Researching mycotoxicosis, biological characteristics and toxicity of some common poisonous mushrooms in cao bang province

AND TRAINING VIETNAM MILITARY MEDICAL ACADEMY NGUYEN TIEN DZUNG RESEARCHING MYCOTOXICOSIS, BIOLOGICAL CHARACTERISTICS AND TOXICITY OF COMMON POISONOUS MUSHROOMS IN CAO BANG... PREAM

AND TRAINING

VIETNAM MILITARY MEDICAL ACADEMY

NGUYEN TIEN DZUNG

RESEARCHING MYCOTOXICOSIS, BIOLOGICAL CHARACTERISTICS AND TOXICITY OF COMMON POISONOUS MUSHROOMS IN CAO BANG

VIETNAM MILITARY MEDICAL ACADEMY

Scientific Supervisors:

• Assc Prof., PhD HOANG CONG MINH

• Assc Prof., PhD PHAM DUE

Opponent 1: Prof., PhD NGUYEN THI DU

Opponent 2: Prof., PhD TRINH TAM KIET

Opponent 3: Assc Prof., PhD NGUYEN TRONG THONG

• Central Medical Library

• Library of Vietnam Military Medical Academy

PREAMBLE

• Necessity of the thesis

The poisonous mushroom includes many kinds of species, each species have different morphological characteristics, toxicity and effects on the body Mycotoxicosis is often due to people’s impossible distinguishing between the poisonous mushrooms and non- poisonous mushrooms

In the world, the mycotoxicosis occupied 2,1% in total poisonings In America, in 11 recent years, there has occurred 85.556 mycotoxicosises In Vietnam, the mycotoxicosis constantly occurs in forested provinces such as

Ha Giang, Cao Bang, Bac Kan, In March 2014, Poison Control Center at Bach Mai Hospital has treated 15 patients with mycotoxicosis from Thai Nguyen, Tuyen Quang, Cao Bang province In which, 10 patients were died (66.7%) Up to now, the research works on poisonous mushroom in Vietnam are very few Until before 2008, posters on mycotoxicosis prevention of the Ministry of Health as well as of the province mainly had relies on images of poisonous mushrooms that grow in the United State, Europe… In which, some mushrooms only grow in temperate climate zones, thus the propaganda effectiveness is not good

Cao Bang is a mountainous province in the North, with diversified and plentiful forest ecosystem According to the Center for Preventive Medicine, Department of Food Safety, Provincial Health Department, in the period from 2003-2009, there had 29 cases of mycotoxicosis leading to 81 poisonous patients with 17 fatal cases Especially, the mycotoxicosis occurred in 8 mortal people in a family Most poisonings have not been identified the species of poisonous mushroom yet

From the above issues, we have studied the subject: “Researching the

mycotoxicosis, biological characteristics and toxicity of some common poisonous mushrooms in Cao Bang Province”

2 Objectives:

2.1 Assessing the mycotoxicosis in Cao Bang Province from 2003 to 2009 and results of the post-intervention mycotoxicosis from 2010 to June 2014 2.2 Identifying the morphological characteristics and allocation of some common poisonous mushrooms in Cao Bang Province

2.3 Identifying the toxicity and changes in biochemical, hematological, cardiovascular and histopathological criteria under the extract effect of 4 common poisonous mushrooms on animals

3 New contributions for the thesis

- The first time a research is conducted to assess the mycotoxicosis in Cao Bang Province from 2003 to 2009 and results of the post-intervention mycotoxicosis from 2010 to June 2014

- Identifying and describing the morphological characteristics and allocation of 13 species of common poisonous mushrooms in Cao Bang Province

- Identifying the toxicity and changes in biochemical, hematological, cardiovascular and histopathological criteria under the extract effect of 4

common poisonous mushrooms on animals In which, the Chlorophyllum

molybdites cause most of poisonings, Amanita virosa can cause mortality, Russula emetica has not been researched and the Coprinus atramentarius is

found out to grow in Cao Bang and not in any other locality

4 Thesis Layout

This thesis is included 156 pages, including the following parts: Introduction (2 pages), Overview (42 pages), Researching Object and Methods (22 pages), Results (44 pages), Discussion (42 pages), Conclusion (3 pages), Recommendations (1 page) This thesis contains 35 tables, 3 diagrams, 46 images, 144 references of which 19 Vietnamese references,

125 English references, 66 references since 2010 up to now

CHAPTER 1- OVERVIEW 1.1 Definition

Poisonous mushrooms are the ones that contain toxin to poison the human body and animals when ingested Previously, these mushrooms were sorted into the flora but today they are separated into the fungi In the world now there have nearly 140,000 species of mushrooms to be identified, of which about 2,000 edible mushrooms, 700 species with active ingredients to

be used in disease treatment and many poisonous mushrooms According to Trinh Tam Kiet (1996), Vietnam has 826 large mushroom species to be recorded, including 512 species newly discovered on the territory of Vietnam Some poisonous mushrooms are listed herein

(monomethylhydrazin), orellanin, muscarin, ibotenic acid and muscimol, coprin, psilocybin and psilocin, as the toxin causing gastrointestinal disorder

1.3 Researches on poisonous mushrooms in the world

1.3.1 Researches on poisonous mushrooms with amatoxin

The poisonous mushrooms with amatoxin cause 90 - 95% fatal cases due to mycotoxicosis in the world, so there have had many researches on these fungi Amatoxin is the common name of toxin contained in the poisonous mushrooms in the genus Amanita, Galerina and Lepiota Amatoxin is contained in entire fruiting body of the mushroom (cap, gill, stalk) and hyphae (mushroom roots)

Amatoxin includes 8 kinds: α-amanitin, β-amanitin, γ-amanitin,

ε-amanitin, amanullin, amanullinic acid, proamanullin, amanin and 7 kinds

phallotoxin: phalloidin, phalloid, prophalloin, phallisin, phallacin,

phallacidin, phallisacin Virotoxin found in these mushrooms

1.3.2 Researches on poisonous mushrooms with muscarin

The group of mushrooms with muscarin often found out in the mushrooms of genus Inocybe, Clitocybe and Omphalotus The genus of

Inocybe: Inocybe patouillardi; Inocybe fastigiata (Inocybe rimosa), The genus of Clitocybe: Clitocybe dealbata, Clitocybe cerussata,… The genus

of Omphalotus: Omphalotus olearius; Omphalotus illudens…

All mushrooms in the genus of Inocybe have toxin Previously, it was thought that the Amanita muscaria caused poisoning symptoms muscarin However, making quantitative analysis of active substances in the

mushroom Amanita muscaria, the muscarin content in the Amanita

muscaria is very low (about 0,0003% fresh weight) so it can not enough to

cause poisoning despite eating the large quantities The mushrooms in genus

of Inocybe and Clitocybe contain high quantity muscarin

1.3.3 Researches on poisonous mushrooms with coprin

The group of poisonous mushrooms with coprin mostly in genus

Coprinus Some fungi can cause poisoning as: Coprinus atramentarius, small Coprinus atramentarius grew into clump (Coprinus disseminatus),

Coprinus micaceus, Coprinus fuscescens, Coprinus insignis Besides, the

mushroom Clitocybe clavipes in genus of Clitocybe may cause poisoning

similar to the mushroom with coprin although coprin has not been found out

in this mushroom

1.3.4 Researches on Chlorophyllum molybdites, the toxin causing

gastrointestinal disorder

Chlorophyllum molybdites, as the fungi cause most cases of poisoning

in many countries in the world and some provinces in Vietnam Until 2004 Kobayashi Y and CS (Japan) conducted to extract and refine from this

mushroom a kind of lectin as N-Glycolylneuraminic acid In 2009 – 2010,

Gong Q.F and CS separated 4 compounds from the hyphae (root) of this

mushroom by 5,6,(22E,24R)-5α,6α-epoxyergosta-8, 22-diene-3β,7α-diol, (22E,24R)-ergosta-7,22-dien-3β-ol Yamada M and CS (2012), has

extracted a kind of toxic protein and named molybdophyllysin Yoshikawa

K (2001), has extracted 2 derivatives steroid as ergosta-4, 6, 8 (14), 22-tetraene and (22E,24R) -5α, 8α-epidioxyergosta- 6,9,22-triene-3β-ol-3-O-β-D-glucopyra-noside

(22E,24R)-3α-ureido-1.4 Researches on poisonous mushroom in Vietnam

1.4.1 Researches on characteristics, allocation, toxicity of poisonous mushrooms

The large studies on mushroom in Vietnam are primarily of biologists and pharmacists on mushroom identification, determining the allocation in different ecological zones and studies on farming breeding of fungi as the food and pharmaceuticals The “List of large mushrooms in Vietnam” (1996) of Trinh Tam Kiet made a list of names and allocations of 826 kinds

of large mushrooms, in which there had names of about 20 kinds of poisonous mushrooms Tran Cong Khanh and Pham Hai (2004) morphologically describe the common poisonous mushrooms From 2007 –

2008, Hoang Công Minh and co-workers conducted to investigate and identify the mushrooms causing poisoning in Ha Giang province In this study, 9 kinds of poisonous mushrooms have been found In which, there have 2 kinds of poisonous mushrooms In Ha Giang province, the

mushroom with amatoxin causing dealth has been detected as the Amanita

verna Hoang Cong Minh (2009) has studied the effect of the fungus extract

on rabbits and found that they have had AST, ALT, billirubin, ure, creatinin increased highly, erythrocytes and hemoglobin decreased, coagulation disorder after poisoning

CHAPTER 2 RESEARCHING OBJECT AND METHODS

2.1 OBJECTS

* 93 patients who were suffered mycotoxicosis in the localities in Cao Bang Province involved in this research In which, 81 patients in the period from 2003 to 2009 as there has no any communication interventions and 12 patients from 2010 to June 2014 after having communication interventions

* Some samples of poisonous mushrooms growing in some representative areas in Cao Bang Province

* Experimental animals:

- White mice in Swiss origination: 1280 mice, healthy, average weight

by 20 ± 2 gam (excluding the white mice used for survey of poisoning dose) The white mice used to identify toxicity (Average lethal dose - LD50) and making histopathological study for four species of poisonous mushrooms

- Rabbit: 60 rabbits, healthy, weight 2,0 ± 0,2 kg (excluding the rabbits

used to identify the minimum lethal dose (LDmin) Rabbits used to study the biochemical and hematologic criteria of 4 kinds of mushroom

- White rat in Wistar origination: 60 rats, healthy, weight 200 ± 20 gam

(excluding the white rats used to identify the minimum lethal dose (LDmin) The white rat used to research the pulse, blood pressure for 4 kinds of

mushroom

2.2 METHODS

2.2.1 Survey methods for the mycotoxicosis

Stage 1 from 2003 to 2009, making survey for the poisonings of poisonous mushroom under method of cross survey, retrospective records, data, interviewing people suffered mycotoxicosis and people in information collection sheet in the family people suffered mycotoxicosis in the localities

in Cao Bang Province Stage 2 from 2010 to June 2014, surveying the mycotoxicosis under statistic report of forest mycotoxicosis, poisonous plants of Food Hygiene and Safety Department, Cao Bang Department of Health (after communication inventions)

2.2.2 Surveying methods of poisonous mushrooms

Making survey of poisonous mushrooms in Surveying Form in the field, where people picked the mushroom as the food and being poisoned In the localities without mycotoxicosis, we came to the areas where grow many kinds of mushrooms as directed by the government, officer of medical station and people in the commune

2.2.3 Identifying methods of kinds of mushroom

Kinds of mushroom are identified under method of Trinh Tam Kiet, Kuo M., identified basing on the morphological characteristics, spores, and chemical reactions when compared with the standard mushroom sample The characteristics to be described as: Cap, gill, ……

2.2.4 Methods to research toxicity, extract effect of 4 kinds of poisonous mushroom on animals

2.2.4.1 Methods to extract poisonous mushroom sample

♦ Extracting methods for dried mushroom:

• Dried mushroom, putting into weighting, crushed into powder and put into the bottle Depending on the kind of fungi to put methanol, water soaking for 24 hours Extracting and taking the whole solvent Continue to put methanol, water into the leaching tank and extract with 2 more with the same way as to totally extract the active ingredient in the dry mushroom samples

• Gather all the solvent into a bottle, aeration to blow the solvent evaporation to collect the residue Residue in the bottle is the total amount

of active ingredients of the fungi, weighting the residue and calculating the equivalent initial weight of the mushroom

• Preparation of residue with distilled water to form extraction solution Before the animals used orally or by injection into abdomen, the extract is boiled in a test tube, leave it cold and to ensure aseptic

♦ Extracting method for fresh mushroom:

• Sample of fresh mushroom is stored in the alcohol 700

(weighting the mushroom before soaking in the alcohol) Sampling the mushroom, alcohol from the soaking bottle to the ceramic bowl, crushing into the suspension Distilling and taking the suspension into the separated bottle Residue left in the bottle is spread with a certain amount of distilled water and placed in a ceramic bowl Continue to put water into the ceramic bowl with mushroom and mashed with water, filtered as above on 2nd, 3rd time to totally extract the active ingredients

• Gathering the whole extract, filtered through filter paper to obtain the extract containing active substances of poisonous mushrooms Aeration for evaporation of alcohol and steam to obtain the active ingredient of the extract residue Weighing the residue and calculating the equivalent initial weight of the mushroom

• Residue of extracts is formulated to research on animals Ensure sterile extracts by boiled and leave it cold before use orally or injecting into the animals

2.2.4.2 Methods of researching acute toxicity of 4 poisonous mushrooms

on the white mice

♦ Method causing poisoning on the white mice:

* Method causing acute poisoning through the digestion: Use a dedicated tool to pump extracts of 4 kinds of poisonous mushroom to be

researched: Amanita virosa, Chlorophyllum molybdites, Russula emetica

and Coprinus atramentarius into the stomach of the white mice

* Method causing acute poisoning by injection into the abdomen just to

be studied for Amanita virosa because the toxicity of these kinds of

mushroom are amatoxin poorly absorbed from the digestion of the white mice, thus out of the digestion, the toxicity by injection into the abdominal

of the white mice will be further researched:

♦ Method identifying the average lethal dose (LD50):

LD50 white mice is identified in method of Karber G

2.2.4.3 Method of conducting biochemical and hematological criteria

The biochemical and hematological criteria are studied at the time before and after being poisoned in the morning at the day 1, 5 and 10 after causing poisoning

Get the vein blood in the rabbit ear, 2 ml for each into the test tube to identify the biochemical and hematological criteria before causing poisoning Causing poisoning rabbit at the dose by 2/3 lethal dose (LDmin)

in each kind of poisonous mushroom (surveyin the dose before experiment)

At this dose, the rabbit is poisoned but not be in death to follow and take blood for experiment at the time of after being poisoned Specifically for each kind of poisonous mushroom causing poisoning on experimental rabbits:

+ Dried Amanita virosa: 0,618 g/kg body weight by abdominal

injection

+ Dried Chlorophyllum molybdites: 5,734 g/kg body weight used

orally

+ Dried Russula emetica: 6,628 g/kg body weight used orally

+ Dried Coprinus atramentarius: 4,504 g/kg body weight used orally

with 5 ml alcohol 400/kg body weight

+ Specifically for Coprinus atramentarius, making group for rabbits

drinking alcohol 5 ml 400/kg body weight

- Centrifugally the blood to take serum Putting the serum tube into the biochemistry analyzer (CHEMIX-180), automatic hematology analyzer (XE 2100) (Japan) at biochemical and hematological laboratory of Research Centre for Military Medicine and Pharmacy of Vietnam Military Medical Academy, to define the testing criteria

2.2.4.4 Researching cardiovascular criteria on the white rat of 4 kinds of poisonous mushroom

♦ Methodology for cardiovascular criteria:

Pulse, blood pressure of the rate measured at the time of before and after being poisoned at the time of 1 hour, 6 hours and 24 hours

Pulse, blood pressure rat tail determined on dedicated devices automatically measuring the pulse, blood pressure of the rat tail of Ugo Basile (Italy) of the subject Military Toxicology and Radioactive Studies of Vietnam Military Medical Academy

2.2.4.5 Researching Methodology of histopathology as liver, kidneys, spleen

Amanita virosa, Chlorophyllum molybdites, Russula emetica and Coprinus atramentarius, researched the histopathology on general situation

of and microbials in the collation group and group causing poisoning on the white mice with a dose by 1 average lethal dose (LD50), specifically:

+ Dried Amanita virosa: 0,322 g/kg body weight by abdominal injection + Dried Chlorophyllum molybdites: 3,718 g/kg body weight used orally + Dried Russula emetica: 4,838 g/kg body weight used orally

+ Dried Coprinus atramentarius: 2,976 g/kg body weight used orally

and with 5 ml alcohol 400/kg body weight

+ Specifically for Coprinus atramentarius, making group for rabbits

drinking alcohol 5 ml 400/kg body weight

The steps as follow:

• Killing rats and dissecting to take liver, kidneys, spleen and putting into the bottle containing a fixed solution Casting paraffin block, slicing with thickness 5-6 microns on a Microtome Dyeing slices under hematoxylin – eosine staining method Conducting morphological observation of specimens on microscope Color photography for illustration The experimental part to cause poisoning, dissecting to take liver, kidneys, spleen and putting into the fixed solution conducted in the Military Toxicology and Radioactive Studies, Vietnam Military Medical Academy Technique to case paraffin block, slicing, staining specimen, observing the lesion morphology in the microscope, reading the results and imaging were conducted at the Department of Disease Anatomy and Forensic Medicine, Military Hospital 103

2.3 Data processing

The data is processed with statistical method, using software as Excel, Epical 2000, EpiInfo 3.5.4

2.4 Ethical issues in research

Assuring of ethics in research

CHAPTER 3: RESEARCHING RESULTS

3.1 The mycotoxicosis in Cao Bang Province

Diagram 3.1: Allocation of number of poisoning, sufferers and mortality

due to eating poisonous mushrooms

Table 3.1: Allocation of number of poisonings, people being poisoned and

mortality due to eating poisonous mushrooms in the districts in Cao Bang

Province

No District and

town Poisonings

Number of people being poisoned

Rate (%)

Number of people in death

The research has detected 13 kinds of poisonous mushroom The poisonous mushrooms growing in many communes and districts are distributed in Cao Bang Province Some mushrooms are detected to be available in all districts

such as Chlorophyllum molybdites Some mushrooms are detected to be

grown in some investigated localities such as Inocybe fastigiata and

Amanita virosa

Table 3.10: List and allocation of poisonous mushrooms in localities

No Vietnamese name (other name) Scientific name; Family Allocation

1 Amanita verna Amanita verna (Bull.:Fr)

Roques Family:

Amanitaceae

Quang Vinh Commune, Tra Linh District Chi Vien Commune, Trung Khanh District

Phan Thanh Commune, Bao Lac District

Hong Nam Commune, Hoa

5 Russula emetica Russula emetica (Schaeff)

Dao Ngan Commune, Ha Quang District

Yen Tho Commune, Bao Lam District

Co Ba Commune, Bao Lac District

9 Panaeolus

papilionaceus

Panaeolus papilionaceus (Bull Ex Fr.) Quel.,

Family: Coprinus atramentarius (Coprinaceae)

Cach Linh, Trieu Au Communes, Phuc Hoa District

Bao Toan, Son Lo – Bao Lac Commune

Coprinus atramentarius (Coprinaceae)

Chi Vien Commune, Trung Khanh District

Le Lai - Thach An Communes Quang Lam - Bao Lam Communes Thi Hoa -

Ha Lang Communes Minh Tam – Nguyen Binh Communes

Minh Khai – Thach An Communes

12 Coprinus

atramentarius

Coprinus atramentarius (Bull.: Fr) Fr Family:

Coprinus atramentarius (Coprinaceae)

Minh Tam, Hung Dao Communes, Nguyen Binh District

Dao Ngan Commune, Ha Quang District

13 Coprinus

atramentarius

Coprinus disseminatus

(Pers ex Fr.) S F Gray Family: Coprinus atramentarius

(Coprinaceae)

Truong Ha Commune, Ha Quang District

Yen Tho Commune, Bao Lam District

An Lac, Thi Hoa, Communes,

Ha Lang District