eChapters

Formulation of meat and sea food products requires various ingredients which are commonly known as food additives. Food additives are those non-meat ingredients which are used to enhance colour, flavour, aroma and texture of the meat and sea food products. These food products can fulfil the demand of modern consumers. As per the definition of Food and Drug Administration (FDA) food additives are those substance which are used to provide a technical effect in foods. The use of food additives has become more prominent in recent years, due to the increased production of prepared, processed, and convenience foods. Additives are used for flavour and appeal, food preparation and processing, freshness, and safety. These additives can be classified as curing ingredients, extenders and binders, flavour enhancers and other additives. For the formulation of different products both raw materials and ingredients should be of food grade and sound hygienic strength. In the starting of this chapter we will discuss about ingredients used then the specific formulations will be discussed for better understanding of meat and sea food processing.

Curing Ingredients

Curing agents such as salt, phosphate, sugars, nitrite and nitrate, sodium ascorbate and erythorbate etc. have traditionally been used in various meat formulations from long back. The details of these ingredients have already been discussed in chapter-8 of this book. So we will try to discuss other ingredients which are relevant for meat and sea food products other than these ingredients. Cure accelerators such as ascorbates and erythorbates are used to speed the curing process. They also stabilize the colour of the final product. The examples are ascorbic acid, erythorbic acid, sodium erythorbate, citric acid, sodium citrate, sodium acid pyrophosphate, glucono delta lactone (GDL) etc. Glucono-delta-lactone (GDL) acts as a curing accelerator in meat and meat products. It is permissible up to 1% in sausages and up to this level it lowers the pH about 0.5 thus inhibits the growth of spoilage micro-organisms. GDL also facilitate the cured colour development.

Food packaging is an essential technique for preserving food quality and minimizing food wastage. It is a scientific technique of containing food against physical, chemical and microbiological contaminants. In addition package also display the product in most appropriate manner and provide product information to the consumers while at the same time allowing convenient handling and efficient distribution. The packaging of meat and meat products are particularly important because meat is highly nutritious food and provides good medium for the growth of micro-organisms. Meat and meat products are also susceptible to bacterial growth due to their high water activity. Exposure of meat and meat products on atmospheric temperature and sunlight leads to various types of deteriorative changes. For example when cut meat surfaces exposed to ambient air provide excellent breeding grounds for most bacteria. Minced meat is even more at risk due to the large exposed surface area. Packaging provides protection of food against external and internal factors but once food gets deteriorated it can not be controlled by packaging. For this reason rigorous hygiene in all processing and pre-packaging steps is vital for packaging of perishable food items like meat and meat products.

Meat processing is the set of methods and techniques used to bring substantial chemical and physical changes in the natural state of meat. These are the methods which are used for making meat and their products attractive, nutritious and marketable as well as for the prolongation of shelf-life. In processing techniques, grinding, chopping, addition of seasoning, alteration techniques for colour, heat treatments etc. are included but cutting, trimming and deboning are not included in processing operations.

History

Meat processing dates back to the prehistoric ages and probably the first processed meat product was sundried meat. Lator it turns to burning of meat over wood fire while salting and smoking of meat was practiced first time in the time of Homer, 850 B.C. In 63B.C.-14 A.D. at the reign of Augustus honey was tried for preservation of meat. Lator various types of cooking such as roasting, smoking, steaming and oven baking came in existance. Salt-preservation was especially common for foods including meat and meat products for warrior and sailor’s diets, until the introduction of canning methods.

Modern food processing technology in the 19th and 20th century was largely developed to serve military needs. In 1809 Nicolus Appert invented a vacuum bottling technique that would supply food for French troops and this contributed to the development of tinning and then canning by Peter Durand in 1810. Although initially expensive and somewhat hazardous due to the lead used in cans. Canned goods would later become a staple around the world. Pasturization discovered by Louis Pasteur in 1862 was a significant advance in ensuring the micro-biological safety of food. In 20^th century, food processing advances with the development of spray drying, freeze drying and the introduction of artificial sweeteners, colouring agents and preservatives such as sodium benzoate. In late 20^th century ready to eat meat food products were introduced to cater the need of consumers.

Ageing of meat is also known as conditioning or tenderising or ripening or maturing and sometime resolution of rigor. It is a process of holding unprocessed meat above freezing point under controlled conditions. By this process we can make muscle soft and pliable thus meat becomes tender. When we keep the meat above freezing point at temperature between 0-3ºC then all those changes occurs which are normally occurs at higher temperature i.e. atmospheric oxidation and enzymatic action of fat, bacterial action etc. When this temperature range becomes below freezing point then it inhibits completely the enzymatic action in the muscles. Ageing is required for marked increase in flavour, juiciness and tenderness of meat. In this process action of enzymes in meat plays key role while bacterial action is not of any importance. Meat tenderness and taste are definitely improved if carcasses or vacuum packed cuts are conditioned after slaughter but paradoxically beef is quite tender just two hours after slaughter and several days of conditioning are required to recover this degree of tenderness.

During the contraction of muscles, thick and thin filaments sliding to each other and decreases the length of sarcomeres. In turn, this decreases the length of fibrils, fibres and the whole muscle. If these muscles remain in contracted length when rigor develops, it causes massive overlap of thick and thin filaments leading to severe meat toughness. It is also necessary to avoid the cooling of meat too rapidly after slaughter, otherwise it cold-shortens and gets tough. If we avoid cold-shortening of muscles during conditioning, it will increase meat tenderness. But with too much conditioning, weight loss, surface spoilage and the cost of refrigerated storage increases.

Ageing of carcasses with little or no fat cover is not recommended by meat specialists. Because these carcasses loose moisture rapidly, loose weight excessively and discolour the meat surface rapidly. In addition, lean meat is also susceptible to deterioration through microbial growth, slime formation by bacteria and mould growth etc. Ageing is important for beef and buffalo meat while pork and lamb do not require ageing because they are slaughtered in the age where they are young and inherently tender. Additionally, the unsaturated fats found in pork fat oxidize during ageing causes rancidity and off flavour. Veal has very little protective fat covering and is high in moisture; thus it does not lend itself to ageing.

Preservation of foods including meat and aquatic foods is a creation of conditions for the food articles in such a way to stop or greatly slow down the activities of micro-organisms, chemical and enzymatic reactions and physical forces to prevent the meat and aquatic foods from spoilage while maintaining its nutritional value and sensory attributes.

Significance of Meat Preservation

1. Meat and aquatic foods are perishable food items and are very prone to spoilage and rancidity development due to high nutrients, moisture and neutral pH.
2. Once meat and aquatic foods is exposed to microbial contamination, chemical and enzymatic reactions then it will be very difficult to attain asepsis.
3. To enhance the keeping quality of meat and aquatic foods so that it will be available for further processing.
4. It protects meat and aquatic foods from shrinkage, sweeting and other physical forces.
5. To protect human health by providing the wholesome meat and aquatic foods to the consumers.
6. By this way we can minimize utilization of energy in processing of meat and aquatic foods.

Fraudulent substitution of meat is the adulteration or mixing of the inferior or cheaper quality meat into superior quality meat. The first case of fraudulent substitution was recorded in thirteenth century A.D. at Florence in Italy. A common fraud in the meat industry in which uninspected meat is substituted for meat that has undergone inspection and been branded as satisfactory. The other frauds are the substitution of meat of another species, e.g. horse for beef especially in Britain and Ireland, beef in kangaroo meat in Australia, cat for chicken or rabbit, goat meat for mutton, mutton for venison, dog meat and cat meat for chevon in other countries including India. As per an estimate about 25-30% of meat sold in India is adulterated. These practices are more common in comminuted meat products. These illegal practices are punishable under PFA act 1955. The choice of method to be applied is depend on the need or condition of meat recovered.

Meat is an animal flesh that is used as food. Most often, this means the skeletal muscle and associated fat but it may also describe other edible tissues such as organs, livers, skin, brains, bone marrow, kidneys or lungs. Meat is highly nutritious food because it contains protein, fat and carbohydrates in relative proportion, supply energy as required by the human body, posses ability to build or replace tissues and the substances present in meat are easily absorbed by the human body. Meat is also a good source of B-complex vitamins but fat soluble vitamins are low in meat. It is a good source of phosphorous, zinc and iron but low in calcium. Meat also contains significant amount of copper, sodium, potassium and magnesium but low in carbohydrate contents. Carbohydrate is found mainly in form of glycogen which disappears completely after completion of rigor mortis. In general lean red meat is particularly good source of protein, niacin, vitamin B6, vitamin B12, phosphorus, zinc and iron. About 100g of meat can provide more than 25% RDI of these nutrients. It also provides more than 10% RDI of riboflavin, pantothenic acid and selenium. Of the four meats, mutton is particularly nutrient-dense and the richest source of thiamine.

Conversion of muscle to meat is a complex process in which several physical and chemical changes takes place over a period of several hours or even days. The major changes which take place during this process include fall in muscle pH and temperature as well as muscle shortening which further leads to proteolysis or ageing. These changes during conversion of muscles to meat are variable and have great effect on meat quality (tenderness, juiciness, flavour, colour etc.) and also on certain processing characteristics like emulsification, binding properties and yield of the products. To know this complex process it is necessary to understand the process of energy production and metabolic pathway shifting in live animals.

Pre Slaughter

In the living animal, aerobic metabolism is used to obtain energy. The source of energy at this stage is either free fatty acids or blood glucose or muscle glycogen. In this process total 38 ATP molecules are produced from single glucose or glycogen molecule by the complex processes known as glycolysis, oxidative decarboxylation and oxidative phosphorylation. However, under stressful situations, anaerobic metabolism may be used and lactic acid is produced. Lactic acid is usually transported from the muscles to the liver, where it is re-synthesized into glucose and glycogen, or to the heart, where it is metabolized to carbon dioxide and water.

The word meat comes from the old english word mete, which referred to food in general. Most often this means the skeletal muscle and associated fat but it may also describe other edible tissues such as organs, livers, skin, brains, bone marrow, kidneys and lungs etc. In other words we can say meat is those animal tissues which are suitable for use as food by the man. It is composed of numerous types of tissues like muscle tissues, epithelial tissues and nervous tissues but the major component of meat is muscle. Particularly skeletal muscle is the principal source of muscle tissue in meat while smooth muscle contributes little to meat. The muscle and connective tissues are the major compositional components and contributes towards qualitative and quantitative characteristics of meat. Broadly muscle can be classified into striated and smooth muscles. Striated muscles have transverse band pattern as seen in skeletal and cardiac muscle. However, smooth muscles do not show such pattern and mainly found as a component of blood vessels.

In spite of big potential of the largest livestock population in the world, meat industry in India is still in nascent stage. It is a business of those who are not trained and very much literate. The industry is primarily based on the raw material that is not reared for meat purpose but serves the secondary role after passing their viable life in primary field. The industry is also facing problem of social prejudice and meat is taboo for certain communities. But scenario is now changing and people are coming forward to organise the sector. Now the society is accepting the facts that meat is an only source of cheap and nutritious food to million of people in India and the good source of animal protein for large segment of Indian population, particularly landless, small and marginal farmers.

India ranks fifth in the world meat production with the annual meat production of about 6.3 million tones which is 2.21% percent of world meat production. In the last two decades meat industry in India has grown @ 4.5% whereas, during the last five years, this segment has been growing very fast at the rate of 27% annually. It has a good future if given proper attention by the Government and Private Entrepreneurs. In total meat production, share of cattle is 17.34%, buffalo 23.3%, sheep 4.6%, goat 9.4%, pigs 5.3%, poultry 36.6% and others 3.4%. The share of various meat types in total Indian meat production accounts buffalo meat 1.5 million tones, beef 1.28 million tones, mutton 0.24 million tones, chevon 0.53 million tones, pork 0.50 million tones, chicken meat 2.20 million tones and duck meat 0.07 million tones. To produce the above quantities, the extraction or slaughter rates in cattle are about 7.9%, buffaloes 11.1%, sheep 47.9%, goat 37.9%, pigs 88.9% and poultry 73.6%. In the world total meat production Indian cattle meat shares 13.1%, buffalo meat 55.8%, mutton 5.7% and chevon 14.9%. However, India is having world’s 15.0% (210 million) cattle, 57.0 %( 111 million) buffaloes, 7.0 %( 74 million) sheep, 17.0 %( 154 million) goat and 1.5% pigs. Overall contribution of meat sector in total output of Livestock sector was Rs 25984 crores in the year 2004-2005.

In sub-tropical agro-climatic conditions of India, a variety of insect-pest, weeds and diseases hamper the economic growth of field crops. For effective control of these pests, a number of pesticides including insecticides, fungicides, herbicides and nematicides are to be applied. Estimation technique for single pesticideonly can not be followed for detection of residues of all categories of pesticides. Hence, this constantly expanding use of pesticides on food crops accentuates the need for rapid, precise and sensitive method for determination of pesticide residues of all the major groups of pesticides. In such situation, multi-residue analytical technique can be efficiently followed for detection and estimation of multiresidues of intra and inter class xenobiotics.

The purpose of multiresidue analysesistodeterminetheresidues ofas manypesticides as possible withina short period of time even if the recoveries of somecompounds are low. In multiresidue methods the recoveries up to 70% are accepted.The recoveriesless than70%have tobe mentionedspecifically. Multiresidue analysis is relativelymore sensitiveand requires extreme vigilance on the part of the analyst during processingof samples. Additionally, instrument, GLC,need to beequipped withcapillary columnsforhigh resolutionand sophisticated detectorslikeECD, NPDand MSD for detectingmulti-residues of the pesticides. Multiresidue analytical methods/techniques havebeen developed for determination of general pesticide residues for some environmental componentsand differentfood commodities.Criteria formulti-pesticides screening as per California Department of Food and Agriculture (CDFA) are:

Crops treated with pesticides invariably contain small amount of these chemicals and the hazard depends on the amount of pesticide residues that remain on the crop and their toxicity. The amount of residue that may remain on crop or food commodity and environmental commodities depend on the nature of pesticides, good agricultural practice, environmental conditions and adoption of safe waiting periods and other perfect methods of storage. As residues are inevitable by product of pesticide use, hence pesticide residue in food commodities and their entry in to food chain has become major cause of concern all over the world. The plant science industry has a responsibility to assist farmers to produce crops that are acceptable to the food chain and consumers alike.

It is clear that pesticide residues in farm produce should be kept as low as practicable, and eliminated whenever possible through Good Agricultural Practices (GAP). However, the use of pesticides can be regulated to ensure minimum residues on food which can be considered safe to human consumption and environment as a whole.

To regulate pesticide residues to safe levels, a concept was introduced by Joint FAO/WHO Expert Committee on Food Additives (1955) and Codex Alimentarius Commissionwas established in 1964 to implement the Joint FAO/ WHO Food Standard Programmes. The Commission has laid down principles for aiming at the maximum residue limits (Tolerance limits) of pesticide on food commodities. The recommendations are based on:

In the present era, residue analyst is exposed to array of analytical choice and as per International regualtions, presence of pesticide residues in environmental components and food commodities are becoming more stringent.Generally, multiresidue methods are based on GC equipped with selective detection either electron capture detector, nitrogen phosphorous detector, flame ionization detector etc. One most widely applied technique in pesticide residue analysis is HPLC, equipped with different detectors. Another up-datemethod for confirmation is gas chromatography-mass spectrometry(GCMS)and HPLC-MS applyingparticlebeam or thermo sprayionisationtechnique. Recently one more analytical technique which has gained ground in the field of pesticide residue analysis, is high performance thin layer chromatography (HPTLC).The criteria of good analytical technique like simple, rapid, flexible, versatile, inexpensive, easily documented and validated has been the inherent advantage of HPTLC. Strictly speaking, HPTLC is not different in principleor application from conventional TLC, but due to improvements in materials, its effectiveness has also enhanced.The technique has a potential to operate upto femtogramlevel. HPTLC is a sophisticated and automated form of TLC. It is a technique which can generate about 5000 or more theoretical plates. The high performance in TLC is achieved by optimized plate coating materials, by improved sample application techniques, and by developing newmethods for the development of mobile phase. Thus HPTLC is an instrumental approach at different stages of TLC. A flow chart for HPLC principle is shown in Figure 8.1. The principle of separation is based on adsorption and this leads to easyseparationof isomers, includingchiralcompounds.Main difference between HPTLC and TLC is given in Table 8.1

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

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

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.

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:

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.

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.

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.

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.

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.

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.

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.

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.

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:

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.

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.

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.

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.

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.

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.

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.

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 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).

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 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.”

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.

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.

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.

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.

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:

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

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

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

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

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

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

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

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.