eChapters

Pfeiffer first isolated and described Haemophilus influenzae as a cause of human influenza (1892). So it is also known as ‘Pfeiffer’s bacillus’. Later other workers isolated and described different species of the genus Haemophilus from animals and birds. In 1903, Friedberger, a research assistant of Pfeiffer, isolated similar kind of organism from prepuce of the dogs. In 1922, Rivers described it as Haemophilus canis. In 1925, Mitchell isolated Haemophilus ovis from the respiratory tract of sheep. In 1931, Lewis and Shope first isolated and illustrated Haemophilus suis from the infected pigs. Glasser found an association between fibrinous serositis, polyarthritis in pigs (Glasser’s disease) and a small gram negative rod as early as 1910, latter became known as Haemophilus parasuis. It was first isolated by Hjarre and Wramby in 1943.  The first recorded isolation of a hemophilic organism from the birds was in 1931 by De Blieck and he called them as Bacillus haemoglobinophilus coryzae gallinarum. He isolated them from the chickens suffering a disease, now known as ‘infectious coryza’.

Taylorella was first isolated by Taylor et al. (1978) as a causative agent of contagious equine metritis (CEM), a venereal disease of horses. The disease (CEM) was first reported in the mares of thoroughbred horses in UK in 1977 breeding season (Crowhurst, 1977). Initially the causative agent was assumed as Haemophilus equigenitalis. Later on the basis of numerical taxonomic characteristics, genomic G+C contents, DNA-DNA hybridisation studies, the new genus Taylorella was proposed in 1983 (Sugimoto et al., 1983). It was approved by the International Committee on Systematic Bacteriology.

Properties of genus

1.   Morphology :  Taylorella are gram negative, small rods or coccobacilli, measuring 5-6 µm by 0.8 µm. They are pleomorphic, piliated, encapsulated, non-sporing and non-motile.

2.   They are microaerophilic (require low O₂ tension for growth), non-fermentative, and non-proteolytic in nature.

3.         Classification : Taylorella belongs to β- subclass of class Proteobacteria (whereas, Haemophilus belongs to the γ- subclass of Proteobacteria). Major important species is Taylorella equigenitalis. The genus was considered as a ‘single species genus’ for a long time. Latter in   1997, National Veterinary Services Laboratories confirmed the identity of another Taylorella species, isolated from a donkey jack (Equus asinus) in California. It was named as T. asinigenitalis. The new species has minor difference in 16S rDNA sequence (97.6% similarity), DNA–DNA hybridization (23% similarity) and (G + C) composition with T. equigenitalis (Matsuda and Moore, 2003)
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Listeria

Murray et al. (1926) first described about Listeria which was followed by Pirie et al. (1927). In India Listeriosis was first reported by Mahajan (1936) in sheep from Andhrapradesh. He recognized it as ‘circling disease’. Latter a number of reports are available from various parts of India.

Lignieres and Spitz first isolated the bacteria from the cattle with Actinomycosis like condition in Argentina in 1902. They referred them as ‘Actinobacilllus’. These findings were also confirmed by Nocard in 1902. Latter, the isolated bacterium from the cattle was named as Actinobacillus lignieresii, in memory of Lignieres. 

Properties of genus

1.   Morphology :  Actinobacillus are gram negative small rods, measuring 1.5 µm in length and 0.4 µm in width, occurring singly, in pairs or more rarely in chains. After isolation in the laboratory they may show pleomorphism varying from coccobacilli to filamentous forms. In gram stained smear they may show both bacillus and coccus form together, known as ‘morse code’ appearance (dots and dashes appearance, Fig.1). They are non-motile (both at room temperature and 37^oC), non-sporing and non-acid fast. The pili are found in A. pleuropneumoniae.

      Some of them (A. pleuropneumoniae) are capsulated. Whereas, slime layer is found in the surface of other species like A. lignieresii, A. suis, A. equuli.  This slime layer is related with stickiness of the colonies in the agar, enhanced resistance of the colonies to the chemical disinfectants, as well as, resistance of the bacterial cell to the antibiotics and desiccation.

2.         Actinobacillus are aerobic, microaerophilic or facultative anaerobic in nature. They are chemoorganothrophic having both respiratory and fermentative types of metabolism.

Cohn (1875) first observed and described a branching filamentous micro-organism in the concretions (coagulated mass) taken from a lacrimal canal, latter identified as Actinomyces israelii. Bollinger first reported the yellow granules in jaw tissues of the cattle in 1877 and established the significance of the organisms as an etiological agent. In 1878, Israel described the first human case. In 1879, Hartz observed the microscopic appearance of granules of Actinomyces infection and suggested the name Actinomyces bovis. Mosselman & Lihnaux (1890) and Wolf &Israel (1891) first isolated the bacteria in artificial laboratory media from lumpy jaw cases of cattle and human Actinomycosis, respectively.

Properties of genus

1.         Morphology :  Actinomyces are gram positive filamentous organisms with branching rods. In artificial media, short bacilli or cocci forms are also observed. During infection, they can produce yellow coloured granules (sulphur granule), commonly found in the pus as ‘opaque speck’ and are visible under naked eye (1-2 mm in diameter). The granules are usually soft and easily broken under light pressure, but they may occasionally be tough or even calcified. They are composed of club shaped bodies, arranged radially with their narrow ends towards the centre. So the organisms are also known as ‘ray fungus’. In the centre of the club, filamentous masses of gram positive organisms with some bacilli or cocci form are found. Together the club like processes and bacterial masses are known as ‘rosette’ (Fig.1). This club is composed of calcium phosphate and inflammatory debris
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The genus Arcanobacterium was first described to include the bacteria originally isolated as Corynebacterium haemolyticum from American wounded soldiers (Collins et al., 1982). The bacteria had different cell wall and lipid composition in comparison to the other members of the genus Corynebacterium. So the new genus Arcanobacterium was created.

Properties of genus

1.         Morphology : Arcanobacterium pyogenes is a Gram positive, non-motile, non- spore forming, short, rod-shaped bacterium, measuring 0.5µm by 2 µm. They are non-capsulated and do not contain any metachromatic granule. Initially (during first 18 hour of growth) the bacterial cells may show characteristic ‘V’-arrangement like Corynebacterium, latter as the growth proceeds, the organisms become granular and segmented so that they resemble small and irregular cocci (whereas, A. hippocoleae and A. pluranimalium appear to be less coccoid). The cell wall is devoid of mycolic acid, arabinogalactan unlike ‘coryneform’ bacterial group (Mycobacterium, Corynebacterium, Rhodococcus, Nocardia). Cell wall peptidoglycan is composed of N-acetyl muramic acid, lysine. Being gram positive in nature, some strains exceptionally possess fimbriae.

Corynebacterium diptheriae was first observed by Klebs in 1883 and described in details by Loeffler in 1884. So it is also known as ‘Klebs-Loeffler bacillus’. In 1888, Nocard isolated a similar type of bacteria from cases of bovine farcy. In 1894, Preisz described it completely and revealed its similarities with the diphtheria bacilli. Latter it was named as Corynebacterium pseudotuberculosis (Preisz-Nocard bacillus). Bollinger (1890) first reported C. renale from the cases of pyelonephritis in cows. Magnusson (1923) first described C. equi (Rhodococcus equi) form suppurative pneumonia of a foal.

Properties of genus

1.   Morphology

Corynebacterium are gram positive, pleomorphic bacteria ranging from coccoid to filamentous rods. The size varies from 0.5-0.6 µm by 1.0- 3.0 µm. Some of the species appear as packets of parallel cells (palisade arrangement) or show ‘V’ shaped form (Chinese letter arrangement) in the microscopic field (Fig.1and Fig.2). The cell wall peptidoglycan contains meso-diaminopimelic acid (meso-DAP). The arabinose and galactose are major cell wall sugars. Short-chain mycolic acids (corynomycolic acids, 22–36 carbon atoms) are present. This kind of cell wall structure is impermeable to the hydrophilic compounds. However, the ‘porin’ present in the cell wall sometimes helps in import of the hydrophilic substances. Mycolic acid containing cell wall helps in intracellular survival within the macrophages. Corynebacterium is non acid fast, non-sporulating, non-capsulated and non-motile bacteria (except C. aquaticum)
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Edmond Nocard (French Veterinarian) first isolated filamentous aerobic bacteria from clinical cases of ‘bovine farcy’ in 1888. In 1889, the genus Nocardia was formed by Trevisan, and the organism described by Nocard was named as Nocardia farcinica. In 1891 another similar organism (Nocardia asteroides) was isolated by Eppinger from a human brain abscess. Initially it was thought that they are the same organisms. Latter on the basis of the molecular studies their separate entity is established.

            However, 80 years later, re-examination of Nocard’s original isolate revealed two different filamentous organisms, one of which was Mycobacterium and the other was Nocardia. The cause of ‘bovine farcy’ has been subsequently attributed to Mycobacterium. Latter, it is proved that Mycobacterium farcinogenes is the actual causative agent of ‘bovine farcy’.

Dermatophilus congolensis was first described from clinical case of exudative dermatitis in cattle in Congo by Van Saceghem (Van Saceghem, 1915). It is primarily known as an animal pathogen causing acute, sub acute or chronic skin disease. The first cases of human infection with D. congolensis were reported in 1961 in four people who developed skin infection after handling an infected deer in a wildlife reserve in USA (Dean et al., 1961).

Properties of genus

1.   Morphology

Dermatophilus is a gram positive, non-acid fast, unusual bacterium having branched filaments. It follows a life cycle with different morphological features. It can be found in smears prepared from clinical samples in any form of its life cycle. Young filaments are 0.5 to 1.5 μm in diameter.  They enlarge within epidermis as they produce layers of transverse and longitudinal septations and reach 5 to 8 μm in diameter, surrounded by a mucoid capsule. The septations result in the formation of a sporangia (a kind of fungal fruiting body, sac like structure with spores) containing up to eight cocci cells. In the presence of liquid, the cocci are released and converted into flagellated motile zoospores, the infective form of the organism. It can infect the new site in the same animal or new host. These zoospores become visible in smear as parallel lines forming a ‘railway-track’ or ‘trum-track’ appearance (Fig.1). Under favourable conditions, the zoospores form germ tubes that develop into branching filaments. These filaments are the invasive form of the organism and the cycle is repeated.

Leptospira 

Antony Van Leeuwenhoek (1681) first observed the Spirochaetes under the microscope and described them. In 1833, Ehrenberg used the name Spirochaeta for free living spiral shaped organisms. Obermeir (1873) first described a Spirochaete of pathogenic significance isolated from human patients of relapsing fever. Inada et al. (1916) first isolated Leptospira icterohaemorrhagiae from human patients of Weil’s disease. In 1980, Nogochi proposed the name Leptospira.

      In India, Taylor and Goyale (1931) first reported Leptospira from human patients suffering from jaundice in Andaman Islands (Srivastava, 2006). Ayyar (1932) first reported Leptospira from dogs in Chennai city. 

Properties of genus

1. Morphology : Leptospira are gram negative, spiral shaped organisms with hooked ends. Length varies from 6-20 µm with 0.1-0.2 μm diameter (Fig.1). They have a typical double membrane covering. The cytoplasmic membrane and peptidoglycan cell wall are closely associated and are surrounded by an outer membrane. Two periplasmic flagella (endoflagella) are found within the cell and are responsible for the motility. No flagella are present as extracellular appendages. One end of the flagella is attached with the pole of the bacterial cell by insertion disc. Other end extends towards the centre of the cell and may overlap the flagella coming from the opposite end. The endoflagella consists of a solid inner core surrounded by tubular sheaths. The FlaA and FlaB proteins constitute these flagellar sheath and core, respectively.

Properties

1. Bacteroides, Dichelobacter and Fusobacterium are major gram negative anaerobes isolated from human and animals. Other gram negative anaerobes are listed below (Table-1).

       2.   They are tolerant to oxygen in comparison to strict anaerobes. They do not die after oxygen exposure

       3.         They naturally inhabit the intestine and mucosal surfaces of human and animals. Majority of them are opportunistic pathogens. Often they cause mixed bacterial infection along with aerobes. Aerobes remove oxygen and create an anaerobic condition favourable for their growth. Aerobes may also supply other growth requirements. For e.g. Porphyromonas asaccharolytica requires vitamin K for its growth, supplied by another bacterium, not the host.

The name Mycoplasma is originated from the Greek word mykes (fungus) and plasma (formed). It was first used by Albert Bernhard Frank, a German Biologist in 1889. He thought it was a fungus. An older name for Mycoplasma was Pleuropneumonia-Like Organisms (PPLO), referring to organisms similar to the causative agent of contagious bovine pleuropneumonia (CBPP). It was later found that the fungus-like growth pattern of M. mycoides is unique to that species. It was first isolated in 1898 by Edmond Nocard and E. Roux from bovine lungs. Whereas, Mycoplasma pneumoniae was considered as a virus previously. Eaton and his co-workers cultured the causative agent of human primary atypical pneumonia (PAP) or "walking pneumonia." It was known as "Eaton's agent." In the early 1960s, Leonard Hayflick, in Philadelphia, was first to isolate and cultivate the causative agent of primary atypical pneumonia and identified the agent as a Mycoplasma and named it Mycoplasma pneumoniae.

History of Rickettsial infections went back to 16^th century and it has long been associated with famine and war. Thus Rickettsial infections played a significant role in the history of Western civilization. Epidemic typhus had major impact to decide over the outcome of several wars. This epidemic alone killed or caused great suffering to over 100,000 people in the two World Wars.  However, the causative agent was not determined until the early part of the 20^th century. Rocky Mountain Spotted Fever (RMSF) was the first detected Rickettsial infection as early as in 1896 in Idaho Valley, USA.  It was originally known as ‘Black Measles’ because of its clinical appearance in the form of characteristic spotted rash appearance throughout the body of the patients. Howard Taylor Ricketts, an American pathologist for the first time described the causative agent of Rocky Mountain spotted fever and was able to isolate it in the laboratory animals during 1902-1909. He was a dedicated scientist and, on several occasions, injected himself with a pathogen for experiment. The causative pathogen of Rocky Mountain spotted fever, Rickettsia rickettsii was named after him. Later, the family Rickettsiaceae as well as the order Rickettsiales was named after him. Ernest William Goodpasture, an American pathologist and physician, invented the methods of isolation and culturing the rickettsial pathogens in chicken embryos and fertilized chicken eggs.

The word ‘chlamydia’ comes from the Greek word ‘chlamys’ which means, “cloak draped around the shoulder”. This describes how the intracytoplasmic inclusions caused by the bacterium are “draped” around the infected cell’s nucleus. Chlamydia was first discovered by a Czech zoologist and parasitologist, Stanislaus von Prowazek in 1907 in Berlin, but scientists believe it was actively infecting people for centuries before its discovery. Because the symptoms of Chlamydia are similar to some other infections it was not identified as a sexually transmitted disease. Originally, because of its structure it was thought to be a virus. In 1963 it was recognized as bacteria rather than a virus.

Properties of genus

           They are poorly stained by Gram’s stain. Chlamydiae are readily stained by Castaneda (blue in appearance), Giemnez and Macchiavello (red in appearance) methods. However, Giemsa staining is preferable to stain the inclusions (Chlamydial micro colony) in the cell culture. Inclusion bodies of the Chlamydiae are basophilic in nature. Again, the staining of the inclusion bodies may differ depending on the species. In C. trachomatis, the inclusion bodies contain glycogen matrix and can be easily stained with iodine solution.

1. 1 Introduction

Irrigation is the artificial application of water needed to the crop field. It is the process of uniformly wetting the root zone depth. The wetting may be done over the land surface or below of it. The application of water in excess to the requirement or accumulation of water in crop field due to rain or unplanned seepage is not considered as irrigation. However, irrigation is supplemental to rainfall. Irrigation water is considered as single most important inputs for crop production.

Measurement of water used in irrigation ensures the application of required amount of water in crops. Knowing the rate of flow the adjustment to period of control at gates, etc. can be done to regulate the water at different branches of canals. To get the total control over the supply of water there should have the provision of water measurement at the critical points in irrigation systems.

Units of Measurement

Water is measured at rest or in motion. The water of reservoir, ponds, tanks, etc. at rest is measured in volumetric unit as liter, cubic meter, hectare-meter and hectare-centimeter. Water flowing in rivers, canals, channel, etc. is measured in liter/s, liter per hour, meter per second and hectare-meter per hour or day.

3. 1 Introduction

Irrigation efficiency evaluates how effectively the water has been distributed for the purpose of crop production. Water is conveyed to crop field through canal water courses and channels. During this process considerable amounts of water get lost through run off, deep percolation, evaporation, etc. The losses of water greatly depend on the skill of the irrigator, degree of land preparation, and design and planning of the system. The extent of water losses starting to diversion of water from its source to crop field though conveyance and ultimate application and distribution of water in crop field which can be estimated separately. The estimation of these efficiencies enables one to identify the step(s) where improvement is required. The method of measurement and evaluation for its efficiency is prerequisite for proper use of water. Irrigation efficiency in developing countries in general is poor particularly in gravity irrigation methods. However, water is become more and more scarce. Due to pressing need of it judicious and efficient management is one of the vital issues to be solved on priority basis.

Scheduling irrigation is the process of determining when to irrigate and how much to irrigate. Selecting the appropriate time span in between two irrigation and applying the required volume of water in each irrigation, ensures saving of water, minimize energy use, higher crop yield and lower production costs. Irrigation applied earlier than due date causes loss of water through evaporation or deep percolation and later causes water stress to plants and thereby affects the growth and yield of the plants. Scheduling of irrigation may be required in two field conditions: (i) when availability of water is adequate, (ii) when availability of water is limited.

In the places where water is adequate scheduling of irrigation follows full irrigation with time sequence of minimum number of irrigation to ensure maximum yield and high water use efficiency. When water is limited its use is rationalized by using at the sensitive stages of the plants and withholding irrigation at other stages. The sensitive stages so selected that by using the available water provides possible maximum yield.

5. 1 Irrigation Channels

Canal or channel is an artificially made conduit through which water is supplied to the field from river, tank, reservoir or wells. The open channel is the most common type of water conveyance system. If the pipes and drains are partially full behave like open channel. The irrigation channel may be lined or unlined and the flow is either uniform or varied.

The canal may be classified in following ways.

(a) Classification based on the source of supply

(i) Permanent or perennial canal

(ii) Inundation canal

The plants collect water by its root system available in the soil medium. A soil rich in nutrients cannot ensure success of crops unless the presence of satisfactory state of water and air, mechanical attributes and thermal regime. The physical properties of soil and plants greatly influence the movement, retention and use of water. The soil should provide sufficient pore spaces and size distribution and decomposition of rocks and for the movement and retention of water for plant needs. Rainwater, irrigation, groundwater, seepage, etc. , contribute in replenishing the water in dried up soils. In all the cases thorough understanding of behavior of water is required to ensure the need of water to plants and optimize the use of irrigation water.

7. 1 General

The major portion of water in a crop field gets lost through evaporation and transpiration. Evaporation is the process by which water is changed from the liquid state to vapor state. It takes place from the adjacent soil, water surface or from the surfaces of leaves of the plants. Transpiration is the removal of water vapour through plant body. Evapotranspiration (ET) is the sum of evaporation and transpiration. The process of evaporation and transpiration goes on simultaneously and difficult to separate them in a crop field.

The term consumptive use is used to refer evapotranspiration together with the water used in the metabolic activities of the crop plants. The actual amount of water used in metabolic activities is insignificant (less then 1% to ET). Therefore, the consumptive use and evapotranspiration are often used as synonymous term. It includes the water consumed by the plants for metabolic activities or transpiration plus the water evaporated from the land and water surfaces of the cropped area.

The factors affecting the rate of evaporation are the vapour pressure at the water surface and air above, air and water temperatures, wind speed, atmosphere pressure and size of the water body. Other factors remaining same, the rate of evaporation increases with an increase in the water temperature. Higher wind velocity causes greater scope for evaporation. Decrease in atmospheric pressure increases evaporation. The solute dissolved in water causes reduction in the rate of evaporation than that of pure water. The evaporation from soil surface occurs in same way to free water surface when the soil particles are surrounded by thin film of water and pore spaces are partially filled. However, in such situation greater resistance has to overcome for evaporation from the soil particles than while evaporating from a free water surface. The evaporation process practically stops when the moisture content in soil goes below a threshold value.

8. 1 Introduction

Water is a precious commodity. Necessity of conservation of water supply is increasingly felt all over the world as the demand of it continues to increase when the sources of supply are increasingly scarce. The conveyance and proportional distribution of water is one of the most important part of any irrigation project. Water available in the stream, reservoirs or wells is carried to the field of use through canals. The economy of any irrigation project largely depends on how efficiently the water is being transported and distributed in respect to water losses and cost.

Water losses in irrigation channels are mainly due to evaporation from the water surface and seepage from the bed and side of the channels. Seepage losses some time become the single important factor which causes unavailability of water particularly in downstream of the channel in canal irrigation system and excess pumping of water in tube wells and river lift irrigation system.

Lining irrigation channels means to cover the channel earthen surface by any substance, which can considerably reduce the seepage loss. The concrete, brick, stone, natural clay of low permeability, bituminous material, plastic compounds, etc. , are used for channel lining.

Poor quality of water is one of the important factors causes gradual accumulation of salts on the ground or in the root zone of crops leads to saline and alkaline condition. This results in loss of permeability, toxicity and anaerobic conditions. In salt affected soils plants face constraint to derive their nutrients at the required rate due to high soil moisture tension, which affects adversely the crop growth and productivity.

10. 1 Land and Water Resource

India has got the world’s 2. 45% area and 16. 87% of population with only the 4% of available fresh water (Table 10. 1). This indicates the necessity of development of water resources and optimal use of it. India at macro level does not have short of water. But the shortage of water is due if not properly managed by conservation and sector wise utilization.

11. 1 Introduction

Agricultural drainage is the removal of excess water from the farmland for the purpose of providing favorable soil air environment for plant growth. Removal of water may take place form the surface or below the surface of the land. The practice of lowering ground water table below the root zone depth to improve the plant growth or to reduce the accumulation of salts in soil is also called as drainage.

Drainage system is broadly classified as surface and subsurface drainage. Removal of excess water over the land surface is known as surface drainage and that of removing water below the surface is known as subsurface drainage. The flow of water in the form of evaporation or evapotranspiration also play important role in improving drainage situation. It gives the idea of the time required to get the wetland workable though it is not called a type of drainage as because this is very slow process and cannot be controlled by the human activities. However, using of certain tree plants e. g. , Eucalytus, Poplar, Casurina or similar species in wet fields, which transpires at high rate and improves the drainage problem by lowering the shallow water table is the new idea known as bio-drainage.

The purpose of a sub-surface drainage system is to lower the ground water table and removal of salts beyond the crop root zone. The ground water table may rise due to commonly practiced flood irrigation method, seepage from unlined channels, percolation of rain and runoff water and sub-surface flow from higher areas. Sub-surface drainage consists of drains laid at a specified depth, spacing and grade below the ground surface. The size, spacing and depth of drains depend largely on the soil-water properties viz. , infiltration, saturated hydraulic conductivity, drainable porosity; and the requirement of removal of water through drains in certain time period fixed on the basis of rainfall and crop cultivated. The drains are usually placed in a pervious stratum. Suitable filter of coarse sand and gravel may be used when it is placed in a low permeable stratum. Sub-surface drainage serves the important job of removal of concentrated salt solution from the crop root zone particularly in arid and semi-arid region.

Plants are used as medicine since time immemorial. One of the area of Ethnobotany, Ethnopharmacology is considered as the scientific evaluation of traditional medicinal plants (Cotton, 1996). Cox (1994) has suggested that the ethno-directed sampling is most likely to succeed in identifying drugs used in the treatment of gastrointestinal, inflammatory and dermatological complaints. The Plant kingdom is a virtual goldmine of potential drug targets and other active molecules awaiting to be discovered. It has been estimated that only 10 - 15 per cent of the 7,50,000 existing species of higher plants have been surveyed for biologically active compounds. Natural products produced by plants, fungi, bacteria, insects and animals have been isolated as biologically active pharmacophores. Approximately one-third of the top-selling drugs in the world are natural products or their derivatives often with ethnopharmacological background. Moreover, natural products are widely recognized in the pharmaceutical industry for their broad structural diversity as well as their wide range of pharmacological activities.

New medicines have been discovered with traditional, empirical and molecular approaches (Harvey, 1999). The traditional approach makes use of material that has been found by trial and error over many years in different cultures and systems of medicine (Cotton, 1996). Examples include drugs such as morphine , quinine and ephedrine that have been in wide spread use for a long time, and more recently adopted compound such as the antimalarial artemisinin. The empirical approach builds on an understanding of a relevant physiological process and often develops a therapeutic agent from a naturally occurring lead molecule (Verpoorte, 2000). Examples include tubocurarine and other muscle relaxants, propranolol and other adrenoreceptor antagonists, cimetidine and other histamine H₂ receptor antagonists. With the development of molecular biological techniques and the advances in genomics, the majority of drug discovery is currently based on the molecular approach.

2.1 Introduction

Dietary supplements from plant sources are sometimes referred to as phytopharmaceuticals. They are produced from fresh, dried or otherwise preserved plants or parts of plants. The active ingredients are usually not completely isolated but rather are obtained along with other naturally occurring components of the plant. These other components are often believed to influence the efficacy of the active ingredient. Sometimes the active ingredients are concentrated, and undesirable substances such as chlorophyll, tannins, or resins, are removed.

2.2 Cultivation

Most of the plants used for dietary supplements or medicinal purposes are cultivated, that is, grown in farms. Some, however, may be collected from the wild. Cultivation allows producers to have more control over quality and purity than does collecting plants from the wild. Cultivars (cultivated varieties) of a number of medicinal plant species have been developed to produce high yields of the desired constituents. Some plants that are grown commercially for medicinal purposes are propagated vegetatively (This means that new plants are grown from cuttings of old plants. Plants grown in this way are genetically identical to the parent plant). Some medicinal plants are grown from selectively bred hybrid seeds, while others are varieties of plants that are unchanged from their natural form.

Phytochemicals from various parts of a plant or tree such as the leaves, the flowers, the seeds, the barks, the roots and the shrubs can be isolated. Once one comes in from collecting plants in the field, it is good idea to freeze them immediately in a freezer at – 20°C or a commercial freezer held at – 80°C (Kaufman et al., 1999). According to Harborne (1984, 1998), fresh plant tissues should be used for phytochemical analysis and the material should be plunged into boiling alcohol within minutes of its collection. Alternatively, plants may be dried before extraction. It is essential that the drying operation is carried out under controlled conditions to avoid too many chemical changes occurring. Once thoroughly dried, plants can be stored before analysis for long periods of time. It is possible to isolate some of the compounds from herbarium samples. One example of the use of the herbarium material is the essential oil analysis that was carried out on type specimens of Mentha leaf, the material being obtained from the original collection of Linneaus made before 1800 (Harborne, 1998). An obvious point to follow by the research worker is freeing of the plant tissue under study from contamination with other plants. While collecting roots, care must be taken that we are collecting roots from one plant. It is essential to use plants which are free from disease, i.e. which are not affected by viral, bacterial or fungal infection. In phytochemical analysis, at some stage in the investigation, a botanist of an acknowledged authority must authenticate the botanical identity of the plants studied and a voucher specimen of the plant may be deposited in a reputed Herbarium for future reference. It is advisable to keep a logbook of the plants that have been collected and used for phytochemical analysis. A backup inventory on the computer is also a good idea.

4.1 Introduction

Extraction is a process whereby the desired constituents of a plant are removed using a solvent. The primary ways for extraction of organic molecules of interest to biologists and medical investigators involve breaking open the cells. Cell rupturing is carried out in a variety of ways. The method used depends on the type of tissue used. For plant cells grown in cell suspension culture or for plant callus tissue, a French press or a sonicator can be used to break open the cell. Plant cells grown in culture can also be ruptured with a glass tissue homogenizer. Silicified or highly lignified plant tissues within the organs such as roots, stems, leaves, fruits and seeds are usually frozen and pulverized using liquid nitrogen in a mortar and a pestle (Kaufman et al., 1999). Softer tissues can be ground in a small volume of buffer in a mortar, washed white sand and a pestle to rupture the cells.

When the cells have been ruptured, the extraction is performed using the appropriate techniques. Water soluble compounds and proteins are extracted in buffers or water. Organically soluble compounds are extracted with organic solvents. Boiling ethanol is a good all-purpose solvent for preliminary extraction (Harborne, 1998). The success of the extraction with alcohol is directly related to the extent chlorophyll (when isolating substances from green tissue) is removed into the solvent. The most common chemical procedure for obtaining organic constituents from dried plant tissues is to continuously extract powdered material in a Soxhlet apparatus with a range of solvents, starting with petroleum ether and chloroform (to separate lipids and terpenoids) and then using alcohol and ethyl acetate (for more polar compounds). This method is useful when working on the gram scale. However, one rarely achieves complete separation of constituents and the same compounds may be recovered (in varying proportions) in several fractions. The extract obtained is clarified by filtration and is then concentrated in vacuo in a rotary evaporator. To prevent fungal growth, a trace of toluene may be added to the concentrated extracts and they should be stored in refrigerator. There are short cuts in extraction procedures which one learns with practice. The concentrated extract may deposit crystals on standing.

5.1 Introduction

The different qualitative chemical tests can be performed for establishing profile of given extract for its chemical composition. The following tests may be performed on extracts to detect various phytoconstituents present in them.

5.2 Detection of Alkaloids (Evans, 1997)

Solvent free extract, 50 mg is stirred with few mL of dilute hydrochloric acid and filtered. The filtrate is tested carefully with various alkaloidal reagents as follows: