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Forensic Pharmacognosy. Forensic: relating to the application of science to decide questions arising from crime or litigation. Litigation or legal action. Poisonous Plants and Abused Drugs. Selection of the studied topics has been based on three considerations:
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Forensic: relating to the application of science to decide questions arising from crime or litigation. • Litigation or legal action.
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.
Course contents • Mycotoxins. • Plants containing toxic alkaloids. • Plants containing toxic glycosides. • Psychoactive drugs. • Criminal investigation, Hair Evidence
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.
Major groups of mycotoxins • ErgotAlkaloids 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 phallotoxinsaretoxins produced by theGenus Amanita (poisonous mushroom عيش غراب ). • Muscarine
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.
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. parasiticus…common in America. • Among various mycotoxins, aflatoxins have assumed significance due to their deleterious effects on human beings, poultry and livestock (domestic animals).
Historical view • The aflatoxin problem was first recognized in 1960, when there was severe outbreak (وباء) 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.
From the economic point of view • Aflatoxins influence: • Health of human being. • Health of livestock and poultry. • The marketability of medicinal herbs • Agricultural products.
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.
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.
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 237°C (G1) and 299°C (M1), • Not destroyed under normal cooking conditions.
Structure • Aflatoxins are normally refers to the • group of difuranocoumarins • coumarin nucleus fused to a difuran units coumarin nucleus furan unit
Structure • They are classified in two broad groups: • 1- Difurocoumarocyclopentenone Series. • (B series) (AFB1 , AFB2 , AFB2A , AFBM1 , AFBM2, AFBM2A and aflatoxicol). • ِِAFB1
2- Difurocoumarolactone series . • (G series) (AFG1 , AFG2 , AFG2A , AFGM1 AFGM2 , AFGM2A). AFG1
Detection and estimation of aflatoxins • Analytical methods ( TLC & HPLC ). • Immunological methods.
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)
Immunological methods (cont.) • RIA and EIA are used for estimation and detection of many drugs, (barbiturates, digoxin, LSD, morphine, nicotine, THC, aflatoxins…………
Immunological methods (cont.) • In the RIA radio isotopes are used. • Radiolabelled ( tagged )drug or antigen. • Drug-protein complex (the immunogen). • The antibody (antiserum)
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.
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.
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 ).
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 > AFG1 > AFB2 > AFG2.
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)
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.
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.
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.
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.
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.
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.
Levels of ochratoxin A in human can be measured by detection of ochratoxin A in human blood and breast milk.
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.
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.
In ruminant animals: rumen microflora • ochratoxin A ochratoxin alpha toxichydrolysisnon-toxic
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.
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.
Nephrotoxicity (from Greek: nephros, "kidney") is a poisonous effect of some substances, (both toxic chemicals and medication), on the kidneys.
The acute toxicity of ochratoxin A is relatively low. • At present, there are no documented cases of acute toxicity reported in humans.
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.
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.
Toxic Mushroom • Less than 1% of mushroom species are poisonous to humans, but these can be extremely dangerous. • *Genus Amanita • Amanita phalloides • Amanita muscaria • *GenusEntoloma • *GenusMycena
Genus Amanita • From the most dangerous toxic mushroom are species of genus Amanita. • Amanita phalloides is fromthe most dangerous spp.
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.
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 O=C- NH bond is called a peptide bond, and the resulting molecule is an amide.
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.
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.
