CYTOTOXICITY TEST

* Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell or some types of venom, e.g. from the puff adder (Bitis arietans) or brown recluse spider (Loxosceles reclusa).


Cytotoxicity Testing

* Examination, evaluation, and interpretation of the harmful effects of a substance by testing it on fish, invertebrate animals or small mammals, and extrapolating the test results to determine the quantity that will produce similar effects in humans or other animals.

* Cytotoxicity testing can be useful as a screening tool for pharmaceuticals before more extensive toxicological testing is performed. Additionally, cytotoxicity testing can be used for quality control purposes for lot release testing of raw materials or of manufactured drug products.

* Cytotoxicity tests are designed to determine the toxicity to cells of compounds either qualitatively or quantitatively.

Atomic Absorption Spectroscopy (AAS)

Atomic Absorption Spectroscopy in analytical chemistry is a technique for determining the concentration of a particular metal element within a sample. Atomic absorption spectroscopy can be used to analyze the concentration of over 62 different metals in a solution.

Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.

Although atomic absorption spectroscopy dates to the nineteenth century, the modern form was largely developed during the 1950s by a team of Australian chemists, lead by Alan Walsh, working at the CSIRO (Commonwealth Science and Industry Research Organization) Division of Chemical Physics, in Melbourne Australia. Typically, the technique makes use of a flame to atomize the sample, but other atomizers such as a graphite furnace are also used. Three steps are involved in turning a liquid sample into an atomic gas:

1. Desolvation – the liquid solvent is evaporated, and the dry sample remains
2. Vaporizations – the solid sample vaporizes to a gas
3. Volatilization – the compounds making up the sample are broken into free atoms. 

Atomic absorption spectroscopy relies on the Beer-Lambert law to determine the concentration of a particular analyte in a sample. The absorption spectrum and molar absorbance of the desired sample element are known, a known amount of energy is passed through the atomized sample, and by then measuring the quantity of light it is possible to determine the concentration of the element being measured.

The process of atomic absorption spectroscopy (AAS) involves two steps:

1.      Atomization of the sample

      2.      The absorption of radiation from a light source by the free atoms

Principles

The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on the Beer-Lambert Law.

In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert Law.

 

PHYTOCHEMICAL ANALYSIS

-- Phytochemical Analysis is a bimonthly peer-reviewed scientific journal established in 1991 and published by John Wiley & Sons. It covers research on the utilization of analytical methodology in botany.

-- Phytochemicals are chemical compounds that occur naturally in plants (phyto means "plant" in Greek).

INDICATORS/REAGENTS FOR PHYTOCHEMICAL SCREENING

*Glycosides

Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with a host of different varieties of organic hydroxy (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-soluble phytoconstituents, found in the cell sap. 

*Alkaloids

These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen. The compounds have basic properties and are alkaline in reaction, turning red litmus paper blue. 

*Phenolics

Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components thatoccur ubiquitously as natural colour pigments responsible for the colour of fruits of plants.

They are very important to plants and have multiple functions.

*Flavonoids 

Flavonoids are important group of polyphenols widely distributed among the plant flora.

The compounds are derived from parent compounds known

as flavans. Quercetin, kaempferol and quercitrin are common flavonoids

present in nearly 70% of plants.

*Saponins

Saponins therefore possess ‘soaplike’ behaviour in water, i.e. they produce foam. Saponins are soluble in water and insoluble in ether, and like glycosides on hydrolysis, they give

aglycones. Saponins are extremely poisonous, as they cause heamolysis of blood and are known to cause cattle poisoning. They possess a bitter and acrid taste, besides causing irritation to mucous membranes. 

*Tannins

These are widely distributed in plant flora. They are phenolic compounds of high molecular

weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and

outer layers of plant tissue. 

*Steroids

Plant steroids also referred to as ‘cardiac glycosides’ are one of the most naturally occurring plant phytoconstituents that have found therapeutic applications as arrow poisons or cardiac drugs. The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action mainly on the cardiac muscle when administered through injection into man or animal.

*Essential Oils

Essential oils have a tendency evaporate on exposure to air even at ambient conditions and are therefore also referred to as volatile oils or ethereal oils. They mostly contribute to the odoriferous constituents or ‘essences’ of the aromatic plants that are used abundantly in enhancing the aroma of some spices. Essential oils have been associated with different plant parts including leaves, stems, flowers, roots or rhizomes.

*Anthraquinones

These are derivatives of phenolic and glycosidic compounds. They are solely derived from

anthracene giving variable oxidized derivatives such as anthrones and anthranols.

*Terpenes

They are flammable unsaturated hydrocarbons, existing in liquid form commonly

found in essential oils, resins or oleoresins. Terpenoids are classified according to the number of isoprene units involved in the formation of these compounds.

PLANT WITH PHYTOCHEMICALS

Lemon Balm

*Lemon balm is a perennial herb which grows up to 1 meter high. Lemon balm has an angular stem which branches. The leaves grow in opposite pairs and have a typical lemon flavour when bruised. The white to cream coloured small flowers bloom the whole summer and partly during autumn.

Parts used

-Mainly the lemon balm leaves, which are harvested just before blooming. 

Phytochemicals

These are typical phytochemicals found in lemon balm: citronellal, triterpenens, geranial, neral, rosmarinic acid, geraniol, flavonoids, polyphenols.

Benefits of Lemon Balm

The Greek Dioscorides used lemon balm as a medicinal herb and describes it as being useful to treat a disordered state of the nervous system. In the 1600s the Swis physisina Paracelsus called it Hearts Delight as it could revivify a man. Lemon balm is known for its calming and soothing properties. Lemon balm is used to treat sleeping problems, stimulation of the appetite and nervous stomach ailments. Tests with mice have shown that lemon balm has sedative properties. 

*The lemon balm flowers are visited by bees which collect honey. The name Melissa is coming from a Greek word meaning bee. Lemon balm is also used in the kitchen as a spice, because of its lemon flavour. The lemon balm leaves are often used to decorate meals, drinks and desserts.

DISEASES THEY CAUSE

Bacterial Diseases, Diseases caused by bacteria. The most common infectious diseases, they range from minor skin infections to bubonic plague and tuberculosis. Until the mid-20th century, bacterial pneumonia was probably the leading cause of death among the elderly. Improved sanitation, vaccines, and antibiotics have all decreased the mortality rates from bacterial infections, though antibiotic-resistant strains have caused a resurgence in some illnesses. Bacteria cause disease by secreting or excreting toxins (as in botulism), by producing toxins internally, which are released when the bacteria disintegrate (as in typhoid), or by inducing sensitivity to their antigenic properties (as in tuberculosis). Other serious bacterial diseases include cholera, diphtheria, bacterial meningitis, and syphilis.

Pathogenic bacteria cause infectious diseases like leprosy, cholera, anthrax, and bubonic plague. They are also responsible for the spread of respiratory infections like tuberculosis. The bacterium legionella causes Legionnaires' disease, a lung infection which is a severe form of pneumonia. The disease spreads by inhaling the bacteria or through contact with a person suffering from the disease. Legionella also cause a flu-like illness called Pontiac fever. Yaws disease is a relatively lesser known disease caused by the bacteria Treponema pertenue. It affects the skin, bones, and cartilage, and spreads through skin contact. Lack of cleanliness and hygiene is the main reason behind this illness. Mostly, children contract the Yaws disease and it commonly occurs in poor communities inhabiting regions that have a tropical, humid climate.

KINDS OF BACTERIA

COCCUS, plural Cocci,  in microbiology, a spherical-shaped bacterium. Many species of bacteria have characteristic arrangements that are useful in identification. Pairs of cocci are called diplococci; rows or chains of such cells are called streptococci; grapelike clusters of cells, staphylococci; packets of eight or more cells, sarcinae; and groups of four cells in a square arrangement, tetrads. These characteristic groupings occur as a result of variations in the reproduction process in bacteria. 

BACILLUS (genus Bacillus), any of a group of rod-shaped, gram-positive, aerobic or (under some conditions) anaerobic bacteria widely found in soil and water. The term bacillus has been applied in a general sense to all cylindrical or rod-like bacteria. 

SPIRILLUM,  genus of spiral-shaped bacteria of the family Spirillaceae, aquatic except for one species (S. minus) that causes a type of rat-bite fever in man. The term spirillum is used generally for any of the corkscrew-like species.

Spirillum is microbiologically characterized as a gram-negative, motile helical cell with tufts of whiplike flagella at each end.

VIBRIO,  (genus Vibrio), any of a group of comma-shaped bacteria in the family Vibrionaceae. Vibrios are aquatic microorganisms, some species of which cause serious diseases in humans and other animals.

Vibrios are microbiologically characterized as gram-negative, highly motile, facultative anaerobes (not requiring oxygen), with one to three whiplike flagella at one end.

SPIROCHETE, also spelled spirochaete,  (order Spirochaetales), any of a group of spiral-shaped bacteria, some of which are serious pathogens for humans, causing such diseases as syphilis,yaws, Lyme disease, and relapsing fever. Spirochetes include the genera Spirochaeta,Treponema, Borrelia, and Leptospira.

Spirochetes are gram-negative, motile, spiral bacteria, from 3 to 500 micrometres long. 

BACTERIA 

Bacteria are single celled microbes. The cell structure is simpler than that of other organisms as there is no nucleus or membrane bound organelles. Instead their control centre containing the genetic information is contained in a single loop of DNA. Some bacteria have an extra circle of genetic material called a plasmid. The plasmid often contains genes that give the bacterium some advantage over other bacteria. For example it may contain a gene that makes the bacterium resistant to a certain antibiotic.

Bacteria are classified into 5 groups according to their basic shapes: spherical (cocci), rod (bacilli), spiral (spirilla), comma (vibrios) or corkscrew (spirochaetes). They can exist as single cells, in pairs, chains or clusters.