Phytochemicals: Protective Plant Compounds and Their Role in Human Health and Medicine, Guías, Proyectos, Investigaciones de Farmacognosia

¡Descarga Phytochemicals: Protective Plant Compounds and Their Role in Human Health and Medicine y más Guías, Proyectos, Investigaciones en PDF de Farmacognosia solo en Docsity! Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents James Hamuel Doughari Department of Microbiology, School of Pure and Applied Sciences, Federal University of Technology, Yola Nigeria 1. Introduction Medicinal plants have been the mainstay of traditional herbal medicine amongst rural dwellers worldwide since antiquity to date. The therapeutic use of plants certainly goes back to the Sumerian and the Akkadian civilizations in about the third millenium BC. Hippocrates (ca. 460-377 BC), one of the ancient authors who described medicinal natural products of plant and animal origins, listed approximately 400 different plant species for medicinal purposes. Natural products have been an integral part of the ancient traditional medicine systems, e.g. Chinese, Ayurvedic and Egyptian (Sarker de Nahar, 2007). Over the years they have assumed a very central stage in modern civilization as natural source of chemotherapy as well as amongst scientist in search for alternative sources of drugs. About 3.4 billion people in the developing world depend on plantbased traditional medicines. This represents about 88 per cent of the world's inhabitants, who rely mainly on traditional medicine for their primary health care. According to the World Health Organization, a medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical semi synthesis. Such a plant will have its parts including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or treatment of a disease condition and therefore contains chemical components that are medically active. These non-nutrient plant chemical compounds or bivactive components are often referred to as phytochemicals (phyto-" from Greek - pluyto meaning plant) or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pests (Abo ct al, 1991; Liu, 2004; Nweze et al, 2004; Doughari ct al, 2009). The study of natural products on the other hand is called phytochemistry. Phytochemicals have been isolated and characterized from fruits such as grapes and apples, vegetables such as broccoli and onion, spices such as turmeric, beverages such as green tea and red wine, as well as many other sources (Doughari é: Obidah, 2008; Doughari et al., 2009). The science of application of these indigenous or local medicinal remedies including plants for treatment of diseases is currently called ethno pharmacology but the practice dates back since antiquity. Etino pharmacology has been the mainstay of traditional medicines the www.intechopen.com 2 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health entire world and currently is being integrated into mainstream medicine. Different catalogues including De 2/ateria l/edica, Historia Plantarum, Species Plantarum have been variously published in attempt to provide scientific information on the medicinal uses of plants. The types of plants and methods of application vary from locality to locality with 80% of rural dwellers relying on them as means of treating various diseases. For example, the use of bearberry (Arciostapluylos uva-ursi) and cranberry juice (Vaccinion macrocarpon) to treat urinary tract infections is reported in different manuals of phytotherapy, while species such as lemon balm (2elissa officinalis), garlic (Allan sativuwn) and tee tree (l/elaleuca alternifolia) are described as broad-spectrum antimicrobial agents (Heinrich ct al., 2004). A single plant may be used for the treatment of various disease conditions depending on the community. Several ailments including fever, astma, constipation, esophageal cancer and hypertension have been treated with traditional medicinal plants (Cousins €: Huffman, 2002; Saganuwan, 2010). The plants are applied in different forms such as poultices, concoctions of different plant mixtures, infusions as teas or tinctures or as component mixtures in porridges and soups administered in different ways including oral, nasal (smoking, snoffing or steaming), topical (lotions, oils or creams), bathing or rectal (enemas). Different plant parts and components (roots, leaves, stem barks, flowers or their combinations, essential oils) have been employed in the treatment of infectious pathologies in the respiratory system, urinary tract, gastrointestinal and biliary systems, as well as on the skin (Rojas et al., 2001; R10s e: Recio, 2005; Adekunle é: Adekunle, 2009). Medicinal plants are increasingly gaining acceptance even among the literates in urban settlements, probably due to the increasing inefficacy of many modern drugs used for the control of many infections such as typhoid fever, gonorrhoea, and tuberculosis as well as increase in resistance by several bacteria to various antibiotics and the increasing cost of prescription drugs, for the maintenance of personal health (Levy, 1993; Van den Bogaard et al,, 2000; Smolinski et al,, 2008). Unfortunately, rapid explosion in human population has made it almostimpossible for modern health facilities to meet health demands all over the world, thus putting more demands on the use of natural herbal health remedies. Current problems associated with the use of antibiotics, increased prevalence of multiple-drug resistant (MDR) strains of a number of pathogenic bacteria such as methicillin resistant Staplaylococcus aureus, Helicobacter pylori, and MDR Klebsiela pnewmonia has revived the interest in plants with antimicrobial properties (Voravuthikunchai de Kitpipit, 2003). In addition, the increase in cases of opportunistic infections and the advent of Acquired Immune Deficiency Syndrome (AIDS) patients and individuals on immunosuppressive chemotherapy, toxicity of many antifungal and antiviral drugs has imposed pressure on the scientific community and pharmaceutical companies to search alternative and novel drug souces. 2. Classes of phytochemicals 2.1 Alkaloids These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen. The compounds have basic properties and are alkaline in reaction, turning red litmus paper blue. In fact, one or more nitrogen atoms that are present in an alkaloid, typically as 1” 2* or 3” amines, contribute to the basicity of the alkaloid. The www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 5 strophanthidin from Strophanthus, digitoxin from Digitalis, barbaloin from Alocs, salicin from Salix, cantharidin from Cantharides, and prunasin from Prunus. However, the systematic names are invariably coined by replacing the “ose” suffix of the parent sugar with “oside”. This group of drugs are usually administered in order to promote appetite and aid digestion. Glycosides are purely bitter principles that are commonly found in plants of the Genitiaceae family and though they are chemically unrelated but possess the common property of an intensely bitter taste. The bitters act on gustatory nerves, which results in increased flow of saliva and gastric juices. Chemically, the bitter principles contain the lactone group that may be diterpene lactones (e.g. andrograplolide) or triterpenoids (e.g, amarogentin). Some of the bitter principles are either used as astringents due to the presence of tannic acid, as antiprotozoan, or to reduce thyroxine and metabolism. Examples include cardiac glycosides (acts on the heart), anthracene glycosides (purgative, and for treatment of skin diseases), chalcone glycoside (anticancer), amarogentin, gentiopicrin, andrographolide, ailanthone and polygalin (Fig. 2). Sarker de Nahar (2007) reported that extracts of plants that contain cyanogenic glycosides are used as fMavouring agents in many pharmaceutical preparations. Amygdalin has been used in the treatment of cancer (HCN liberated in stomach kills malignant cells), and also as a cough suppressant in various preparations. Excessive ingestion of cyanogenic glycosides can be fatal. Some foodstufís containing cyanogenic glycosides can cause poisoning (severe gastric irritations and damage) if not properly handled (Sarker de Nahar, 2007). To test for O-glycosides, the plant samples are boiled with HCI/B2O to hydrolyse the anthraquinone glycosides to respective aglycones, and an aqueous base, e.g. NaOH or NH¿OH solution, is added to it. For C-glycosides, the plant samples are hydrolysed using FeCl/HCI, and and an aqueous base, e.g, NaOH or NH0H solution, is added to it. In both cases a pink or violet colour in the base layer after addition of the aqueous base indicates the presence of glycosides in the plant sample. om Ó ON ok CY o ( e O Y Dv «Terpineol Cinnamyl acetate — Eugenol Taxifolin-7-O- 10 Fig, 2. Basic structures of some pharmacologically important plant derived glycossides 2.3 Flavonoids Flavonoids re important group of polyphenols widely distributed among the plant flora. Stucturally, they are made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. Over four thousand flavonoids are known to exist and some of them are pigments in higher plants. Quercetin, kaempferol and quercitrin are common flavonoids present in nearly 70% of plants. Other group of flavonoids include flavones, dihydroflavons, flavans, flavonols, anthocyanidins (Fig, 3), proanthocyanidins, calchones and catechin and leucoanthocyanidins. www.intechopen.com 6 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health 4 ol 7 3 . (OH) H(Oglyc) FLAVONE (FLAVONOU) ANTHOCYANIDIN (ANTHOCYANIN) O. O. (0H) (OH) DIHYDROFLAVONE (DIHYDROFLAVONOL) FLAVAN (FLAVANOL) Fig, 3. Basic structures of some pharmacologically important plant derived lavonoids 2.4 Phenolics Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that occur ubiquitously as natural colour pigments responsible for the colour of fruits of plants. Phenolics in plants are mostly synthesized from phenylalanine via the action of phenylalanine ammonia lyase (PAL). They are very important to plants and have multiple functions. The most important role may be in plant defence against pathogens and herbivore predators, and thus are applied in the control of human pathogenic infections (Puupponen- Pimiá ct al,, 2008). They are classified into (i) phenolic acids and (ii) flavonoid polyphenolics (£lavonones, flavones, xanthones and catechins) and (iii) non-flavoncid polyphenolies. Caffeic acid is regarded as the most common of phenolic compounds distributed in the plant flora followed by chlorogenic acid known to cause allergic dermatitis among humans (Kar, 2007). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples, green-tea, and red-wine for their enormous ability to combat cancer and are also thought to prevent heart ailments to an apreciable degree and sometimes are antiinflammatory agents. Other examples include flavones, rutin, naringin, hesperidin and chlorogenic (Fig, 4). 2.5 Saponins The term saponin is derived from Saponaria vaccaria (Quillaja saponaria), a plant, which abounds in saponins and was once used as soap. Saponins therefore possess “soaplike' behaviour in water, Le. they produce foam. On hydrolysis, an aglycone is produced, which is called sapogenin. There are two types of sapogenin: steroidal and triterpenoidal. Usually, the sugar is attached at C-3 in saponins, because in most sapogenins there is a hydroxyl group at C-3. Quillaja saponaria is known to contain toxic glycosides quillajic acid and the sapogenin senegin. Quillajic acid is strenutatory and senegin is toxic. Senegin is also present in Polygala senega. Saponins are regarded as high molecular weight compounds in which, a www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 7 Flavone Rutin OH Resveratrol HO, , Ñ O—naringenin (0H HO HO, HO ÓH Naringin Hesperidin Ho CcooH HO Caffeic acid HOO OH oH ño “0H O (O) on Chlorogenic acid Fig, 4. Basic structures of some pharmacologically important plant derived phenolics sugar molecule is combined with triterpene or steroid aglycone. There are two major groups of saponins and these include: steroid saponins and triterpene saponins. Saponins are soluble in water and insoluble in ether, and like glycosides on hydrolysis, they give aglycones. Saponins are extremely poisonous, as they cause heamolysis of blood and are known to cause catile poisoning (Kar, 2007). They possess a bitter and acrid taste, besides causing irritation to mucous membranes. They are mostly amorphous in nature, soluble in alcohol and water, but insoluble in non-polar organic solvents like benzene and n-hexane. www.intechopen.com 10 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health pink or violet colour in the base layer indicates the presence of anthraquinones in the plant sample (Sarker de Nahar, 2007). ol óR 0 Salinos poramide Luteolin Fig, 7. Basic structures of some pharmacologically important plant derived anthraquinones 2.9 Essential oils Essential oils are the odorous and volatile products of various plant and animal species. Essential oils have a tendency evaporate on exposure to air even at ambient conditions and are therefore also referred to as volatile oils or ethereal oils. They mostly contribute to the odoriferous constituents or essences” of the aromatic plants that are used abundantly in enhancing the aroma of some spices (Martinez et al, 2008). Essential oils are either secreted either directly by the plant protoplasm or by the hydrolysis of some glycosides and structures such as directly Plant structures associated with the secretion of essential oils include: Glandular hairs (Lamiaceae e.g. Lavandula angustifolia), Oil tubes (or vittae) (Apiaceae eg, Foeniculun vulgare, and Pimpinela anisum (Aniseed), modified parenchymal cells (Piperaceae e.g. Piper nigrum - Black pepper), Schizogenous or lysigenum passages (Rutaceae e.g, Pinus palustris - Pine oil. Essential oils have been associated with different plant parts including leaves, stems, flowers, roots or rhizomes. Chemically, a single volatile oil comprises of more than 200 different chemical components, and mostly the trace constituents are solely responsible for attributing its characteristic flavour and odour (Firm, 2010). Essential oils can be prepared from various plant sources either by direct steam distillation, expression, extraction or by enzymatic hydrolysis. Direct steam distillation involves the boiling of plant part in a distillation flask and passing the generated steam and volatile oil through a water condenser and subsequently collecting the oil in florentine flasks. Depending on the nature of the plant source the distillation process can be either water distillation, water and steam distillation or direct distillation. Expression or extrusion of volatile oils is accomplished by either by sponge method, scarification, rasping or by a mechanical process. In the sponge method, the washed plant part e.g. citrous fruit (2.2, orange, lemon, grape fruit, bergamot) is cut into halves to remove the juice completely, rind tumed inside out by hand and squeezed when the secretary glands rupture. The oozed volatile oil is collected by means of the sponge and subsequently squeezed in a vessel. The vil floating on the surface is separated. For the the scarification process the apparatus Ecuelle a Piquer (a large bowl meant for pricking the outer surface of citrus fruits) is used. It is a large funnel made of copper having its inner layer tinned properly. The inner layer has numerous pointed metal needles just long enough to penetrate the epidermis. The lower stem of the apparatus serve two purposes; first, as a receiver for the oil; and secondly, as a www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 11 handle. Now, the freshly washed lemons are placed in the bowl and rotated repeatedly when the oil glands are punctured (scarified) thereby discharging the oil right into the handle. The liquid, thus collected, is transferred to another vessel, where on keeping the clear oil may be decanted and filtered. For the rasping process, the outer surface of the peel oÉ citrus fruits containing the oil gland is skilfully removed by a grater. The “raspings” are now placed in horsehair bags and pressed strongly so as to ooze out the oil stored in the oil glands. Initially, the liquid has a turbid appearance but on allowing it to stand the oil separates out which may be decanted and filtered subsequently. The mechanical process involves the use of heavy duty centrifugal devices so as to ease the separation of oil/water emulsions invariably formed and with the advent of modern mechanical devices the oil output has increased impressively. The extraction processes can be carried out with either volatile solvents (e.g hexane, petroleum ether or benzene) resulting into the production of “foral concretes”- oils with solid consistency and partly soluble in 95% alcohol, or non volatile solvents (tallow, lard or olive oil) which results in the production of perfumes. Examples of volatile oils include amygdaline (volatile oil of bitter almond), sinigrin (volatile oil of black mustard), and eugenol occurring as gein (volatile oil of Gevn 20tanon) (Fig, 8). NC, OC,H,O HO pr Cato, oH att en, ocn O «Y O” N OH OSO,K'H,O ¿Ha CH,.CH CH, CH,cH-cn, Gein Eugenol Fig, 8. Basic structures of some pharmacolo gically important plant derived essential oils Amygdalin Sinigrin 2.10 Steroids Plant steroids (or steroid glycosides) also referred to as “cardiac glycosides” are one of the most naturally occurring plant phytoconstituents that have found therapeutic applications as arrow poisons or cardiac drugs (Firn, 2010). The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action mainly on the cardiac muscle when administered through injection into man or animal. Steroids (anabolic steroids) have been observed to promote nitro gen retention in osteoporosis and in animals with wasting illness (Maurya ct al, 2008; Madziga ct al, 2010). Caution should be taken when using steroidal glycosides as small amounts would exhibit the much needed stimulation on a diseased heart, whereas excessive dose may cause even death. Diosgenin and cevadine (from Veratrun veride) are examples of plant steroids (Fig, 9). 3. Mechanism of action of phytochemicals Different mechanisms of action of phytochemicals have been suggested They may inhibit microorganisms, interfere with some metabolic processes or may modulate gene expression and signal transduction pathways (Kris-Etherton et al, 2002; Manson 2003; Surh 2003). Phytochemicals may either be used as chemotherapentic or chemo preventive agents with chemoprevention referring to the use of agents to inhibit, reverse, or retard tumorigenesis. In this sense chemo preventive phytochemicals are applicable to cancer therapy, since www.intechopen.com 12 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health CH, Ho ==cc00 CH, Y OH Cevadine Diosgenin Fig, 9. Basic structures of some pharmacologically important plant derived steroids molecular mechanisms may be common to both chemoprevention and cancer therapy (D'Incalci et al., 2005; Sarkar €e Li, 2006). Plant extracts and essential oils may exhibit different modes of action against bacterial strains, such as interference with the phospholipids bilayer of the cell membrane which has as a consequence a permeability increase and loss of cellular constituents, damage of the enzymes involved in the production of cellular energy and synthesis of structural components, and destruction or inactivation of genetic material In general, the mechanism of action is considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport, and coagulation of cell contents (Kotzekidou et al., 2008). Some specific modes of actions are discussed below. 3.1 Antioxidants Antioxidants protect cells against the damaging effects of reactive oxygen species otherwise called, free radicals such as singlet oxy gen, super oxide, peroxyl radicals, hydroxy] radicals and peroxynite which results in oxidative stress leading to cellular damage (Mattson de Cheng, 2006). Natural antioxidants play a key role in health maintenance and prevention of the chronic and degenerative diseases, such as atherosclerosis, cardiac and cerebral ischema, carcinogenesis, neurodegenerative disorders, diabetic pregnancy, rheumatic disorder, DNA damage and ageing (Uddin ct al., 2008; Jayasri et al, 2009). Antioxidants exert their activity by scavenging the “free-oxygen radicals' thereby giving rise to a fairly “stable radical”. The free radicals are metastable chemical species, which tend to trap electrons from the molecules in the immediate surroundings. These radicals if not scavenged effectively in time, they may damage crucial bio molecules like lipids, proteins including those present in all membranes, mitochondria and, the DNA resulting in abnormalities leading to disease conditions (Uddin et al 2008). Thus, free radicals are involved in a number of diseases including; tumour inflammation, hemorrhagic shock, atherosclerosis, diabetes, infertility, gastrointestinal ulcerogenesis, asthma, rheumatoid arthritis, cardiovascular disorders, cystic fibrosis, neurodegenerative diseases (e.g. parkinsonism, Alzheimer's diseases), AIDS and even early senescence (Chen ct al, 2006; Uddin ct al, 2008). The human body produces insufficient amount of antioxidants which are essential for preventing oxidative stress. Free radicals generated in the body can be removed by the body's own natural antioxidant defences such as glutathione or catalases (Sen, 1995). Therefore this deficiency had to be www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 15 the amount of TTP (Thrombotic Thrombocytopenic Purpura), IR (Insulin Resistance), and GLUTA (Glucose Transporter-4) in 3T3-L1 Adipocytes. It was suggested that the mechanism of Cinnamon's insulin-like activity may be in part due to increase in the amounts of TP, IRP, and GLUTA and that Cinmamon polyphenols may have additional roles as anti-inflammatory and/or anti-angio genesis agents (Jakhetia et al,, 2010). 3.6 Anti-inflammatory Essential oil of C. osmophloewn twigs has excellent anti- inflammatory activities and cytotoxicity against HepG2 (Human Hepatocellular Liver Carcinoma Cell Line) cells. Previous reports also indicated that the constituents of C. osmophloewmn twig exhibited excellent antiinflammatory activities in suppressing nitric oxide production by LPS (Lipopolysaccharide)-stimulated macrophages (Jakhetia et al., 2010). 3.7 Multifunctional targets Multiple molecular targets of dietary phytochemicals have been identified, from pro- and antiapoptotic proteins, cell cycle proteins, cell adhesion molecules, protein kinases, transcription factors to metastasis and cell growth pathways (Awad €: Bradford, 2005; Aggarwal de Shishodia, 2006; Choi €: Friso, 2006). Phytochemicals such as epigallocatechin- 3-gallate (EGCG) from green tea, curcumin from turmeric, and resveratrol from red wine tend to aim at a multitude of molecular targets. It is because of these characteristics that definitive mechanisms of action are not available despite decades of research (Francis ct al, 2002). The multi-target nature of phytochemicals may be beneficial in overcoming cancer drug resistance. This multi-faceted mode of action probably hinders the cancer cell's ability to develop resistance to the phytochemicals. It has also been demonstrated that EGCG has inhibitory effects on the extracellular release of verotoxin (VT) from E. col 0157: H7 (Voravuthikunchai de Kitpipit, 2003). Ethanol pericarp extracts from Punica granatum was also reported to inhibited VT production in periplasmic space and cell supernatant. Mechanisms responsible for this are yet to be understood, however the active compounds from the plant are thought to interfere with the transcriptional and translational processes of the bacterial cell (Voravuthikunchai €: Kitpipit, 2003). More work is needed to be done in order to establish this assumption. Phytochemicals may also modulate transcription factors (Andreadi et al, 2006), redox-sensitive transcription factors (Surh ct al., 2005), redox signalling, and inflammation. As an example, nitric oxide (NO), a signalling molecule of importance in inflammation, is modulated by plant polyphenols and other botanical extracts (Chan é: Fong, 1999; Shanmugam et al,, 2008). Many phytochemicals have been classified as Phytoestrogens, with health-promoting effects resulting in the phytochemicals to be marketed as nutraceuticals (Moutsatsou, 2007). 4. Methods of studying phytochemicals No single method is sufficient to study the bioactivity of phytochemicals from a given plant. An appropriate assay is required to first screen for the presence of the source material, to purify and subsequently identify the compounds therein. Assay methods vary depending on what bioactivity is targeted and these may include antimicrobial, anti-malarial, anticancer, seed germination, and mammalian toxicity activities. The assay method however www.intechopen.com 16 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health should be as simple, specific, and rapid as possible. An in vitro test is more desirable than a bioassay using small laboratory animals, which, in turn, is more desirable than feeding large amounts of valuable and hard to obtain extract to larger domestic or laboratory animals. In addition, in vivo tests in mammals are often variable and are highly constrained by ethical considerations of animal welfare. Extraction from the plant is an empirical exercise in which different solvents are utilized under a variety of conditions such as time and temperature of extraction. The success or failure of the extraction process depends on the most appropriate assay. Once extracted from the plant, the bioactive component then has to be separated from the co extractives. Further purification steps may involve simple crystallization of the compound from the crude extract, further solvent partition of the co extractives or chromatographic methods in order to fractionate the compounds based on their acidity, polarity or molecular size. Final purification, to provide compounds of suitable purity for such structural analysis, may be accomplished by appropriate techniques such as recrystallization, sublimation, or distillation. 4.1 Extraction of phytochemicals 4.1.1 Solvent extraction Various solvents have been used to extract different phytoconstituents. The plant parts are dried immediately either in an artificial environment at low temperature (50-60*C) or dried preferably in shade so as to bring down the initial large moisture content to enable its prolonged storage life and. The dried berries are pulverised by mechanical grinders and the oil is removed by solvent extraction. The defatted material is then extracted in a soxhlet apparatus or by soaking in water or alcohol (95% v/v). The resulting alcoholic extract is filtered, concentrated in vacuo or by evaporation, treated with HCl (12N) and refluxed for at least six hours. This can then be concentrated and used to determine the presence of Pphytoconstituents. Generally, the saponins do have high molecular weight and hence their isolation in the purest form poses some practical difficulties. The plant parts (tubers, roots, stems, leave etc) are washed sliced and extracted with hot water or ethanol (95% v/v) for several hours. The resulting extractis filtered, concentrated in wcuo and the desired constituent is precipitated with ether. Exhaustive extraction (EE) is usually carried out with different solvents of increasing polarity in order to extract as much as possible the most active components with highest biological activity. 4.1.2 Supercritical fluid extraction (SFE) This is the most technolo gically advanced extraction system (Patil €: Shettigar, 2010). Super Critical Fluid Extraction (SFE) involves use of gases, usually CO», and compressing them into a dense liquid. This liquid is then pumped through a cylinder containing the material to be extracted. From there, the extractladen liquid is pumped into a separation chamber where the extract is separated from the gas and the gas is recovered for re-use. Solvent properties of CO, can be manipulated and adjusted by varying the pressure and temperature that one works at. The advantages of SFE are, the versatility it offers in pinpointing the constituents you want to extract from a given material and the fact that your www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 17 end product has virtually no solvent residues left in it (CO, evaporates completely). The downside is that this technology is quite expensive. There are many other gases and liquids that are highly efficient as extraction solvents when put under pressure (Patil de Shettigar, 2010). a. Conpled SFE-SFC System in which a sample is extracted with a supercritical fluid which then places the extracted material in the inlet part of a supercritical fluid chromatographic system. The extract is than chromatographed directly using supercritical fluid. b. Coupled SFE-GC and SFE-LC System in which a sample is extracted using a supercritical fluid which is then depressurized to deposit the extracted material in the inlet part or a column of gas or liquid chromatographic system respectively. SFE is characterized by robustness of sample preparation, reliability, less time consuming, high yield and also has potential for coupling with number of chromatographic methods. 4.1.3 Microwave-Assisted extraction Patil de Shettigar (2010) reported an innovative, microwave-assisted solvent-extraction technology known as Micro wave-Assisted Processing (MAP). MAP applications include the extraction of high-value compounds from natural sources including phytonutrients, mutraceutical and functional food ingredients and pharmaceutical actives from biomass. Compared to conventional solvent extraction methods, MAP technology offers some combination of the following advantages: 1 Improved products, increased purity of crude extracts, improved stability of marker compounds, possibility to use less toxic solvents; 2. Reduced processing costs, increased recovery and purity of marker compounds, very fast extraction rates, reduced energy and solvent usage. With micro wave-derived extraction as opposed to diffusion, very fast extraction rates and greater solvent flexibility can be achieved. Many variables, including the micro wave power and energy density, can be tuned to deliver desired product attributes and optimize process economics. The process can be customized to optimize for commercial/cost reasons and excellent extracts are produced from widely varying substrates. Examples include, but are not limited to, antioxidants from dried herbs, carotenoids from single cells and plant sources, taxanes from taxus biomass, essential fatty acids from microalgae and oilseeds, phytosterols from medicinal plants, polyphenols from green tea, flavor constituents from vanilla and black pepper, essential oils from various sources, and many more (Patil € Shettigar, 2010). 4.1.4 Solid phase extraction This involves sorption of solutes from a liquid medium onto a solid adsorbent by the same mechanisms by which molecules are retained on chromatographic stationary phases. These adsorbents, like chromatographic media, come in the form of beads or resins that can be used in column or in batch form. They are often used in the commercially available form of syringes packed with medium (typically a few hundred milligrams to a few grams) through which the sample can be gently forced with the plunger or by vacuum. Solid phase extraction media include reverse phase, normal phase, and ion-exchange media. This is method for sample purification that separates and concentrates the analyte from solution of crude extracts by adsorption onto a disposable solid-phase cartridge. The analyte is www.intechopen.com 20 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health B. Gas Chromatograplny-1 ass Spectroscopy Gas chromatography equipment can be directly interfaced with rapid scan mass spectrometer of various types. The flow rate from capillary column is generally low enough that the column output can be fed directly into ionization chamber of MS. The simplest mass detector in GC is the Ion Trap Detector (ITD). In this instrument, ions are created from the eluted sample by electron impact or chemical ionization and stored in a radio frequency field; the trapped ions are then ejected from the storage area to an electron multiplier detector. The ejection is controlled so that scanning on the basis of mass-to-charge ratio is possible. The ions trap detector is remarkably compact and less expensive than quadrapole instruments.GC-MS instruments have been used for identification of hundreds of components that are present in natural and biological system (Oleszek 4: Marston, 2000; Philipson, 2007; Daffre ct al., 2008). 4.1.6.3 Supercritical Fluid Chromatography (SFC) Supercritical fluid chromatography is a hybrid of gas and liquid chromatography that combines some of the best features of each. This technique is an important third kind of column chromatography that is beginning to find use in many industrial, regulatory and academic laboratories. SFC is important because it permits the separation and determination of a group of compounds that are not conveniently handled by either gas or liquid chromatography. These compounds are either non-volatile or thermally labile so that GC procedures are inaplicable or contain no functional group that makes possible detection by the spectroscopic or electrochemical technique employed in LC. SFC has been applied to a wide variety of materials including natural products, drugs, foods and pesticides. 4.2 Other Chromato-Spectrometric studies The NMR techniques are employed for establishing connectivities between neighbouring protons and establishing C-H bonds. INEPT is also being used for long range heteronuclear correlations over multiple bondings. The application of Thin Layer chromatography (TLC), High Performance Chromatography (HPLC) and HPLC coupled with Ultra violate (UV) photodiode array detection, Liquid Chromatography-Ultraviolet (LC-UV), Liquid Chromatography-Mass Spectrophotometry (LCMS), electrospray (ES) and Liquid Chromatography-Nuclear Magnetic Resonance (LC-NMR) techniques for the separation and structure determination of antifungal and antibacterial plant compounds is on the increase frequently (Oleszek é Marston, 2000; Bohlin and Bruhm, 1999). Currently available chromatographic and spectroscopic techniques in new drug discovery from natural products Currently, computer modeling has also been introduced in spectrum interpretation and the generation of chemical structures meeting the spectral properties of bioactive compounds obtained from plants (Vlietinck, 2000). The computer systems utilise 1H, 15C, 2D-NMR, IR and MS spectral properties (Philipson, 2007). Libraries of spectra can be searched for comparison with complete or partial chemical structures. Hyphenated chromatographic and spectroscopic techniques are powerful analytical tools that are combined with high trroughput biological screening in order to avoid re-isolation of known compounds as well as for structure determination of novel compounds. Hyphenated chromatographic and spectrosco pic techniques include LC-UV-MS, LC-UV-NMR, LC-UV- ES-MS and GC-MS (Oleszek de Marston, 2000; Philipson, 2007). www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 21 4.3 Simple assay methods 4.3.1 Antimicrobial assay Common methods used in the evaluation of the antibacterial and antifungal activities of plant extracts and essential oils, include the agar diffusion method (paper disc and well), the dilution method (agar and liquid broth) (Yagoub, 2008; Okigbo et al., 2009 El Mahmood, 2009; Aiyegoro et al,, 2009), and the turbidimetric and impedimetric monitoring of microbial growth (Rios de Recio, 2005). These methods are simple to carry out under laboratory conditions. 4.3.2 Antioxidant assays Most common spectrophotometric assay method applied is the DPPH radical scavenging system in which the hydrogen or electrons donation ability of plant extracts are measured from bleaching of purple methanol solution of 2, 2'-diphenyl-1 picrylhydrazyl (DPPH) free radical (Changwei ct al,, 2006). This spectrophotometric assay uses the stable radical DPPH as a reagent. DPPH absorbs at 517 nm, and as its concentration is reduced by existence of an antioxidant, the absorption gradually disappears with time. A 2-ml aliquot of a suspension of the ethanol extracts is mixed with 1 ml of 0.5 mM 2,2. diphyenyl-1-picrylhydrazyl (DPPH) solution and 2 ml of 0.1 M sodium acetate buffer (pH 5.5). After shaking, the mixture is incubated at ambient temperature in the dark for 30 minutes, following which the absorbance is measured at 517 nm using a UV-160A spectrometer. A solvent such as ethanol can be used as negative control. Radical scavenging activity is often expressed as percentage inhibition and is often calculated using the formula: % radical scavenging activity = [(Avw -Avo)/ Acro] x 100 Where Acontror is the absorbance of the control (DPPH solution without test sample) and Ayest is the absorbance of the test sample (DPPH solution plus antioxidant). Phenolics content and reducing power of extracts is often determined using the Folin- Ciocalteu method. Equal volumes of Folin-Ciocalteu reagent and given quantity (mg) of plant extracts of different concentrations (e.g. 0.4, 0.3, 0.2, 0.1 and 0.05 mg/ml) are often mixed in different sets of test tubes shaken thoroughly, and left to stand for 1 min. Ten percent of NaHCO» is then added and the mixture once again allowed to stand for 30 minutes, after which the absorbance (725 nm) is measured spectrophotometrically. Gallic acid (0.05-0.5 mg/ml) is often used to produce standard calibration curve and the total phenolic content expressed as mg equivalent of gallic acid (mg GAE) per gram dry weight of the extract by computing with standard calibration curve (Djeridane et al,, 2006). For determination of reducing power of plant extracts, the ferric reducing/ antioxidant power (FRAP) assay method can be applied. The assay is based on the reducing power of a compound (antioxidant). A potential antioxidant reduces the ferric ion (Fe**) to the ferrous ion (Fe2»); the later forms a blue complex (Fe?*/2, 4, 6, tripyridyLs-triazine (TPTZ)), which increases the absorption at 593 nm. Stronger absorption at this wavelength indicates higher reducing power of the phytochemical, tus higher antioxidant activity. Reaction mixture containing test extract sample at different concentrations (10-10041) in phosphate buffer (0.2 M, pH 6.6) and equal amounts of 1% (w/v) potassium ferricyanide are incubated at 50*C for www.intechopen.com 22 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health 20 minutes and then the reaction terminated by the addition of equal volumes of 10% (w/v) tricarboxyllic acid (TCA) solution and the mixture centrifuged at 3000rpm for 20 minutes. The supernatant is mixed with equal volume of distilled water and 0.1 % (w/v) ferric chloride solution and the absorbance measured at 700 nm. Increased absorbance of the mixture with concentration indicates the reducing power of extract (Jayasri, 2009). 4.3.3 Toxicological studies These are often carried out to determine the toxicity of a plant part. Usually animal models such as mice, guinea pigs or rabbits are often employed. In these procedures, the LD:o of the extracts in the experimental animal is often determined via either oral or intradermal administration. The toxic response of experimental animals to the administration of plant alkaloids is usually detected by assay of the serum ALT and AST of the animal as sensitive indicators of hepatocelular damage (Chapatwala ct al., 1982). Any toxicity usually results in distortion of hepatocytes membrane integrity due to hepatocelular injury and plasma levels rise, as a consequence of high toxin levels present within hepatocytes. 5. Safety concerns for phytochemicals Plants are natural reservoir of medicinal agents almost free from the side effects normally caused by synthetic chemicals (Fennel et al, 2004). The World Health Organization estimates that herbal medicine is still the main stay of about 75-80% of the world population, mainly in the developing countries for primary health care because of better cultural acceptability, better compatibility with the human body, and lesser side-effects (Kamboj, 2000; Yadav de Dixit, 2008). The over use of synthetic drugs with impurities resulting in higher incidence of adverse drug reactions, has motivated mankind to go back to nature for safer remedies. Due to varied locations where these plants grow, coupled with the problem of different vanacular names, the World Health Organization published standards for herbal safety to minimize adultartion and abuse (WHO, 1999). A number of modern drugs have been isolated from natural sources and many of these isolations were based on the uses of the agents in traditional medicine (Rizvi et a1,, 2009). Antimicrobial properties of crude extracts prepared from plants have been described and such reports had attracted the attention of scientists worldwide (Falodun et al., 2006; El- Mahmood t: Amey, 2007, El-Mahmood, 2009). Herbs have been used for food and medicinal purposes for centuries and this knowledge have been passed on from generation to generation (Adedapo et al, 2005). This is particularly evident in the rural areas where infectious diseases are endemic and modern health care facilities are few and far thus, compelling the people to nurse their ailments using local herbs. Herbal treatments have been adjudged to be relatively safe (WHO, 1999). For instance, daily oral doses of epigallocatechin-3-gallate (EGCG) for 4 weeks at 800 mg/day in 40 volunteers only caused minor adverse effects (Phillipson, 2007). In a 90-day study of polyphenon E (a formulation of green tea extract with 53% EGCG), the oral no effect level (NOEL) values are 90 mg/kg/ day for rats and 600 mg/kg/day for dogs (Boocock et al., 2007). For curcumin given to cancer patients at 3600 mg/day for 4 months or 800 mg/day for 3 months, only minor adverse effects are seen. For resveratrol, a single oral dose at 5 gin 10 volunteers only causes minor adverse effects (Boocock ct al., 2007). Though herbs are relatively safe to use, their combined www.intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 25 appropriate pharmacological activity (McGaw et al., 2005; Doughari et al., 2009; Okigbo ct al,, 2009). Another approach is the use of “chemical- genetics profiling' (Doughari et al., 2009). In this method, by building up a database of the effects of a wide range of known compounds, itis possible to interrogate drugs with unknown mechanisms or mixtures of compounds such as natural product mixtures. The technique highlighted unexpected similarities in molecular effects of unrelated drugs (e.g, amiodarone and tamoxifen) and also revealed potential anti-fungal activity of crude extracts. This activity was confirmed by isolation and testing of defined compounds, stichloroside and theopalauamide (Fig, 10). Because these compounds are not structurally similar, they would not have been expected to act via the same biological target, thus providing more chances for a very versatile drug, component with high efficacy against antibiotic resistant bacteria. I's been reported that despite the popularity of chemical drugs, herbal medicine in Africa and the rest of the world, continued to be practiced due to richness of certain plants in varieties of secondary metabolites such as alkaloids, flavonoids, tannins and terpenoids (Cowan, 1999; Lewis de Ausubel, 2006; Adekunle é: Adekunle, 2009). Stapleton et al. (2004) reported that aqueous extracts of tea (Camelha sinensis) reversed methicillin resistance in methicillin resistant Staphylococcus aureus (MRSA) and also to some extent reduced penicillin resistance in beta- lactamase-producing Staphylococcus aureus. Also, Betoni ct al. (2006) reported synergistic interactions between extracts of guaco (l/ikania glomerata), guava (Psidium guajava), clove (Syzyguim aromaticum), garlic (Allin sativwn) lemon grass (Cymbopogon citratus) ginger (Zingiber officinale) cargueja (Baccharis trimera), and mint (1/entha pieria) and some antibiotics against S. aureus. However, these are preliminary investigations and more works are needed to actually determine the active ingredients in these plants extracts and this may help in improving management of the different infectious diseases that are developing resistance to commonly used antibiotics and possibly to verocytotoxuic bacteria. Furthermore, toxicological studies can also be carried out to determine the reliance on these herbs without many side effects. Researchers have also devised cluster of chemically related scaffolds which are very useful in guiding the synthesis of new compounds. In an attempt to combine the advantages of virtual screening of chemically diverse natural products and their synthetic analogues (scaffolds) with the rapid availability of physical samples for testing, an academic collaboration has established the Drug Discovery Portal (http: / /www.ddp.strath.ac.uk/). This brings together a wide variety of compounds from academic laboratories in many different institutions in a database that can be used for virtual screening, Academic biology groups can also propose structures as targets for virtual screening with the Portal's database (and with conventional commercially available databases). Access to the Portal is free for academic groups and the continued expansion of the chemical database means that there is a valuable and growing coverage of chemical space through many novel chemical compounds (Feher €: Schmidt, 2003; Galm e: Shen, 2007). Despite all of the advances made by the pharmaceutical industry in the development of novel and highly effective medicines for the treatment of a wide range of diseases, there has been a marked increase in the use of herbal medicines even including the more affluent countries of the world Germany has the largest share of the market in Europe and it was reported that the sales of herbal medicinal products (HMPs) in 1997 were US$ 158 billion (Barnes et al., 2007).Numerous scientific medical/pharmacentical books have been www.intechopen.com 26 Phytochemicals — A Global Perspective of Their Role in Nutrition and Health Fig, 10. Natural products - recently discovered and/or in development. (1) Salinosporamide A; (2) curacin A; (3) dolastatin 10; (4) tarbomycin A; (5) cryptophicin; (6) vancomycin; (7) platensimycin; (8) platencin; (9) stichloroside; (10) theopalanamide (Source; Doughari et al,, 2009). www. intechopen.com Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 27 published in recent years aiming to provide the general public and healthcare professionals with evidence of the benefits and risks of herbal medicines (Barnes ct al, 2007; Phillipson, 2007). The pharmaceutical industry has met the increased demand for herbal medicines by manufacturing a range of HMPs many of which contain standardized amounts of specific natural products. In the 1950s, it would not have been possible to predict that in 50 years time there would be a thriving industry producing HMPs based on the public demand for herbal medicines. To date European Pharmacopoeia has even published up to 125 monographs on specific medicinal herbs with another 84 currently in preparation (Mijajlovic et al,, 2006; Phillipson, 2007. The monographs are meant to provide up-to-date knowledge of phytochemistry for defining the chemical profiles of medicinal herbs and an understanding of analytical tests for identification of the herbs and for the quantitative assessment of any known active ingredients (Phillipson, 2007). Several regulatory bodies incuding Traditional Medicines Boards (TMBs, in Nigeria and other African Countries) Medicines and Healthcare products Regulatory Agency (MHRA), Herbal Medicines Advisory Committee (HMAC) (Uk) and American Herbal Products Association (AHPA) and several other pharmacopoeia (British, Chinese, German, Japanese) provide guidelines and advice on the safety, quality and utilization of the plant herbal products in several countries (Yadav de Dixit, 2008). Scientific and Research communities are currently engaged in phytochemical research, and pharmacognosy, phytomedicine or traditional medicine are various disciplines in higher institutions of learning that deals specifically with research in herbal medicines. It is estimated that >5000 individual phytochemicals have been identified in fruits, vegetables, and grains, but a large percentage still remain unknown and need to be identified before we can fully understand the health benefits of phytochemicals (Liu, 2004). 7. Concluding remarks With the increasing interest and so many promising drug candidates in the current development pipeline that are of natural origin, and with the lessening of technical drawbacks associated with natural product research, there are better opportunities to explore the biological activity of previously inaccesible sources of natural products. 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