Developments in Phytochemistry

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Introduction
Carbohydrates, proteins, fats and oils are utilized as food by man and animals. Other chemical compounds in plants apart from these listed above are phytochemical. Such compounds usually exert peculiar, unique and specific active physiological effects responsible for their therapeutic and pharmacological functions. Activities of such naturally occurring compounds are generally responsible for changes, which are utilized to satisfy man's desires. Phytochemical studies afford revelation and understanding of phytoconstituents, as much as possible conserving their bioactivities, and are on how to standardize them; compared with the crude herbal methods that are not easily standardized. These complex substances of diverse nature occur mostly in plant based foods; they are in very small amounts in grams or mg or μg/Kg of samples. They do not add to body calorie and are numerous in types. These phytochemical are applied mostly for preventive and healing purposes. About 25% of prescribed drugs are obtained from phytochemical in higher plants. Plants are safe means of obtaining drugs. About 250,000 higher plants have promising phytochemical, half of which are located in tropical forests; 60% of these have their biological activities established, while about 15% of them have their phyto-compounds isolated and reported [Hamburger and Hostettman, 1991].
Studies and researches into medicinal constituents of plants, involve qualitative and quantitative analyses. There is rationale behind each experimental work involving definite steps and processes; having in mind properties of compounds analyzed in conjunction with procedures utilized. Also our desired active metabolite to be isolated and studied as interested lead compound, many times is in very complex mixtures of many unwanted and undesired materials [known as contaminants], which have close properties to our desired bioactive molecules. the aim of isolations, characterizations and better establish bioactivities of active metabolites.
Phytochemical methods mainly involve EXTRACTIONS, PURIFICATIONS and ISOLATIONS of the active compounds in plants. Procedures are ways of carrying out the methods and techniques. There are numerous methods some specific for interested compounds one is looking for, duly modified to meet the required aim and focus of work. There are daily modifications of techniques and procedures to suit individual purposes of having the phytochemical compound(s) of interest. It is important to say that some natural product may (to variable extent) or may not possess their pharmacological properties and activities when in isolation compared to when among mixture of compounds (synergy) in the natural setting in organism. Recently in genomics whole plant is analyzed which afford easier and truthful analyses of contents in the whole plant as in their natural state.
It is important to first establish proper botanical taxonomic identifications and classification of plant of study. Scientific names must be established, common and local names must be sought. Right choice of study plants or part of plant to study may be from local or traditional surveys i.e. ethno-medicine, ethno-pharmacology or ethno-botanical uses and applications. Geographical location and environmental effects [time and period of plant collection], must be considered also, which may be responsible for variations. Voucher samples of plant of study may be filed in local and national herbaria for accurate authentications. Usually plants are richer in active metabolites during their flowering and fruiting stages [Mendonca-Filho, 2006].
Procedures involve first the analytical stages. Most times our desired active metabolite to be isolated and studied as interested lead compound, is in very complex mixtures of many unwanted and undesired materials [known as contaminants], more so they usually have close properties to our desired bioactive molecules.
Preliminary tests and screenings on plant extracts are faster and easily done following standard procedures and methods in manuals and literature. They detect the presence and amount of basic phytoconstituents like terpenoids, alkaloids, flavonoids, saponin, glycosides, steroids, tannins, phlobatannins and anthraquinones to mention few.
More common and familiar separation and isolation techniques in phytochemical studies are distillation, crystallization, solvent extractions, continuous and liquid-liquid extractions, partitioning using separatory funnels, and chromatography. For accuracy characterizations follow side by side with the above techniques. Bioactivities can also be tested along the above, such as antibacterial, antifungal and antioxidant. The followings are important to consider during choice of procedures for separations and isolation of interested biomolecules of interest: • Availability of necessary materials, equipments and chemicals. Also economically cheap methods.
• Ease and simplicity of procedures, and risk involved.
• Compatibility of interested solute with phases or solvents such as it distilling out [more volatile phyto-compound], crystallizing and recrystallizing out from phases/ solvents etc.
• Possibility of retrieval methods. www.intechopen.com • Selectivity and sensitivity of chosen method(s) and equipments.
• Feasibility of chromatographying out our desired biomolecules from stationary phase using appropriate and suitable solvent or mixture of solvents for elution. Also consider factors like polarity, temperature, agitations etc.
• Provision of rapid online information on activities and structures of the phytocompound. [Specific methods can be better studied by consulting literature and appropriate textbooks].

Isolation, characterization and identification of phytochemical in natural products
Phytochemical are active metabolites that necessarily require extraction and isolation from their natural sources with many unwanted materials. The phytochemical can come singly or as a mixture of important substances to form active principle responsible for its activity (synergy). When singly active, the processes of their separations are of great practical advantages, which in many cases the isolated phytochemical have better and higher activity. We will consider genomics and metabolomics later as more efficient methods of rapid phytochemical screening and characterizations of plant extracts to study their chemical constituents, using NMR-based metabolomics. It utilizes mathematical data; NMR is used directly on extracts before commencing detai l e d w o r k o n p l a n t . I t m a k e s i t e a s y t o determine which plants are more promising to research into. A large number of variables are collected, then choices on which are important are made, followed by selection procedures.

More modern spectroscopy utilized in phytochemical studies
Once preliminary separations and detections have confirmed presence of active secondary metabolites, their characterizations as they are separated follows. Chromatographic techniques are utilized in separations and purifications to isolate bioactive constituents based on polarity or other gradient factors. The isolated compound is characterized by spectroscopic methods. The four basic types of spectroscopy are utilized in the characterizations of purified natural product compounds. They are ultraviolet (UV), infrared (IR), mass-spectroscopy (MS) and nuclear magnetic resonance (NMR) techniques. MS is an instrumental technique, while the other three utilizes different parts of the broad electromagnetic radiation spectrum. UV spectroscopy discovered and utilized in the 1930s gives detailed information on detecting presence of conjugation in molecules and the extents of conjugation. NMR is a type of absorption chromatography which reveals connectivity of nuclei in the metabolite. Superficially and most common, 1 H and 13

Techniques of establishing phytochemical bio-activities by bio-assay
Bio-assay of extracts or fractions and bioactivity guided fractionations are important and are major steps in phytochemical studies. Bioassay combines biological and chemical screenings to obtain important information on and about plant constituents and chemical compositions. It investigates, establish and estimate biological activities of biomolecules, involving chemical screening techniques. The amount of material to be tested is important www.intechopen.com determinant of method to be used. There are many methods of in-vitro assays for assessing different activities like antimicrobial or cytotoxic activities. Such have advantages of easy automations with robotics and miniaturized techniques resulting in rapid through-put screening of large numbers of samples. They are more common as their materials are easy to get. Other assays which utilize affected organisms or living cells directly, give more reliable results, though may not be cheap. Such include use of brine shrimps, ants and insects like Drosophila sp., cell lines in different media, tissue culturing, ligand bindings, use of rabbits and rats. Assays may identify promising molecular structures.
Usually many biological activities are screened and tested during a particular bioassay. It is important to note that results of bioassays are not strong enough to establish uses and dosage of compounds found to be bioactive; also they cannot replace pharmacological discovery and establishment of potent drugs in development of lead compounds to consumables and marketed substances. They may be seen as alternatives. Also one must be extremely careful when interpreting in bioassays to get results, especially in cases of clinical studies and investigations. It is best and more reliable for effective results to perform and run bioassays alongside with chemical separations and characterizations. These in modern times are achieved by using hyphenated processes. Particular constituents of extracts or plants as they are separated are characterized as well as assessing their activities side by side. The information obtained from the bioassay and chemical analyses (separations and characterizations) give full description of the bioactive compound, and afford easy and appropriate detection of specific targeted bioactive metabolite(s).
To get pure constituents, modify structures, and carry out toxicological tests bioassay results are very important.

General biosynthetic relationships between primary metabolites with interlink precursors and secondary metabolites
The whole plant or organism serves as an active laboratory for the production of natural products from primary metabolites such as proteins, amino acids, carbohydrates, fats and oils, which are mostly obtained from food items. The primary metabolites are basic biological molecules also called biochemist molecules, which are functional compounds found virtually in all plants and organisms. Secondary metabolites are varieties of simple to sophisticated bizarre molecules also called natural products. They are fascinating chemical molecules, very useful and of great importance in nature, as well as highly diversified in structures, properties, uses, chemistry etc. These varied properties and characters emerge from their biological generation, production and formation from basic primary metabolite sources and origin. Natural products are in restricted taxonomic groups and species of organisms. They are from secondary metabolic processes and express individualities of organisms. These are the areas of interest in phytochemistry and pharmacognosy.
We will be examining the underlining principles behind formation, production and generation of natural products syntheses in plants. Primary metabolites are first formed in the first phase (primary metabolism), which is followed by secondary metabolism processes to give the more sophisticated and complicated more specific secondary metabolites.
Successive enzymes which are proteinous organic biocatalysts are utilized in catalyzing specific metabolic reactions and processes, all coded by specific genes in plant's DNA in the www.intechopen.com nucleus which controls all activities leading to creation of new substances and new organisms. Organelles in cells of the plant carry out specific biochemical functions. Transcriptive processes are involved to get particular enzymes; common reactions in the plant include syntheses, breakdowns, isomerizations, cyclizations, regulations, hydrolysis etc; the key energy molecules in the cell are ATP, ADP, AMP, GRP and derivatives. There are many biosynthetic pathways occurring in plants, initial pathways such as carbon-reduction cycles, pentose phosphate pathways, glycolysis, Krebs cycle, shikimic acid pathway and tricarboxylic acid cycles lead to biosyntheses of primary metabolites, which are precursors of the diverse secondary metabolites. There is need for continuous supply and flow of energy for the ordered transformations of substances in cells. Metabolic activities in specific pathways occur vegetative in cells producing precursors for components of cells to further react and produce simple to complex natural product metabolites. Generally biosynthetic procedures can be viewed as starting (primary metabolism) from biosynthetic activities to produce carbohydrates. From it more complex metabolites are formed. Main metabolites as precursors of specific secondary metabolites include fatty acids and lipids to give the polyketides, amino acids and sulphur containing metabolites to form the peptides and alkaloids, phenyl propanoid and cinnamic acid metabolites, isoprenoids which yield terpenoids, carotenoids, steroids etc. There are now genomic approaches to studies of biosyntheses of natural products, which will be discussed shortly.

Importance of the phytochemical to plant producing them and man
Plants are energetic organisms that carry out specific oriented processes to produce useful compounds. They do not waste time to form substances that have no use to the plant. The wide categories of phytochemical produced have their importances to the plant generating them. Some of these are as follows- www.intechopen.com 8. Inulins and fructans are soluble polysaccharides made mostly of fructose sugar with some glucose in the chain. Hydrolyses of the stored fructans in plants at spring, provide energy to plant for commencement of its growth in early spring. 9. Lignin are hydrophobic complex polymers in secondary cell walls made of units of aromatics like phenylpropanoids, coumaryl, coniferyl and sinapyl alcohols via shikimic acid pathway. It provides additional supports with rigidity, impermeability to water and prevent water loss from plant, also give compressive strength to cell walls. Ligins resist intrusion of herbivores, but prevent growth and bending of plant tissues. 10. Anthocyanins are flavonoids responsible for colored pigments like blue, red, pink, purple in plant parts like flowers, fruits, stems, leaves, roots, seeds etc. They are flower attractants for birds and insect pollinators, as well as attractants to animals and birds that disperse their fruits and seeds. They also protect plant from UV irradiation. 11. Alkaloids are nitrogen-containing heterocyclic organic bases with complicated structures usually with specific physiological functions. They are biosynthesized and derived from amino acids through mevalonic acid pathways. Most alkaloids are toxic to man and animals, hence prevent herbivores from consuming them; so acts as defensive compounds in plants. 12

Aspects of biotechnology in biochemical and molecular regulations in the industrial development of plant phytochemical with the syntheses of metabolites
Biotechnology in phytochemical studies involve bio-reactions and manipulations in plants for producing better and healthier plant growth, developments, protections, expansions and improved potentials of its phytochemical constituents with higher productivity. It applies www.intechopen.com recent areas of studies like genomics, metabolomics, system-biology and proteomics for producing beneficial natural products. Generally development of plant cells biotechnologically is for economic and industrial purposes. Current methods and techniques utilize highthroughput applications on genomic modifications by homologous and recombinations at specific sites. It applies basic principles of plant and molecular biology, involving recombinant DNA technology modifying functional genes in natural product biosyntheses. The DNA and molecular biomarkers are involved; favourable traits are recognized, identified and isolated, then the selection of the genotype. Results from these assist in appropriate creation of transgenic plants that yield important and economic natural products using plant cells and tissues to get genetically modified plants and natural products. Such results are also utilized in assessments of biodiversities and chemotaxonomy. It is explorative applying foreign genes into plant genomics, so creating improved metabolic biosyntheses with genetic modifications to have faster and better production of active secondary metabolites, than from the conventional ways. But some are opposed to it because of the risk involved, and suggested it should not be applied to food developments. Well it requires first understanding details of genetic information on the plant and its natural product also know and identify the marker genes to be able to successfully transfer its genetic culture, so positively manipulating the plant with beneficial characters which last longer, and so affords better strategies in natural product formation and studies.
Incorporation methods (in-vivo) can be used as well as in-vitro cultivation and regeneration of excised or cultured whole plant provided necessary nutrients and hormones are available. Phytochemical researches in most part of the world utilize wild field cultivated plants or plants in the wilderness. It is more tasking to form plants and natural products from biotechnology, and the yield may even be too low, making it to be more costly and uneconomical. But biotechnology methods are very appropriate for endangered plant species and their natural products. Stereo-and regiospecific bio-transformations and bio-conversions afford in cultured cell suspension cultivations the discovery of new biomolecules which are not in the intact plant, so need to identify the particular enzymes causing this synthesis of new natural products which can be applied on large scale productions.
Molecular farming afford massive production of phytochemical from bioreactor plants to give cheaper and safer ways of forming recombinant proteins of higher values to give more valuable natural products and pharmaceuticals. It is a new area of bioengineering. It has the advantage of expressing gene at specific organs like leaves, fruits, roots or seeds. Gene of the host plant is modified so it forms stable products. These methods are important in fermentation processes, commercial proteins and products, in the generation of therapeutics and vaccines, as well as in diagnostics.
Biotechnology in natural products research afford discovery of bioactive natural products from sources outside the already known conventional plants, so reveals wider diversities of phyto-compounds. This is an important aspect of bio-prospecting.

Practical involvements of specific phytochemical in health and treatments as for example antimalaria, antibacterial, anti-fungal, antioxidant, anti-ulcer, anticancer
Active metabolites from natural sources usually have very minimal or no toxic effect on organism using them; hence they are more useful and promising. The great diversity of tropical forest plants are good sources of great number of bioactive substances with many therapeutic uses from which drugs can be discovered and processed. Many lead compounds have been isolated and derived from plants, which are now very useful drugs.

Bioinformatics, genomics, and synergies of phytochemical with pathogens
We face challenges on how do plants with its enzymes and regulatory genes biosynthesize specific natural product compounds. It gives deeper and clearer understanding of processes involved in generating our highly interested natural products from perspectives of www.intechopen.com molecular biology. Studies here are on characterization of functional genes and sequencing of genome because they express and regulate syntheses of natural product which is our main concern in phytochemistry and pharmacognosy. Important areas for molecular biology techniques of studying genomics of natural products in plants include identifying and expressing genes, functional genes and silencing. DNA and RNA isolations and clones with proteins derived from them. The important aspect of phytochemistry is the interest in processes occurring in each plant species for the biosyntheses of the metabolites. Techniques here are very sensitive high-through put plant metabolomics screenings with better separation, purification and structure characterization methods and instruments, as well as very sensitive methods of detections. These will reveal total and detailed natural product constituents in plants, which is our focus, and due to the great importances of natural products in isolated form and as synergies. With great diversities of natural product compounds, this method is highly reliable. The genes which govern the regulations for the different metabolic pathways and biosynthetic enzymes involved are identified and studied. Understanding families or categories of different biosynthetic genes leading to the production of many specific secondary metabolites is the main focus of natural product metabolomics and its processes. Genomics and metabolomics studies will be able to reveal and decipher detail phytochemical in a particular medicinal plant species. Probably in the near future, transgenic methods may also provide good alternative biosynthetic methods of forming new active secondary metabolites not known now. Therefore this will demonstrate biotechnology in natural product studies, and it may even be a potential application in natural product researches, moreso plants are easy organisms to manipulate their genetics so as to obtain diverse natural products. These will also lead to safe nutraceutical natural products.