Forensic Pharmacognosy

Poisonous Plants and Abused Drugs

Plants containing toxic glycosides

II -Cyanogenic glycosides

• Cyanogenic glycosides occur naturally in plants . (at least 2000 spp.).

• Family Rosaceae (Prunus spp.) such as:• P. persica peach ……………. ……………….amygdalin• P. domestica plum ……….. ………………….amygdalin• P. amygdalus var.amara (bitter almond) ……amygdalin• P. armeniaca apricot …………………………. amygdalin• P. laurocerasus (cherry-laurel leaves)….……prulaurasin• The leaves and seeds contain cyanogenic glycosides• P. serotina barks………………………………..prunasin

• 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.

• 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.

• 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.

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.

• Cassava roots and leaves should not be consumed raw because they contain two cyanogenic glucosides, 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.

• 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 .

• 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).

• 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.

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 nutnut, almond is the seed of a drupe (a botanic name for a type of fruit),

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.

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

• Mandelic acid

• Mandelonitrile

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.

• 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.

Cyanogenic glycosides in edible plants

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.

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.

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).

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.

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.

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.

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).

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.

Toxicity

Cyanide 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).

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.

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.

Toxicology• The cyanide anion is an inhibitor of the complex enzyme

cytochrome 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.

• 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.

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.

Toxicity

• Acute poisoning• Chronic exposure

�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.

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.

• 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

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.

Treatment of cyanide poisoningA-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.

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.

• 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.

Treatment of cyanide poisoning(cont.)

B- Hydroxycobalamin Cyanokit:• 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.

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 Nazi regime in Germany for mass murder in some gas chambers.

Criminal investigationHair Evidence

• Hairs can provide investigators with valuable information for potential leads .

• Identification and comparison of human and animal hairs .

Tools for the identification and comparison of hairs

• The microscopic characteristics found in the different type of hairs.

• Nuclear DNA testing.

• Microscopical measurement ( Micrometry )

•When the microscope(microscopic characteristics and micrometry) is coupled with DNA technologies,

this combination of these technologies profoundly affects hair evidence .

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.

Structure of Hair

• The hair has three regions:

• the cuticle, medulla, and cortex .

•

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

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.

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.

scale structure

• There are three basic scale structures that make up the cuticle:

• coronal (crown-like),

• spinous (petal-like), and

• imbricate (flattened).

• Combinations of these types are possible.

Coronal (crown-like) scales

• Coronal scales are commonly found in the hairs of small rodents and bats but rarely in human hairs.

Spinous (petal-like) scales

• Spinous scales are triangular in shape and protrude from the hair shaft. They are found at the proximal region of mink hairs and on the fur hairs of cats, and some other animals. They are never found in human hairs.

Imbricate (flattened) scales

• The imbricate scales type consists of overlapping scales with narrow margins. They are commonly found in human hairs.

Bat Hair (crown-like scale)

Mink Hair (petal-like scales)

Human Hair (flattened scales)

Medulla

• The medulla is a central core of cells that may be present in the hair.

• . In human hairs, the medulla is generally amorphous in appearance, whereas in animal hairs, its structure is frequently very regular and well defined.

• When the medulla is present in human hairs, its structure can be described as—fragmentary or trace, discontinuous or broken, or continuous.

Diagram of the three basic medullary types in Human Hair.

Cortex

• The cortex is the main body of the hair composed of elongated and fusiform (spindle-shaped) cells.

• It contains:

• Cortical fusi, they are irregular-shaped air spaces of varying sizes.

Cortical Fusi in Human Hair

Cortex (cont.)

• Pigment granules, are small, dark, and solid structures that are granular in appearance and considerably smaller than cortical fusi. They vary in color, size, and distribution in a single hair.

• In human hairs the pigment granules are commonly distributed toward the cuticle.

• except in red-haired individuals • Animal hairs have the pigment granules

commonly distributed toward the medulla.

Pigment Distribution Human Hair

Pigment Distribution in Animal Hair

Cortex (cont.)

• Ovoid bodies are large (larger than pigment granules), solid structures , spherical to oval in shape, with regular margins. They are abundant in cattle and dog hairs.

Animal Versus Human Hairs

• Human hairs are distinguishable from hairs of other mammals.

• Animal hairs are classified into the following three basic types.

• Guard hairs that form the outer coat of an animal and provide protection.

• Fur or wool hairs that form the inner coat of an animal and provide insulation.

• Tactile hairs (whiskers الشارب أو that are ( اللحيةfound on the head of animals provide sensory functions.

• Other types of hairs found on animals include tail hair and mane hair (horse). الفرس عرف

• Human hair is not so differentiated and might be described as a modified combination of the characteristics of guard hairs and fur hairs.

Animal Versus Human Hairs

Human hairAnimal hair

ColourConsistent in colour and pigmentation throughout the length of the hair shaft.

Exhibit colour changes.

Pigmentationevenly distributed, or slightly more dense toward the cuticle.

more dense toward the medulla.

Animal Versus Human Hairs

MedullaIf present, amorphous.The width is less than one-third the overall diameter of the hair shaft.

Continuous, structured and occupies an area of greater than one-third the overall diameter of the hair shaft.

Root Hairclub-shaped highly variable .

Animal Versus Human Hairs

Human hairAnimal hair

Scale pattern of the cuticle.

routinely imbricate

variable scale patterns

Human Hair Root

Hair Microscopy

• The regions of the body that are primarily used in forensic comparisons are the hairs of head and pubic areas.

• The examination of human hairs in the forensic laboratory involves a two-step process:

• The identification of questioned hairs.

• The direct comparison of questioned and known hairs.

• The comparison microscope consists of two microscopes connected by an optical bridge that allows for the viewing of questioned hairs and known hairs at the same time.

DNA Examinations

• Hairs that have been matched or associated through a microscopic examination should also be examined for DNA sequencing.

• The combined procedures add credibility to each.

Microscopical measurements ( Micrometry )

• Microscopical measurements are used for identification purposes and are particularly useful in determining the dimensions of different types of Hairs and Fibers.

• They are useful in determining the purity and botanical sources of certain powdered drugs .

• The most helpful dimensions are those of prominent structures such as stone cells, fibers , pollen grains, starch granules and crystals of calcium oxalate.

Hairs and FibersFiber width

Cotton9 – 24 μm

Animal Hair13 – 40 μm

Human Hair40 – 50 μm (very fine)

110 – 120 μm (very coarse)

Micrometry

• Microscopical measurements may be made either by means of:

• a stage micrometer and an eye piece micrometer (MICROMETERS).

• OR

• by means of a stage micrometer and camera lucida .

Eye piece micrometer

• It is a glass disc of suitable diameter.

• fitted within the eye lens of the microscope

• Engraved on the center of the disc a linear scale of 50 or 100 small equal divisions.

• It is not a standard scale .

Stage micrometer

• It is a glass slide with scale engraved on it.

• It is a standard scale of 1 or 2 mm long.

• It is divided into 0.1 and 0.01 parts of mm .

• The stage micrometer is used for the calibration of the eye piece micrometer.

Calibration of eye piece micrometer using stage micrometer

• To find out how each small division of E.P.M. is equal :

• Make the two scales parallel to each other and zero of both scales is coincident .

• Determine the line of E.P. which coincide with the line of stage micrometer .

Low Power High Power EPM SM EPM SM

Calibration of the eye piece micrometer

• Using low power• 6 large divisions of E.P. = 0.9 mm S.M.• 60 small divisions of E.P. = 0.9 mm S.M.• = 900µ• So, each small division of E.P.= 900/60 = 15µ • Using high power• 34 small divisions of E.P. = 0.1mm S.M.• = 100µ• So, each small division of E.P.=100/34 = 2.9µ

Determination of fiber or hair width using eye piece micrometer

• Determine the number of small divisions of the E.P. micrometer that cover the fiber or hair .

• The hair or fiber width =

number of small divisions of E.P. x equivalence of each division in µ .