Evaluation of Acute and Chronic Toxicity of the ethanolic extract of seeds of Tragia involucrata Linn.

Abstract: The present study is mainly aimed to discuss the acute and chronic toxic effects of ethanolic extract of seeds of Tragia involucrata. The toxicity profile of the ethanolic extracts of seeds of Tragia involucrata was evaluated in the male rats treated daily for 90 days using 100-400mg/kg p. o. doses. No adverse effect were observed. There was no significant changes were observed in feed intake, body weight and relative organs weight except the significant (p<0.05) reduction in kidney and increase in relative weight of testes were seen at doses of 200 and 400mg/kg p. o. No significant result was seen in the hematological indices, hepatological function as well as renal function indices (urea and creatinine). Uric acid was however reduced significantly (p<0.05). Study of effect on serum lipid showed no significant effect on cholesterol but a significant reduction of triglyceride at 200mg/kg p. o. dose. The results suggested that T. involucrata seed ethanolic extract is safe to use as powder, paste or

decoction for medicinal purposes.

Key words: Chronic toxicity, acute toxicity, Tragia involucrata.

Introduction: Man kind have been using plants as therapeutic agents for thousand of years and continues to rely on them for their health care. According to WHO report around 80% of the world population depend on traditional medicine for their primary health care, majority of which used plants  in the form of water extracts, paste, powder or both or their active principles. Plants used in traditional medicine contain various types of ingredients that can be used to treat chronic as well as infectious diseases. A vast knowledge of how to use the plants against different ailments may be expected to have accumulated in areas where the use of plants is still of great importance. The medicinal values of plants lies in the chemical substances that produce a definite physiological action on human body. The most important of these biologically active compounds of plants are alkaloids, flavonoids, tannins and phenolic compounds (Arora et al, 2003, Chopra et al, 1956, Chopra et. al,1982, Kirtikar and Basu, 1975 Nadkarni, 1976,). Folk people of the remote areas of the developing countries depends upon the plant sources for their herbal remedies, food and forage. Traditional healers claim that their medicine is cheaper, more effective and impart less side effects as compared to the synthetic medicine in the developing countries. Low income people such as farmers,people of small isolate villages and native communities use folk medicine for the treatment of their common ailments (Rojas et al, 2006). Medicinal plants are used as a source of many potent and powerful drugs (Srivastava, et al,1996). The different parts of the medicinal plants used are root, stem, stem bark, leaf, flower, fruits, twigs, gum exudate etc.The herbal medicines are a great source of economic value in the Indian subcontinent. Nature has bestowed on us a very rich botanical health and a large

number of diverse types of plants in different parts of the country. The toxicity study of the diseases. The local and tribal people commonly use the plant Tragia involucrata

for the treatment of their ailments.The modern system of medicine had always been enthusiastic to evoke non-specific defense mechanisms of human physiology, which led to the discovery of active immunization using microbial preparations to enhance the host defense against infection. Gutali et. al (2002) reported the novel group of substances from natural sources that modulate the immune response of living systems. In the herbal medicine T. involucrata is used in symptoms like anxiety, restlessness, different kinds of fevers specially intermittent and bilious fevers, headache,

gastritis, hepatitis, abdominal colic, constipation, diarrhoea, dry cough, rheumatism and also useful in clinical conditions like vertigo, dysmenorrhoea, bronchitis and seasonal fevers. T. involucrata is also used in tribal medicine for the treatment of many kinds of acute and chronic inflammatory diseases. The rationale for the utilization of medicinal plants has largely rested on the long term clinical experience with little or no scientific data on their efficacy and safety. It has also been observed that the possibility of toxicity is associated with long term low dose exposure of medicinal products. This possibility explains the need of thorough scientific investigation of herbal medicines for the validation of their folkloric usages including benefits and toxicity. Presently the study involves the toxicological report of the ethanolic extract of the seeds of T.involucrata

The study also involves the evaluation of acute and sub acute toxicity of the ethanolic extract of the seeds. This study will provide the information that would be useful in

the drug development and subsequent clinical uses.

Materials and Methods:

Plant Materials: The fruits of the selected plant T.involucrata were collected in the appropriate season. The fruits were collected and identified by our professor from the Dept. of Botany and the voucher specimen kept the deposit in our Herbarium. The Seeds were taken out of the fruit and crushed to coarse powder. 100 g of the powder of

the seeds of T. involucrata is taken in the soxhlet and extracted with 450 ml 95% ethanol at controlled temperature. The collected extract was concentrated under reduced pressure below 450C using rotary evaporator. The complete removal of the solvent from the extract was carried out in the rotary evaporator. The material thus

obtained was stored at 4-50C until used.

Experimental Animals: Charles Foster strain male and female albino rats (weighing 150-200g) were used through out the experiment. Animals were procured from the

National Animal House, Lucknow. Animals had ad libitum access of standard laboratory diet and water, except during the previous night of the experiment. The animals were

grouped randomly into control and treated group containing five rats in each group. They were housed under standard environmental conditions of temperature and were allowed to free access to drinking water and pallet diet. The rats were kept in experimental facilities for one week to allow them to be acclimated prior to dosing. Animals were put on fasting except water up to 16-18 hours prior to giving them

medicine at day zero. As per the WHO guidelines (2000) and the Organization of Economic Co-operation and Development guidelines for the testing of chemicals 420 (OECD,2001), the extract of the dose of 5000mg/kg was given orally to test group of rats while the control group was given only water in the same volume by canula fitted steel feeding needle.

Observation of toxicity sign: Body weight, sign of toxicity (general behaviour, respiratory pattern, cardiovascular signs, motor activities, reflexes, change in skin and fur, mortality were observed after the administration at the 1st, 2nd, 4th, and 6th hour and once daily for 14 days (Chan et al, 1982). On the 15th day, all rats were kept fasted overnight and then anesthetized. The internal organs were excised and weighed.

Evaluation of Chronic toxicity: Charles Foster strain male and female albino rats (weighing 150-200g) were divided into five groups of 10 animals each. At the onset of dosing or dose administration animals should be between 170-

190g.

Dose administration: According to WHO,2000 and OECD, 2001, the ethanolic seeds extract of T. involucrata was orally administered at concentration of 300, 600 and 1200mg/kg three subsequently treated groups for 90 days, the control group was given distilled water only.

Observation of toxicity sign: General behaviour, respiratory pattern, cardiovascular signs, motor activities, reflexes, change in skin and fur, mortality and the body weight changes were monitored daily (Chan et. al, 1982). the time of onset, intensity and duration of these signs, if any,was recorded.

Hematological and Blood Chemical Analyses: At the end of the study of experiment , all animals were kept for 16-18 hours and then anesthetized. Blood samples were collected for the hematological and blood chemical analysis from carotid artery. Heperinized blood samples were taken for complete blood count by the blood analyzer. The serum from non-heparinized blood was carefully collected for blood chemistry and enzyme analysis (glucose, blood urea nitrogen (BUN), creatinine, total protein, albumn, total and direct billirubins, serum glutamate – oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP).

These levels were automatically determined using the blood analyzer of Systronics make.

Necropsy: All rats were sacrificed after blood collection. The position, shapes, sizes and colours of internal organs were evaluated. Heart, lungs, thymus, liver, pancreas, spleen, kidneys, adrenals, small intestine, stomach and duodenum, muscle with sciatic nerve, thoracic spines, brain, eyes, sex organs, uterus and epididymis were removed from all rats to visually detect gross lesions and weighed to determine relative organs weights. All tissues were preserved in 1% neutral buffered formaldehyde solution for histopathological examination.

Statistical analysis: Results were expressed as mean ± standard error of mean (SEM). Statistical significance was determined by one way of variance (ANOVA) and post hoc least – significant difference (LSD) test. The data obtained from acute toxicity study were analyzed using student t-test. P values less than 0.05 were considered significantly.

Result and Discussion: Both female and male rats (Acute toxicity) given the seed extract of T. involucrata at a dose of 5000mg/kg did not show any toxicity during experimental period. In both sexes, body weight gain of the treated rats was not changed significantly relatively to that of control. For the male treated group the brain and lungs weight were slightly but significantly lower than those of the control group (p<0.05). The internal organs of the treated rats such as brain, heart, liver, spleen, pancreas, adrenal gland, kidneys, and sex organs showed no pathological 3 Journal of Recent Advances in Science , 2011, Vol 1 No .1 abnormalities relative to these organs of the control. Thus the standardized ethanolic extract of seeds of T. involucrata with an LD50 > 5000 mg is considered to be non-toxic

through acute exposure in rats.

Chronic toxicity: For evaluation of chronic toxicity, the doses of the seeds extract (300, 600, and 1200 mg/kg) did not show any change either in behaviour or toxic sign during the experimental period. Body weight and the body weight gain in treatment groups of female and male rats were significantly lower than those of the their control

groups. In cases of 600 and 1200mg/kg doses, body weight in male treated group significantly decreased on the 90th day.

Hematological Analysis: The hematopoietic system is very sensitive to toxic compounds and serves as an important index of the physiological status for both animals and humans (Adeneye et al. 2006). Hematological parameters provide vital information regarding the status of bone marrow activity and intra vascular effects such as hemolysis and anemia (Gregg and Voigt, 2000). In hematological

examinations, significant increase in platelet counts in female treatment rats was observed for 600mg/kg body weight (Table 1) while no change in the male treatment rats (Table 2). The differential white blood counts are listed in Table 3 & 4. significant increase in eosonophils and neutrophils was found at 300mg/kg body weight dose in the

female and male treatment group respectively. However, these values were also within the normal range (Feldman et al., 2000; Inala et al, 2002) indicating that ethanol extract of seed of T. involucrata does not effect hematopoiesis and

leukopoiesis in rats.

Blood Chemical Analysis: Clinical blood chemistry was carried out in order to evaluate any toxic effects on the pancreas functions, kidney functions, creatinne, and liver

functions. In general if the clinical blood chemistry values differ more or less than one fold from the normal values, abnormalities should be noted in pancreas, kidney and liver

functions (Caisey, and King, 1980, Sacher and McPherson, 2000). Determination of plasma protein like albumin can act as a criterion for asseying synthetic capacity of liver

(Woodman, 1996). The increased level of those enzymes more than one fold would then be highly significant for clinical pathological with abnormalities in physical

appearances. The results in table 5 & 6 show significant differences among the experimental groups in albumin, SGOT,and ALP. Nevertheless, these significant values also fell within the normal ranges (Caisey and King, 1980, Sacher and Mcpherson, 2000, Angkhasirisap et al, 2002; Levine, 2002). This indicates the healthy status of liver and kidney in the treatment groups.

Organs Weights: As shown in the Table 7 kidney and heart weights of the female treatment group with 300 and 600 mg/ kg body weight doses respectively were significantly lower than that of their control. The organs weight of the male group are listed in the Table 7. Significant weight decrease in brain and spleen was observed for 300mg/kg/body dose. At the dose of 1200mg/kg/day significant weight decrease

in brain was found.

Conclusion: In conclusion, the standardized ethanolic extract of seeds of T. involucrata did not produce any oral acute or chronic toxicity in both male and female rats which

could be considered as a no-observed-adverse effect levels (NOAEL) crude drugs that acts safely under the current normal usage (WHO,1987; Copplestone, 1988). NOAEL of the extract was found to be 1200mg/kg/day which are considerably higher than the traditionally used dose of N.arbortristis seeds ethanolic extract (100-200mg/kg/day).

The extrapolation of the these results to human suggests that T. involucrata seed rthanolic extract should be accepted safety level for usage at the doses of 300, 600 and 1200 mg/ kg/day.

Table – 1. Hematological examinations of female rats under chronic doses of the standardized ethanol extract of seeds of N.

arbortristis.

…..................................................................................................................................................................................................

Control Treated Animal

…..................................................................................................................................................................................................

300mg 600mg 1200mg .

Red blood cell (Х106/μl) 6.98 ±0.13 7.26 ±0.10 7.46± 0.21 7.11± 0.22

Hemoglobin (g/dl) 14.12 ±0.18 14.46± 0.45 15.65± 0.64 14.65 ±1.32

Hematocrit (%) 45.67 ±0.93 45.50±0.80 45.20±0.93 44.65±0.78

Mean corpuscular

volume (fl) 61.24±0.48 61.11±0.51 60.59±0.40 60.42±0.49

Mean corpuscular

hemoglobin (pg) 19.89±0.83 19.86±0.41 20.85±1.35 19.87±0.28

Mean corpuscular hemoglobin

concentration (g/dl) 32.20±0.35 31.86±0.15 35.56±3.12 32.50±0.18

Platelet (Х105/μl) 7.09±0.51 7.01±0.26 8.01±0.31* 8.04±0.58

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10. *significantly different from control. P<0.05.

4 Journal of Recent Advances in Science , 2011, Vol 1 No .1

Table – 2. Hematological examinations of male rats under chronic doses of the standardized ethanol extract of seeds of N.

arbortristis.

…..................................................................................................................................................................................................

Control …......... . Treated Animal............................................. .

300mg 600mg 1200mg

…..................................................................................................................................................................................................

Red blood cell (Х106/μl) 7.98±0.22 7.86±0.11 7.91±0.31 7.89±0.12

Hemoglobin (g/dl) 14.96 ±0.18 14.94± 0.19 15.00± 0.20 14.85 ±0.11

Hematocrit (%) 46.67 ±0.93 45.80±0.70 46.40±1.93 46.45±0.98

Mean corpuscular

volume (fl) 59.34±0.58 59.11±0.51 58.69±0.30 59.62±0.69

Mean corpuscular

hemoglobin (pg) 18.89±0.83 18.86±0.41 18.11±0.13 18.25±0.11

Mean corpuscular hemoglobin

concentration (g/dl) 31.20±0.35 31.26±0.15 31.56±3.12 31.50±0.18

Platelet (Х105/μl) 8.49±0.51 8.31±0.26 9.21±0.31 9.04±0.58

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10

*significantly different from control. P<0.05.

Table – 3. Differential white blood cell counts of female rats under chronic doses of the standardized ethanol extract of seeds

of T. involucrata.

….................................................................................................................................................................................................

Control …......... . Treated Animal............................................. .

300mg 600mg 1200mg

White blood cell (Х103/μl) 2.45 ±0.45 2.21 ±0.35 3.5 ±0.56 2.35 ±0.34

Neutrophil (%) 23.03 ±1.50 20.10 ±1.65 25.60 ±2.30 25.60 ±2.00

Lymphocyte (%) 64.30 ±3.01 64.50 ±2.98 63.30 ±1.65 63.50 ±2.11

Monocyte (%) 7.90 ±0.87 8.60 ±0.91 7.50 ±0.58 7.36 ±0.64

Eosinophil (%) 3.60 ±0.85 5.11 ±0.01* 2.36 ±0.57 3.40 ±0.67

Basophil (%) 0.00± 0.00 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10

*significantly different from control. P<0.05.

Table – 4. Differential white blood cell counts of male rats under chronic doses of the standardized ethanol extract of seeds of

T. involucrata.

…..................................................................................................................................................................................................

Control …......... . Treated Animal............................................. .

300mg 600mg 1200mg

White blood cell (Х103/μl) 3.51±0.46 3.18±0.26 4.17 ±0.49 3.65±0.28

Neutrophil (%) 25.20±1.65 32.40±2.5* 29.10 ±2.98 29.60±1.86

Lymphocyte (%) 64.70±1.98 56.50±2.17* 60.70 ±3.01 59.70±2.35

Monocyte (%) 6.98±0.61 7.68±1.21 6.80 ±0.79 7.50 ±0.82

Eosinophil (%) 3.40±0.72 3.20±0.60 2.30 ±0.67 2.00 ±0.38

Basophil (%) 0.00±0.00 0.00±0.00 0.00 ±0.00 0.00 ±0.00

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10

*significantly different from control. P<0.05.

5 Journal of Recent Advances in Science , 2011, Vol 1 No .1

Table – 5. Clinical blood chemistry examination of female rats under chronic doses of the standardized ethanol extract of

seeds of T. involucrata.

…..................................................................................................................................................................................................

Control …......... . Treated Animal............................................. .

300mg 600mg 1200mg

Glucose (dl) 137.50 ±3.86 130.31±3.01 135.75±3.75 130.01±4.53

BUN (mg/dl) 17.40 ± 0.51 17.20 ±0.37 18.36±0.61 17.50±0.83

Creatinine (mg/dl) 0.39 ± 0.03 0.36 ±0.01 0.36±0.03 0.38±0.01

Total protein (mg/dl) 6.80 ± 0.12 6.71 ±0.09 6.84±0.12 6.93±0.10

Albumin (mg/dl) 3.52 ± 0.08 3.42 ±0.07 3.64±.010 3.84±0.08

Total bilirubin (mg/dl) 0.23 ± 0.02 0.22 ±0.01 0.24±0.03 0.23±0.01

Direct bilirubin (mg/dl) 0.08 ± 0.01 0.09 ±0.01 0.06±0.01 0.10±0.01

SGOT (U/L) 90.80 ± 4.56 95.00±6.14 95.40±4.11 91.50± 5.01

SGPT (U/L) 36.21 ± 3.46 36.30±4.01 39.60±2.39 37.80 ±3.28

ALP (U/L) 23.10 ± 1.16 27.60±2.65 30.98±2.34 34.00 ±4.68*

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10

*significantly different from control. P<0.05.

Table – 6. Clinical blood chemistry examination of male rats under chronic doses of the standardized ethanol extract of seeds

of T. involucrata.

…..................................................................................................................................................................................................

Control …......... . Treated Animal............................................. .

300mg 600mg 1200mg

Glucose (dl) 128.46±3.58 127.90±3.56 136.80±3.45 136.00±3.46

BUN (mg/dl) 20.80±0.66 21.50±0.78 20.00±0.70 21.30±0.63

Creatinine (mg/dl) 0.51 ±0.02 0.54±0.03 0.53±0.02 0.51±0.01

Total protein (mg/dl) 6.53 ±0.12 6.47±0.11 6.78±0.19 6.81±0.26

Albumin (mg/dl) 3.68 ±0.03 3.48±0.05 3.34±0.06 3.63±0.08

Total bilirubin (mg/dl) 0.12 ±0.01 0.10±0.01 0.12±0.01 0.15±0.03

Direct bilirubin (mg/dl) 0.02 ±0.01 0.02±0.01 0.03±0.01 0.05±0.03

SGOT (U/L) 106.50 ±5.18 102.70±6.27 99.01±8.08 111.70±6.03

SGPT (U/L) 50.80 ±4.23 42.60±3.56 48.20±6.02 46.38±8.75

ALP (U/L) 51.30 ±1.87 47.70 ±0.86 55.80±3.98 49.58±1.56*

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10

*significantly different from control. P<0.05.

Table – 7. Organ weights (in grams) of female rats under chronic doses of the standardized ethanol extract of seeds of N.

arbortristis.

…..................................................................................................................................................................................................

Control …......... . Treated Animal............................................. .

300mg 600mg 1200mg

Brain 1.90±0.02 1.90±0.03 1.89±0.01 1.88±0.02

Lungs 1.70±0.06 1.67±0.04 1.66±0.06 1.67±0.03

Heart 1.32±0.05 1.21±0.03 1.14±0.04* 1.17±0.03

Liver 8.12±0.23 8.00±0.32 7.85±0.29 7.98±0.35

Pancreas 1.40±0.08 1.32±0.05 1.43±0.07 1.51±0.06

Spleen 0.90±0.03 0.91±0.04 0.82±0.04 0.81±0.04

Adrenal glands 0.06±0.02 0.04±0.00 0.04±0.00 0.06±0.03

Kidneys 1.13±0.02 1.15±0.02* 1.17±0.02 1.19±0.01

Ovary 0.09±0.00 0.08±0.00 0.08±0.00 0.11±0.03

Uterus 0.72±0.07 0.72±0.07 0.60±0.04 0.80±0.11

…..................................................................................................................................................................................................

Values are expressed as mean ± SEM, n=10

*significantly different from control. P<0.05.

6 Journal of Recent Advances in Science , 2011, Vol 1 No .1

References:

1. Angkhasirisap W, Inala P, Sirimontaporn A,

Inpunkaew R, Rungrojejinda K, Kengkoom K,

Ratanasak W, Buripadi Lawson D. 2002. Blood

chemistry profiles of out bred Sprague-Dawley rat

in the Facility of National Laboratory Animal

Centre. 28th Congress on Science and Technology

of Thailand.

2. Arora, S. Kaur, K a,d Kaur, S.2003, Indian

medicinal plants as a reservoir of protective

phytochemicals, Teratogenesis Carcinog Mutagen

Suppl. 1; 295-300.1.

3. Chan PK, O’Hara GP, Hayes AW. 1982. Principles

and methods for acute and subchronic toxicity. In:

Hayes AW, editor. Principles and Methods of

Toxicology. New York: Raven Press. P 17-19.

4. Chopra, R.N., Handa,K.D.Kapur, L.D (eds)1958,

Indigous Drugs of India. V. N. Dhar and Sons Pvt.

Ltd., Calcutta.

5. Chopra, R.N., Handa,K.D.Kapur, L.D (eds)1982,

Indigous Drugs of India. V. N. Dhar and Sons Pvt.

Ltd., Calcutta.

6. Caisey JD, King DJ. 1980. Clinical chemical

values for some common laboratory animals. Clin

Chem 26:1877-1879.

7. Copplestone JF. 1988. The development of the

WHO recommended classification of pesticides by

hazard.Bulletin of the World Organization 66:545-

551.

8. Feldman BF, Zinkl JG, Jain NC, Moor DM. 2000.

Schalm’s Veterinary Hematology. 5th ed.

Philadelphia:Lippincott Williams & Wilkins.

9. Gregg L, Voigt DVM. 2000. Anemias and

Polychythenias. In: Hematology Techniques and

Concepts forVeterinary Technicians. U.S.A: Iowa

State University Press. P 95-101.

10. Inala P, Sirimontaporn A, Inpunkaew R,

Rungrojejinda K, Kengkoom K, Ratanasak W,

Buripakdi Lawson D. 2002. Hematological

analysis of outbred Sprague-Dawley rat in the

Facility of National Laboratory Animal Centre.

28th Congress on Science and Technology of

Thailand.

11. Levine BS. 2002. Animal Clinical Pathology. In:

Derelanko MJ, Hollinger MA, editors. Handbook

of Toxicology. 2nd ed. U.S.A: CRC Press. P 742-

768.

12. Kirtikar, K.R., Basu, B.D. (eds) 1975, Indian

medicinal Plants, Vol.1 2nd ed, New Delhi

Connaught Place, Dehra Dun; M/S Bishen Singh,

Mahendra Pal Singh.

13. Nadkarni, K.M., Nadkarni, A. K. (eds) 1976,

Indian Materia Medica Vol.1 3rd ed. Mumbai: M/S

Popular Prakasan Pvt. Ltd.

14. Nadkarni, K.M., Nadkarni, A. K. (eds) 1994, Dr.

K.M. Nadkarni's Indian Materia Medica Vol.1 3rd

ed. Popular Prakasan Mumbai.

15. The Organization of Economic Co-operation and

Development (OECD). 2001. The OECD

guidelines for testing of chemical: 420 Acute Oral

Toxicity – Fixed Dose Procedure. France.

16. The Organization of Economic Co-operation and

Development (OECD). 1981. The OECD guideline

for testing of chemical: 452 Chronic Toxicity

Studies. France.

17. Rojas, J. J, Ochoa, V.J., Ocampo, S.A., and Munoz,

J.F.,2006, Screening for antimicrobial activity of

ten medicinal plants used in Colambian folkloric

medicine. A possible alternative in the treatment of

nonosocomial infections BMC Comlimentary and

Alternative Medicine, 6:2.

18. Srivastava, J, Lambert, J. and Vetmeyer, 1996,

Medicinal Plants. An expending role in

development, World Bank Technical, p. - 320.

19. Sacher RA, McPherson RA. 2000. Test of liver

function. In: Sacher RA, McPherson RA, Campos

JM,

20. Widmann FK, editors. Widmann’s Clinical

Interpretation of Laboratory Test. 11th ed. U.S.A:

F.A. Davis Company. P 562-599.

21. Woodman DD. 1996. Assessment of hepatotoxicity.

In: Evans GO, editor. Animal Clinical Chemistry: a

Primer for Toxicologist. London: Taylor & Francis.

P 71-86.

22. World Health Organization (WHO). 1987.

Principles for the safety assessment of food

additives andContaminants in food. IPCS

Environmental Health Criteria 70, World Health

Organization, Geneva, p 39-59.

23. World Health Organization (WHO). 2000. General

guidelines for methodologies on research and

evaluation of traditional medicine. Switzerland.