collection and Preparation of Modulator:

The entire plant of Bidens pilosa was collected from Sengottai of Tirunelveli (Dist) Tamil Nadu, India. The plant material was taxonomically identified and authenticated by the Department of Botany, Annamalai University, Annamalai Nagar, India. The samples were cleaned, dried under shade, powdered by a mechanical grinder and stored in an airtight container. Methanol and petroleum ether was used as solvent in the extraction process and extracts were prepared by using soxhlet apparatus. The percentage of yield in methanolic and petroleum ether extract was found to be 6.46% w/w and 7.57% w/w.

Animals:

Both the sexes of mice (8 weeks old) weighing 25-30 g were procured from the central animal house of the institute. They were kept in standard polypropylene cages maintained under standard room temperature (22 ± 2°C; relative humidity 60-70%) in 12 hr. The animals were fed with standard pellet diet and water ad libitum. All animals and experimental design was approved by the Institutional Animal Ethical Committee (IAEC) [Register No: 160/1999/CPCSEA & Proposal No: 241].

Phytochemical Studies:

The MEBP and PEBP were subjected to preliminary phytochemical screening for various plant constituents^{6, 7}.

Acute toxicity studies:

Swiss albino mice of either sex with the body weights ranging from 18-30g were selected. Since the compound was insoluble in water, it was mixed evenly in 4 % v/v Tween 80 solution and administered orally in the doses of 3.0, 3.5, 4.0, 4.5, 5.0 and 5.5 g/kg in different group (2, 3, 4, 5, 6 and 7) containing three mice each. Group 1 was treated with the vehicle, served as solvent control. Observations for toxic symptoms and mortality were observed up to 72 hours. Clinical signs of toxicity as excitation, tremors, and twitches, motor coordination, righting reflex and respiratory changes also monitored⁸.

Sub acute toxicity studies:

Thirty mice of average range between 26-30g were acclimatized for a week in cleaned cages and randomly divided into five groups of six animals each. Group I solvent control, Group II and III were administered MEBP (200 and 400 mg/kg, b.wt, p.o) Group IV and V animals were administered PEBP (200 and 400 mg/kg, b.wt, p.o). Before administration of 1^st dose animals were fasted overnight. All the drugs were administered once a day for 28 days. Body weight and food intake were monitored at the end of every week throughout the duration of study⁹. All the animals were sacrificed on the day 29, blood sample were collected from abdominal aorta and frozen in polypropylene tube for evaluating haematological parameters like haemoglobin (Hb), white blood cells count (WBC), red blood cells count (RBC), packed cell volume (PCV) and differential leucocytes count (DLC)^10-12. Total protein¹³ was additionally evaluated.

Histopathological study:

Liver, kidney and small intestine of sacrificed animals were cut in to small pieces were collected and immediately fixed in 10% formalin buffered saline, dehydrated in ethanol, cleared in xylene and embedded in paraffin. Sections 4-5mm thick were prepared and then stained with hematoxylin and eosin (H and E) dye for photo microscopic observation.

Statistical analysis:

Results were expressed as mean ± standard error of mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA) and post hoc least-significant difference (LSD) test. The data obtained from acute toxicity studies was analyzed using Student's paired t-test. p<0.05 were considered significant.

RESULTS:

The preliminary phytochemical evaluation showed the entire plants of B. pilosa are rich in Flavonoids and terpenes. Results are shown in Table 1. Preliminary chemical investigation of the MEBP showed the presence of alkaloids, carbohydrates, fixed oils and fats, tannins and phenolic compounds, whereas the PEBP showed the presence of carbohydrates, fixed oils and fats, tannins and phenolic compounds, flavonoids and terpenes.

Mortality determination for LD₅₀ estimation:

In acute toxicity study, MEBP result exhibited 100% death for all the animals that received 5.5 g/kg b.wt of the extract while 10, 40, 70 and 90% deaths were recorded for the animals that received 3.5, 4.0, 4.5 and 5.0 g/kg b.wt of the extract, respectively. There was no death in animals administered doses up to 3.0 g/kg b.wt. LD₅₀ of the MEBP was found to be 3.5 g/kg b.wt. PEBP extract result showed 100% death for all the animals that received 5.5 g/kg b.wt of the extract while 30, 60 and 80% deaths were recorded for the animals that received 4.0, 4.5 and 5.0 g/kg b.wt of the extract, respectively. There was no death in the animals that received 3.0 and 3.5 g/kg b.wt. LD₅₀ of the PEBP was calculated to be 4 g/kg b.wt. Results are shown in Table 2.

Clinical signs of toxicity observed in all the cases include initial excitement, restlessness, and difficulty in breathing, loss of appetite, general weakness and depression which was seen as the doses increased. There were variable changes in the body weight of the animals in all the treated groups. The Group I gained body weight during the period of experiment, while the treated Group (II, III, IV and V) showed significant reduction (p<0.05) in body weight when compared with Group I (data not shown).

Table 1. Preliminary phytochemical evaluation of MEBP and PEBP

Name of chemical tests	MEBP	PEBP
Test for Alkaloids	+	-
Test for Carbohydrates	+	+
Test for Glycosides	-	-
Test for Phytcosterols	-	-
Test for fixed oils and fats	+	+
Test for saponins	-	-
Test for tannins and phenolic comounds	+	+
Test for proteins and free amino acids	-	-
Test for Flavanoids	-	+
Test for Liginin	-	-
Test for Terpenes	-	+

+ indicates presence and - indicates absence

Table 2. Methods of determination of LD₅₀ dose of MEBP and PEBP

Group	Number of mice	MEBP		PEBP
		No of death	% mortality	No of death	% mortality
1	10	0	0	0	0
2	10	0	0	0	0
3	10	1	10	0	0
4	10	4	40	3	30
5	10	7	70	6	60
6	10	9	90	8	80
7	10	10	100	10	100

The relative weights of the organs (liver, kidney and intestine) were not significantly different from the Group I. However, the lungs of the group treated at 3.0 g/kg, b.wt were significantly different (p<0.05) (data not shown). Histopathological changes were not observed in any of the organs except congestion in the lungs. Histopathological examination of the organs did not reveal any abnormalities.

The PCV, DLC (Lymphocytes, Monocytes and neutrophils), Hb, TRBC, TLC and total plasma protein were significantly (p<0.001) reduced when compared with Group I. However, the white blood cell and total plasma protein of group treated with 400 mg/kg body weight showed decreased values. Results were shown in Table 3.

DISCUSSION:

Herbal medicines have received greater attention as an alternative to clinical therapy leading to increasing demand¹⁴. The exclusive use of herbal remedies are formulated and distributed by unqualified trained herbalists, for treatment of certain changes in physiological and pathological conditions is very common in all over world. Experimental screening method is important in order to ascertain the safety of herbal products as well as to establish the bioactive component of these herbal medicines.

The extracts of B. pilosa showed the presence of flavanoids, alkaloids, carbohydrates, fixed oils and fats, tannins and phenolic compounds and terpenes. The safety pharmacology of MEBP and PEBP were monitored as safety dose administration to animals. In the present acute toxicity study was revealed that LD₅₀ levels of MEBP and PEBP were high and apparently showed the safety of those extracts. The zero percent mortality for MEBP and PEBP of both extracts were found in Group III and IV respectively. The animals were observed continuously for 4 hr at first, then at an interval of two hours up to 24 hrs to examine any change in the mice behaviour, lacrimation, respiration, writhing, CNS stimulation or depression, fighting, aggressiveness, reflexes, muscle weakness, salivation, nasal secretion and congestion, diarrhoea, skin eruption, food and water intake and mortality. The causes of death may be due to pulmonary congestion and edema, mild hydrothorax or intestinal hemorrhage.

In this current study of acute toxicity of the extract, there were no changes in behavior, sensory nervous system responses and gastrointestinal effects were observed in the experimental animals. All the mice were administered the extracts in a group wise survived beyond the 24 h of observation. The LD₅₀ of the extracts must be above 3.5 g/Kg b.wt^{9, 15.} The extract can be classified as non-toxic, since the LD₅₀ was found to be more than 3.5 g/Kg b.wt.

Acute toxicity studies of B. pilosa were screened through the oral administration of a single dose of a substance or multiple doses given within 24 hrs^15,16. The zero percent mortality for MEBP and PEBP were found to be 3 g/kg b.wt and 4.5 g/kg b.wt respectively. Optimally, studies were terminated when animals exhibited clinical signs and symptoms of toxicity. Such endpoints are generally death or morbidity of animals, since they minimize pain and distress¹⁷.

The results showed that lower concentrations of both extracts did not show any significant difference than higher concentration with respect to RBC, hemoglobin and hematocrit parameters. Administration of 400 mg/kg b.wt led to a significant increase in hemoglobin when compared to that of the control group. In the groups treated with the MEBP of the plants, the hemoglobin concentrations significantly increased, but red blood cells decreased. These changes were not seen in the PEBP treated animals. In mice treated with 400 mg/kg b.wt of MEBP, lymphocytes and eosinophils were significantly decreased and neutrophils are increased when compared with Group I. However, such changes of these values were minor, and most importantly the alteration of hematological and white blood cell count values were insignificant and remained within the normal range¹⁸.

The double dose elevations in TLC could be an attestation of the fact that MEBP and PEBP may contain biologically active principles has found to be the ability to stimulate the immune system through increasing numbers of defensive leucocytes in blood. Lymphocyte count was Reduced and compensated by increased in neutrophils count was suggested that MEBP and PEBP may possess anti-lymphocytic activity¹⁹.

Table 3: Hematological parameters and total protein levels of entire plant extracts of MEBP and PEBP

Group	Hemoglobin (g %)	RBC (millions cells/mm^3)	WBC (thousands cells/mm^3)	Packed Cell Volume (pg)	Differential leucocytes count (%)			Total protein (mg/dl)
					Lymph ocytes	Neutr ophils	Mono cytes
I	14.97 ± 0.12	6.84 ± 0.19	10.23 ± 0.26	20.46 ± 0.22	80.61 ± 0.17	35.49 ± 0.24	4.78 ± 0.1	8.15 ± 0.23
II	11.28 ± 0.20***	3.49 ± 0.30***	7.52 ± 0.35**	12.34 ± 0.70**	55.13 ± 0.51***	30.29 ± 0.20**	2.71 ± 0.5**	5.98 ± 0.64***
III	13.32 ± 0.52**	4.44 ± 0.36**	8.42 ± 0.31**	17.29 ± 0.46**	65.26 ± 0.30***	36.08 ± 0.23*	1.75 ± 0.2***	7.19 ± 0.44*
IV	10.43 ± 0.26***	2.42 ± 0.20***	5.97 ± 0.69***	14.73 ± 0.21***	41.14 ± 0.60***	15.48 ± 0.31***	4.08 ± 0.4*	5.29 ± 0.96***
V	12.37 ± 0.25**	3.38 ± 0.28***	7.62 ± 0.33**	15.43 ± 0.41***	75.58 ± 0.34**	23.40 ± 0.23***	2.22 ± 0.5**	6.65 ± 0.49**

Comparisons were made between: Group I vs. II, II, III, IV and V, Data are expressed as mean ± SEM of 6 animals in each group.

Symbols represent statistical significance: *p<0.05, **p<0.01 and ***p<0.001.

There were no significant changes in the hematological parameters between the control and extracts treated mice at a dose of 400 mg/kg b.wt, suggesting that the MEBP treated mice even at a dose of 400 mg/kg b.wt may not be toxic as they do not affect the circulating red cells, nor the haemopoiesis and leucopoiesis that could otherwise have caused a megaloblastic anemia, nor changes in PCV and eosinophils. Plasma levels of total proteins were not affected when MEBP administered orally at a dose of 400 mg/kg b.wt. All values were found to be within the normal range for mice and there were no significance between the groups. The above finding indicates that the normal metabolism of the animals was not affected²⁰.

The biochemical markers are the index of kidney and liver function^21,22. It is found that the MEBP the plant did not induce toxicity in the internal organs such as kidneys and liver. To conclude, oral treatment of MEBP entire plants did not cause acute toxicity in mice.

In the acute toxicity study of the extract, did not show any alteration in behavior and sensory nervous system responses. The congruency of the results of acute toxicity study for two extracts, indicated its reproducibility and on the other hand the results of 2 extracts in the toxicity study showed the validity of the experiments, coinciding with the results of previous studies^23,24. The validity of the experiments allows affirming that acute toxicity of the MEBP and PEBP of the plants in study is over 5.5 g/kg, b.wt, being considered as nontoxic substances under experimental conditions of this study.

Regarding histological studies of liver section of Group I showed normal hepatic cells with well-preserved cytoplasm, prominent nucleus and nucleolus and conspicuous central vein (Fig 1a) Group III showed mild congestion of endothelial and kuffer cells as compared to control (Fig 1b) Group V having highly differentiated hepatocytes and periportal chronic inflammatory infiltrate were identified (Fig 1c). Since kuffer cells were affected this may be the cause of increase in RBC and hemoglobin 400mg/kg b.wt dose.

Whereas, Small intestines section of Group I showed normal cellular architecture with the villi and mucinous epithelium appears spindles shaped and inter connected (Fig 2a). Group III showed in accumulation of inflammatory cells in villi of small intestines (Fig 2b). Group V had inflammatory cells in villi of small intestine and damages in tissue (Fig 2c).

Group I showed normal glomeruli and tubules of kidney (Fig 3a). Group III showed congestion and mild inflammation was observed around the blood vessels of tubular epithelium cells (Fig 3b). Group III showed congestion and mild inflammation around the blood vessels and also moderate inflammation around tubular epithelial cells and mild tissue damages (Fig 3b and 3c respectively), which are not apparent.

CONCLUSIONS:

It is evident from the haematological, biochemical markers and histopathological study that animals treated only with 400mg/kg b.wt, p.o., on acute toxicity confirms the depression in CNS and respiratory activity. Regarding histopathological observation no organs showed any significant change except insignificant changes observed in the higher dosed group. From this it is evident that the plant used for the present study is quiet safe for medicinal use considering the toxicological profile.

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Received on 19.07.2010

Accepted on 04.08.2010

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Research Journal of Pharmacognosy and Phytochemistry. 2(5): Sept.-Oct. 2010, 397-402