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Acute intraperitoneal and oral toxicity of the leaf glycosidic extract of Trigonella foenum-graecum in mice
Journal of Ethnopharmacology 70 (2000) 65 – 68 www.elsevier.com/locate/jethpharm
Short communication
Acute intraperitoneal and oral toxicity of the leaf glycosidic extract of Trigonella foenum-graecum in mice Jamal Ahmed Abdel-Barry a,*, Mohammad H.H. Al-Hakiem b a
Department of Biochemistry, College of Medicine, Uni6ersity of Basrah, Basrah, Iraq b College of Pharmacy, Uni6ersity of Basrah, Basrah, Iraq
Received 24 November 1998; received in revised form 28 April 1999; accepted 19 June 1999
Abstract The present study was carried out to determine the acute toxicity of the leaf glycosidic extract of Trigonealla foenum-graecum by estimation of its medium lethal dose (LD50) after oral and intraperitoneal administration to mice and also to identify the target organs for its possible toxic effects. The main target organ affected among the four organs studied (liver, kidney, stomach, small and large intestine) was the liver, where early degeneration with infiltration of mononuclear and mild hepatitis was found in some animals treated with toxic doses of glycosidic extract. It is concluded that the glycosidic extract of T. foenum-graecum leaves is considered to be safe and have minimal adverse effect. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acute toxicity; Hepatitis; Trigonella foenum-graecum leaves
1. Introduction Trigonella foenum-graecum is a herb plant from the family Leguminacae (Townsend and Evan, 1974), locally known by its arabic name ‘Helba’. It has been mentioned that the seed of T. foenumgraecum has many pharmacological actions — hypoglycaemic, cardiotonic and antihypertensive (Kirtikar et al., 1935).
* Corresponding author.
A series of studies was carried out on the leaf extracts (aqueous, alkaloidal and glycosidic) of this plant to investigate their hypoglycaemic and antihyperglycaemic actions in both animals and human (Abdel-Barry et al., 1997a,b). It has been found that the hypoglycaemic and antihyperglycaemic actions of T. foenum-graecum leaf are due to its glycosidic compounds (Abdel-Barry et al., 1998a,b). The present paper aimed to study the oral and intraperitoneal acute toxicity (LD50) of the glycosidic extract of T. foenum-graecum leaf in animal and, much more importantly, to investigate its target organ effects.
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 0 9 - 9
66
J.A. Abdel-Barry, M.H.H. Al-Hakiem / Journal of Ethnopharmacology 70 (2000) 65–68
2. Materials and methods
2.1. Plant material T. foenum-graecum was collected from Abu AlKhasseeb region (south of Basrah) in winter (January 1995). The plant was botanically authenticated and voucher (2114) specimens were deposited in the herbarium of BASRA (Basrah, Iraq, College of Science, University of Basrah). The plant was shed-dried at 25°C. The dried leaves of the plant were ground with a blender, and the powdered part was kept in nylon bags in a deep freezer until the time of use.
2.2. Preparation of glycosidic extract Eighty grams (80 g) of powdered leaves were
refluxed with 400 ml of (2% acetic acid–water) for 8 h in a waterbath at 70°C. The residue was removed by filtration and the filtered material was extracted with saturated n-butanol and sodium chloride. The alcoholic fraction was evaporated under vaccum in RotaVapor, and the dried residue (12 g) was kept in an air-tight glass bottle in a deep freezer until the time of use (Gayon, 1972).
2.3. Experimental animals Wistar albino mice weighing (20–30 g) from a colony bred at the College of Science animal house were employed. They were housed in plastic cages according to group and sex under controlled conditions of temperature (2091°C), humidity (70%) and light (12:12 h, light:dark cycle). They were fed pellets with free access to tap water. The animals were divided into one control group and six treatment groups, each group consisting of ten animals. They were placed in separate cages for 3 days before the experiment to establish their baseline values of body weight, water and diet consumption, and daily up to 7 days after dosing. The means of body weights as well as water and dietary consumption were calculated.
2.4. Route of administration Graded doses of glycosidic extract in 0.2 ml of Tween 80 (10%) were injected intraperitoneally (i.p.) into the animals in the treatment groups to have a dose of 0.2, 0.4, 0.5, 0.8 and 1 g/kg, respectively; 0.2 ml of Tween 80 (10%) alone was injected intraperitoneally into the animals in the control group. In addition, the glycosidic extract in 1 ml of Tween 80 (10%) was directly administered to the stomach of the animals using a gastric tube in a dose of 1, 2, 4, 6, 8 and 10 g/kg, respectively; 1 ml of Tween 80 (10%) alone was fed orally to the animals in the control group.
2.5. Signs and obser6ations Fig. 1. Effects of intraperitoneal administration of a lethal dose (1 g/kg i.p.) of glycosidic extract. (A) Liver of control group (H&E, × 25). (B) Infiltration of mononuclear cells and mild hepatitis (H&E, × 25).
The animals were observed continuously for 1 h intermittently for 6 h and 14 h, and then after 1 day, 2 days, 3 days and 7 days (Twaij et al., 1983).
J.A. Abdel-Barry, M.H.H. Al-Hakiem / Journal of Ethnopharmacology 70 (2000) 65–68
The animals dying during the period of observation and those that survived up to the end of the period (7 days) were killed by neck dislocation and inspected for histopathological analysis.
2.6. Histopathological analysis Histopathological studies were made on four organs: liver, kidney, stomach, small and large intestine. The organs were fixed in 10% formal saline, and dehydrated in different ethanol concentrations of 50, 70, 80 and 90%. Later the samples were dehydrated in ethanol (100%) and paraffin blocks were prepared. Paraffin sections (6 mm thick) were stained with haematoxylin and eosin (H&E) (Wasfi et al., 1994). Sections were evaluated for pathological changes using light microscopy.
2.7. Determination of LD50 The number of mice that died within the period of study were noted for each group and subsequently the LD50 calculated by probit analysis (Finney, 1971; Goodman et al., 1980). Statistical analysis was performed using the paired and unpaired forms of Student’s t-test as appropriate.
67
examined no significant changes in motor activity, rectal temperature and skin colour were found.
3.2. Body weight, water and dietary consumption The water and dietary consumption of the animals in the treatment groups increased with increasing doses of glycosidic extract but was statistically not significant when compared with the control group. On the other hand, there were no noticeable changes in the body weights of the animals that tolerated fair amounts of glycosidic extract by both routes compared with the control group.
3.3. Target organ effects The light microscopy of liver, stomach, kidney, small and large intestine of both control and glycosidic-extract-treated animals orally and intraperitoneally revealed that: (1) no gross abnormality was observed in any organ of the animals that received low or moderate doses of the glycosidic extract (groups 1–3); (2) animals that received the lethal dose of the glycosidic extract intraperitoneally showed early degeneration in the liver with mild hepatitis (Fig. 1); (3) animals surviving to the end of the observation period were killed and no histopathological findings were observed in the structure of the organs examined and no alteration in the weights of those organs.
3. Results
4. Discussion
3.1. Beha6ioural effects and toxicity
The difference of LD50 values obtained may be due to difference in the rate of absorption. The rate of absorption after oral administration is variable and depends on many factors, such as the chemical nature of the drug (acidic or basic), degree of solubility, degree of ionization and the rate of gastric emptying (Waynforth, 1980). This result is in agreement with LD50 values obtained for the aqueous leaf extract of Trigonella foenum-graecum (Abdel-Barry et al., 1997b). No significant difference in LD50 values calculated for males and females was found. On the other hand, the reverse was found in our previous study (Abdel-Barry et al., 1997b), in which we
The method used for the determination of LD50 employs cumulative values. The LD50 computed is 0.65 g/kg after the intraperitoneal administration of different doses of glycosidic extract, while its value after oral adminisration was 7 g/kg. Oral and intraperitoneal administration of low and moderate doses of glycosidic extract showed only mild central nervous system (CNS) stimulation. However, higher doses caused rapid respiration, twitches, writhing, strabtail, tremor and generalized convulsions; and clumping together at the corners of the cages was noticed. At all doses
68
J.A. Abdel-Barry, M.H.H. Al-Hakiem / Journal of Ethnopharmacology 70 (2000) 65–68
found that female mice were better able to tolerate the lethal effect of the aqueous leaf extract of Trigonell foenum-graecum than males after intraperitoneal administration (LD50 values of 3.1 and 1.4 g/kg for females and males, respectively). Both oral and intraperitoneal administration of the glycosidic extract appears to exert their toxic effects on the CNS. Increasing doses of the glycosidic extract have caused graded CNS effects, ranging from twitches, writhing, tremor to loss of painful stimuli, paralysis and finally death. Animals that died following intraperitoneal administration of lethal doses of the glycosidic extract showed only a mild degree of pathological changes in the liver, where early degeneration with mild hepatitis was found.
Acknowledgements We are grateful to Dr Ahmed H. Ali, lecturer in the Department of Pathology and Forensic Medicine and to Dr Jassim M. AiDiab, consultant pathologist in the Department of Pathology and Forensic Medicine, College of Medicine, University of Basrah, for their help in performing the histopathological part of this study.
References Abdel-Barry, J.A., Abdel-Hassan, I.A., Al-Hakiem, M.H.H., 1997a. Hypoglycaemic and antihyperglycaemic effects of
.
Trigonella foenum-graecum leaf in normal and alloxan induced diabetic rats. Journal of Ethnopharmacology 58, 149 – 155. Abdel-Barry, J.A., Al-Hakiem, M.H.H., Abdel-Hassan, I.A., 1997b. Acute intraperitoneal toxicity (LD50) and target organ effects of aqueous extract of Trigonella foenum-graecum leaf in the mouse. Basrah Journal of Science Section B Biology 58, C58 – C65. Abdel-Barry, J.A., Abdel-Hassan, I.A., Jawad, A.M., AlHakiem, M.H.H., 1998a. Hypoglycaemic effect of aqueous extract of the leaves of Trigonella foenum-graecum in healthy volunteers. Eastern Mediteranean Health Journal (in press). Abdel-Barry, J.A., Al-Hakiem, M.H.H., Abdel-Hassan, I.A., 1998b. The hypoglycaemic action of Trigonella foenumgraecum leaves is due to its glycosidic component. Basrah Journal of Science (in press). Finney, D.G., 1971. Probit Analysis, 3rd ed. Cambridge University Press, London. Gayon, P.R., 1972. Plant Phenolics. Oliver and Boyd, Edinburgh. Goodman, A.G., Goodman, L.S., Gilman, A., 1980. Principles of toxicology. In: Goodman and Gilman: The Pharmacological Basis of Therapeutics, sixth ed. Macmillan, New York, pp. 1602 – 1603. Kirtikar, K.R., Basu, B.D., An, I.C.S., 1935. Indian Medicinal Plants, second ed. Periodicals Experts, Delhi. Townsend, C.C., Evan, G., 1974. Flora of Iraq, vol. 3. Ministry of Agriculture and Agrarian Reform, Iraq. Twaij, H.A.A., Kory, A., Al-Khazraji, N.K., 1983. Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala. Journal of Ethnopharmacology 9, 299 – 314. Wasfi, I.A., Bashir, A.K., Amiri, M.H., Abdalla, A.A., 1994. The effect of Rhazya stricta on glucose haemeostasis in normal and streptozocin diabetic rats. Journal of Ethnopharmacology 43, 141 – 147. Waynforth, H.B., 1980. Experimental and Surgical Technique in the Rat. Academic Press, London.
Short communication
Acute intraperitoneal and oral toxicity of the leaf glycosidic extract of Trigonella foenum-graecum in mice Jamal Ahmed Abdel-Barry a,*, Mohammad H.H. Al-Hakiem b a
Department of Biochemistry, College of Medicine, Uni6ersity of Basrah, Basrah, Iraq b College of Pharmacy, Uni6ersity of Basrah, Basrah, Iraq
Received 24 November 1998; received in revised form 28 April 1999; accepted 19 June 1999
Abstract The present study was carried out to determine the acute toxicity of the leaf glycosidic extract of Trigonealla foenum-graecum by estimation of its medium lethal dose (LD50) after oral and intraperitoneal administration to mice and also to identify the target organs for its possible toxic effects. The main target organ affected among the four organs studied (liver, kidney, stomach, small and large intestine) was the liver, where early degeneration with infiltration of mononuclear and mild hepatitis was found in some animals treated with toxic doses of glycosidic extract. It is concluded that the glycosidic extract of T. foenum-graecum leaves is considered to be safe and have minimal adverse effect. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acute toxicity; Hepatitis; Trigonella foenum-graecum leaves
1. Introduction Trigonella foenum-graecum is a herb plant from the family Leguminacae (Townsend and Evan, 1974), locally known by its arabic name ‘Helba’. It has been mentioned that the seed of T. foenumgraecum has many pharmacological actions — hypoglycaemic, cardiotonic and antihypertensive (Kirtikar et al., 1935).
* Corresponding author.
A series of studies was carried out on the leaf extracts (aqueous, alkaloidal and glycosidic) of this plant to investigate their hypoglycaemic and antihyperglycaemic actions in both animals and human (Abdel-Barry et al., 1997a,b). It has been found that the hypoglycaemic and antihyperglycaemic actions of T. foenum-graecum leaf are due to its glycosidic compounds (Abdel-Barry et al., 1998a,b). The present paper aimed to study the oral and intraperitoneal acute toxicity (LD50) of the glycosidic extract of T. foenum-graecum leaf in animal and, much more importantly, to investigate its target organ effects.
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 0 9 - 9
66
J.A. Abdel-Barry, M.H.H. Al-Hakiem / Journal of Ethnopharmacology 70 (2000) 65–68
2. Materials and methods
2.1. Plant material T. foenum-graecum was collected from Abu AlKhasseeb region (south of Basrah) in winter (January 1995). The plant was botanically authenticated and voucher (2114) specimens were deposited in the herbarium of BASRA (Basrah, Iraq, College of Science, University of Basrah). The plant was shed-dried at 25°C. The dried leaves of the plant were ground with a blender, and the powdered part was kept in nylon bags in a deep freezer until the time of use.
2.2. Preparation of glycosidic extract Eighty grams (80 g) of powdered leaves were
refluxed with 400 ml of (2% acetic acid–water) for 8 h in a waterbath at 70°C. The residue was removed by filtration and the filtered material was extracted with saturated n-butanol and sodium chloride. The alcoholic fraction was evaporated under vaccum in RotaVapor, and the dried residue (12 g) was kept in an air-tight glass bottle in a deep freezer until the time of use (Gayon, 1972).
2.3. Experimental animals Wistar albino mice weighing (20–30 g) from a colony bred at the College of Science animal house were employed. They were housed in plastic cages according to group and sex under controlled conditions of temperature (2091°C), humidity (70%) and light (12:12 h, light:dark cycle). They were fed pellets with free access to tap water. The animals were divided into one control group and six treatment groups, each group consisting of ten animals. They were placed in separate cages for 3 days before the experiment to establish their baseline values of body weight, water and diet consumption, and daily up to 7 days after dosing. The means of body weights as well as water and dietary consumption were calculated.
2.4. Route of administration Graded doses of glycosidic extract in 0.2 ml of Tween 80 (10%) were injected intraperitoneally (i.p.) into the animals in the treatment groups to have a dose of 0.2, 0.4, 0.5, 0.8 and 1 g/kg, respectively; 0.2 ml of Tween 80 (10%) alone was injected intraperitoneally into the animals in the control group. In addition, the glycosidic extract in 1 ml of Tween 80 (10%) was directly administered to the stomach of the animals using a gastric tube in a dose of 1, 2, 4, 6, 8 and 10 g/kg, respectively; 1 ml of Tween 80 (10%) alone was fed orally to the animals in the control group.
2.5. Signs and obser6ations Fig. 1. Effects of intraperitoneal administration of a lethal dose (1 g/kg i.p.) of glycosidic extract. (A) Liver of control group (H&E, × 25). (B) Infiltration of mononuclear cells and mild hepatitis (H&E, × 25).
The animals were observed continuously for 1 h intermittently for 6 h and 14 h, and then after 1 day, 2 days, 3 days and 7 days (Twaij et al., 1983).
J.A. Abdel-Barry, M.H.H. Al-Hakiem / Journal of Ethnopharmacology 70 (2000) 65–68
The animals dying during the period of observation and those that survived up to the end of the period (7 days) were killed by neck dislocation and inspected for histopathological analysis.
2.6. Histopathological analysis Histopathological studies were made on four organs: liver, kidney, stomach, small and large intestine. The organs were fixed in 10% formal saline, and dehydrated in different ethanol concentrations of 50, 70, 80 and 90%. Later the samples were dehydrated in ethanol (100%) and paraffin blocks were prepared. Paraffin sections (6 mm thick) were stained with haematoxylin and eosin (H&E) (Wasfi et al., 1994). Sections were evaluated for pathological changes using light microscopy.
2.7. Determination of LD50 The number of mice that died within the period of study were noted for each group and subsequently the LD50 calculated by probit analysis (Finney, 1971; Goodman et al., 1980). Statistical analysis was performed using the paired and unpaired forms of Student’s t-test as appropriate.
67
examined no significant changes in motor activity, rectal temperature and skin colour were found.
3.2. Body weight, water and dietary consumption The water and dietary consumption of the animals in the treatment groups increased with increasing doses of glycosidic extract but was statistically not significant when compared with the control group. On the other hand, there were no noticeable changes in the body weights of the animals that tolerated fair amounts of glycosidic extract by both routes compared with the control group.
3.3. Target organ effects The light microscopy of liver, stomach, kidney, small and large intestine of both control and glycosidic-extract-treated animals orally and intraperitoneally revealed that: (1) no gross abnormality was observed in any organ of the animals that received low or moderate doses of the glycosidic extract (groups 1–3); (2) animals that received the lethal dose of the glycosidic extract intraperitoneally showed early degeneration in the liver with mild hepatitis (Fig. 1); (3) animals surviving to the end of the observation period were killed and no histopathological findings were observed in the structure of the organs examined and no alteration in the weights of those organs.
3. Results
4. Discussion
3.1. Beha6ioural effects and toxicity
The difference of LD50 values obtained may be due to difference in the rate of absorption. The rate of absorption after oral administration is variable and depends on many factors, such as the chemical nature of the drug (acidic or basic), degree of solubility, degree of ionization and the rate of gastric emptying (Waynforth, 1980). This result is in agreement with LD50 values obtained for the aqueous leaf extract of Trigonella foenum-graecum (Abdel-Barry et al., 1997b). No significant difference in LD50 values calculated for males and females was found. On the other hand, the reverse was found in our previous study (Abdel-Barry et al., 1997b), in which we
The method used for the determination of LD50 employs cumulative values. The LD50 computed is 0.65 g/kg after the intraperitoneal administration of different doses of glycosidic extract, while its value after oral adminisration was 7 g/kg. Oral and intraperitoneal administration of low and moderate doses of glycosidic extract showed only mild central nervous system (CNS) stimulation. However, higher doses caused rapid respiration, twitches, writhing, strabtail, tremor and generalized convulsions; and clumping together at the corners of the cages was noticed. At all doses
68
J.A. Abdel-Barry, M.H.H. Al-Hakiem / Journal of Ethnopharmacology 70 (2000) 65–68
found that female mice were better able to tolerate the lethal effect of the aqueous leaf extract of Trigonell foenum-graecum than males after intraperitoneal administration (LD50 values of 3.1 and 1.4 g/kg for females and males, respectively). Both oral and intraperitoneal administration of the glycosidic extract appears to exert their toxic effects on the CNS. Increasing doses of the glycosidic extract have caused graded CNS effects, ranging from twitches, writhing, tremor to loss of painful stimuli, paralysis and finally death. Animals that died following intraperitoneal administration of lethal doses of the glycosidic extract showed only a mild degree of pathological changes in the liver, where early degeneration with mild hepatitis was found.
Acknowledgements We are grateful to Dr Ahmed H. Ali, lecturer in the Department of Pathology and Forensic Medicine and to Dr Jassim M. AiDiab, consultant pathologist in the Department of Pathology and Forensic Medicine, College of Medicine, University of Basrah, for their help in performing the histopathological part of this study.
References Abdel-Barry, J.A., Abdel-Hassan, I.A., Al-Hakiem, M.H.H., 1997a. Hypoglycaemic and antihyperglycaemic effects of
.
Trigonella foenum-graecum leaf in normal and alloxan induced diabetic rats. Journal of Ethnopharmacology 58, 149 – 155. Abdel-Barry, J.A., Al-Hakiem, M.H.H., Abdel-Hassan, I.A., 1997b. Acute intraperitoneal toxicity (LD50) and target organ effects of aqueous extract of Trigonella foenum-graecum leaf in the mouse. Basrah Journal of Science Section B Biology 58, C58 – C65. Abdel-Barry, J.A., Abdel-Hassan, I.A., Jawad, A.M., AlHakiem, M.H.H., 1998a. Hypoglycaemic effect of aqueous extract of the leaves of Trigonella foenum-graecum in healthy volunteers. Eastern Mediteranean Health Journal (in press). Abdel-Barry, J.A., Al-Hakiem, M.H.H., Abdel-Hassan, I.A., 1998b. The hypoglycaemic action of Trigonella foenumgraecum leaves is due to its glycosidic component. Basrah Journal of Science (in press). Finney, D.G., 1971. Probit Analysis, 3rd ed. Cambridge University Press, London. Gayon, P.R., 1972. Plant Phenolics. Oliver and Boyd, Edinburgh. Goodman, A.G., Goodman, L.S., Gilman, A., 1980. Principles of toxicology. In: Goodman and Gilman: The Pharmacological Basis of Therapeutics, sixth ed. Macmillan, New York, pp. 1602 – 1603. Kirtikar, K.R., Basu, B.D., An, I.C.S., 1935. Indian Medicinal Plants, second ed. Periodicals Experts, Delhi. Townsend, C.C., Evan, G., 1974. Flora of Iraq, vol. 3. Ministry of Agriculture and Agrarian Reform, Iraq. Twaij, H.A.A., Kory, A., Al-Khazraji, N.K., 1983. Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala. Journal of Ethnopharmacology 9, 299 – 314. Wasfi, I.A., Bashir, A.K., Amiri, M.H., Abdalla, A.A., 1994. The effect of Rhazya stricta on glucose haemeostasis in normal and streptozocin diabetic rats. Journal of Ethnopharmacology 43, 141 – 147. Waynforth, H.B., 1980. Experimental and Surgical Technique in the Rat. Academic Press, London.