eChapters

1. Introduction

Lichens are the most fascinating and widely distributed organisms on earth. The lichens are unique in having two microorganism in a single plant- a phycobiont (alga) and a mycobiont (fungus) forming a thallus that does not resemble either symbionts in the free living (non- lichenized) state. By virtue of the peculiar structure and physiology, lichens have high tolerance of drought and cold and are able to grow in the diverse geographical regions from icy expanses to tropical and subtropical parts and from drier, hot deserts to moist humid climate. Lichens grow on any substratum that provides a convenient foot hold to them. This may be soil (terricolous), humus (humicolous), stones, rocks, brick (saxicolous), lime plaster (calcicolous), leaves (folicolous), tree trunk (corticolous), decaying wood (lignicolous) and other man made substratum like iron pipes, asbestos sheet and glass panes. Sometimes lichens also grow on some insects and animals. Lichens which are bigger in size and shape can be easily recognized as leaf like (foliose) and thread like (fruticose) (commonly called macrolichens), while those which forming a crust over the substratum and quite smaller in size are categorized under microlichens.

Apart from altitudinal variation, the vegetation and forest types of higher plants also play an important role in determining the type of lichen flora of the region. Based on the forest types six lichen vegetation zones are known in India such as: Moist tropical evergreen forest, Cold deserts in the Himalayas, South Peninsular region, Mid Eastern Indian & Peninsular Plateau, Dry and arid regions and Indo-gangetic plains of central India, Coastal regions of India and Andaman & Nicobar Islands. The cold deserts in the Himalayas exhibit some unique group of lichens having restricted distribution only in such habitats.

1. Introduction

The word ‘lichen’ has a Greek origin, which was referred to the superficial growth of fungus like organism on the bark of olive trees. Theophrastus, the Father of Botany coined the term ‘lichen’ during 300 BC and introduced this group of plants to the scientific world.

Lichen is a combination of two organisms, an alga and a fungus, living together in symbiotic association. Sometimes instead of an alga a cyanobacterium (blue-green alga) may be present in the lichen thallus. The algal component in the lichen is generally called as ‘photobiont’ as it contains photosynthetic pigment. The fungal part is called as ‘mycobiont’ (myco = fungus). The photobiont and the mycobiont loose their original identity in symbiotic association and the resulting organism is the lichen which behaves as a single organism, both morphologically and physiologically. Hence, the lichen is called as a composite organism. The lichen thallus is made 90% of mycobiont which provides shape, structure and colour to the lichen. In a lichen thallus whatever visible from outside is fungal part, which holds algae inside its body (Fig. 1) and therefore, the lichens are placed within the Kingdom - Fungi. The fungi present in lichens are called as ‘lichenized fungi’. Among the 20,000 lichen species present in the world, 98.9% of them belongs to Ascomycetes group while Bacidiomycetes and Deuteriomycetes groups are represented by only 0.1% and 1%, respectively.

1. Introduction

India, as a party to the Convention on Biological Diversity (CBD), has obliged to document a whole range of organism diversity within her territorial boundaries, make all attempts to conserve these bio-resources and monitor the efficacy of the conservation measures adopted. With its diverse ecological conditions and unique geographical location, Indian landmass supports rich diversity of algal flora. However, this algal wealth needs to be systematically surveyed and documented from all possible habitats. Ecological patterns emerged from recent studies within India are assessed with the identification of knowledge gaps and conservation implications in algal systematics.

During last one decade, great advances have been made in the field of Algology/Phycology throughout the world. Over the years algae has attained a greater reputation and popularity because of its economic importance, particularly as food, for commercial products, water contamination, toxin production, sewage oxidation, municipal water purification in fisheries and above all for the study of fundamental scientific information. But before the algae put in to such important use, it is imperative to have a baseline data about the material in question. Without knowing the status of algal resources of any area one cannot have projections for their utilization or gain knowledge of their ecological role and functions. The taxonomy of algae is, therefore, a prerequisite for all applied fields of algae apart from academic interest. Recent global surveys have revealed that there is lack of knowledge about fresh water algal diversity of tropical countries including India.

1. Introduction

The objective of algal taxonomic collections is to represent the natural population, flora and diversity in size and form. Therefore, it is important to collect the representative samples seasonally from various habitats. Collection of information about the habitats and other details are also very important. For algal sampling the first thing to consider is how to collect the specimens. The freshwater algae show an ability to tolerate a wide range of environmental condition. Under natural conditions, they usually grow in mixed communities which may include many species and genera. Field study provides information about the site, environment, season and size and shape of the living algal thallus in its natural surroundings. Many algae can be tentatively identified by their appearance, shape and colour of thallus in a particular habitat. Algal population can be sampled in a number of different ways, depending on the nature of the habitat and the location of the algae within the water body. The specimens must be prepared carefully so that important morphological characters are displayed as fully and completely for further researches.

1. Introduction

Bryophytes are atracheate, archegoniate non-flowering group of highly diversified plants. Among plants they form the second largest group comprising of 15000 – 25,000 species (Gradstein et al. 2001, Crum 2001). They occur in every continent on globe, in every location habitable by photosynthetic plants and grow luxuriantly between altitude of 1000-3500m. In India, nearly 850 species of liverworts, 41 species of hornworts and 2000 species of mosses, such as are known so far (Singh 1997, Vohra and Aziz 1997). These plants exhibit a variable range of size as some of them are only 0.5-2 mm in size, while on the other end mosses Polytrichum commune and Dawsonia superba can attain a height of 50 and 70 cm, respectively. These plants play a vital role in soil conservation and formation of fertile substrata for other plants in forest ecosystem. On account of unique moisture retaining capacity, Sphagnum and other pleurocarpous mosses are being used as mulching substances, moss grass, moss sticks and bags in horticulture industry. These plants are very sensitive to environmental pollution due to simple structure and devoid of cuticle, and accumulate heavy metals too, hence serve as good bio-monitor of pollution. Some novel compounds viz., marchantin, riccardin, lunularic acid, terpenes and other phenolic compounds have been detected in several thalloid as well as leafy liverworts.

Bryophytes exhibit heteromorphic alternation of generations in which the gametophytic phase (haploid phase) is a prominent and nutritionally self- sufficient independent phase in the life cycle while the sporophyte (diploid phase) is attached and dependent on the gametophyte. The main plant body (Gametophyte) is either thalloid or leafy (differentiated into axis and leaves). They are attached to substratum by simple hair like structures called as rhizoids. The rhizoids are usually unicellular, simple (smooth walled) in hornworts; simple, tuberculate or sinuate in liverworts or multicellular, oblique septate in mosses. Sex organs, antheridia and archegonia produce antherozoids and eggs respectively. After fertilization the zygote is formed which develops into the sporophyte. The sporophyte consists of foot, seta and capsule. The foot is embedded in the gametophytic tissue and draws nourishment for the development of sporophyte. Seta is generally stalk like may be of variable length which holds the capsule. The capsule is the main fertile portion of the sporophytic generation. The spore mother cells, after meiotic division, form spore tetrads having haploid spores. The elater mother cells form elaters, which are sterile and help in the dispersal of spores. Bryophytes are homosporous. Spores germinate to form young gametophyte in liverworts and hornworts, while in mosses, spores first give rise to protonema then buds develop on the protonema to form new gametophytes.

1. Introduction

Pteridophytes consist of ferns and their allies and are one of the oldest groups of plants on the earth, and they are commonly known as vascular cryptogams. Pteridophytes have a long history of evolution and were the dominant group of plants during the carboniferous period. The ferns, which constitute major element of the pteridophytes, preferentially grow in shade, moist habitats with moderate temperature but also occur throughout from high altitude in arctic-alpine to rain forests of tropic regions of the world. The most favoured habitat of these plants, however, is tropical mesic environment, where 65% of the living species of fernsthrive vigorously and grow luxuriantly (Page, 1979). Ferns are distinct in having rhizomes, stipes, fronds and are homosporous, whereas the fern allies exhibit scale like leaves and are heterosporous (mega and micro). In ferns, the spore matures into gametophyte that bears both male and female gametangia in a definite pattern. In fern allies, both the mega and microspore mature into female and male gametophyte that bears female and male gametangia, respectively. Pteridophytes are unique in having two different phases-the gametophyte and sporophyte. There has been a wide range of variation in spatial distribution, habitat, growth pattern and morphology of pteridophytes. Fern and fern allies, are comprised of six distantly related classes viz. Lycopodiopsida, Selaginellopsida, Isoetopsida, Equisetopsida, Psiloptopsida and Polypodiopsida (Copeland 1947; Pichi Sermoli 1987).

There are about 12000 species of the pteridophytes belonging to 300 genera in the world, whereas India is represented by about 1200 species (Chandra, 2000). Beddome (1892) during the first quarter of the twentieth century provided a comprehensive account on ferns of British India, and subsequently, Clarke (1880) and Hope (1899, 1900, 1902, 1903, 1904) made significant contributions in taxonomic studies of pteridophytes mainly the ferns of north western India. Several workers (Nayar and Kaur 1974; Dixit 1984; Chandra and Kaur 1987, 1994; Manickam et al. 1992; Khullar 1994, 2000; Chandra 2000; Singh 2003; Singh and Panigrahi 2005a, 2005b) have significantly contributed towards the taxonomy of Indian pteridophytes.

1. Introduction

The gymnosperms constitute a distinct subdivision of spermatophytes, the seed bearing plants. The term ‘gymnosperm’ is derived from Greek words, gymnos, naked and sperma, seed, which indicates that these are naked-seeded plants, i.e., those in which the seeds are not enclosed within the fruits but are borne freely exposed on an open carpel. In contrast in the angiosperms the seeds are enclosed within a closed carpel (ovary). The first known use of the term gymnosperm was given by Theophrastus in 300 BC in his book “Enquiry into Plants” to describe “plants with unprotected seeds”. Goebel (1905) rightly described gymnosperms as “Phanerogams without ovary”. The Scottish botanist, Robert Brown in 1825 first distinguished gymnosperms from angiosperms. At one time they were considered to be single class of seed plants, called Gymnospermae, but taxonomists are now tend to recognise four distinct divisions of extant gymnospermous plants - coniferophyta (Pinophyta), Cycadophyta, Ginkgophyta and Gnetophyta. Geological history reveals that all divisions of gymnosperms have been distinct groups since their origin.

Among the gymnosperms are plants with stems that may barely project above the ground and others develop into the largest trees on the earth. The extant gymnosperms are a small group of plants comprising ca. 1026 species belonging to 83 genera and 12 families (Christenhusz et al. 2011). They are distributed throughout the world, with extensive latitudinal and longitudinal ranges. Economically, gymnosperms are of outstanding value, fulfilling the greater proportion of our timber as well as resin requirements. They are also the dominant forest makers of the world and in addition they play a leading role in the preservation of the environment (Sahni 1990). By far the largest group of living gymnosperms is the conifers (pines, cypresses and relatives), followed by cycads and Gnetales (Gnetum, Ephedra and Welwitschia).

1. Introduction

Among three major functions identification, classification and nomenclature of taxonomy (Fig. 1), identification is a core activity and one of the major objectives of taxonomy, which determines whether other two elements are the same or different. Classification and nomenclature are two separate methods in taxonomy, however, both are totally dependent on the correct identification of the objects. The identification of an unidentified plant with a correctly identified specimen also involves classification. So, both identification and classification, involve comparison and judgment of similarities and differences. Therefore, the identification is a primary function in classification involving nomenclature which performs an essential role as a means of communication. According to Blackwelder (1967) “identification enables us to retrieve the appropriate facts from the system (classification) to be associated with some specimen at hand” and is “better described as the recovery side of taxonomy.” The identification of plants is very important for proper assessment and characterization of biodiversity on which the whole life of human beings is dependent starting from birth to death. The use of biodiversity for various purposes like food, nutrition, pharmacognosy etc. requires accurate and reliable identification and naming of plants (Nesbitt et al. 2010).

1. Introduction

Herbarium is simply a dried and pressed collection of plants arranged in an accepted sequence of classification (Lawrence 1951, Shetler 1969). It can be a very useful teaching aid or an absorbing hobby (Cronquist 1966). According to Fosberg and Sachet (1965) “A modern herbarium is a great filing system for information about plants, both primarily in the form of actual specimens of plants and secondarily in the form of published information, pictures and recorded notes”. It is also a research, training and service institution that serves as a reference centre, documentation facility and data store house (Radford 1986). It is now well recognized that a herbarium is a vast reservoir of facts about plants. The word ‘Herbarium’ was originally applied, not to a collection of plants but to a book dealing with medicinal herbs. Tournefort in about 1700 used the term as an equivalent to ‘Hortus Siccus’ which was later on adopted by Linnaeus (Stearn 1957). The present concept and development of herbarium is due to efforts of botanists for more than four centuries. The concept of herbarium has been changing continuously, which in turn, has affected the gradual development of herbarium and herbarium practices.

For practical reasons, the classification of the world’s flora is primarily based on herbarium material and the literature associated with it. Despite its limitations, a herbarium has certain advantages over living collections. It is usually only in the herbarium that we can compare all the related species of a genus in the same place, in the same state and at the same time (Davis 1961).

The herbarium material, therefore, formed the foundation of all botanical studies. We should, therefore, realise that herbaria are the custodians of enormous data pertaining to plants. They are usually seen as old-fashioned and expensive “stamp collection” without realising their paramount role in biological sciences. However, the world-wide rise of interest in the field of conservation, biodiversity and bioprospection offer a new chance for herbaria. Furthermore, computers offer a golden opportunity to raise the status of herbarium taxonomy by making routine curatorial procedures more efficient.

Although Jain and Rao (1976) elaborately discussed the methods of field collections, identification and preservation in the herbarium, the salient features of herbarium in up dated form with various procedures followed in Indian context are discussed in this chapter.

1. Introduction

The increasing size and activities of humans have mounted severe pressure on the survival of other life forms on the earth. This is a matter of great concern for the humanity. Therefore, a critical study to inventory all plant species has become very essential and urgent before they are lost or become extinct. In the face of extinction of many of these species, we need to learn as much about them as quickly as possible. In the recent years there has been a great concern for biological diversity and conservation of biological resources. Although much emphasis has been given to the ways and means for inventories (Flora) of biological diversity, there is also need for more in depth studies that deal with basic relationship of species. Monographic, revisionary and systematic studies that provide basic descriptive information about organisms and their relationship are required to document the information and knowledge about the plant wealth of a country. This chapter is to highlights the usefulness of the Flora and revisionary studies, and provides the relevant information to the beginners and the budding plant taxonomists about the preparation of an ‘Ideal Flora’ and ‘Creative Revision’ to meet the requirement of the present time.

1. Introduction

The scientific naming of plants is called botanical nomenclature. Although, nomenclature is included in taxonomy, but it distinctly differs from taxonomy. Plant taxonomy is concerned with grouping and classifying plants, however, the botanical nomenclature provides names for the results of this process. Names are a means of reference to all living and non-living things. The common names of pants and animals vary from place to place and language to language. But, scientific names of plants and animals are universal. For uniformity and consistency, the scientific names are given to plants according to certain internationally accepted rules provided in International Code of Nomenclature. It was Linnaeus who for the first time proposed some rules for generic names of plants in his Fundamenta Botanica, 1736 and Critica Botanica, 1737. However, the first proper set of rules of plant nomenclature was drafted by A. P. de Candolle and passed by the International Botanical Congress at Paris in 1867. The present Botanical nomenclature is governed by the International Code of Nomenclature for algae, fungi, and plants (ICN), which replaces the International Code of Botanical Nomenclature (ICBN) in 2012.

Botanical nomenclature has a long history, going back to the period when Latin was the scientific language throughout Europe, and perhaps further back to Theophrastus. The starting point for modern botanical nomenclature is Linnaeus’ Species Plantarum (1753). In the nineteenth century it became increasingly clear that there was a need for rules to govern scientific nomenclature and initiatives were taken to produce a body of laws. These rules were published in successively more sophisticated editions after every six years. The most recent is the Melbourne Code, adopted in 2012.

All formal botanical names are governed by the ICN, and within the limits set by that code there is another set of rules, the International Code of Nomenclature for Cultivated Plants (ICNCP). The latter Code applies to plant cultivars that have been deliberately altered or selected by humans and require separate recognition

1. Introduction

Plant taxonomy and the cultivated plants have a close relationship of great antiquity. This ancient relationship can be traced back to the invention of incipient agriculture (neolithic revolution), about 10-13000 years BCE (Stearn 1965; Balter 2007). The early foundation of plant taxonomy was thus laid largely on humans’ knowledge on cultivated plants. This body of rudimentary knowledge on identifying the properties of different types of plants for food, medicine, clothing, shelter, and other uses led to the development of plant taxonomy- the first aspect of plant science to emerge as botany (Constance 1957). The fundamental principles of modern plant taxonomy with regard to the natural vegetation and flora were originally derived from cultivated plants (Li 1974).

The active process of cultivation of a plant, primarily through the intentional activity of humans has been considered the main criterion for categorising cultivated plants (Mansfeld 1959). According to Mansfeld’s approach, the number of cultivated plant species of the world was estimated to be about 7000 species, excluding partly the forestry plants, ornamentals, and plants used in amenity horticulture (Henelt and IPK 2001). Khoshbakht and Hammer (2008) expanded this list to include a total of 35000 species of cultivated plants, including 28000 ornamental plant species connected to gardening and landscaping. About 2500 plant species have undergone domestication worldwide, with over 160 families contributing one or more crop species (Zeven and de Wet 1982; Dirzo and Raven 2003).The growing numbers of cultivated plant species and the enormous diversity found in each of these species offer plenty of challenges as well as opportunities to the taxonomists to take up cultivated plant taxonomy as an active scientific pursuit. Despite this, there has been a lack of interest among taxonomists towards cultivated plant taxonomy.

1. Introduction

Among the various well associated morphological units in plants, the reproductive units demand maximum protection which is achieved in pollen grains (also in spores) by encasing the germplasm with a well organized wall with a unique structure and ornamentation. Apart from its functional significance, the wall bears important characteristics which are of immense diagnostic and phylogenetic values. The morphological diversities in pollen and spore organization are greatly exemplified in the vascular plants. The term ‘Palynology’ was coined by Hyde and Williams in 1944 for the science of pollen studies, which include the study of pollen as well as spores. Although some investigators tend to limit the scope of palynology to morphological investigations but others include pollen analyses of peats, coals, honey, and rocks as well as the resulting conclusions drawn from this information, and in a wider sense it is also connected with the pollen chemistry.

The wall of pollen and spores is a very peculiar substance which is resistant to the destructive action of corrosive acids and alkalis and is readily preserved in nature under suitable conditions. The shape, size, and ornamentation or sculpturing of pollen has been the subject of study since the invention of the microscope. Pollen morphology is therefore greatly utilized in interpretation of ancient floras, for determining authentic species phylogeny, distribution and climatic conditions under which they grew, based upon the pollen content of the matrices. Pollen which lodges in moss tufts and lichen mats, or falls in water and gradually sink, or those that fall on sphagnum and clays particularly under anaerobic conditions, are well preserved. This pollen is not destroyed in the transformation of peats to lignite or clay in shale and thus has been preserved for hundreds of thousands of years.

During the present century pollen morphological studies are substantiated and elaborated magnanimously with the aid of Electron microscopy. The surface configuration and finer details are being accurately analyzed for their proper application in plant taxonomy as well as in microfossil diagnosis.

1. Introduction

Seeds have complex and remarkable diversity in shape, size, colour and morphological and anatomical sculpturing details of seed coat, which are valuable for taxonomic significance and can be used as an additional parameter combined with other parameters during taxa identification and classification. Distinctive seed coat morphology usually allowing identification of species even solely on the basis of seed characters. All members of a particular taxon possess variation within acceptable limits of their description and can be used for identification purposes. High structural diversity provides most valuable criteria for classification between species and family level. Takhtajan (1959) pointed out that even for the phylogenetic correlation between families and genera, the structure of the seed coat might be important. Most taxonomists agree that data concerning the structure and microstructure are of great significance mainly for the classification of angiosperm taxa. Consequently, in addition to vegetative and reproductive characters, seed characters are being used in most of the studies for the analysis of taxonomic relationship in wide variety of plant families. It was observed that seed morphology and anatomical features are rather conservatives, which makes them taxonomically important (Esau 1977; Barthlott 1984).

Gaertner (1788-1805) was the first botanist to use seeds and fruit morphology for comparison and identification of different taxa. Later on, Harz (1885) presented a comprehensive account of seed structures and their individual characters of different taxa. In the 20^th century interest in legume seed morphology was renewed. Capitaine (1912) studied the seed morphology of entire family and concluded that seed morphological characters are helpful in legume classification and identification at the tribal, generic and specific levels. After 40 years of Capitaine, Jensen (1998) documented 27 publications on seed morphology. Gunn (1981) summerized the seed characteristics for 510 legume genera. Seed characters of seventeen families were compared by Isley (1947). Duke (1961) indicated that seed shape, sculpturing and colour provide a critical indication of the systematic position of species of Drymaria (Caryophyllaceae). Obermeyer (1962) pointed out that the only reliable distinction between the two genera Chlorophytum and Anthericum of Liliaceae appears the number and shape of seeds. Hairy outgrowth on the testa, their length and colour provide useful characters in the distinction of genera/species in Malvaceae, Convolvulaceae, Asclepiadaceae and Acanthaceae. Gunn (1970, 1971) divided one hundred species of Vicia into major and minor groups on the basis of seed size, shape and length of the hilum, lens position and seed coat pattern and sculpturing. He successfully employed these characters up to specific level. Gunn and Gaffney (1974) studied the seeds of 42 economically important taxa of Solanaceae, including hilum and surface pattern as key character. Later on Gunn and Barnes (1977) had also used seed characters in the delimitation of Erythrina species. Seavey et al. (1977) studied the seeds of 210 species of Onagraceae, where seven seed groups were recognized and their evolutionary implications were also discussed. Matthews and Levinus (1986) demonstrated fairly constant seed characters in those species of Portulacaceae which have restricted geographical distribution, whereas the seeds of species with wider geographical distribution show varied morphological structures.

1. Introduction

The study of systematics and genetic diversity between or within different species, populations, and individuals is a central task for many disciplines of biological sciences. Systematic studies can indicate which genomes in the plant kingdom to search, sample, and study for the answers to questions relating to the evolution of chemical and physical structures and their synthesis or ontogeny as well as questions about the kinship of the individuals. Such studies would help find new and useful genes and provide information about the phylogenetic relationships among crop species and their wild relatives. The application of molecular markers has been the most active area of research in both animal and plant systematics. The analysis of the genetic variability within and among populations of the species is crucial for understanding their future maintenance and developing improvement as well as conservation programs.

Classical phenotypic characters, such as morphological traits, are still extremely useful but they can sometimes be influenced by environmental conditions. Therefore, during the past decade, classical strategies of evaluating genetic variability such as comparative anatomy, morphology, embryology and physiology have been increasingly complemented by molecular techniques. These include the analysis of chemical constituents (e.g., plant secondary metabolites) and, most importantly, the characterization of macromolecules. The development of molecular markers, which are based on polymorphisms found in proteins or DNA, has greatly facilitated research in a variety of disciplines such as taxonomy, phylogeny, ecology, genetics, and plant breeding (Weising et al. 1995). The greater utility of molecular markers derives from following five inherent properties that distinguish them from morphological markers.

1. Introduction

Biological systematics (biosystematics) deals with the grouping of biological organisms in discrete taxonomic units based on their character states. These character states are determined through morphological, anatomical, ecological, biochemical and molecular biological analyses of specific parameters. If these character states are converted into numerical parameters, we can apply several statistical and numerical methods to achieve appropriate biosystematic inferences. In fact this particular application of statistical and numerical methods has given rise to what we may describe as Numerical Taxonomy, a concept first developed by Robert R. Sokal and Peter H. A. Sneath nearly a half century ago. In recent years the application of numerical methods to taxonomy has received a great deal of attention with the development of the computer and computer technology to the extent that even classical taxonomists are now resorting to the use of such numerical taxonomy methods with the help of computers. In this article the various methods commonly used, their application and specific limitations or otherwise are discussed in the context of their usefulness for classification and taxonomic disposition of the plants.

1. Introduction

If we want to know what kind of plants and animals are in a particular habitat, and how many they are of each species, it is obviously (usually) impossible to go and count each and every one present. It would be like trying to count different sizes and colors of grains of sand on the beach. This problem is usually solved by taking a number of samples from around the habitat, making the necessary assumption that these samples are representative of the habitat in general. In order to be reasonably sure that the results from the samples do represent the habitat as closely as possible, careful planning beforehand is essential.

The usual sampling unit is a quadrat. Quadrats normally consist of a square frame, the most frequently used size being 20×20 m² for trees, 5×5 m² for shrubs, 1×1 m² for herbs. The purpose of using a quadrat is to enable comparable samples to be obtained from areas of consistent size and shape. Rectangular quadrats and even circular quadrats have been used in some surveys. It does not really matter what shape of quadrat is used, provided it is a standard sampling unit and its shape and measurements are stated in any write-up. It may however be better to stick to the traditional square frame unless there are very good reasons, because this yields data that are more readily comparable to other published research. (For instance, you cannot compare data obtained using a circular quadrat, with data obtained using a square quadrat. The difference in shape of the sampling units will introduce variations in the results obtained.)

1. Introduction

Plant diversity is vital for the survival and well-being of humanity. A number of domesticated plant species are critical to food security. In addition to the cultivated species, many wild plants also play an important role in meeting local needs for food, fuel, medicine and construction materials; crop wild relatives are also of special interest for crop breeding programmes. There are currently hundreds of underutilized plant species and varieties displaying traits of interest to meet present and future needs, while the value of many other plant species is yet to be discovered (Stohlgren et al. 1997). Researchers have confirmed the importance of the contributions and interdisciplinary understanding in relation to taxonomy, physiology, reproductive biology, conservation biology, hydrology, soils, as well as socioeconomic and climate change for holistic understanding of biodiversity patterns (Dale 1999; Myers et al. 2000). It is thus necessary that the professionals studying plant diversity require a synoptic view on the regulators of biodiversity and the tools through which the processes could be understood.

Spatial analysis can contribute significantly for improved understanding and monitoring of plant diversity. Results obtained from spatial analysis make it possible to formulate and implement targeted conservation strategies, which can be more effective (Escudero et al. 2003; Dale 1999). Outputs from spatial studies can provide critical information like evaluation of the current status of plant species and prioritize areas for conservation on the plant diversity, in particular geographic region. Spatial information, combined with available characterization and/or evaluation data, is very useful for effective genebank management (e.g. definition of core collections, identification of collection gaps, etc.). This type of analysis is conducted using Geographic Information System (GIS) tools, which allows one to carry out complex analyses combining different (spatial) data sources and generate maps, to facilitate the development and implementation of conservation policies (Goodchild 1992; Crosetto et al. 2000; McCarthy 2001).

1. Introduction

In the last few decades there has been worldwide revival on the use of herbal drugs/phytochemical for diverse purposes including medicinal, nutritional and as cosmetic. The revival of interest in natural drugs and the herbal products was due to the widespread belief that ‘green’ medicine is healthier than synthetic products. This has led to the rapid spurt of demand for health products like herbal tea, ginseng and such products of traditional medicine during the 1980s. The health promotion and disease prevention strategy in treatment is widely prevalent in oriental systems, especially the Indian (‘Ayurveda’, ‘Siddha’, ‘Unani and ‘Amchi’) and the Chinese systems of medicine, which are finding increasing popularity and acceptance in the world over. Because of this sweeping ‘greenwave’ a large number of herbal drugs and the plant derived herbal products are sold in the health food shops all over the developed countries. Export– Import Bank reports revealed that the global trade of plant-derived and plant originated products is around US $60 billion (with growth of 7% per annum) where India holds stake of US $1 billion which is expected to reach 3 trillion US$ by the end of 2015 (Pandey et al. 2008). These targets can be achieved by providing scientifically validated, safe and standardized herbal products in domestic and international markets.

Standardization is an important step for the establishment of a consistent biological activity, a consistent chemical profile, or simply a quality assurance programme for production and manufacturing of herbal drugs. It is now well known that the pharmacological activity of a medicinal plants is due to the presence of certain bioactive phytochemicals, which are either primary or secondary metabolites. Amount of these compounds are controlled and conditioned by a variety of factors such as its genetic predisposition, habitat of the plant agro climatic conditions, season and also the stage of growth and development of the plant etc. Therefore, it is desirable to study the effect of these on secondary metabolites to get the right/optimum amount of secondary metabolites in the plant part/raw drug. The formation of herbal drug based on the traditional knowledge using modern tools is the requirement of the present day. WHO’s specific guidelines for the assessment of the safety, efficacy and quality of herbal medicines as a prerequisite for global harmonization are of utmost importance. Considering the importance of the herbal drugs/products, quality assurance of raw drug/formulation and developing botanical and chemical parameters for assuring their quality, have become major riders on the pharma or drug research and industry.

1. Introduction

Ornamentals include plants which have either attractive foliage or flowers. They are used for various purposes like bedding plants in the garden or as cut-flowers and have huge commercial importance in terms of nursery business. New cultivars of ornamentals, which have been developed by various plant improvement techniques (hybridization, mutation breeding and colchiploidy) are always an added attraction to the plant lovers and a highly prized commodity for commercialization.

New cultivars of ornamentals usually inherit characters of both the parents singly or in combination and produce an entirely new trait. The progenies, thus, express a range of phenotypic characters. One has to screen these plants and identify elite plant(s) having desired phenotypic characters.

Second part of this process is characterization of the selected plants. Various characters of these plants are studied as per laid down procedures, which include vegetative, floral, seed and rhizome characters. The recorded observations are then compared with the parental characters to establish their distinctiveness. Subsequently, these plants are subjected to field trials for studying their uniformity and stability of the desired phenotypic characters.

New cultivars of ornamentals have enough potential for commercialization and good source of earning on the part of breeders who may be farmers, nurseryman, and researchers. However, for claiming right over the new cultivars developed, it is now mandatory to get those new varieties registered with appropriate agencies. The registration authorities ask for every morphological details as well as parental history. Therefore, characterization is a must for the newly developed cultivars/varieties.

1. Introduction

Medicinal plants play an important role in human life to combat various diseases. The rural folks and tribals in India even now depend largely on the surrounding plants/forests for their day-to-day needs. Majority of the medicinal and aromatic plants are still collected from wild for preparation of herbal drugs and perfumes/cosmetics. Rapid population growth and rising popularity of herbal drugs and natural essential oils have brought into focus the acute scarcity in availability of some of the plants due to indiscriminate and unregulated collection, habitat destruction through expanding agricultural lands, deforestation and urbanization. Fall in the supply of good quality, genuine raw material has resulted in price rise and deterioration in the quality of formulations. With growing demand and use of medicinal and aromatic plants, the important species have to be introduced into commercial agriculture.

The Indian annual turnover of herbal material has crossed Rs. 4000 crores mark, of which Rs. 300 to 400 crores worth is being the export market. The share of Indian trade in medicinal and aromatic plants is only 1.5 percent of the world market. India is one of the major exporters of crude drugs mainly to the developed countries like the USA, the UK, Germany, Japan, France, Switzerland, etc. The main crude drugs having good export opportunities are Aconite, Aloe, Dioscorea, Ephedra, Digitalis, Bach, Belladona, Cincona, Ergot, Isabgol, Opium, Vinca, Senna, Sarpagandha, etc. Of these senna leaves, isabgol husk/seed, and Cassia tora seeds are in maximum demand. China is the major producer of herbal plant material in the world. China earns US$ 5 billion per year from herbal trade besides meeting its domestic requirements. Though India has more potential of production of number of medicinal and aromatic plants than China, the contribution of India in the world market is very low because of the production of poor quality produce (Chadha and Gupta 2005).

1. Introduction

Present day botanic gardens have their origins from medicinal plant gardens established during the middle of the 16^th century meant for the cultivation of medicinal plants. Therefore, the roots of botany are traced from the medicine. The modern botanic gardens are considered as an ordered collection of plants, assembled primarily for scientific and educative purposes. They serve as the living repositories and offer excellent opportunities for ex-situ conservation, taxonomy, and studies on various other botanical aspects as mentioned below:

Plants are used for many purposes apart from food such as industrial products like perfumes, pharmaceuticals, agrochemicals, food additives, cosmetics and also for rectifying the side effects of allopathic medicines, etc. Interest was taken in the chemical constituents of plants in the very early days of modern science. Plants were used for very many purposes besides providing food, such as for treating medical ailments and for dyeing in textiles.

To the agricultural chemists, any biological organism is ultimately constituted from the elements like C, H, O, N, P, Ca, Mg, S etc. Two or more elements combine to form complex organic compounds such as carbohydrates, fats, proteins, enzymes, alkaloids, glycosides, organic acids and many more complex compounds. These biomolecules can be classified into two main major metabolites. (1) Primary metabolites and (2) Secondary metabolites.

Nowhere in the plant kingdom do ecology, evolution, and human affairs come together as intricately as phenomena involving secondary metabolites. The importance of these compounds is apparent in some of the earliest annals of human history. For example, Papaver somniferum, the original source of morphine and codeine, was known to the Sumerians in 4000 B.C. as hul gil (joy-plant). These two alkaloids, so important in human affairs for many years, probably evolved within the genus Papaver originally as protective compounds against herbivores. As a testament to the importance of secondary metabolites,

P. somniferum was one the first plants to be cultivated for a reason other than its caloric or nutritive value. Sadly, its importance has only increased over the time. Organic chemists of the nineteenth century were among the first to recognize and study the diversity of plant secondary metabolites and to analyze them both in terms of structure and function. The German organic chemists developed techniques to acetylate morphine and produce what they claimed at the time to be a non addictive version of the drug. However, acetylation product of morphine trade name Heroin, was vastly more addictive. The economies of several developing nations in Asia still depend on the acetylation of this plant secondary metabolite, and tens of thousands of lives each year are destroyed by its commercial importance. In another striking example of profitable secondary metabolites, acetylation techniques to salicylic acid, derived originally from the bark of willows, Salix, to create aspirin. The modern appreciation of these secondary metabolites from ecological and evolutionary perspectives had its spread outside of the plant-insect ecology community. The overwhelming array of chemicals found in plants was more than simply by-products of primary metabolism that served as waste products for these organisms that lacked excretory systems.

Ecologists and evolutionary biologists soon took up the cudgels, and the past 60 years have seen the development, testing, refinement, and, in some cases, rejection of models that describe the ecology and evolution of these “secondary” substances. With the functional roles of these compounds broadly acknowledged, several challenges presented themselves to scientists interested in their ecological and evolutionary dynamics. Most obviously, the details of the processes underlying the interactions between each of these compounds and their target pest or pathogen became one of the most vibrant topics in ecological and physiological research.

Plant cells contain far more compounds than are produced by the basic metabolism. Terpenes, waxes, alkaloids and pigments are just some key words that illustrate what is meant. The term basic metabolism comprises all pathways necessary for the survival of the cells, while secondary plant products are such that occur usually only in special, differentiated cells and are not necessary for the cells themselves but may be useful for the plant as a whole. Examples are flower pigments and scents or stabilizing elements. Both metabolisms are overlapping since it is often not understood why a certain compound is produced. Furthermore, no typical cell exists so that some compounds may be necessary for the survival of some cells but not for that of others.

Arguments in support of important functions for plant secondary products are underlined by the organizational complexity and specificity which characterizes many aspects of their biosynthesis. Furthermore – with the very important exception of substances produced in response to stress or pathogen attack – much of secondary metabolism is reproducible from one individual of a species to another and, to a remarkable degree, produces the same principal compounds in broadly the same relative proportions. For example, an Atropa plant extracted for alkaloids can confidently be predicted to contain predominantly atropine, rather than its immediate precursor, tropine, or a more distantly-related alkaloid, nicotine – just as the passenger boarding a train for Mumbai at Anand can be reassured that his train will not terminate prematurely at a “precursor station” (Surat), or even adopt an “alternative pathway” (to Poona). On the other hand, the evolutionary circumstances leading to the development of atropine formation in Atropa, rather than to the formation of other alkaloids with comparable activity (for example, nicotine) remain substantially unknown. A similar question has been discussed regarding two families of plant pigments with apparently similar functions, the anthocyanins and the betalains. With regard to defense (and similarly but perhaps less obviously protection against ionizing radiation), a general hypothesis argues that plants evolved a broad defensive strategy based upon a diversity of antimicrobial and anti feedant substances from which, as a result of chance mutation, more potent substances conferring additional selective advantage from time to time arose. It is conceivable that in some cases enzymes evolved or became adapted to catalyse reactions of benefit which could already occur to some extent (though in a non-stereospecific manner) non-enzymically. Mutation will then have led to the development of modified enzymes able to catalyse reactions with new or different substrates, or to catalyse alternative reactions with existing substrates.

The functional diversity of chemicals within plants is best demonstrated by terpenoids. More than 30,000 terpenoids have been identified, each one composed of 5-carbon (prenyl diphosphate) building blocks added together to make 10-carbon, 15-carbon, and even 2000–500,000-carbon chains, as is the case for rubber. Terpenes are functionally diverse, and many are integral to primary metabolism, e.g., hormones such as gibberellins and abscisic acid, electron carriers such as plastoquinone and ubiquinone, terpene-derived compounds that form structural parts of membranes such as phytosterols, and photosynthetic pigments such as carotenoids.

Plant terpenes are also available as essential oil or ethereal oil. Essential oil is defined as the volatile oil obtained by the steam distillation of plants. There is a distinction between fixed or fatty oils and volatile oils. Their volatility (boiling point) and plant origin are characteristic properties of these oils.

The alkaloids represent yet another large group of secondary plant products. Many, but not all, exhibit important pharmacological properties & some of these have been known for hundred of years. For e.g. Cinchona alkaloids, present in bark of Cinchona spp. have quinine as their main constituent, has been known since 1639 as an effective antimalarial agent.

An alkaloid contains nitrogen, frequently as a part of a heterocyclic ring, and most are as the general name implies, basic. Alkaloids are the naturally occurring basic, nitrogenous, organic, heterocyclic ring compounds mostly of plant origin with physiological and pharmacological properties. At present, the definition is much more pragmatic and includes all nitrogen containing natural products which are not otherwise classified as peptides, amines, non-protein amino acids, cyanogenic glycosides, glucosinolates, cofactors, phytohormones or primary metabolites (such as purines and pyrimidines bases). Even a number of antibiotics produced by bacteria or fungi are therefore included as alkaloids. Alkaloids evolved early in evolution and were already present (200 million years ago) when the angiosperms began to radiate. Today, more than 12,000 alkaloids are known to occur from more than 4000 species of Angiosperms. Ladenberg defined alkaloid as :

“Naturally occurring basic, complex, nitrogenous organic, heterocyclic ring compound of plant origin with physiological and pharmacological properties.”

Glycosides are compounds containing a carbohydrate and a non carbohydrate residue in the same molecule. They are the complex polysaccharides which are chemically heterogeneous collection of substances, which have in common one property that the molecules are linked together and contain at least one sugar unit. The term glycoside represents a group of compound containing glucose and one or more basic or acidic organic compound in chemical combination. When the sugar other than glucose is present, it is called glycoside. Each sugar is linked through a glycosidic bond to either a non sugar component, aglycones, (aglycone or aglucone is the nonsugar molecule in glycosides).

The plant gums are the neutral salts and complex polysaccharide acids which contain methyl pentose, pentose, hexose and uronic acid residues joined glycosidically by linkages involving various carbon atoms within one molecule to form branched chain structure. Uronic acid is generally present in all cases. The sugars include L-fructose, D-xylose, L-arabinose, D-rhamnose, and D-galactose.

General Properties of Gums

They have high molecular weight. Some are linear while some are branched. They are generally viscous in nature. Some forms a gel also. They have tremendous commercial application in food industry. Gums may be an exudates gum produced by the plant or may be a seed gum, which is accumulated during the seed maturity. Gums of polysaccharide origin are obtained from Acacia spp. (gum Arabic), Cymopsis tetragonolobus (guar gum), Guggle gum, Ficus indica, chia gum etc (Table 7.1).

Plant oleoresins, often referred to as resins, are sticky plant secretions synthesized in large quantities by about 10 % of plant families. Resin is secreted when a plant is disturbed and the sticky substances produced contain many toxic compounds used to defend the plant from herbivores and pathogens. A major component of resin is the terpenoid compounds. Although terpenoid compounds can be quite complex, they are all composed of five-carbon units, with an isoprene unit as the base. The simplest of these terpenes are the monoterpenes that possess 10- carbon atoms derived from two fused isoprene units. Monoterpenes are an important component of the volatile portion of terpenes and often emit very strong odors.

The term rare amino acid is misleading in some respects. It refers to all those amino acids that are not incorporated into proteins. We already got to know some of them like ornithine or citrulline that are rather common intermediates of the basic metabolism. Roughly 220 different structures are known, most of which occur in plant cells in a free state though glutamate-, oxalate- or acetyl- derivatives can also sometimes be found.

Isolation and Purification of Secondary Metabolites

Isolation

The act of isolating something; setting something apart from others.

Purification

The process of making pure, free from anything that debases, pollutes, or contaminates, the process of removing impurities.

Role of Biotechnological Approaches

In our fore-going chapters, we have studied variety of plant products such as alkaloids, glycosides, volatile or essential oils, gums and mucilage and oleoresins with varied role to play to the plants as well as to human.Knowledge of plants and knowledge of healing have been closely linked. It is well established that industries have many direct and indirect effects on human population. Increased stress is most evident at the same time health awareness goes parallel in the recent times. Most of the natural plant products are compounds biosynthetically derived from primary metabolites such as amino acids, carbohydrate metabolic intermediates and fatty acids. These secondary metabolites are the major sources of pharmaceuticals, food additives, fragrance and pesticides.

Application of biotechnology know how helps to synthesize the novel molecules and also could be beneficial to enhance the production of secondary metabolites. Classical definition of biotechnology states that it is “The use of living organisms or their products to enhance our lives and our environment.” The word Pharming is commonly use to describe genetically engineered plants or livestock to produce medically useful products –predominantly protein drugs. Plants or animals are genetically engineered to contain genes capable of producing a biologic or drug compound. Therapeutic proteins are designed to treat illness includes serum proteins, cytokines, growth regulators, anti coagulants, antbiotics, lysosomal enzymes and plant secondary metabolites.

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:

6.1 Introduction

All separation procedures depend mainly on some physical characteristics of the compounds. It is relatively easy to separate compounds that have significantly different physical characteristics by simple techniques such as solvent extraction. If the various compounds are similar to each other in their molecular size, any slight difference in any one of their physical properties is exploited to achieve separation. The separation and purification of plant constituents is mainly carried out using one or other, or a combination of chromatographic techniques: Paper Chromatography (PC), Thin Layer Chromatography (TLC), Column Chromatography (CC), High Performance Liquid Chromatography (HPTLC), Optimum Performance Laminar Chromatography (OPLC), Gas Liquid Chromatography (GLC) and High Performance Liquid Chromatography (HPLC). One further technique, which has fairly wide application in phytochemistry, is electrophoresis.

7.1 Introduction

For qualitative and quantitative analysis of phytochemicals, chromatography can be used. Gas chromatography is a powerful means of performing qualitative and quantitative measurements of complex mixtures of volatile substances, which are vaporizable without decomposition.Gas Chromatography-Mass Spectrometry (GCMS) system is a widely used instrument for qualitative and quantitative analysis of solid, liquid, and gaseous samples. Samples are converted into gaseous ions and then separated on the basis of their mass-tocharge ratio. This technique is capable of providing information about 1. the qualitative and quantitative composition of both inorganic and organic analytes in complex mixtures; 2. the structure of a wide variety of complex molecular structures; 3. isotopic ratios of atoms in samples; and 4. the structure and composition of solid surfaces. High performance liquid chromatography (HPLC) is a physico-chemical separation technique for the qualitative and quantitative analysis of mixtures consisting of compounds soluble in solvents, e.g. all organic compounds and inorganic ions. Advances in the applications of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) for the separation, detection, and qualitative and quantitative determination of compounds has also increased.

8.1 Introduction

After isolation and purification of the compound, it has to be identified. It is necessary to find out the class of the compound first and then to find out the specific compound within the class. The class of compound is usually determined from the colour tests, solubility, Rf properties and UV spectral characteristics of the compound. Further identification within the class depends on measuring physical properties and spectral measurements and then comparing these data with those in the literature. The physical properties include melting point (for solids), boiling point (for liquids), optical rotation (for optically active compounds) and Rf. The spectral measurements include Ultra Violet (UV) , Infra Red (IR) , Mass Spectrum (MS) and Nuclear Magnetic Resonance (NMR). Data on X – ray crystallography of the compound are also useful for identification of the compound.

On completion of the analysis of the spectra obtained, the pieces that have been identified are listed. Then the pieces, the MW and / or molecular formula are checked and the pieces are refined to fit. Finally, the pieces are assembled paying particular attention to ¹H-NMR chemical shifts and coupling patterns. The answer should be checked with the ¹H-NMR data very carefully and it is probably the most critical test to pass. The ¹H-NMR spectrum is potentially the most useful for assembling the structure of the compound.

A known compound can usually be identified on the basis of the above mentioned physical and spectral characteristics. For final confirmation, direct comparison with authentic compound (if available) should be carried out Authentic compounds can be obtained from commercial companies. In the absence of authentic compound, the data from literature can be carefully compared for the identification of the isolated compound. If a new compound is present, all the above data are sufficient enough to identify it. However, data on chemical degradation or laboratory synthesis of the compound are necessary to confirm the identification of the new compound.

9.1 Introduction

Phytochemicals are the individual chemicals from which the plants are made. Phytochemical is simply a word that means plant chemicals. Plants have the ability to synthesize mixtures of structurally diverse bioactive compounds with multiple and mutually potential therapeutic effects. The plants have the capacity of manufacturing the secondary products. Phytochemicals with antioxidant properties tend to be brightly colored because they contain chromophores, ie, a series of alternating single-bonded and double-bonded carbons. Isoprene is often the building block of such units. The darkest green vegetables contain the most chlorophyll, and vegetables with the most chlorophyll require the most antioxidants. Green will mask the other colors, when other-colored antioxidant phytochemicals are present. Hundreds of phytochemicals are currently being studied. Many are believed to have a major positive impact on human health (Duke and Sternberg, 2005). Important plant secondary metabolites have been isolated over a period of time from natural sources. The phytochemical may belong to the following categories : Terpenoids, Phenolic compounds, Alkaloids, Glycosides, Carbohydrates, Lipids, Proteins, Nucleic acids, etc. Some of the phytochemicals from the plants are listed in Table 9.1 and structures of some of them are given in Fig.9.1.

10.1 Introduction

The following plants have been worked out in the laboratory of Prof. N. Raaman: Cynodon dactylon, Artemisia pallens, Leptadenia reticulata, Clausena dentata, Tylophora indica, Aristolochia bracteolata, A. indica, Cissus quadrangularis, Hypericum mysorense. and Solanum trilobatum. Out of some of the data of these plants have been included in the previous chapters. In this chapter, compound isolation (CD1) from Clausena dentata (worked by Dr. R. Kamaraj) and characterization of the compound are given in detail.

10.2 Preparation of extracts from Clausena dentata

The dry powder of stem bark (2.5 kg) was first soaked, at room temperature, in hexane (1:4 w/v) for 24 h. The extract was suction filtered using Whatmann filter paper. This is repeated for two more days and similar extracts were pooled together and concentrated at 40°C under reduced pressure using Buchi R - 153 Rotavapor. The residual plant material was extracted successively with chloroform and methanol in the same manner as followed for hexane. All the concentrates were subjected to column chromatography to isolate the active principles.

Part A

1. Pesticides

A pesticideis defined as anysubstance intended for preventing, destroying, attracting, repelling or controlling pests including unwanted species of plant, or animals duringthe production, storage,transportation, distributionand processing of the food, agricultural commodities or animal's feed or which may be administered to the animals for the control of ectoparasites. Pesticides include insecticides, herbicides, fungicides, acaricides, nematicides, etc.

According to food and agricultural organization (FAO) 1986, the term pesticidehas been defined as“any substance or mixture of substances intended for preventing or controlling any pest and includes anysubstance or mixture of substances intended for use as plant growth regulator (PGR), defoliant or desiccant. The term excludes fertilizers and antibiotics or other chemicals administered to animals to stimulate their growth or to modify their reproductive behaviour”. Pesticides are the largest group of poisonous substances that are widely broadcast today.

The knowledge on the fate of pesticides, after application in the environment is useful to research workers associated with their safe and effective use. It is important to know whether a xenobioticwillpersist orwill be inactivated. Frequently persistence is desirable for sustained control. However, for many cases, rapid detoxificationof the compound may be required to avoid cumulative effects. Most of the pesticides are not very soluble in water andtheir oxidation orhydrolysis (or both)leads to formation of polar groups. Such reactions are called primary metabolic reactions, often, but not always. Theproducts ofthese primary reactions undergo secondary changes whereby they are conjugated with endogenous substances to form molecules that are more readily excreted. In higher animals, these changes facilitate the transportation of metabolites of pesticides in blood, which in turn leads to their eventual excretion.

After having performed their function, pesticides in the parent form or in some converted form reach the soil, the ultimate sink for these moieties which are collectively termed as residues. According to FAO 1986, pesticide residue is defined as any substance or mixture of substances in food, agricultural commodities or animal feed resulting from the use of pesticides and includes any specified derivatives such as degradation products, metabolism, reaction products and impurities that are considered to be of toxicological significance. From the soil they move into other segments of the environment. The pesticide residues are absorbed by plants and enter the food chain, where they get concentrated in animal fat. Some residues are carried away by wind in the vapor form, which enables them to be transported to long distances from the source of application and then get collected by dust and rain before they are deposited on the ground again. Pesticide residues also move to aquatic environments through runoff, leaching, and atmosphere disposition.

Chromatography is an important analytical technique for the separation, isolation and identification of the constituents of a mixture. It is a physical method of separation in which the components to be separated are distributed between two phases; one of these phases consisting a stationary bed of large surface area, the other being a fluid that percolates through or along the stationary bed. All forms of chromatography do have the common features given in this definition. All of them involve the physical movements of the sample across a flat surface (tube like) or through a tube. The stationary phases in all of these possess a large surface area. In all the techniques, the movement of the sample is due to the motion of the fluid/mobile phase. The principle underlying all these methods is the selective retardation of the movement of the components by the stationary phase. These methods result in the production of bands of concentrated components. The basis of naming various chromatographic methods are different:

Chromatography was first employed by Ramsay (1905) to separate mixtures of gases and vapours. The first scientist to recognize chromatography as an efficient method of separation was the Russian botanist Tswett (1906), who used a simple form of liquid-solid chromatography to separate a number of plant pigments. The colored bands he produced on the adsorbent bed evoked the term chromatography for this type of separation (color writing).Althoughcolor haslittletodo with modern chromatography, the name has persisted and, despite its irrelevance, is still used for all separation techniques that employs the essential requisites for a chromatographic separation,viz. a mobile phase and a stationary phase.

Gas-liquid chromatography (GC) was invented by James and Martin (1952) and is a separation technique in which the mobile phase is a gas (usually helium or nitrogen) and the stationary phase is a liquid. In the original columns used by James and Martin, the liquid stationary phase was adsorbed on the surface of an inert support such as Celite (a diatomateous earth) or calcined Celite (a form of brick dust). The support was usually deactivated before use by acid treatment and subsequent reaction with hexamethyldisilazane. The technique was extensively used for the separation of a wide range of volatile substances. Thus, chromatography has been defined as a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. As a consequence, solutes are eluted from the system as local concentrations in the mobile phase in the order of their increasing distribution coefficients with respect to the stationary phase.By separating the sample into individual components, it is easier to identify (qualitate) and measure the amount (quantitate) of the various sample components.

High performance liquid chromatography (HPLC) was developed during late 1960s and early 1970s. Today it is a widely accepted separation technique for both sample analysis and purification in a variety of areas. HPLC, basically a highly improved form of column chromatography and popularly known as liquid chromatography, like other forms of chromatography, whether paper, thin layer or gas chromatography, needs two essential components, the stationary and the mobile phase. It has the unique ability to separate and quantitate residues of polar, non volatile and heat labile chemical compounds. HPLC uses high pressure pumps up to 400 atmospheres that makes it much faster, short and narrow columns packed with micro particulate phase and a detector. To affect a separation, mixture to be analysed is dissolved in a solvent (ideally this solvent is identical to mobile phase) and then forced to flow through analytical column along with mobile phase. In the column the constituents of mixture get resolved and are detected with a suitable detector by a proper choice of column, mobile phase and detector. HPLC is a technique having high degree of versatility. There are several excellent texts published over the last decade which may be consulted for general information on the instrumentation in liquid chromatography

The phenomenon and behavior of supercritical fluid (SCF) has been the subject of research right from 1800's and suggestion of supercritical fluid chromatography (SFC) came in to exixtence in 1958. Supercritical fluid chromatography (SFC) was first developed in 1960, and that time, it was considered as a science fiction chromatography and a revolutionary separation technique. But its reputation has slowly ebbed over the years and it is now moving forward by leaps and bounds as a stable analytical method with many advantages over the existing chromatographic methods. It is an unit operation that exploits the high dissolving power of fluids at temperature and pressurre above their critical values for extracting analytes from sample matrices.The first commercial packed column of SFC was made available in 1981 and the first commercial capillary column SFC instrument was introduced in 1985. Several refrences are useful for understanding the super critical fluid extraction and super critical fluid chromatography