Preface Various techniques are available in different disciplines of Science such as Botany, Chemistry, Physics, Mathematics etc. The isolation, characterization and identification of phytochemicals from plants involve the knowledge of all disciplines of Science. With the growing interest in the field of medicinal plants by researchers and industries, it is necessary to have a book which will give information on different interdisciplinary techniques. The aim of this book is to give a self-contained detail of all aspects of phytochemical techniques. It is designed as a textbook for those students who use phytochemical techniques and for researchers working on medicinal plants. Important plant secondary metabolites such as glycosides, flavonoids, lignins, terpenoids and alkaloids have been isolated over a period of time from natural sources. To encourage many students and researchers to carry out work on phytochemicals and to find out some novel compounds from plants, various techniques are given in a nutshell. This book contains 10 chapters. Chapter 1 provides a general introduction. In chapter 2, the production processes for herbals and botanicals are given. The selection of plant parts for extraction is briefed in chapter 3. The chapter 4 provides the various methods of extraction such as organic solvent extraction, extraction with supercritical gases, steam distillation, purification and concentration of miscella and schemes for extraction. Different procedures of qualitative phytochemical screening are presented in chapter 5. The methods for separation of phytochemicals including paper chromatography, thin layer chromatography and column chromatography are discussed in chapter 6. In chapter 7, qualitative and quantitative estimation of phytochemicals with gas and liquid chromatography, HPTLC and OPLC are given. The methods of identification using physical characteristics and spectroscopy which includes UV, IR, NIR, Mass and NMR are presented in chapter 8. In chapter 9, details and structures of different categories of phytochemicals are considered. Finally in chapter 10, a case study of isolation and identification of compounds from Clausena dentata in my laboratory is discussed. I would like to thank all those who helped me in the preparation of the text. A special mention is to Prof. Muraleedharan G. Nair, Bioactive Natural Products and Phytoceuticals, National Food Safety and Toxicology Center, Michigan State University, USA who extended the facilities to me during my visit to USA. My sincere thank to Prof. R. Raghunathan, Department of Organic Chemistry, University of Madras, Chennai, India, Dr.A. Banerji (Former Head), Dr.S.Chattopadhyay (Head) and Dr.G.J. Chintalwar of Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai, India for their help in identification of some of our compounds. Prof. P.T. Manoharan (Former Head) and Prof. A.K. Mishra (Head) of Sophisticated Analytical Instrument Facility, Indian Institute of Technology, Chennai, India were kind enough to allow us to use the facility. The help rendered by Prof. D. Velmurugan, Department of Crystallography & Biophysics, University of Madras, Chennai, India for crystallographic studies and ANCHORM, Mumbai, India for HPTLC studies is of worth mentioning. I am thankful to the staff of New India Publishing Agency, New Delhi, India for bringing out this book in time.

Plants are used as medicine since time immemorial. One of the area of Ethnobotany, Ethnopharmacology is considered as the scientific evaluation of traditional medicinal plants (Cotton, 1996). Cox (1994) has suggested that the ethno-directed sampling is most likely to succeed in identifying drugs used in the treatment of gastrointestinal, inflammatory and dermatological complaints. The Plant kingdom is a virtual goldmine of potential drug targets and other active molecules awaiting to be discovered. It has been estimated that only 10 - 15 per cent of the 7,50,000 existing species of higher plants have been surveyed for biologically active compounds. Natural products produced by plants, fungi, bacteria, insects and animals have been isolated as biologically active pharmacophores. Approximately one-third of the top-selling drugs in the world are natural products or their derivatives often with ethnopharmacological background. Moreover, natural products are widely recognized in the pharmaceutical industry for their broad structural diversity as well as their wide range of pharmacological activities. New medicines have been discovered with traditional, empirical and molecular approaches (Harvey, 1999). The traditional approach makes use of material that has been found by trial and error over many years in different cultures and systems of medicine (Cotton, 1996). Examples include drugs such as morphine , quinine and ephedrine that have been in wide spread use for a long time, and more recently adopted compound such as the antimalarial artemisinin. The empirical approach builds on an understanding of a relevant physiological process and often develops a therapeutic agent from a naturally occurring lead molecule (Verpoorte, 2000). Examples include tubocurarine and other muscle relaxants, propranolol and other adrenoreceptor antagonists, cimetidine and other histamine H2 receptor antagonists. With the development of molecular biological techniques and the advances in genomics, the majority of drug discovery is currently based on the molecular approach.

2.1 Introduction Dietary supplements from plant sources are sometimes referred to as phytopharmaceuticals. They are produced from fresh, dried or otherwise preserved plants or parts of plants. The active ingredients are usually not completely isolated but rather are obtained along with other naturally occurring components of the plant. These other components are often believed to influence the efficacy of the active ingredient. Sometimes the active ingredients are concentrated, and undesirable substances such as chlorophyll, tannins, or resins, are removed. 2.2 Cultivation Most of the plants used for dietary supplements or medicinal purposes are cultivated, that is, grown in farms. Some, however, may be collected from the wild. Cultivation allows producers to have more control over quality and purity than does collecting plants from the wild. Cultivars (cultivated varieties) of a number of medicinal plant species have been developed to produce high yields of the desired constituents. Some plants that are grown commercially for medicinal purposes are propagated vegetatively (This means that new plants are grown from cuttings of old plants. Plants grown in this way are genetically identical to the parent plant). Some medicinal plants are grown from selectively bred hybrid seeds, while others are varieties of plants that are unchanged from their natural form.

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

4.1 Introduction Extraction is a process whereby the desired constituents of a plant are removed using a solvent. The primary ways for extraction of organic molecules of interest to biologists and medical investigators involve breaking open the cells. Cell rupturing is carried out in a variety of ways. The method used depends on the type of tissue used. For plant cells grown in cell suspension culture or for plant callus tissue, a French press or a sonicator can be used to break open the cell. Plant cells grown in culture can also be ruptured with a glass tissue homogenizer. Silicified or highly lignified plant tissues within the organs such as roots, stems, leaves, fruits and seeds are usually frozen and pulverized using liquid nitrogen in a mortar and a pestle (Kaufman et al., 1999). Softer tissues can be ground in a small volume of buffer in a mortar, washed white sand and a pestle to rupture the cells. When the cells have been ruptured, the extraction is performed using the appropriate techniques. Water soluble compounds and proteins are extracted in buffers or water. Organically soluble compounds are extracted with organic solvents. Boiling ethanol is a good all-purpose solvent for preliminary extraction (Harborne, 1998). The success of the extraction with alcohol is directly related to the extent chlorophyll (when isolating substances from green tissue) is removed into the solvent. The most common chemical procedure for obtaining organic constituents from dried plant tissues is to continuously extract powdered material in a Soxhlet apparatus with a range of solvents, starting with petroleum ether and chloroform (to separate lipids and terpenoids) and then using alcohol and ethyl acetate (for more polar compounds). This method is useful when working on the gram scale. However, one rarely achieves complete separation of constituents and the same compounds may be recovered (in varying proportions) in several fractions. The extract obtained is clarified by filtration and is then concentrated in vacuo in a rotary evaporator. To prevent fungal growth, a trace of toluene may be added to the concentrated extracts and they should be stored in refrigerator. There are short cuts in extraction procedures which one learns with practice. The concentrated extract may deposit crystals on standing.

5.1 Introduction The different qualitative chemical tests can be performed for establishing profile of given extract for its chemical composition. The following tests may be performed on extracts to detect various phytoconstituents present in them. 5.2 Detection of Alkaloids (Evans, 1997) Solvent free extract, 50 mg is stirred with few mL of dilute hydrochloric acid and filtered. The filtrate is tested carefully with various alkaloidal reagents as follows:

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

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

8.1 Introduction After isolation and purification of the compound, it has to be identified. It is necessary to find out the class of the compound first and then to find out the specific compound within the class. The class of compound is usually determined from the colour tests, solubility, Rf properties and UV spectral characteristics of the compound. Further identification within the class depends on measuring physical properties and spectral measurements and then comparing these data with those in the literature. The physical properties include melting point (for solids), boiling point (for liquids), optical rotation (for optically active compounds) and Rf. The spectral measurements include Ultra Violet (UV) , Infra Red (IR) , Mass Spectrum (MS) and Nuclear Magnetic Resonance (NMR). Data on X – ray crystallography of the compound are also useful for identification of the compound. On completion of the analysis of the spectra obtained, the pieces that have been identified are listed. Then the pieces, the MW and / or molecular formula are checked and the pieces are refined to fit. Finally, the pieces are assembled paying particular attention to 1H-NMR chemical shifts and coupling patterns. The answer should be checked with the 1H-NMR data very carefully and it is probably the most critical test to pass. The 1H-NMR spectrum is potentially the most useful for assembling the structure of the compound. A known compound can usually be identified on the basis of the above mentioned physical and spectral characteristics. For final confirmation, direct comparison with authentic compound (if available) should be carried out Authentic compounds can be obtained from commercial companies. In the absence of authentic compound, the data from literature can be carefully compared for the identification of the isolated compound. If a new compound is present, all the above data are sufficient enough to identify it. However, data on chemical degradation or laboratory synthesis of the compound are necessary to confirm the identification of the new compound.

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

10.1 Introduction The following plants have been worked out in the laboratory of Prof. N. Raaman: Cynodon dactylon, Artemisia pallens, Leptadenia reticulata, Clausena dentata, Tylophora indica, Aristolochia bracteolata, A. indica, Cissus quadrangularis, Hypericum mysorense. and Solanum trilobatum. Out of some of the data of these plants have been included in the previous chapters. In this chapter, compound isolation (CD1) from Clausena dentata (worked by Dr. R. Kamaraj) and characterization of the compound are given in detail. 10.2 Preparation of extracts from Clausena dentata The dry powder of stem bark (2.5 kg) was first soaked, at room temperature, in hexane (1:4 w/v) for 24 h. The extract was suction filtered using Whatmann filter paper. This is repeated for two more days and similar extracts were pooled together and concentrated at 40°C under reduced pressure using Buchi R - 153 Rotavapor. The residual plant material was extracted successively with chloroform and methanol in the same manner as followed for hexane. All the concentrates were subjected to column chromatography to isolate the active principles.

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