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Assessing The Efficacy Of Aqueous Garlic Extract Against Cyanide Toxicity In Mice Using Righting Reflex Recovery

By: Sajid Ali | Dr. Muhammad Avais.
Contributor(s): Dr. Aneela Zameer Durrani | Prof. Dr.
Material type: materialTypeLabelBookPublisher: 2013Subject(s): Department of Clinical Medicine & SurgeryDDC classification: 1776,T Dissertation note: The KCN intake has been linked to central nervous system (CNS) syndromes and thyroid in animals as well as humans. It is very potent toxin even at very narrow dose ranges. Prolonged KCN exposure has also been associated with reduced growth rate in animals, disturbance in thyroid metabolism, lesions in liver, kidneys, lungs and also CNS pathology. Male mice weighting about 20-40 grams were purchased from Veterinary Research Institute, Lahore, National Institute of Health, Islamabad, Animal house, University of Veterinary and Animal Sciences, Lahore and divided into 30 treatment groups. After acclimatization of mice, carefully, the mice were grouped as follows: Control group A group of 6 mice were treated with potassium cyanide (KCN) intra-peritoneally at 5.5mg/kg that induced a knock down state with recovery period of approximately 1 hour. Prior to each experiment, three or more mice were treated with KCN at this dose to verify that the recovery time was close to 1 hour. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 250 mg/kg: Group 1 (5-min pre-KCN) A group of 6 mice was given AGE intraperitoneally (i.p) at 250 mg/kg. After 5 minutes of AGE injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 250 mg/kg: Group 2 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 250 mg/kg i.p. and righting reflex recovery time was noted. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 500 mg/kg: Group 3 (5-min pre-KCN) A group of 6 mice was given AGE intraperitoneally (i.p) at 500 mg/kg BW. After 5 minutes of AGE injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 500 mg/kg: Group 4 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 500 mg/kg i.p. and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 750 mg/kg: Group 5 (5-min pre-KCN) A group of 6 mice was treated with AGE intraperitoneally (i.p) at 750 mg/kg. After 5 minutes of AGE injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 750 mg/kg: Group 6 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 750 mg/kg i.p. and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 250 mg/kg: Group 7 (30-min post-KCN) One more group of 6 mice was given AGE at 250 mg/kg orally. After 30 minutes of AGE administration, an intraperitoneal (i.p) injection of KCN at 5.5 mg/kg was given and righting refelx recovery time was noted. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 500 mg/kg: Group 8 (30-min post-KCN) A group 6 mice was given AGE at 500 mg/kg orally. After 30 minutes of AGE administration, an intraperitoneal (i.p) injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was noted. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 750 mg/kg: Group 9 (30-min post-KCN) One more group of 6 mice was given AGE at 750 mg/kg orally. After 30 minutes of AGE administration, an intraperitoneal (i.p) injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Garlic Extract Group: (Group 10-15) Three groups 10 - 12 each with 6 mice were injected intraperitoneally (i.p) with AGE at 250, 500 and 750 mg/kg respectively and righting reflex recovery time of each mouse in respective dose group was noted to observe any effects of AGE on righting reflex recovery time. Three groups 13 - 15 each with 6 mice were treated with AGE orally at 250, 500 and 750 mg/kg respectively and righting reflex recovery time of each mouse in respective dose group was noted to observe any effects of AGE on righting reflex recovery time. Potassium Cyanide (KCN) + Sodium Nitrite (SN): Group 16 (5-min pre-KCN) A group of 6 mice was given SN at 20 mg/kg intraperitoneally (i.p). After 5 minutes of SN injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Sodium Nitrite (SN): Group 17 (5-min post-KCN) Another group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with SN at 20 mg/kg i.p. and righting reflex recovery time was noted. Potassium Cyanide (KCN) + Sodium Thiosulphate (ST): Group 18 (5-min pre-KCN) A group of 6 mice was given ST at 600 mg/kg intraperitoneally (i.p). After 5 minutes of ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Sodium Thiosulphate (ST): Group 19 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with ST at 600 mg/kg i.p. and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Sodium Nitrite (SN) & Sodium Thiosulphate (ST): Group 20 (5-min pre-KCN) A group of 6 mice was given SN and ST at 20 mg/kg and 600 mg/kg intraperitoneally (i.p) respectively. After 5 minutes SN and ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Sodium Nitrite (SN) & Sodium Thiosulphate (ST): Group 21 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with SN and ST at 20 mg/kg and 600 mg/kg i.p. respectively and righting reflex recovery time was noted. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Nitrite (SN): Group 22 (5-min pre-KCN) A group of 6 mice was given AGE at 750 mg/kg and SN at 20mg/kg intraperitoneally (i.p). After 5 minutes of AGE and ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was noted. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Nitrite (SN): Group 23 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 750 mg/kg and SN at 20 mg/kg i.p. and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Thiosulphate (ST): Group 24 (5-min pre-KCN) A group of 6 mice was given AGE at 750 mg/kg and ST at 600mg/kg intraperitoneally (i.p). After 5 minutes of AGE and ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded. Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Thiosulphate (ST): Group 25 (5-min post-KCN) A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse will be treated with AGE at 750 mg/kg and ST at 600 mg/kg i.p. and righting reflex recovery time was noted. Sodium Nitrite (SN) alone (Group 26) 6 mice in this group were treated with SN at 20 mg/kg intraperitoneally (i.p) and any effects on righting reflex recovery time were noted. Sodium Thiosulphate (ST) alone (Group 27) A group of 6 mice in group 27 were treated with ST at 600 mg/kg intraperitoneally (i.p) and any effects on righting reflex recovery time were noted. Aqueous Garlic Extract (AGE) + Sodium Nitrite (SN) (Group 28) The mice in group 28 were treated with AGE + SN at 750 mg/kg and 20 mg/kg intraperitoneally (i.p) respectively and righting reflex recovery time was noted. Aqueous Garlic Extract (AGE) + Sodium Thiosulphate (ST) (Group 29) A group of 6 mice in this group were treated with AGE at 750 mg/kg and ST at 600 mg/kg intraperitoneally (i.p) and righting reflex recovery time was recorded. Sodium Nitrite (SN) + Sodium Thiosulphate (ST) (Group 30) A group of 6 mice in group 30 were treated with SN and ST at 20 mg/kg and 600 mg/kg intraperitoneally (i.p) respectively and righting reflex recovery time was recorded. The efficacy of the antidote estimated was based upon the reduction in righting reflex recovery time. The righting reflex recovery time that was close to 1 hour was determined against KCN regimen. AGE was tested as potential antidote against sub-lethal KCN toxicity at dose regimens 250, 500 and 750 mg/kg intraperitoneally as well as orally at dose regimens 250, 500 and 750 mg/kg 5 minutes before and 5 minutes after KCN administration. SN and ST were tested at 20, and 600 mg/kg respectively as antidotes against sub-lethal KCN toxicity, 5 minutes before and 5 minutes after KCN administration. These were also tested in comparison and a dose with minimum recovery time was noted. A comparison was made between the efficacy of AGE and SN or ST or SN + ST as potential antidote against sub-lethal KCN toxicity. After 48 hours 3 mice from each group was sacrificed for postmortem examination. For this purpose the righting reflex recovery time and survival data was collected for each mouse challenged with KCN. A group of 3 mice was tested with each increasing dose of KCN to create dose response curves for both righting reflex recovery times and percent survival. Increasing doses of KCN increased the recovery time of the righting reflex. The dose 5.5 mg/kg of KCN showed the righting reflex recocery time mean 64.66 ± .333 min and was selected for trials. AGE (750 mg/kg) showed the most significant results as compared to ST and SN alone as well as in combination. Second most effective drug was ST as it showed better results than SN. Treatment results were more pronounced in 5 min pre-KCN groups as compared to 5 min post-KCN groups. From this study it was concluded that the aqueous AGE is an effective antidote against sub-lethal KCN toxicity as the recovery times indicated that its effects are more pronounced than SN and ST, secondly, as it is easily available and very much cheaper in Pakistan and due to easy unavailability of SN and ST, it is effective antidote that could be used in the field against sub-lethal KCN toxicity with comparatively better results both orally and intraperitoneally.
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The KCN intake has been linked to central nervous system (CNS) syndromes and thyroid in animals as well as humans. It is very potent toxin even at very narrow dose ranges. Prolonged KCN exposure has also been associated with reduced growth rate in animals, disturbance in thyroid metabolism, lesions in liver, kidneys, lungs and also CNS pathology.
Male mice weighting about 20-40 grams were purchased from Veterinary Research Institute, Lahore, National Institute of Health, Islamabad, Animal house, University of Veterinary and Animal Sciences, Lahore and divided into 30 treatment groups. After acclimatization of mice, carefully, the mice were grouped as follows:
Control group
A group of 6 mice were treated with potassium cyanide (KCN) intra-peritoneally at 5.5mg/kg that induced a knock down state with recovery period of approximately 1 hour. Prior to each experiment, three or more mice were treated with KCN at this dose to verify that the recovery time was close to 1 hour.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 250 mg/kg: Group 1 (5-min pre-KCN)
A group of 6 mice was given AGE intraperitoneally (i.p) at 250 mg/kg. After 5 minutes of AGE injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.

Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 250 mg/kg: Group 2 (5-min post-KCN)

A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 250 mg/kg i.p. and righting reflex recovery time was noted.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 500 mg/kg: Group 3 (5-min pre-KCN)
A group of 6 mice was given AGE intraperitoneally (i.p) at 500 mg/kg BW. After 5 minutes of AGE injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 500 mg/kg: Group 4 (5-min post-KCN)
A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 500 mg/kg i.p. and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 750 mg/kg: Group 5 (5-min pre-KCN)
A group of 6 mice was treated with AGE intraperitoneally (i.p) at 750 mg/kg. After 5 minutes of AGE injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.


Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 750 mg/kg: Group 6 (5-min post-KCN)
A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 750 mg/kg i.p. and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 250 mg/kg: Group 7 (30-min post-KCN)
One more group of 6 mice was given AGE at 250 mg/kg orally. After 30 minutes of AGE administration, an intraperitoneal (i.p) injection of KCN at 5.5 mg/kg was given and righting refelx recovery time was noted.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 500 mg/kg: Group 8 (30-min post-KCN)
A group 6 mice was given AGE at 500 mg/kg orally. After 30 minutes of AGE administration, an intraperitoneal (i.p) injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was noted.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) at 750 mg/kg: Group 9 (30-min post-KCN)
One more group of 6 mice was given AGE at 750 mg/kg orally. After 30 minutes of AGE administration, an intraperitoneal (i.p) injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.



Garlic Extract Group: (Group 10-15)
Three groups 10 - 12 each with 6 mice were injected intraperitoneally (i.p) with AGE at 250, 500 and 750 mg/kg respectively and righting reflex recovery time of each mouse in respective dose group was noted to observe any effects of AGE on righting reflex recovery time. Three groups 13 - 15 each with 6 mice were treated with AGE orally at 250, 500 and 750 mg/kg respectively and righting reflex recovery time of each mouse in respective dose group was noted to observe any effects of AGE on righting reflex recovery time.
Potassium Cyanide (KCN) + Sodium Nitrite (SN): Group 16 (5-min pre-KCN)
A group of 6 mice was given SN at 20 mg/kg intraperitoneally (i.p). After 5 minutes of SN injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Sodium Nitrite (SN): Group 17 (5-min post-KCN)
Another group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with SN at 20 mg/kg i.p. and righting reflex recovery time was noted.
Potassium Cyanide (KCN) + Sodium Thiosulphate (ST): Group 18 (5-min pre-KCN)
A group of 6 mice was given ST at 600 mg/kg intraperitoneally (i.p). After 5 minutes of ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Sodium Thiosulphate (ST): Group 19 (5-min post-KCN)
A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with ST at 600 mg/kg i.p. and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Sodium Nitrite (SN) & Sodium Thiosulphate (ST): Group 20 (5-min pre-KCN)
A group of 6 mice was given SN and ST at 20 mg/kg and 600 mg/kg intraperitoneally (i.p) respectively. After 5 minutes SN and ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Sodium Nitrite (SN) & Sodium Thiosulphate (ST): Group 21 (5-min post-KCN)
A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with SN and ST at 20 mg/kg and 600 mg/kg i.p. respectively and righting reflex recovery time was noted.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Nitrite (SN): Group 22 (5-min pre-KCN)
A group of 6 mice was given AGE at 750 mg/kg and SN at 20mg/kg intraperitoneally (i.p). After 5 minutes of AGE and ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was noted.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Nitrite (SN): Group 23 (5-min post-KCN)
A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse was treated with AGE at 750 mg/kg and SN at 20 mg/kg i.p. and righting reflex recovery time was recorded.


Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Thiosulphate (ST): Group 24 (5-min pre-KCN)
A group of 6 mice was given AGE at 750 mg/kg and ST at 600mg/kg intraperitoneally (i.p). After 5 minutes of AGE and ST injection, an i.p. injection of KCN at 5.5 mg/kg was given and righting reflex recovery time was recorded.
Potassium Cyanide (KCN) + Aqueous Garlic Extract (AGE) & Sodium Thiosulphate (ST): Group 25 (5-min post-KCN)
A group of 6 mice was administered with KCN intraperitoneally (i.p) at 5.5 mg/kg. After 5 minutes of KCN injection, each mouse will be treated with AGE at 750 mg/kg and ST at 600 mg/kg i.p. and righting reflex recovery time was noted.
Sodium Nitrite (SN) alone (Group 26)
6 mice in this group were treated with SN at 20 mg/kg intraperitoneally (i.p) and any effects on righting reflex recovery time were noted.
Sodium Thiosulphate (ST) alone (Group 27)
A group of 6 mice in group 27 were treated with ST at 600 mg/kg intraperitoneally (i.p) and any effects on righting reflex recovery time were noted.
Aqueous Garlic Extract (AGE) + Sodium Nitrite (SN) (Group 28)
The mice in group 28 were treated with AGE + SN at 750 mg/kg and 20 mg/kg intraperitoneally (i.p) respectively and righting reflex recovery time was noted.
Aqueous Garlic Extract (AGE) + Sodium Thiosulphate (ST) (Group 29)
A group of 6 mice in this group were treated with AGE at 750 mg/kg and ST at 600 mg/kg intraperitoneally (i.p) and righting reflex recovery time was recorded.

Sodium Nitrite (SN) + Sodium Thiosulphate (ST) (Group 30)
A group of 6 mice in group 30 were treated with SN and ST at 20 mg/kg and 600 mg/kg intraperitoneally (i.p) respectively and righting reflex recovery time was recorded.
The efficacy of the antidote estimated was based upon the reduction in righting reflex recovery time.
The righting reflex recovery time that was close to 1 hour was determined against KCN regimen. AGE was tested as potential antidote against sub-lethal KCN toxicity at dose regimens 250, 500 and 750 mg/kg intraperitoneally as well as orally at dose regimens 250, 500 and 750 mg/kg 5 minutes before and 5 minutes after KCN administration. SN and ST were tested at 20, and 600 mg/kg respectively as antidotes against sub-lethal KCN toxicity, 5 minutes before and 5 minutes after KCN administration. These were also tested in comparison and a dose with minimum recovery time was noted. A comparison was made between the efficacy of AGE and SN or ST or SN + ST as potential antidote against sub-lethal KCN toxicity. After 48 hours 3 mice from each group was sacrificed for postmortem examination.
For this purpose the righting reflex recovery time and survival data was collected for each mouse challenged with KCN. A group of 3 mice was tested with each increasing dose of KCN to create dose response curves for both righting reflex recovery times and percent survival. Increasing doses of KCN increased the recovery time of the righting reflex. The dose 5.5 mg/kg of KCN showed the righting reflex recocery time mean 64.66 ± .333 min and was selected for trials.
AGE (750 mg/kg) showed the most significant results as compared to ST and SN alone as well as in combination. Second most effective drug was ST as it showed better results than SN. Treatment results were more pronounced in 5 min pre-KCN groups as compared to 5 min post-KCN groups.
From this study it was concluded that the aqueous AGE is an effective antidote against sub-lethal KCN toxicity as the recovery times indicated that its effects are more pronounced than SN and ST, secondly, as it is easily available and very much cheaper in Pakistan and due to easy unavailability of SN and ST, it is effective antidote that could be used in the field against sub-lethal KCN toxicity with comparatively better results both orally and intraperitoneally.

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