Forensic Pharmacognosy

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Forensic Pharmacognosy
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• Forensic relating to the application of science
  to decide questions arising from crime or
  litigation.
• Litigation or legal action.

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Poisonous Plants and Abused Drugs

• Selection of the studied topics has been based on
  three considerations
• The frequency of using different botanicals.
• The importance and seriousness of the toxic
  effect of these botanicals.
• The scientifically interesting nature of the
  action of these botanical drugs (pharmacological
  and physiological point of view).
• In adjusted doses many plant poisons are highly
  active substances as medicines.

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Course contents

• Mycotoxins.
• Plants containing toxic alkaloids.
• Plants containing toxic glycosides.
• Psychoactive drugs.
• Criminal investigation, Hair Evidence

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Mycotoxins
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Mycotoxins

• The term 'mycotoxin' is usually reserved for the
  toxic chemical products produced by fungi.
• A mycotoxin from Greek (mukos) "fungus" and Latin
  (toxicum) "poison "
• The fungus consume organic matter wherever
  humidity and temperature are sufficient.
• The reason for the production of mycotoxins is
  not yet known they are neither necessary for
  growth nor the development of the fungi.

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Major groups of mycotoxins

• Ergot Alkaloids are compounds produced as a toxic
  mixture of alkaloids in the sclerotia of species
  of Claviceps.
• Aflatoxins are a type of mycotoxin produced by
  Aspergillus species of fungi, such as A. flavus
  and A. parasiticus .
• Amatoxins and phallotoxins are toxins produced by
  the Genus Amanita (poisonous mushroom ??? ????
  ).
• Muscarine

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Major groups of mycotoxins (cont.)

• Ochratoxin is a mycotoxin produced by Penicillium
  and Aspergillus species.
• Citrinin is a toxin that was first isolated from
  Penicillium citrinum, but has been identified in
  over a dozen species of Penicillium and several
  species of Aspergillus
• Patulin is a toxin produced by the Aspergillus,
  Penicillium, and Paecilomyces fungal species.
• Fusarium toxins are produced by over 50 species
  of Fusarium.

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Aflatoxins

• are a type of mycotoxin
• produced by Aspergillus species of fungi,
• such as A. flavus and A. parasiticus .
• A. flavus. common in Asia Africa.
• A. parasiticuscommon in America.
• Among various mycotoxins, aflatoxins have assumed
  significance due to their deleterious effects on
  human beings, poultry and livestock (domestic
  animals).

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Aspergillus
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Historical view

• The aflatoxin problem was first recognized in
  1960, when there was severe outbreak (????) of a
  disease referred as "Turkey 'X' Disease" in UK,
  in which over 100,000 turkey poults were died.
• The cause of the disease was shown due to toxins
  in peanut meal infected with Aspergillus flavus
  and the toxins were named as aflatoxins.

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From the economic point of view

• Aflatoxins influence
• Health of human being.
• Health of livestock and poultry.
• The marketability of medicinal herbs
• Agricultural products.

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Natural occurrence

• Agricultural products contaminated with
  aflatoxins include
• Cereals (maize, rice, wheat).
• Oil seeds (groundnut, soybean, sunflower,
  cotton).
• Spices (black pepper, coriander, turmeric,
  zinger).
• Tree nuts (almonds, pistachio, walnuts, coconut).
• Milk.

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Properties

• 18 different types of aflatoxins were identified.
  
• The major members are aflatoxin B1, B2, G1 and
  G2.
• Based on their fluorescent color when exposed to
  ultraviolet light.
• (B series blue fluorescence)
• (G series yellow-green fluorescence).
• The aflatoxins fluoresce strongly in UV (365 nm).
• Aflatoxin M1and M2 are major metabolites of
  aflatoxin B1 and B2 respectively, found in milk
  of animals that have consumed feed contaminated
  with aflatoxins.

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Properties (cont.)

• Aflatoxins M1, M2 may be found in the absence of
  other aflatoxins.
• Aflatoxin B1 (AFB1) is normally predominant in
  cultures as well as in food products.
• Aflatoxins are slightly soluble in water.
• Soluble in moderately polar organic solvents.
• Insoluble in non polar solvents.
• Aflatoxins decompose at their melting points,
  which are between 237C (G1) and 299C (M1),
• Not destroyed under normal cooking conditions.

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Structure

• Aflatoxins are normally refers to the
• group of difuranocoumarins
• coumarin nucleus fused to a difuran units
• 
  
• coumarin nucleus furan unit

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Structure

• They are classified in two broad groups
• 1- Difurocoumarocyclopentenone Series.
• (B series) (AFB1 , AFB2 , AFB2A , AFBM1 ,
  AFBM2, AFBM2A and aflatoxicol).
• ??AFB1

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• 2- Difurocoumarolactone series .
• (G series) (AFG1 , AFG2 , AFG2A , AFGM1
• AFGM2 , AFGM2A).
• AFG1

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Detection and estimation of aflatoxins

• Analytical methods ( TLC HPLC ).
• Immunological methods.

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Immunological methods

• The Immunological method is a biochemical
  technique.
• Can be performed to evaluate either the
• presence of antigen or the presence of
  antibody.
• Radio Immuno Assay (RIA)
• Enzyme Immuno Assay (EIA)
• or
• Enzyme-Linked Immuno Sorbent Assay (ELISA)

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Immunological methods (cont.)

• RIA and EIA are used for estimation and detection
  of many drugs, (barbiturates, digoxin, LSD,
  morphine, nicotine, THC, aflatoxins

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Immunological methods (cont.)

• In the RIA radio isotopes are used.
• Radiolabelled ( tagged )drug or antigen.
• Drug-protein complex (the immunogen).
• The antibody (antiserum)

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Immunological methods (cont.)

• AFB1 BSA a conjugate between the aflatoxin
  and bovine serum albumin and this is required to
  induce antibodies.
• To render the small molecules drugs immunogenic.

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Immunological methods (cont.)

• Disadvantages of RIA
• Restricted to license (approved) laboratories.
• Problems of radioactive waste.
• Scintillation counter is very expensive.
• EIA or ELISA is an alternative method enjoying
  the sensitivity and selectivity of RIA without
  any disadvantages.

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Immunological methods (cont.)

• In EIA the tagged drug or antigen is an enzyme -
  labeled antigen rather than a radioactive labeled
  antigen.
• The enzymatic activity is indicated by adding a
  suitable substrate capable of producing optical
  signals.
• The enzyme reaction can be measured photometrical
  ( the measurement of the intensity of light ).

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Toxicity

• Aflatoxins are carcinogenic
• ( cause hepatocellular carcinoma )
• teratogenic
• mutagenic
• Immunosuppressive. (suppresses the immune
  system).
• The aflatoxins display potency of toxicity,
  carcinogenicity, mutagenicity in the order of
  AFB1 gt AFG1 gt AFB2 gt AFG2.

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Carcinogenicity

• Aflatoxins are known to be human carcinogens
  (hepatocellular carcinoma, or primary liver-cell
  cancer).
• The risk of liver cancer increased significantly
  with increasing aflatoxin consumption.
• Biomarkers for aflatoxin exposure (aflatoxin
  metabolites in the urine and aflatoxin albumin
  adducts in the blood)

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Preventing exposure to aflatoxin

• The traditional approach to preventing exposure
  to aflatoxin has been to ensure that foods
  consumed have the lowest practical aflatoxin
  concentrations.
• Chemoprotection and enterosorption to limit
  biologically effective exposure.

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• Chemoprotection is based on manipulating the
  biochemical processing of aflatoxin to ensure
  detoxification rather than preventing biological
  exposure.
• Enterosorption is based on the approach of adding
  a binding agent to food to prevent the absorption
  of the toxin while the food is in the digestive
  tract the combined toxin-sorbent is then
  excreted in the feces.
• This approach has been used extensively and with
  great success in animal feeding food.

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Ochratoxin

• Ochratoxins are a group of mycotoxins produced as
  secondary metabolites by several fungi of the
  Aspergillus and Penicillium spp.
• They are weak organic acids consisting of
  isocoumarin derivatives
• coupled to phenylalanine.

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Penicillium
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• The family of ochratoxins consists of three
  members, A, B, and C.
• Ochratoxin A is the most abundant , the most
  commonly detected and the most toxic one.
• It is a potent toxin affecting mainly the kidney.
• As in other mycotoxins, ochratoxin A can
  contaminate a wide variety of foods as a result
  of fungal infection in crops, in the field during
  growth, at harvest, in storage and in shipment.

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Occurrence in common food products

• Ochratoxin A is found mainly in cereal and cereal
  products.
• In coffee, spices, dried fruits, grape juice.
• Meat and meat products of non-ruminant animals
  (chickens, rabbits) exposed to feedstuffs
  contaminated with this mycotoxin.

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• Aspergillus ochraceus is the best known species
  of ochratoxin producing Aspergillus.
• It grows at moderate temperatures and at a high
  water activity and is a significant source of
  ochratoxin A in cereals.

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• Levels of ochratoxin A in human can be measured
  by detection of ochratoxin A in human blood and
  breast milk.

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Toxicity and health implications

• Ochratoxin A is a toxic carcinogenic fungal toxin
  found in a variety of food.
• Ochratoxin A is absorbed from the
  gastrointestinal tract.
• The absorbed ochratoxin A is distributed via
  blood, mainly to the kidneys, and at lower
  concentrations to the liver, muscle and fat.

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• In non-ruminant animals such as pigs, chickens,
  rabbits and rats, around half of the ingested
  ochratoxin A may be absorbed.
• In ruminant animals such as cow and sheep
  effective hydrolysis of ochratoxin A to the
  non-toxic ochratoxin alpha takes place in the
  stomach before absorption into the blood, this is
  due to the presence of the ruminant protozoa in
  the stomach rendering the species resistant to
  the effects of the toxin.

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• In ruminant animals
• rumen microflora
• ochratoxin A ochratoxin alpha
• toxic hydrolysis
  non-toxic

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• Transfer to the milk has been demonstrated in
  rats, rabbits and humans. In contrast, little
  ochratoxin A is transferred to the milk of
  ruminants, again due to metabolism of this
  mycotoxin by the rumen microflora.

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Toxicity

• The subchronic and chronic effects of ochratoxin
  A are of greatest concern. Ochratoxin A has been
  shown to be nephrotoxic, hepatotoxic, teratogenic
  and immunotoxic to several species of animals and
  carcinogenic in mice and rats causing tumours of
  the kidney and liver.

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• Nephrotoxicity (from Greek nephros, "kidney") is
  a poisonous effect of some substances, (both
  toxic chemicals and medication), on the kidneys.

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• The acute toxicity of ochratoxin A is relatively
  low.
• At present, there are no documented cases of
  acute toxicity reported in humans.

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• In 1993, the International Agency for Research on
  Cancer (IARC) classified ochratoxin A as possible
  human carcinogen and concluded that there was
  sufficient
• evidence in experimental animals for the
  carcinogenicity of ochratoxin A.

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• Ochratoxin A was found more frequently and in
  high concentrations in blood samples obtained
  from people living in regions where the fatal
  human kidney disease, Balkan Endemic Nephropathy,
  occurs.
• Nephropathy (a disease or medical disorder of the
  kidney),
• A highly significant relationship has been
  observed between Balkan endemic nephropathy and
  tumours of the urinary tract, particularly with
  tumours of the renal pelvis and ureters.

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Toxic Mushroom

• Less than 1 of mushroom species are poisonous to
  humans, but these can be extremely dangerous.
• Genus Amanita
• Amanita phalloides
• Amanita muscaria
• Genus Entoloma
• Genus Mycena

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Genus Amanita

• From the most dangerous toxic mushroom are
  species of genus Amanita.
• Amanita phalloides is from the most dangerous
  spp.

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Amanita phalloides (death cap)

• A single Amanita phalloides mushroom may be
  lethal.
• Amatoxins and phallotoxins are the mycotoxins of
  Amanita phalloides.
• They are cyclic olegopeptide toxins .
• oligopeptide (oligo-, "few") consists of between
  2 and 20 amino acids.

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The Peptide Bond

• A peptide bond is a covalent bond that is formed
  between two molecules of amino acids.
• When the COOH gp. of one molecule reacts with
  the NH2 gp. of the another molecule, releasing a
  molecule of water.
• This is a condensation reaction and usually
  occurs between amino acids.
• The resulting OC - NH bond is called a peptide
  bond, and the resulting molecule is an amide.

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Formation of the peptide bond

• The carboxylic acid group of one amino acid
  reacts with the amine group of the other. For
  example, two amino acids (glycine) combining
  through the formation of a peptide bond to form a
  dipeptide.

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Amatoxins and phallotoxins

• They are cyclic olegopeptide toxins .
• Amatoxins are cyclic octapeptides.
• Phallotoxins are cyclic heptapeptides (seven
  amino acids)
• Amatoxins are 20 times more toxic than the
  phallotoxins.

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Amanita phalloides
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Toxicity

• Amatoxins and phallotoxins
• are Hepatotoxic.
• There is hepatic necrosis and death in 20 of
  intoxicated persons.
• Amatoxins inhibit the production of specific
  proteins within liver and kidney cells. Without
  these proteins, cells cease to function and
• cell death occurs.
• Phallotoxins have little input into the death
  cap's toxicity as it is not absorbed through the
  gut.

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Symptoms

• A major problem in diagnosing and treating
  amanita poisoning
• Following ingestionfive to twenty-four hours
  (average, 15h) pass before nausea, vomiting,
  abdominal pain, and diarrhea begin (regardless of
  dose).
• These initial symptoms are followed by a brief
  period of apparent improvement, but without
  treatment, severe liver damage and kidney failure
  often result in coma and death."

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Treatment

• In cases where early diagnosis is accomplished,
  effective therapies include
• The use of milk-thistle extract
• The milk-thistle extract inhibits the
  amatoxins from effecting their most severe liver
  damage and promote regrowth of damaged cells.
• Albumin dialysis.
• In more severe cases, especially those in which
  diagnosis is delayed,
• Liver transplant often becomes the only therapy.

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Silybum marianum (milk thistle)

• Fam. Asteraceae
• Its extract is used in medicine under the name
  silymarin
• (a flavonolignane complex consisting of
  (sylimarin / silybin / silibinin).

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Silybum marianum??????? ??????- ???? ?????
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Amanita muscaria

• The Alkaloidal toxin muscarine, was isolated for
  the first time from Amanita muscaria in
  1869.(historical point of view)
• It was the first parasympathomimetic substance
  studied.
• It is used in classifying cholinergic receptors.
• Muscarinic receptors.
• Nicotinic receptors.

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• Muscarine is only a trace compound in Amanita
  muscaria.
• It is also found in harmless trace amounts in
  other mushroom genera.
• Entoloma and Mycena genera have been found to
  contain levels of muscarine which can be
  dangerous if ingested.

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Muscarine poisoning

• Muscarine mimics the action of the
  neurotransmitter acetylcholine at muscarinic
  acetylcholine receptors.
• Parasympathetic nervous system contains nicotinic
  muscarinic receptors (cholinergic receptors)
  Ach acts on both.
• Muscarine, causes profound activation of the
  peripheral parasympathetic nervous system.

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Muscarine poisoning

• Muscarine poisoning is characterized by increased
  salivation, sweating (perspiration), and tearflow
  (lacrimation) within 15 to 30 minutes after
  ingestion of the mushroom.
• With large doses, these symptoms may be followed
  by abdominal pain, severe nausea, diarrhea,
  blurred vision, and dificult breathing.
  Intoxication generally subsides within 2 hours.
  Death is rare, but may result from cardiac or
  respiratory failure .

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Treatment

• Specific antidotes muscarinic antagonist
  atropine.

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