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Forensic Pharmacognosy

Forensic Pharmacognosy

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Forensic Pharmacognosy

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  1. Forensic Pharmacognosy Poisonous Plants and Abused Drugs

  2. Plants containing toxic glycosides

  3. II- Cyanogenic glycosides • Cyanogenic glycosides occur naturally in plants . (at least 2000 spp.). • Family Rosaceae (Prunus spp.) such as: • P. persicapeach ……………. ……………….amygdalin • P. domesticaplum ……….. ………………….amygdalin • P. amygdalus var.amara (bitteralmond) ……amygdalin • P. armeniacaapricot …………………………. amygdalin • P. laurocerasus (cherry-laurel leaves)….……prulaurasin • The leaves and seeds contain cyanogenic glycosides • P. serotina barks………………………………..prunasin

  4. Family Linaceae • Linum usitatissimum (linseed)…..linamarin • Family Leguminosae • Phaseolus beans……………phaseolunatin • Family Gramineae • Sorghum vulgare…………...............dhurrin • Family Euphorbiaceae • Manihot esculenta (manioc tubers or Cassava)………. linamarin and lotaustralin.

  5. The aglycone of the cyanogenic glycosides contains a cyanide group. • In plants, these glycosides are stored in the cell sap. • but if the plant is attacked, they are released and become activated by enzymes plant. • Contact between enzyme and substrate. • These remove the sugar part of the molecule and release toxic hydrogen cyanide.

  6. A cyanide is any chemical compound that contains the cyano group (C≡N). • Inorganic cyanides are generally salts of the anion CN−. • Organic compounds that have a –C≡N functional group are called nitriles. • Those that can release the cyanide ion (CN−) are highly toxic.

  7. Examples 1.Cassava (Manihot esculenta, Euphorbiaceae), is an important food plant in Africa and South America, (the plant is reach in starch and carbohydrates). The plant contains cyanogenic glycosides and therefore has to be washed and ground under running water before to be conssumed. • In people of undeveloped countries where food containing cyanogenic glycosides is a major part of the diet, these peoples develop ataxic neuropathy.

  8. Cassava roots and leaves should not be consumed raw because they contain two cyanogenicglucosides, linamarin and lotaustralin. • These are decomposed by linamarase, a naturally occurring enzyme in cassava, liberating hydrogen cyanide. • The resulting hydrogen cyanide escapes to the atmosphere.

  9. During cassava starch production, large amounts of cyanoglycosides are released and hydrolysed by plant-borne enzymes, leading to cyanide concentrations in wastewater as high as 200 mg/litre .

  10. 2-Sorghum vulgare (Gramineae) contains cyanogenic glycosides (dhurrin) . • Sorghum contains levels of hydrogen cyanide, lethal to grazing animals. • Sorghum is resistant to pests such as rootworms that plague maize. • (cyanogenic contents).

  11. 3- Sweet and bitter almonds • Prunus amygdalus var.dulcis and P. amygdalus var.amara. (chemotypes). • The kernel contains a fixed oil and emulsion. • The sweet almond oil is used internally in medicine, it must not be adulterated with that of the bitter almond. • It is used in alternative medicine as a carrier oil in aromatherapy.

  12. Botanical terms • The kernel: this is not a scientific term but is usually used to indicate all the stuctures of the seed enclosed within the testa. • Although popularly referred to as a nut, almond is the seed of a drupe (a botanic name for a type of fruit),

  13. Amygdalin • As an example of the cyaogenetic glycosides is amygdalin from bitter almonds and other Prunus spp. • The bitter almond contains the enzyme emulsin (an enzyme mixture containing mainly β-glucosidase activity) which, in the presence of water, acts on amygdalin, yielding 2 glucose, cyanide and the essential oil of bitter almonds, which is nearly pure benzaldehyde. • The effects of bitter almond, even in small doses, are severe and in larger doses can be deadly. • Hydrolytic enzymes occur in the bitter almond, and consequently the seeds invariably contain free cyanide and benzaldehyde.

  14. Hydrolysis of amygdalin *Acid hydrolysis: ▪ Amygdalin→2 glucose + mandelonitrile ▪ Mandelonitrile (spont.) → benzaldehyde+HCN *Enzymatic hydrolysis: ▪ Amygdalin(amygdalase) → prunasin ▪ Prunasin(prunase)→glu.+benz.+HCN ▪ Amygdalin(prunase)→gentiobi.+benz.+HCN ▪ Amygdalin(emulsin or acid)→glu.+benz.+HCN

  15. Mandelic acid • Mandelonitrile

  16. Conclusion • There is three decomposition products of amygdalin: sugar, benzaldehyde, and prussic acid (hydrogen cyanide). • This decomposition may occur by the action of inorganic acids or by enzymatic hydrolysis.

  17. Amygdalin might have anti-cancer properties, but this idea was disproven. • It is sometimes sold as a cancer cure under the name "Vitamin B17", it is not a vitamin. • It was claimed to be a vitamin in the hope that if classified as a nutritional supplement it would escape the federal legislation regarding the marketing of drugs. * Humans taking amygdalin orally in the hope of preventing cancer are likely to be exposed to levels of cyanide in excess.

  18. Cyanogenic glycosides in edible plants

  19. Natural occurrence • Hydrogen cyanide is present in nature. • Cyanide occurs naturally as cyanogenic glycosides in at least 2000 plants. • Amygdalin has been found in about 1000 species of plants. • It is released into the atmosphere from biomass burning, volcanoes, and natural biological processes from higher plants, bacteria, algae, and fungi.

  20. Release to the environment • 1- Cyanide release in Water: The major sources of cyanide release to water are discharges from wastewater treatment plants (During cassava starch production), iron and steel production, and the organic chemical industries. • 2- Cyanide release in air: The major sources of cyanide released to air are exhaust from vehicle emissions, metallurgical industries, extraction of gold and silver from low-grade ores , fumigation operations, combustion of polyurethanes, acrylonitrile, and polyamide plastics & smoking.

  21. Cyanide is present in air as gas, and has the potential to be transported over long distances from their respective emission sources. (One cigarette without a filter liberates 500 µg hydrogen cyanide, while filter cigarettes liberate only 100 µg).

  22. Determination of cyanides • Determination of cyanides in environmental media. • Determination of cyanides in biological tissue and fluids. • Test and Determination of cyanogenic glycosides in plants.

  23. Determination of cyanides in environmental media • Cyanides in environmental media are measured by spectrophotometry , colorimetry, or by gas chromatography. • Since many cyanides are unstable and emit volatile hydrogen cyanide gas, sampling, storage, and analysis must be done with caution, preferably immediately upon collection.

  24. Determination of cyanides in biological tissue and fluids • Cyanide in biological tissue and fluids can be measured spectrophotometrically after reaction with methemoglobin. (Slide no. 39) • A chromatographic technique with fluorescence detection is used to detect trace amounts of cyanide in blood cells.

  25. Test for cyanogenetic glycosides in plants • Mix about 1 gram of the crushed seeds in 1 c.c. of water in a flask. • A strip of filter paper impregnated with Na picrate (Guignard’s paper, filter paper soaked in 1% picric acid solution, dried and then soaked in 10%, Na carbonate solution and again dried) is then suspended in the neck of the flask. • The flask is stoppered, and then warm in a water-bath (not exceeding 45°C) for about 30-60`. • The co-existing enzymes act upon the glycosides with the liberation of HCN which turns the Na picrate paper brick-red in colour. (Na purpurate).

  26. Determination of cyanogenic glycosides in plants • The determination of cyanogenic glycosides in plants is often performed by HPLC. • However, in this analysis, interferences due to compounds in the matrix, such as tannins and other pigments, are encountered.

  27. ToxicityCyanide poisoning • Many cyanide-containing compounds are highly toxic, but some are not. • Nitriles (which do not release cyanide ions) and • hexacyanoferrates (ferrocyanide and ferricyanide, where the cyanide is already tightly bound to the iron ion) have low toxicities, while most other cyanides are deadly poisonous. • The most dangerous cyanides are hydrogen cyanide (HCN) and salts derived from it, such as potassium cyanide (KCN) and sodium cyanide (NaCN).

  28. Cyanide poisoning (cont) *Significant amounts of cyanide are released when amygdalin, is given orally. *The amount of cyanide liberated following oral administration is dependent on the bacterial flora of the human small intestine. • Beta-glucosidase, one of the enzymes that catalyzes the release of the cyanide from amygdalin, is present in human small intestine and in a variety of common foods. • This leads to an unpredictable and potentially lethal toxicity when amygdalin is taken orally. • The lethal dose is 1 to 1.5 mg/Kg body weight.

  29. Toxicology,Mitochondria • Mitochondria are described as "cellular power " because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of energy.

  30. Toxicology • The cyanide anion is an inhibitor of the complex enzymecytochrome c oxidase ( known as aa3 ). • The complex enzyme ( cytochrome c oxidase ) contains two heme centers , cytochrome a and cytochrome a3, and two copper centers, the CuA and CuB centers. • This complex enzyme is found in the membrane of the mitochondria and has a role in the electron transport chain . • The electron transport chain(ETC) is a series of redox reactions. • The redox reactions are chemical reactions in which electrons are transferred from a donor molecule to an acceptor molecule.

  31. An electron transport chain(ETC) couples a chemical reaction between an electron donor (such as NADH) and an electron acceptor (such as O2) to the transfer of electrons, through a series of redox biochemical reactions. These electrons are used to produce adenosine triphosphate (ATP), the main source of energy in living organisms.

  32. Toxicology • Cyanide attaches to the iron within this protein (aa3 enzyme). • The binding of cyanide to the cytochrome c oxidase prevents the transport of electrons . • As a result, the electron transport chain is disrupted, meaning that the cell can no longer produce ATP for energy. • And this results in asphyxiation of cells. • Tissues such as the central nervous system and the heart, are particularly affected.

  33. Toxicity • Acute poisoning • Chronic exposure

  34. ِAcute poisoning • Inhalation of high concentrations of cyanide: • Clinical signs occur in rapid succession. • Initially there is excitement and muscle tremors. • Rapid and difficult breathing, coma, apnea. • Convulsions may occur then cardiac arrest. • Then death following in a matter of minutes. • At lower doses: • general weakness. • headaches, dizziness, confusion. • loss of consciousness, and difficulty in breathing.

  35. Acute poisoning (cont.) • The pupils are dilated and mucous membranes are bright pink. • A characteristic sign of HCN toxicity is a bright cherry-red color of the blood, a symptom that persists for several hours after death. • Although blood is oxygenated, HCN interferes with the release of oxygen from oxyhemoglobin to other tissues.

  36. Skin color goes pink from cyanide-hemoglobin complexes. • A fatal dose for human can be as low as 1-1.5 mg/kg body weight

  37. Chronic exposure • Exposure to lower levels of cyanide over a long period results in: • increased blood cyanide levels. • which can result in weakness. • and a variety of symptoms, including ataxic neuropathy and permanent paralysis.

  38. Treatment of cyanide poisoning A-The United States standard cyanide antidote kit: • Inhaled dose of amyl nitrite, intravenous sodium nitrite. Followed by intravenous sodium thiosulfate • first uses a small inhaled dose of amyl nitrite.As an inhalant, it has a psychoactive effect and induces a brief euphoria state. It also induces the formation of methemoglobin. • Followed by intravenous sodium nitrite. • The nitrites oxidize some of the hemoglobin's iron from the ferrous state to the ferric state, converting the hemoglobin into methemoglobin. Cyanide preferentially bonds to methemoglobin rather than the cytochrome c oxidase, converting methemoglobin into cyanomethemoglobin.

  39. Treatment of cyanide poisoning(cont.) • Followed by intravenous sodium thiosulfate • In the last step, the intravenous sodium thiosulfate converts the cyanomethemoglobin to thiocyanate, sulfite, and methemoglobin. • The thiocyanate is then excreted in urine. • Disadvantage: • Treatment with nitrites is not recomended as methemoglobin cannot carry oxygen, and methemoglobinemia needs to be treated with methylene blue.

  40. Methylene blue accelerates the methemoglobin reduction pathway. • In the absence of further accumulation of methemoglobin, these methemoglobin reduction pathways can clear methemoglobin at a rate of approximately 15% per hour.

  41. Treatment of cyanide poisoning(cont.) B- HydroxycobalaminCyanokit: • a form of vitamin B12. • Hydroxycobalamin reacts with cyanide to form cyanocobalamin. • Cyanocobalamin can be eliminated by the kidneys. • Administration of sodium thiosulfate improves the ability of the hydroxycobalamin to detoxify cyanide poisoning. • This treatment is considered so effective that it is administered routinely in Paris to victims of smoke inhalation to detoxify any associated cyanide intoxication. • However it is relatively expensive and not universally available. • This antidote kit is sold under the brand name Cyanokit and was approved by the FDA in 2006.

  42. Notable cyanide deaths • Cyanides have been used as poison many times throughout history. • The most infamous application was the use of hydrogen cyanide gas by the Naziregime in Germany for mass murder in some gas chambers.

  43. Criminal investigationHair Evidence • Hairs can provide investigators with valuable information for potential leads . • Identification and comparison of human and animal hairs .

  44. Tools for the identification and comparison of hairs • The microscopic characteristics found in the different type of hairs. • Nuclear DNA testing. • Microscopicalmeasurement ( Micrometry ) • When the microscope(microscopic characteristics and micrometry) is coupled with DNA technologies, this combination of these technologies profoundly affects hair evidence.

  45. Basic Structure of Hair • A hair can be defined as a slender, thread-like outgrowth from a follicle in the skin of mammals. • Its basic components are: • keratin (a protein), • Melanin (a pigment), and • Trace quantities of metallic elements.

  46. Structure of Hair • The hair has three regions: • the cuticle, medulla, and cortex .

  47. Glossary • Cuticle: Translucent outer layer of the hair shaft consisting of overlapping scales. • Medulla: Central portion of hair, the core area. • Cortex: Middle portion of hair extending from the cuticle to the medulla and containing the pigment granules, cortical fusi, and ovoid bodies

  48. Glossary (cont.) • Cortical fusi: Air spaces located in the cortex of hairs. • Ovoid bodies: Dark bodies of unknown origin that are a useful discriminatory characteristic in their pattern of appearance. • proximal is the root end of the hair. • distal is the tip end of the hair.

  49. Cuticle The cuticle is a translucent outer layer of the hair shaft consisting of scales that cover the shaft. The cuticular scales point from root end (proximal) of the hair to the tip end (distal) of the hair.