Acute and sub acute toxicity studies on ethanolic fraction of Thuja occidentalis Linn.

 

S. K. Dubey*, A. Batra1

*Sanjeevan College of Pharmacy, Dausa (Raj.) India.

1Dept. of Botany, University of Rajasthan, Jaipur (Raj.) India.

* Corresponding Author E-mail: skdpharma_cognosy@yahoo.co.in

 

ABSTRACT

The present study was carried out to evaluate acute and subacute toxicity of ethanolic fraction of Thuja occidentalis (EFTO). Acute toxicity of this extract was evaluated in Swiss albino mice. The animals were fed with EFTO between the doses of 1.0 to 20.0 g/kg body weight and were observed continuously for the first 4 h and for every hour for the next 24 h, then 6 hourly for 48 h. Wistar rats were also fed with different doses of EFTO for 30 days and effects on biochemical parameters evaluated (sub acute toxicity model). The LD50 of EFTO was found to be above 20.0 g/Kg body weight. There was reduction in the plasma glucose and low-density lipoprotein (LDL)-cholesterol levels, and increase in high-density lipoprotein (HDL)-cholesterol level in the treated animals. A significant increase in the body weight was observed for groups treated with lower doses of EFTO while groups treat with higher doses showed no significant weight increase. Aspartate aminotransferases (AST) and alanine amino transferases (ALT) levels were not affected at lower doses of EFTO but there was increase in creatinine levels in all the treated animals. The extract demonstrated good hypoglycaemic effects by lowering the plasma sugar level and also had some beneficial and reduction effects on cardiovascular risk factors. There was no evidence of drug-induced symptoms or death at all the doses of EFTO administered in acute study but subacute results revealed a tendency to cause kidney problems on a long-term use.

 

KEY WORDS Acute, sub acute toxicity, Thuja occidentalis

 

INTRODUCTION:

Thuja occidentalis, family- cupressaceae, commonly known as Arbor vitae or white cedar, is indigenous to North America and is grown in Europe as an ornamental tree. In folk medicine, Thuja occidentalis has been used to treat bronchial catarrh, enuresis, cystitis, psoriasis, uterine carcinomas, amenorrhea and rheumatism1. Extract of this plant has shown anti oxidant, anti viral, anti diarrhoeal activity2,3. It has been reported to increase the proliferation of spleen cells as well as increase in TNF-α, IL-6 and IL-1 activity in serum and also have protective effect against radiation- induced toxicity4. Today it is mainly used in homeopathy as mother tincture or dilution.

 

Plant-based medicaments had served from the outset as the most important therapeutic weapon available to man to fight various human and animal diseases. The exclusive use of herbal remedies to treat and manage ailments continued until the introduction of modern synthetic medicines. The advent of synthetic medicines in the health care system coupled with industrialization, urbanization in the most developed countries, and embracing of western culture by the developing countries, made the use of herbal products to decline from about the beginning of the 20th century up to the 1970s. However, in recent times there is a renewal and growing interest in the use of plant-derived biologically active compounds as drugs or as leads in the manufacture of more potent medicaments5. Plants, therefore, remain the main source of the active drugs from a natural source and are still indispensable in the traditional medicine for treating a number of diseases.

 

Traditional medicines are used by about 60% of the world population both in the developing countries and developed countries where modern medicines are predominantly used. They are administered in most disease conditions over a long period without a proper dosage monitoring and consideration of toxic effects that might result from such a prolonged use6. The warning regarding the potential toxicity of these therapies means that the practitioners should be kept abreast of the reported incidence of renal and hepatic toxicity associated with the ingestion of medicinal herbs. For a plant or herbal preparation containing active organic principles to be identified for use in the traditional medicine, a systemic approach is required for the evaluation of efficacy and safety through experiment and clinical findings7. The aim of this study was to evaluate the safety of ethanolic fraction of Thuja occidentalis by carrying out toxicity studies in animals. Acute and subacute toxicity evaluations are required to establish potential adverse effects of this valuable herbal preparation.

Table-1.The effect of EFTO (ethanolic fraction of Thuja occidentalis) on average weight in control and treated rats of sub acute toxicity studies.

Treatment

Day 1

Day4

Day 8

Day12

Day16

Day20

DAY24

DAY28

DAY30

Control

160.01

2.7

162.03

2.18*

164.06

1.80

165.51

1.10

167.60

2.5

168.75

2.7

168.75

2.2

170.05

2.0

170.72

2.70

EFTO

(50mg/kg)

150.01

0.1

150.20

0.4*

151.3

1.2*

151.6

1.82

152.51

0.20

154.50

0.2

155.3

0.3

156.3

6.3

160.02

0.01

EFTO 100 mg/kg)

150.02

0.12

152.40

2.80

152.7

0.79**

153.04

1.4

155.41

1.30

160.25

2.36

160.20

2.3

165.06

2.01

165.50

3.18

EFTO(250mg/kg)

150.01

0.30

150.20

1.50*5**

148.02

7.5

148.20

6.32

153.75

4.97

148.61

11.2

150.04

1.12*

150.50

0.36

151.02

2.01

EFTO(500mg/kg)

160.20

6.12

161.25

6.92**

155.10

4.8

153.75

4.97

153.75

4.97

163.05

2.25*

163.5

1.2

163.7

3.21

163.83

5.21

Mean SEM (n = 5). *p < 0.05; ** p < 0.01 vs control group. Control group received 0.5 mL 2% Tween 80 solution

MATERIALS AND METHODS:

Plant material

Fresh aerial part (twigs) of Thuja occidentalis were collected from Jaipur, Rajasthan, India, in October-2007 and were authenticated by experts of Deptt. Of Botany University of Rajasthan, Jaipur. The voucher specimen is preserved for further research in our laboratory.

 

Preparation of extract

Shade dried and powdered twigs (40-mesh size, 1kg) were soxhlet extracted with 90% ethyl alcohol (Dept. of Botany), the solvent was removed and the residue was triturated with hot (650C) petroleum ether (60-800C). Solvent was evaporated from the petroleum ether soluble portion and the residue dissolved in ethanol. On removal of the ethanol by evaporation, a semi solid reddish brown mass (12.76g) was obtained. Phytochemical investigations showed the presence of flavonoids (quercetin, kaempherol), tannic acids, polysaccharides and proteins.

 

Animals

Wistar albino rats and swiss albino mice were used. Animals were maintained under standard environmental conditions and had free access to standard pellet food (Hindustan lever, India) and water. Rats of either sex, weighing 175-220 g, and mice of either sex weighing 18-22g were provided by the Sanjeevan college of Pharmacy, Dausa, Rajasthan, India, were used. The animals were maintained as per the norms of IAEC and the experiments were cleared by IAEC and the local institutional ethical committee.

 

Acute Toxicity Test

Thirty five (35) male and female Swiss albino mice weighing 20 25 g were used for the acute toxicity study. They were randomly distributed into one control group and six treated groups, containing five animals per group and were maintained on standard animal diet (Hindustan Lever Ltd) and provided with water ad libitum. They were allowed to acclimatize for seven days to the laboratory conditions before the experiment. After fasting the animals over-night, the control group received 0.3 ml of 2% Tween 80 solutions orally. Solution of the extract was prepared by dispersing 8.0 g of the dried extract (EFTO)with 10 ml of 2% tween 80 solution and each treated group received the extract doses as follows: 1.0, 2.50, 5.0, 10.0, 15.0 and 20.0 g/kg orally. The animals were observed continuously for the first 4 h and for every hour for the next 24 h, then 6 hourly for 48 h after administering the extract to observe any changes in general behavior or other physiological activities8,9.

 

Table 2. The effects of EFTO (ethanolic fraction of Thuja occidentalis) on weight of kidney, heart, liver and brain in control and the treated groups.

 

Organ

Control

50mg/

kg

100mg

/kg

250mg

/kg

500mg

/kg

Heart (g)

0.49

0.03

0.41

0.08*

0.49

0.02**

0.49

0.01**

0.45

0.06

Kidney (g)

0.93

0.10

0.77

0.18*

0.960

.051**

0.93

0.06

0.85

0.07

Liver (g)

0.85

0.07

4.22

0.53

5.140

.57

5.10

0.63*

4.70

0.43**

Brain (g)

1.17

0.14

0.970

.08*

1.07

0.081*

1.17

0.04

1.07

0.08*

 

Mean SEM (n = 5). *p < 0.05; ** p < 0.01 vs control group. Control group received 0.5 mL 2% Tween 80 solution.

 

Sub Acute Toxicity Test

Male and female Wistar albino rats weighing 160 10 g were used. They were allowed to acclimatize to the laboratory conditions for seven days and were maintained on standard animal feeds and provided with water ad libitum. The animals were weighed and divided into five groups of five animals each. After fasting the rats overnight, the control group received a dose of 0.5 ml of 2% Tween 80 solution orally once a day for 30 days. The four treated groups respectively received the following doses: 50, 100,

250 and 500 mg/kg of the EFTO dispersed with 2% Tween 80 solutions orally once a day for 30 days10,11. The animals were then weighed every four days, from the start of the treatment, to note weight variation. At the end of the experiment, they were anaesthetized with warm urethane and chloralose (25%: 1%v/v) at a dose of 5 ml/kg body weight and blood collected via cardiac puncture in two tubes: one with EDTA for immediate analysis of haematological parameters and to separate plasma for biochemical estimations. The collected blood was centrifuged within 5 min of collection at 4000 g for 10 min to obtain plasma, which was analyzed for total cholesterol, total triglyceride, HDL-cholesterol levels by precipitation and modified enzymatic procedures from Sigma Diagnostics12. LDL-cholesterol levels were calculated using Friedwald equation13. Plasma was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine by standard enzymatic assay methods14. Plasma glucose contents and protein contents were determined using enzymatic spectroscopic methods15. Haematocrit was estimated using the methods of Ekaidem et al. Haemoglobin contents were determined using Cyanmethaemoglobin (Drabkin) method16.

 

Table 3. Effect of daily administration of EFTO (ethanolic fraction of Thuja occidentalis) for 30 days on biochemical profiles of control and treated rats.

Parameters

Control

50mg

/kg

100mg

/kg

250mg

/kg

500mg

/kg

Glucose

(mg/dl)

103.96

0.2

90.85

0.12

81.18

0.32

58.83

0.52

52.03

1.50

Cholesterol

(mg/dl)

58.46

0.60

45.00

0.03**

62.58

0.42

42.58

1.4

19.35

0.7

Triglyceride

(mg/dl)

56.87

0.45

66.69

1.50

83.29

0.02

118.37

0.02

103.31

0.04

HDL

(mg/dl)

95.31

0.002

104.5

0.007

163.67

0.07

150.39

0.49

140.030

.39

LDL

(mg/dl)

81.44

2,50

32.48

4.75

31.60

0.60

30.77

3.50

26.70

0.50*

Protein

(g/dl)

6.89

0.32

7.75

0.25

7.26

0.60

5.90

0.20

3.310.1

5**

Creatinine

(mg/dl)

0.15

0.003

0.17

.001*

0.22

0.05

0.50

0.01

0.52

0.001

AST

IU/L

57.18

1.20

55.69

2.50**

53.14

0.02

65.48

2.3

62.83

0.60

ALT

IU/L

24.54

1.80

28.15

2.52**

29.79

2.6 3

4.72

3.6

55.85

2.40

 

Mean SEM (n = 5). *p < 0.05; ** p < 0.01 vs control group. Control group received 0.5 mL 2% Tween 80 solution

 

Statistical analysis

All data collected were summarized as mean SEM. Significant differences were determined using a Students t- test and the differences were considered significant if p < 0.05.

 

RESULTS AND DISCUSSION:

There is, in recent times, a growing and increasing interest in herbal medicines. Consequently, herbal medicines have received greater attention as an alternative to clinical therapy leading to increasing demand. The exclusive use of herbal drugs, prepared and dispensed by unscientifically trained herbalists, for treatment of diseases is very common. Experimental screening method is, therefore, important in order to ascertain the safety and efficacy of herbal products as well as to establish the active component of these herbal remedies. In the acute toxicity study of the extract, no changes in the behavior and in the sensory nervous system responses were observed. In addition, no adverse gastrointestinal effects were observed in male and female mice used in the experiment. All the mice that received 20.0 g/Kg dose of the EFTO survived beyond the 24 h of observation. The median acute toxicity value (LD50) of the extract must be above 20.0 g/Kg body weight. The extract can be classified as non-toxic, since the LD50 was found to be more than 15.0 g/Kg17. In the acute toxicity study, EFTO up to the dose level of 20g/kg of body weight did not exhibit any lethality or toxic symptoms. Further, dosing to estimate the LD50 of EFTO was not performed. According to Organization for Economic Cooperation and Development (OECD) guidelines for acute oral toxicity, an LD50 dose of 2000mg/kg and above is categorized as unclassified and hence the drug is found to be safe18.

 

Table 4.

Effect of EFTO (ethanolic fraction of Thuja occidentalis) on Haematological parameters of the control and treated rats.

Parameters

Control

50mg

/kg

100mg

/kg

250mg

/kg

500mg

/kg

Hemoglobin

(mMol/L)

10.60

0.3

10.80

0.25**

11.30

0.40

11.60

0.40

11.60

0.40

PCV %

45.80

2.20

46.20

2.3*

46.85

1.52

44.85

2.50

45.55

0.50**

 

There was a significant increase (p < 0.05) in the body weights of the animals treated with the lower doses of the extract (50 and 100 mg/kg) compared with the control while at higher doses (250 and 500 mg/kg) there was neither decrease nor a significant increase in the weights of the animals (Table 1). The constant body weight of the animals treated with higher doses of the extract might be because of suppression of fats accumulation and deposition in the organs and muscles of the animals.

 

The effects of the extract on the organs are summarized in Table 2. The macroscopic examinations of the organs of the animals treated with various doses of the extract did not show any changes in color compared with the control. Also, there were no significant changes in the organs weight of the treated animals compared with the control. Since no death was recorded in the acute toxicity study, and no changes in animal behavior and in organs weight were observed at all doses used, the EFTO can be claimed to be non-toxic. The effects of the extract on the biochemical parameters are summarized in Table 3. The significant decrease (p < 0.05) in the plasma glucose level especially at higher doses in the treated rats compared with control might be due to the presence of hypoglycemic components in EFTO. This observation gives credence to the use of the herbal product as a hypoglycemic agent. There was no significant change observed in the protein levels of the rats treated with lower doses of the extract (50 and 100 mg/kg) compared with control, while an observed significant decrease in the protein levels of the rats treated with a high dose (500 mg/kg) may be a sign of impaired renal function. Also, there was a significant increase (p < 0.05) in the plasma creatinine levels of all the treated groups. The elevation in the plasma creatinine concentration indirectly suggests kidney damage specifically renal filtration mechanism. The liver releases alanine aminotransferase (ALT) and an elevation in plasma concentration are an indicator of liver damage. The liver and heart release AST and ALT, and an elevation in plasma concentration are an indicator of liver and heart damage. There was no significant increase in AST and ALT in the animals treated with lower doses of the extract compared with control but a significant increase in ALT was observed in the group treated with a high dose of the extract (500 mg/kg). This implies that the extract at the doses used had no effects on the heart tissue but at a high dose could have some deleterious effects on the liver tissue. The observed decrease in plasma total cholesterol (TC) level might be due to the presence of hypolipidemic agents in the extract while the increase in the triglyceride (TG) levels could be secondary to a variety of disorders that might be induced by the extract19. A significant increase in HDLcholesterol levels in all the treated animals and reduction in LDL-cholesterol levels in some treated animals were observed. This showed that the extract had some beneficial effects by reducing cardiovascular risk factors, which contribute to death of diabetic patient20, and establishes the use of the formulation as a hypoglycaemic agent. Phytochemical screening (result not tabulated) indicated the presence of alkaloids and polyphenols. Polyphenols such as flavonoids and tannins have been shown to have numerous health protective benefits, which include lowering of blood lipids. Furthermore recent reports have suggested that several plant sterols reduce serum cholesterol by the inhibition of intestinal cholesterol absorption. Thus, it can be suggested that the synergistic interaction of poly phenols and tannins contents in the extract may impart hypo lipidemic property to the herbal preparation. There were no significant changes observed in the hemoglobin contents and in the packed cell volume (PCV) of the treated animals compared with the control (p > 0.01) (Table 4). The EFTO did neither improve nor produced any deleterious effects on the hematological parameters. The little increase in the hemoglobin levels might be due to the increased absorption of iron21.

 

REFERENCES:

1.         Chang LC, Song LL, Park EJ, Luyengi L, Lee K J and Norman R. Bioactive Constituents of Thuja occidentalis Journal of Natural Product. 2000; 63:1235-1238.

2.        Nam SH and Kang MY. Antioxidant activity of Medicinal Plants. Pharma. Biotech. 2005; 42: 409.

3.        Deb L, Dubey SK, Jain AK, Jain A, Pandian GS and Rout SP. Antidiarrhoeal activity of Thuja occidentalis Linn. ethanol extract on experimental animal. Indian Drugs. 2007; 44:319-321.

4.        Belal N, Cornelia B, Martin T and Ulrike L. Thuja occidentalis (Arbor vitae): A Review of its Pharmaceutical, Pharmacological and Clinical Properties. Advance Access Pub. 2005; 2:69-78.

5.        Houghton PJ, Raman A Laboratory handbook for fractionation of natural extracts. Chapman and Hall, London.1998; 199-203.

6.        Mythilypriya R, Shanthi P and Sachdanandam P. Oral acute and subacute toxicity studies with Kalpaamruthaa ,a modified indigenous preparation, on rats . J.Health Sci. 2007; 53(4): 351-358.

7.        Pieme CA, Penlap VN, Nkegoum B, Taziebou CL Tekwu EM, Etoa FX, and Ngongang. J. Evaluation of acute and subacute toxicities of aqueous ethanolic extract of leaves of (L) Roxb(Ceasalpiniaceae). African J. Biotech. 2006; 5(3): 283-289.

8.        Tdong L, Dzeufiet PDD, Dimo T, Asongalem EA, Sokeng SN, Flejou JF, Callard P and Kamtchouing P. Acute and Subchronic toxicity of Anacardium occidentale Linn (Anacardiaceae) leaves hexane extract in mice. African J. Traditional Alternative Med. 2007; 4(2): 140-147.

9.        Shah Ayub MA, Garg SK and Garg KM . Subacute toxicity studies on Pendimethalin in rats. Ind. J. Pharmacol. 1997; 29: 322-324.

10.     Bhrger C, Fischer DR, Cordenunzzi DA, Batschauer de Borba AP, Filho VC and Soares dos Santos AR. Acute and subacute toxicity of the hydroalcoholic extract from Wedelia paludosa (Acmela brasilinsis) (Asteraceae) in mice. J. Pharma. Sci. 2005; 8(2): 370-373.

11.     Joshi CS, Priya ES and Venkataraman S. Acute and subacute studies on the polyherbal antidiabetic formulation Diakyur in experimental animal model. J.Health Sci. 2007; 53(2): 245-249.

12.     Wasan KM, NajafiS, Wong J and Kwong M . Assessing plasma lipid levels body weight and hepatic and renal toxicity following chronic oral administration of a water soluble phytostanol compound FMVP4, to gerbils. J. Pharma. Sci. 2001; 4: 228-234.

13.     Crook MA. Clinical Chemistry and Metabolic Medicine. Hodder Arnold Publication, Oxford University Press, 7thEdition ,2006 ; 426-428.

14.     Sushruta K, Satyanarayana S, Srinivas N and Sekhar Raja J. Evaluation of the bloodglucose reducing effects of aqueous extracts of the selected Umbellifereous fruits used in culinary practice. Tropical J. Pharma. Research. 2006; 5(2): 613-617.

15.     Hussain A and Eshrat HM. Hypoglycemic hypoipidemic and antioxidant properties of combination of Curcumin from Curcuma longa, Linn and partially purified product from Abroma augusta, Linn. in streptozotocin induced diabetes. Ind. J. Clin. Biochem. 2002; 17(2): 33-43.

16.     Ekaidem IS, Akpanabiatu MI, Uboh FE and Eka OU . Vitamin B12 supplementation: effects on some biochemical and haematological indices of rats on phenytoin administration. Biokemistri. 2006; 18(1): 31-37.

17.     Klassen CD, Amdur MO, Doull J Casarett and Doulls. Toxicology: The basic science of poison, Mc Graw Hill, USA, 8th Edition 1995;. 13-33.

18.     OECD guidelines for the testing of chemicals. Revised Draft Guidelines 423; acute oral toxicity-acute toxic class method revised document, October 2000.

19.     Ellefson DR and Caraway TW. Lipids and Lipoproteins. In Fundamentals of Clinical Chemistry. Tietz WN (ed.) WB, Saunders Company Philadelphia USA, 2nd Edition,1982; 474-541.

20.     Ogbonnia S, Adekunle AA, Bosa MK and Enwuru VN. Evaluation of acute and sub acute toxicit of Alstonia congensis Engler (Apocynaceae) bark and Xylopia aethiopica (Dunal) A.Rich (Annonaceae) fruit mixtures used in treatment of diabetes. African J. Biotech. 2008; 7(6); 701-705.

21.   Ameyaw Y and Owusu-Ansah E. Morphohistological studies of two plant species used in Ethnomedicine. J. Herbs, Spices Medicinal Plants. 1998; 5(4): 60-85.

 

Received on 06.07.2008 Modified on 21.07.2008

Accepted on 25.08.2008 RJPT All right reserved

Research J. Pharm. and Tech. 1(3): July-Sept. 2008; Page 245-248