Deactivation Study of α-Amanitin Toxicity in Poisonous Amanita spp. Mushrooms by the Common Substances In Vitro

The purpose of this research was to find out the substance which deactivate α-Amanitin Toxicity The materials and methods used in the study include analysis with high performance liquid chromatography (HPLC) to: 1.Demonstrate the standard α-amanitin at concentrations of 25, 50 and 100 μg/ml 2.Determine the deactivation of α-amanitin with 1) 18% acetic acid 2), calcium hydroxide 40 mg/ml, 3) potassium permanganate 20 mg/ml, 4) sodium bicarbonate 20 mg/ml 3.Report the statistical analysis as the mean ± standard deviation (SD) and paired t-test. The result revealed that potassium permanganate could eliminate 100 percent of the α-amanitin at 25, 50 and 100 μg/ml. Calcium hydroxide, sodium bicarbonate and acetic acid had lower elimination rates at those concentrations: 68.43 ± 2.58 (-71.4, -67.2, -66.7%), 21.48 ± 10.23 (-29.4, -25.2, -9.9%) and 3.21 ± 0.02% (-3.2, -3.2, +1.1%), respectively. The conclusion of this study was suggested that potassium permanganate could be applied as an absorbent substance during gastric lavage in patients with mushroom poisoning. It also might be effective as a cleansing wash for uncooked mushrooms. Investigation of potassium permanganate’s ability to absorb α-amanitin in animal models and humans should be considered. . Patients with α-amanitin poisoning can develop severe toxic hepatitis, centrilobular necrosis, liver steatosis, and acute tubulointerstitial necrosis leading to hepatorenal syndrome all of which have a high mortality rate. The most effective treatment is emptying the stomach promptly by performing gastric lavage with 1:2,000 tannic acid or 1:10,000 potassium permanganase plus triggering emesis. A warm saline solution of potassium permaganase can be used as an emergency treatment to reduce toxins in the intestines and stomach. Thus, KMnO4, NaHCO3, Ca(OH)2 and acetic acid should be considered as a cleanser for uncooked mushrooms to deactivate α-amanitin toxicity and potentially reduce the mortality rate from liver failure, acute renal failure, respiratory failure and gastro-intestinal haemorrhage[9]. Materials and Methods In this study, experiments were performed on α-amanitin at concentrations of 25, 50 and 100 μg/ml eached mixed with 1) 18% acetic acid 2), calcium hydroxide 40 mg/ml, 3) potassium permanganate 20 mg/ml, 4) sodium bicarbonate 20 mg/ml (results analyzed with high performance liquid chromatography (HPLC) with Luna C18 (150 × 4.6 mm I.D., 5 micron) from Phenomenex®, USA), or 5) mobile phase (a mixture of 0.02 M aqueous ammonium acetate and acetonitrile (88/12, v/v) pH 5.0 at an absorbance of 280 nm). Glacial acetic acid was used to Citation: Narongchai P, Narongchai S (2017) Deactivation Study of α-Amanitin Toxicity in Poisonous Amanita spp. Mushrooms by the Common Substances In Vitro. J Forensic Res 8: 396. doi: 10.4172/2157-7145.1000396


Introduction
The poisonous mushrooms which are very dangerous toxicity are Amanita mushroom species: A. phalloides, (death cap) A. virosa, (destroying angel), A. verna, A. bisporigera, Galerina marginata and Conocybe filaris ( Figures 1A and 1B). Alpha-amanitin is the most potent of the toxins occurring in poisonous mushrooms in the genus Amanita [1,2]. The lethal oral dose (LD50) of α-amanitin in mice is 0.1-0.3 mg/kg. One cap of A. phalloides, A. virosa or A. verna contains ten amatoxins. Ingestion of A. phalloides results in the highest mortality rate of all poisonous mushrooms, ranging from 50 to 90%. Unfortunately, distinguishing poisonous and non-poisonous mushrooms by morphology alone is challenging due to their similar gross appearance. Theoretically, there are ten important amatoxins: [3] -amanitin, β-amanitin, γ-amanitin, ε-amanitin, ð-amanitin, amanullin, amanullinic acid, amaninamide, amanin and proamanullin [4] These differ from the other amatoxins in that they are heat resisitant, alcohol and lipid soluable [5] and indigestible in gastric and small intestinal enzymes. In addition, they can be absorbed rapidly by both gastric and duodenal tissues. One hundred grams of of A. phalloides contain eight milligrams of α-amanitin [1] The RNA polymerase II and III inhibitor α-amanitin [2] affects many organs, especially liver cells, resulting in necrotic hepatic cytolysis [3]. The mortality rate of α-amanitin poisoning is very high because of the difficulty diagnosis, the delayed toxicity (average 24 hrs.), the severity of the toxicity and the administration of an ineffective antidote. The cycle peptide chain, α-amanitin, has eight amino acids: asparagine, hydroxyl proline, dihydroxy isoleucine, hydroxyl-mercapto-tryptophan, glycine, isoleucine, glycine and alanine. Hydroxyl proline and dihydroxy isoleucine activate RNA polymerase II and III by binding to the enzyme molecule, enhancing the activating effect by means of glycine and isoleucine binding [6][7][8]. The immediate cause of death from mushroom poisoning is kidney, liver and respiratory failure which occur within one week of ingestion.
Patients with α-amanitin poisoning can develop severe toxic hepatitis, centrilobular necrosis, liver steatosis, and acute tubulointerstitial necrosis leading to hepatorenal syndrome all of which have a high mortality rate. The most effective treatment is emptying the stomach promptly by performing gastric lavage with 1:2,000 tannic acid or 1:10,000 potassium permanganase plus triggering emesis. A warm saline solution of potassium permaganase can be used as an emergency treatment to reduce toxins in the intestines and stomach. Thus, KMnO 4 , NaHCO 3 , Ca(OH) 2 and acetic acid should be considered as a cleanser for uncooked mushrooms to deactivate α-amanitin toxicity and potentially reduce the mortality rate from liver failure, acute renal failure, respiratory failure and gastro-intestinal haemorrhage [9].

Analysis of the effectiveness of α-amanitin deactivation using sodium bicarbonate
The levels of deactivation of α-amanitin at concentrations of 25, 50 and 100 ug/ml by sodium bicarbonate (NaHCO 3 ) are shown in Figure  8 and Table 3. Deactivation was statistically significant when compared with the control group (p<0.01).

Analysis of the effectiveness of α-amanitin deactivation by potassium permanganate (KMnO 4 )
The results of deactivation of α-amanitin at concentrations of 25, 50 and 100 ug/ml by potassium permanganate (KMnO 4 ) are shown in Figures 9-11 and Table 4. The results showed statistically significant deactivation when compared with the control group (p<0.05) ( Figure 12).

Analysis of the effectiveness of α-amanitin deactivation by calcium hydroxide (Ca (OH) 2 ) 40 mg/ml
The effectiveness of deactivation of α-amanitin at concentrations of 25, 50 and 100 ug/ml by calcium hydroxide (Ca (OH) 2 ) is shown in Figure 13-16 and in Table 5. Deactivation was statistically significant when compared with the control group (p<0.01).
adjust the acid-base condition. Each of the mixtures was injected into the HPLC three separate times [10].
Results of statistical analysis of the α-amanitin concentrations are reported as the mean ± standard deviation (SD) for each group. The levels of α-amanitin concentration inhibited or destroyed by each substance were analyzed by paired t-test.

α-amanitin concentrations as determined by HPLC
The concentrations of standard α-amanitin solution (25, 50 and 100 µg/ml) were injected into the HPLC to measure α-amanitin retention time of 10.2 minutes. The results of the chromatography is shown in Figure 2 and Table 1. The equation of the linear correlation is Y=1723.8X+1228.6 (R 2 =0.9999) (Figure 3).

Analysis of the effectiveness of α-amanitin deactivation by 18% acetic acid
The results of deactivation of α-amanitin at concentrations of 25, 50 and 100 µg/ml by 18% acetic acid are shown in the Figure 4-7 and in Table 2. The deactivation levels were not statistically significant.  280 potassium permanganate 20 mg/ml, 4) sodium bicarbonate 20 mg/ml (results analyzed with high performance liquid chromatography (HPLC) with Luna C18 (150×4.6 mm I.D., 5 micron) from Phenomenex ® , USA), or 5) mobile phase (a mixture of 0.02 M aqueous ammonium acetate and acetonitrile (88/12, v/v) pH 5.0 at an absorbance of 280 nm). Glacial acetic acid was used to adjust the acid-base condition. Each of the mixtures was injected into the HPLC three separate times. (10) Results of statistical analysis of the -amanitin concentrations are reported as the mean ± standard deviation (SD) for each group. The levels of -amanitin concentration inhibited or destroyed by each substance were analyzed by paired t-test.

Discussion and Conclusions
Four common substances, Potassium permanganate (KMnO 4 ), calcium hydroxide (Ca (OH) 2 ), sodium carbonate (NaHCO 3 ) and acetic acid are regularly used for vegetable detoxification. As mushrooms are one of the most popular ingredients in Thai food, especially in rural areas, routine detoxification of potential toxins in uncooked mushrooms is one solution that should be considered to avoid mushroom poisoning, particularly α-amanitin toxicity.      Potassium permanganate (KMnO 4 ) is an antiseptic, alkali, and highly soluble detoxification substance. It contains potassium (K + ) and permanganate ions which are strong oxidizing and organic substance destroying agents. Manganese oxide (MnO 2 ) destroys the cell wall and cell membrain of bacteria, protozoa, parasites and algae. This study revealed that potassium permanganate (KMnO 4 ) 20 mg/ml completely destroyed α-amanitin at concentrations of 25, 50 and 100 µg/ml. We found that potassium permanganate (KMnO 4 ) is the strongest of the oxidizing agents tested and that it has the ability to destroy or deactivate peptides of α-amanitin molecules in vitro [11,12]. Potassium permanganate (KMnO 4 ) could potentially be used for gastric lavage in patients with mushroom poisoning. Additional experiments in animal models should be conducted to evaluate the effectiveness of these four substances.
Calcium hydroxide (Ca (OH) 2 ) and sodium carbonate (NaHCO 3 ) have an alkaline property which can significantly deactivate alkaline hydrolysis [13,14] caused by α-amanitin. Acetic acid has an antiseptic acid property but it cannot destroy the cell membranes or cell walls of any vegetable including α-amanitin because it has a low pKa value. α-amanitin in poisonous mushrooms cannot be destroyed by acids or by gastric or duodenal enzymes [15].
Potassium permanganate (KMnO 4 ) is the most effective deactivation substance for α-amanitin. It completely inhibits α-amanitin activation. Both calcium hydroxide (Ca (OH) 2 ) and sodium carbonate (NaHCO 3 ) (to a lesser degree) inhibit α-amanitin, but they do not do so completely. Acetic acid only very weakly inhibits α-amanitin. Based on these findings, washing uncooked poisonous Amanita spp. mushrooms with KMnO 4 to detoxify or deactivate α-amanitin before ingestion would definitely be beneficial to health, especially in rural areas where many people gather their own mushrooms from forested areas rather than purchasing them at a market.

Ethical Approval
This research did not involve any human participants or animals.