The effectiveness of silibinin on the change in some blood biochemical indices and liver histopathology of rabbit experimentally poisoned with amanita virosa mushroom

Journal of military pharmaco-medicine n06-2018<br /> <br /> THE EFFECTIVENESS OF SILIBININ ON THE CHANGE IN SOME<br /> BLOOD BIOCHEMICAL INDICES AND LIVER HISTOPATHOLOGY<br /> OF RABBIT EXPERIMENTALLY POISONED WITH<br /> AMANITA VIROSA MUSHROOM<br /> Ngo Thi Thanh Hai*; Nguyen Thanh Binh*; Tran Van Tung*; Be Hong Thu**<br /> SUMMARY<br /> Objectives: To evaluate the effectiveness of silibinin on the change in some blood biochemical<br /> indices and liver histopathology of rabbit poisoned with Amanita virosa mushroom in order to<br /> get evidence of protective effect of the drug against the toxicity of amatoxin. Subjects and<br /> methods: Rabbits were randomly grouped then poisoned with Amanita virosa mushroom.<br /> 12 hour after the intoxication, the rabbits were given silibinin then blood samples were collected<br /> for the testing of GOT, GPT, GGT, urea, glucose, total bilirubin and direct bilirubin on a<br /> automated system. Liver histology was processed and examined followed standard HE staining<br /> procedure. Results: GOT, GPT, GGT concentrations in blood of rabbits poisoned with Amanita<br /> rd<br /> th<br /> virosa in the group with silibinin treatment decreased on the 3 and 5 days after poisoning<br /> compared to the group without treatment. Silibinin treatment also decreased hepatocellular<br /> damages caused by Amanita virosa mushroom poisoning. Conclusion: Silibinin has protective<br /> effect against the toxicity of Amanita virosa mushroom.<br /> * Keywords: Silibinin; Amatoxin; Amanita virosa; Blood biochemical indices; Liver histopathology;<br /> Rabbit.<br /> <br /> INTRODUCTION<br /> According to statistics from the Poison<br /> Control Center of Vietnam Military Medical<br /> University from 2004 to 2011, in Backan<br /> province there were 28 incidences of<br /> mushroom poisoning with the total number<br /> of 94 infected people and 14 deaths.<br /> The results of investigation showed that<br /> the deaths from poisoning of poisonous<br /> mushrooms in Backan province were<br /> caused by Amanita virosa [1].<br /> Amanita virosa is pure white, fleshy,<br /> very beautiful and attractive. Amanita<br /> <br /> virosa contains amanitines that cause<br /> slow and sustainable poisoning to heat<br /> and has a very high toxicity. Characteristics<br /> of these toxins are to cause hepatocellular<br /> necrosis leading to liver failure and death<br /> [3, 4, 5].<br /> The principle of treatment Amanita<br /> virosa poisoning includes the limited<br /> absorption and increased excretion of<br /> amatoxin, and use liver protection drugs<br /> as soon as possible accompanied by<br /> maintaining vital functions and symptomatic<br /> treatment [5, 6].<br /> <br /> * Vietnam Military Medical University<br /> ** Bachmai Hospital<br /> Corresponding author: Ngo Thi Thanh Hai (ngohaixpnoi@gmail.com)<br /> Date received: 18/05/2018<br /> Date accepted: 20/06/2018<br /> <br /> 146<br /> <br /> Journal of military pharmaco-medicine n06-2018<br /> Silibinin dihemisusccinate prevents<br /> amatoxin from entering hepatocytes,<br /> reducing amatoxin in the intestinal and<br /> hepatic circulation, thereby increasing<br /> amatoxin excretion in bile and increasing<br /> the synthesis of ARN polymerase 1 to<br /> reduce hepatocellular injury [4 ,6, 7]. It is<br /> recommended that this drug should be<br /> used as an antidote to amantoxin. This study<br /> was conducted to: Investigate the role of<br /> silibinin in the change of biochemical indices<br /> of rabbits poisoned with of amanita virosa<br /> to provide the experimental evidence on<br /> the ability of hepatocellular protection of<br /> silibinin against toxic effect of amatoxin.<br /> SUBJECTS AND METHODS<br /> 1. Subjects.<br /> - Amanita virosa mushroom samples<br /> were collected in Backan province. After<br /> collection, the mushroom samples were<br /> weighed, then preserved in ethanol till<br /> next experiment.<br /> - Rabbit: 30 rabbits, regardless of male<br /> or female, healthy, weight: 2.0 ± 0.2 kg.<br /> The rabbits were raised in the same mode<br /> during the experimental period.<br /> ®<br /> <br /> - Legalon SIL (silibinin dihemisusccinate)<br /> 528.5 mg.<br /> 2. Research methods.<br /> * Method of poisoning on rabbits:<br /> Amanita virosa preserved samples<br /> were processed to evaporate ethanol.<br /> The ethanol free samples were then<br /> homogenized with a tissue homogenizer.<br /> Water was then added to dilute the<br /> sample then filtered with a filter paper.<br /> The homogenizing and filtering procedure<br /> <br /> were repeated three times to remove fiber<br /> and particulate material from the mushroom.<br /> The filtered solution was then administered<br /> orally into rabbit stomach by specialized<br /> equipment with a dosage equal to 2/3 of<br /> the minimum lethal dose (LDmin) which<br /> was pre-determined.<br /> * Method of conducting the indicators:<br /> - 30 rabbits were divided into 3 groups,<br /> each group had 10 rabbits marked separately.<br /> - Collect blood from vein in rabbit ears<br /> before and after poisoning, on the first,<br /> third and fifth days to test biochemical and<br /> hematological indicators.<br /> - Group 2 is not treated by drug after<br /> poisoning.<br /> - Group 3: After poisoning 1 day, inject<br /> silibinin into rabbit vein with a maintenance<br /> dose of 60 mg/kg/day for 4 days.<br /> - Biochemical and hematological indicators<br /> include: GOT, GPT, GGT, and urea<br /> concentrations; creatinine, electrolytes,<br /> blood sugar, total and bilirubin, etc. are<br /> directly performed on automatic biochemical<br /> analyzer.<br /> - Liver histology was processed and<br /> examined followed standard HE staining<br /> procedure at Department of Pathology,<br /> 103 Military Hospital.<br /> - Evaluation of clinical criteria: Skip meals,<br /> diarrhea, mischievous level, etc.<br /> * Statistical processing method:<br /> The data is averaged (X), standard<br /> deviation (SD) and compare 2 average<br /> values. Calculate p before and after<br /> poisoning, compare between the experimental<br /> group and control group by t-test.<br /> <br /> 147<br /> <br /> Journal of military pharmaco-medicine n06-2018<br /> RESULTS<br /> 1. Changes in GOT, GPT and GGT activities in rabbits poisoned with Aminata<br /> virosa treated or untreated with silibinin.<br /> Group 1<br /> <br /> UI/L<br /> <br /> Group 2<br /> <br /> 800<br /> <br /> Group 3<br /> 748.51<br /> <br /> 700<br /> 600<br /> 500<br /> 351.27<br /> <br /> 400<br /> 300<br /> 200<br /> 100<br /> 0<br /> <br /> 44.29<br /> 51.48<br /> <br /> 49.93<br /> 42.9<br /> <br /> 44.02<br /> <br /> 153.71<br /> <br /> 47.72<br /> <br /> 182.13<br /> <br /> 46.79<br /> Time<br /> <br /> 43.21<br /> Before poisoning<br /> <br /> After 12h<br /> <br /> 3rd days after<br /> <br /> 5th days after<br /> <br /> Figure 1: Changes in GOT activity.<br /> GOT activity in serum of rabbits poisoned with Aminata virosa increased highly on the 3rd<br /> and 5th days after poisoning. However, GOT activity in serum of poisoned rabbits untreated<br /> with sibilinin was higher than that in poisoned rabbits treated with sibilinin (p < 0.05).<br /> Group 1<br /> <br /> UI/L<br /> <br /> Group 2<br /> <br /> Group 3<br /> <br /> 700<br /> <br /> 598.33<br /> 600<br /> 500<br /> <br /> 333.75<br /> <br /> 400<br /> 300<br /> 200<br /> 100<br /> <br /> 54.56<br /> 60.32<br /> 62.94<br /> <br /> 59.19<br /> 63.1<br /> 70.39<br /> <br /> 101.64<br /> <br /> 66.3<br /> <br /> 54.14<br /> <br /> 0<br /> <br /> Before poisoning<br /> <br /> 181.42<br /> <br /> After 12h<br /> <br /> 3rd days after<br /> <br /> 5th days after<br /> <br /> Time<br /> <br /> Figure 2: Changes in GPT activity.<br /> GPT activity in serum of rabbits poisoned with Aminata virosa increased highly on<br /> the 3rd and 5th days after poisoning. However, GPT activity in serum of rabbits untreated<br /> with sibilinin was higher than that of rabbits treated with sibilinin (p < 0.05).<br /> 148<br /> <br /> Journal of military pharmaco-medicine n06-2018<br /> Before poisoning<br /> <br /> UI/L<br /> <br /> After 12h<br /> <br /> 3rd days after<br /> 192.5<br /> <br /> 5th days after<br /> <br /> 200<br /> 180<br /> <br /> 167.14<br /> <br /> 160<br /> <br /> 138.83<br /> <br /> 140<br /> <br /> 126.05<br /> <br /> 120<br /> 100<br /> 80<br /> <br /> 44.05<br /> <br /> 60<br /> 40<br /> <br /> 24.29<br /> <br /> 27.86<br /> <br /> 30.29<br /> <br /> 11.46<br /> 11.49<br /> <br /> 20<br /> <br /> 12.31 11.68<br /> <br /> 0<br /> <br /> Group 1<br /> <br /> Group 2<br /> <br /> Group 3<br /> <br /> Group<br /> <br /> Figure 3: Changes in GGT activity in 3 groups.<br /> GGT activity in non-poisoned groups did not differ between time points. In the<br /> untreated group (group 2), GGT activity increased significantly from day 3. In the treated<br /> group (group 3), GGT activity also increased from day 3 after poisoning. However,<br /> GGT activity in group 3 was significantly lower than group 1.<br /> 2. Changes on CPK, glucose and urea levels in serum of rabbits poisoned with<br /> Aminata virosa treated and untreated with silibinin.<br /> UI/L<br /> <br /> Group 1<br /> <br /> Group 2<br /> <br /> Group 3<br /> <br /> 1400<br /> 1167<br /> <br /> 1200<br /> 1000<br /> 716.8<br /> <br /> 800<br /> 600<br /> 266<br /> 400<br /> 200<br /> <br /> 635.5<br /> <br /> 287.8<br /> 284.8<br /> <br /> 271.4<br /> <br /> 491.7<br /> <br /> 0<br /> <br /> Before poisoning<br /> <br /> After 12h<br /> <br /> 3rd days after<br /> <br /> 5th days after<br /> <br /> Figure 4: Changes in CPK level in 3 groups.<br /> Before poisoning and after poisoning 1 day, CPK level did not differ between groups<br /> with p > 0.05. CPK level increased at 3 days after poisoning and CPK in group 1 increased<br /> significantly higher than group 2 with p < 0.05.<br /> 149<br /> <br /> Journal of military pharmaco-medicine n06-2018<br /> <br /> mmol/L<br /> <br /> Before poisoning<br /> <br /> After 12h<br /> 7.6<br /> <br /> 8<br /> 7<br /> <br /> 3rd days after<br /> <br /> 7.2<br /> <br /> 7.1<br /> 6.5<br /> <br /> 6.3 6.1<br /> <br /> 6<br /> <br /> 5.7<br /> <br /> 6<br /> <br /> 6.2<br /> <br /> 5th days after<br /> <br /> 6.1<br /> <br /> 6<br /> <br /> 6.4<br /> <br /> 5<br /> 4<br /> 3<br /> 2<br /> 1<br /> 0<br /> Group 1<br /> <br /> Group 2<br /> <br /> Group 3<br /> <br /> Group<br /> <br /> Figure 5: Changes in glucose level.<br /> Average blood glucose level in groups 2 and 3 increased slightly at 12 hours after<br /> poisoning compared with before poisoning. Plasma glucose concentrations on days 3<br /> and 5 after poisoning did not differ between three groups.<br /> mmol/L<br /> <br /> Group 1<br /> <br /> Group 2<br /> <br /> Group 3<br /> <br /> 9<br /> 8<br /> 7<br /> 6<br /> 5<br /> 4<br /> 3<br /> 2<br /> 1<br /> 0<br /> <br /> Before poisoning<br /> <br /> After 12h<br /> <br /> 3rd days after<br /> <br /> 5th days after Time<br /> <br /> Figure 6: Changes in urea concentration.<br /> The plasma concentrations of uremia in rabbit blood at the time before and after<br /> poisoning were not statistically significant.<br /> 150<br /> <br />