NATURAL PRODUCTS

1. NATURAL PRODUCTS

2. NATURAL PRODUCTS • Compounds that occur naturally in plants and animals • Ubiquitous compounds are usually not classified as • natural products. Ubiquitous = occurs everywhere in nature, universal excludes amino acids, nucleic acids, all compounds found in major metabolic pathways. • Natural products are unique to only one species or family • of organisms.

3. Natural Product - how did the term arise? 1769-85 Scheele showed that tartaric acid in grapes, citric acid in lemons, malic acid in apples, gallic acid in galls, lactic acid in milk, uric acid in urine. 1772-7 Lavoisier burned sugar, ethanol and acetic acid in oxygen and found only CO2 and H2O, thus the burned chemicals must have been made of carbon and hydrogen only. Quantification showed that they must also contain oxygen. Later studies by others found that some natural substances when burned also gave off nitrogen hence must also contain nitrogen.

4. 1807 Berzelius introduced the terms organic and inorganic , to refer to chemicals made by living organisms and found in minerals respectively 1828 Wöhler showed that he could make urea hence the “vital force” of living organisms was not needed to make organic chemicals. 1833 Persoz and Payen first noted enzyme activity. The concept of vitalism now transferred to enzymes in whole cells. However in 1897 Büchner showed enzyme activity in cell-free solutions so vitalism suffered a final blow. • 19th C. The blossoming of the study of organic molecules gradually split into the study of man-made organic chemicals (the organic chemicals and organic chemistry of today) and organic chemicals made by organisms (=Natural Products and Natural Product Chemistry of today).

5. 1891 Kössel, a German physiological chemist, proposed that the metabolism of organisms could be divided into two type. Primary metabolism was the basic biochemistry common to all cells. Secondary metabolism was the type of biochemistry found only in some species. Thus to physiological chemists, later to be called biochemists, Secondary Metabolites are what chemists call Natural Products. 20th C Physiological chemistry split off from Chemistry Departments and became Biochemistry ... but that split normally left Natural Products being studied in Chemistry Departments and now ignored by biochemists. None of the major introductory biochemistry texts in the library give the term Natural Product in their indices • Synthetic chemicals are made by the use of chemically reactive reagents. The chemicals tend to fairly crude in bring about changes to structures – addition, subtraction, substitution and rearrangements

6. Natural Products are made by enzymes which can be much more selective – they can target their action on parts of the molecule by bringing the active site of the enzyme into close proximity with one part of the molecule to be changed. A primary metaboliteis a kind of metabolite that is directly involved in normal growth, development, and reproduction. It usually performs a physiological function in the organism (i.e. an intrinsic function). A primary metabolite is typically present in many organism or cell. It is also referred to as a central metabolite, which has an even more restricted meaning (present in any autonomously growing cell or organism). Conversely, a secondary metabolite is not directly involved in those processes, but usually has an important ecological function (i.e. a relational function). A secondary metabolite is typically present in a taxonomically restricted set of organisms or cells (Plants, Fungi, Bacteria...).

7. Secondary metabolitesare organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism's survivability, fecundity ( الخصوبة), or aesthetics ((جماليات, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic (النشوء والتطور) group. Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenss. Humans use secondary metabolites as medicines, flavorings, and recreational drugs ( العقاقير المنشطة ).

8. 1803-Derosne extracted a mixture of narcotine and morphine 1806-Serturner recognised the alkaline nature of the principle content of opium However the structures of these complex molecules were made known only in the 2nd half of the 20th century NMR & X-ray- the tedious task has become much easier. Many drugs were discovered from natural products and their complex structures have intrigued many chemists and due to this, synthesis of them were of great challenge. 1952-morphine was synthesised. 1950’s –reserpine was synthesized.

9. PRELIMINARY BIOLOGICAL SCREENING OF SAMPLES • Plant collection • Identify the botanical name • Prepare a herbarium sample • Dry and grind sample • Extract the samples with various solvents • Send extracts for biological screening

10. Natural products are expected to play an important role as one of the major sources of new drugs in the years to come because of • their incomparable structural diversity, • (ii) the relatively small dimensions of many of them (<2000 Da, 1 kilogram is equal to 6.022 E+26 dalton), and • (iii) their ‘‘drug-like’’ properties, i.e. their ability to be absorbed and metabolized. • Isolation of natural products from higher plants, marine organisms and microorganisms is therefore still urgently needed, calling for state-of-the-art methodologies for separation and isolation procedures. • Taking into consideration that a plant may contain thousands of constituents, the separation and isolation process can be long and tedious. • Isolation of natural products generally combines various separation techniques, which depend on the solubility, volatility and stability of the • compounds to be separated.

11. Several sample preparation, pre-purification and clean-up steps are used prior to isolation and/or analysis of natural products. Initial extraction with low-polarity solvents yields the more lipophilic components, while ethanolic solvents obtain a larger spectrum of non-polar and polar material. If a more polar solvent is used for the first extraction step subsequent solvent partition allows a finer division into different polarity fractions.

12. Extraction methods are therefore used as a pre-purification step to selectively remove interfering components and/or to isolate the active compounds. Other pre-purification methods are filtration, precipitation, removal of chlorophyll, waxes and tannins, solid-phase extraction (SPE) using pre-packed cartridges with a variety of packing material, both normal- and reversed-phase silica gel, or short columns with other suitable packing material such as alumina, Celite, Amberlite resins and Sephadex LH-20. Pre-packed cartridges for SPE operate on the principle of liquid–solid extraction and may be used in one of two modes: a) the interfering matrix elements of a sample are retained on the cartridge while the components of interest are eluted; b) the components of interest are retained while the interfering matrix elements are eluted. In the latter case, a concentration effect can be achieved. The required compounds are then eluted from the cartridge by changing the solvent.

13. Extraction techniques used for separation and isolation The first step in the analysis and isolation of natural products is extraction to separate the compounds from the cellular matrix. Extraction and recovery of a solute from a solid matrix may be regarded as a five-stage process: (i) desorption of the compound from the active sites of the matrix; (ii) diffusion into the matrix itself; (iii) solubilisation of the analyte in the extractant; (iv) diffusion of the compound in the extractant; (v) collection of the extracted solutes. Ideally, an extraction process should be exhaustive with respect to the constituents to be analysed or isolated, rapid, simple, inexpensive, and – at least for routine analysis – amenable for automation.

14. Conventional methods for the extraction of natural products include Soxhlet extraction, maceration, percolation, turbo-extraction and sonication. These traditional methods present major drawbacks, including long extraction times, labour-intensive procedures, large amounts of organic solvents, unsatisfactory extraction efficiency, and potential degradation of labile compounds. Example of pre-packed cartridges

15. Soxhlet extractor Normally a solid material containing some of the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet extractor. The extraction solvent to be used is taken into a distillation flask and the Soxhlet extractor is now placed onto this flask. The Soxhlet is then equipped with a condenser. The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material. The chamber containing the solid material is slowly filled with warm solvent. Some of the desired compound will then dissolve in the warm solvent. When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. The thimble ensures that the rapid motion of the solvent does not transport any solid material to the still pot. This cycle may be allowed to repeat many times, over hours or days. During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded.

16. A schematic representation of a Soxhlet extractor1: Stirrer bar 2: Still pot (the still pot should not be overfilled and the volume of solvent in the still pot should be 3 to 4 times the volume of the soxhlet chamber) 3: Distillation path 4: Thimble 5: Solid 6: Siphon top 7: Siphon exit 8: Expansion adapter 9: Condenser 10: Cooling water in 11: Cooling water out

17. Maceration Processes (Steady – State Extraction) • Maceration : • This simple widely used procedure involves leaving the pulverized plant to soak in a suitable solvent in a closed container .simple maceration is performed at room temperature by mixing the ground drug with the solvent (drug solvent ratio : 1:5 or 1:10) and leaving the mixture for several days with occasional shaking or stirring. The extract is then repeated from the plant particles by straining . The process is repeated for once or twice with fresh solvent .Finally the last residue of extract is pressed out of the plant particles using a mechanical press or a centrifuge.kinetic maceration differe from simple one by continous stirring. • -The method is suitable for both initial and bulk extraction. •  -The main disadvantage of maceration is that the process can be quite time-consuming, taking from a few hours up to several weeks

18. Percolation : the powdered plant material is soaked initially in a solvent in a percolator . Additional solvent is then poured on top of the plant material and allowed to percolate slowly (dropwise) out of the bottom of the percolator. Additional filtration of the extract is not required because there is a filter at the outlet of the percolator. -Percolation is adequate for both initial and large-scale extraction. -The main disadvantages are : 1-fine powders and materials such as resins and plants that swell excessively (e.g., those containing mucilages) can clog the percolator.  2-if the material is not distributed homogenously in the container, the solvent may not reach all areas and the extraction will be incomplete.

19. Simple percolator

20. Turbo Distillation Extraction: Turbo distillation is suitable for hard-to-extract or coarse plant material, such as bark, roots, and seeds. In this process, the plants soak in water and steam is circulated through this plant and water mixture. Throughout the entire process, the same water is continually recycled through the plant material. This method allows faster extraction of essential oils from hard-to-extract plant materials

21. In recent years new extraction techniques with significant advantages over conventional methods have been developed for extracting analytes from solid matrices, e.g. reduction in organic solvent consumption and in sample degradation, reduction of extraction and clean-up times or even elimination of additional sample clean-up and concentration steps, improvement in extraction efficiency, selectivity, and/or kinetics, ease of automation. These recent extraction techniques include supercritical fluid extraction (SFE), pressurised liquid extraction (PLE), microwave-assisted extraction (MAE), solid-phase microextraction (SPME), ultrasound- assisted extraction (UAE), superheated liquid extraction, and extraction with supercritical or subcritical water. Most of these methods have similar pros and cons with regard to solvent volume, extraction time and extraction efficiency.

22. Supercritical fluid extraction (SFE) Supercritical fluid extraction (SFE) represents an interesting alternative technique to conventional solid–liquid extraction (e.g. Soxhlet extraction) with lower solvent consumption and lower working temperature. It is a form of liquid extraction where the usual liquid solvent phase has been replaced by a supercritical fluid—a substance that is above its critical point. Amongst a wide variety of supercritical fluids, carbon dioxide is essentially the only convenient supercritical extraction solvent used because of its comparatively low critical temperature (31.1C) and pressure (73.8 bar/7.38 MPa).

24. An organic solvent (also called modifier) may be added to the supercritical fluid to enhance its solvating properties. In case of CO2, volatile polar solvents such as ethanol, methanol or acetonitrile are preferred. By using CO2 as the supercritical fluid, extractions can be performed under mild conditions, thus reducing both the risks of thermal degradation and the poor collection efficiencies of volatile analytes. CO2 is most effective for dissolving organic compounds, particularly molecules displaying some degree of lipophilicity, such as esters, ethers and lactones. The modifier component may be introduced into the fluid either using a separate pump and suitable mixing device or may be added to the sample matrix in the extraction cell prior to pressuring with CO2. Frequently, an off-line valve is incorporated between the pump and the extraction vessel and between the vessel and the restrictor. In this set-up static or dynamic extraction or a combination of the two may be performed. The restrictor maintains the pressure within the extraction vessel by flow control.

25. The use of SFE both at the analytical and processing scales is quite widespread in the food industry for the extraction of fats and oils from seeds, foodstuffs, and other materials. The technique has also been applied to the extraction of active compounds from medicinal plants, such as steroids, terpenes, alkaloids, various oxygen containing heterocyclic compounds, as well as aromatic and phenolic compounds.

26. Pressurised liquid extraction (PLE) PLE is a solid–liquid extraction process using organic solvents at an elevated temperature (usually between 50 and 200 C) and applying higher pressure (between 10–15 MPa) to extract samples in an extraction cell. Extractions are carried out under pressure in order to maintain the solvent in its liquid state, even at temperatures above boiling point. Moreover, pressure allows the extraction cell to be filled more quickly, and helps to force the solvent into the matrix pores. Thus, the efficiency of the extraction process is improved. Extraction at elevated temperatures increases solubility, diffusion rate, and mass transfer, coupled with the ability of the solvent to disrupt the analyte– matrix interactions

27. PLE thus allows fast extraction owing to increased solubility, better desorption and enhanced diffusion, and the extraction is generally completed within a few minutes. PLE was developed especially for laboratories with increased sample throughput. Comparison with conventional extraction methods has demonstrated faster extraction, higher extraction efficiency, and lower solvent consumption, along with comparable recoveries in most cases. No evidence was seen for thermal degradation during the extraction of temperature-sensitive compounds

28. Scheme for PLE System

29. There are two ways to perform PLE. The first is the static mode in which the extraction cell is filled with a solvent, followed by heating to generate pressure in the cell. After a period of time (5–10 min is usually sufficient), the system is rinsed with fresh solvent to ensure that all of the extract reaches the collection vials, and is purged with gas to avoid any losses or ‘memory’ effects. In the second method, the dynamic mode, fresh solvent is continuously percolated through the cartridge under pressure at a constant flow rate for a fixed period of time. The extraction cell is placed in a thermostatted oven. In both cases, under conditions of elevated pressure and temperature, the mass transfer rates are accelerated. The typical volume collected depends on the cell size. Volumes between 10 and 100 ml may be required, and hence repeated evaporation steps are needed to concentrate the final extracts. Both commercially available and laboratory-assembled PLE systems are used.

30. Microwave-assisted extraction (MAE) Microwave-assisted extraction (MAE) is a recent technology for extracting soluble products into a fluid from a wide range of materials using microwave energy. It provides a technique which allows one to extract compounds more selectively and more rapidly (usually in less than 30 min) with similar or better recovery than traditional extraction processes. Microwaves directly heat the solvent or solvent mixture, thus accelerating the speed of heating. Besides the advantage of high extraction speed, MAE also enables a significant reduction in the consumption of organic solvent.

31. The application of microwave energy to the samples may be performed using two technologies: either closed vessels under controlled pressure and temperature, or open vessels at atmospheric pressure. The two technologies are commonly named pressurized MAE (PMAE), with a multi-mode cavity, or focused MAE (FMAE) using the waveguide as a single-mode cavity, respectively. Both systems are shown in Fig. 6. Whereas in open vessels the temperature is limited by the boiling point of the solvent at atmospheric pressure, in closed vessels the solvent can be heated above its boiling point at atmospheric pressure by simply applying suitable pressure, thus enhancing both extraction speed and efficiency. However, after extraction with closed vessels, one needs to wait for the temperature to decrease before opening the vessel, thereby increasing the overall extraction time (by approximately 20 min). Open systems use focused microwaves, resulting in homogenous and very efficient heating of the sample. In closed systems using diffuse microwaves, the electric field in the cavity is non-homogenous, and therefore the vessels are placed on a turntable.

32. Scheme of the two microwave systems, using diffused or focused microwaves.

33. MOST COMMON CHROMATOGRAPHIC TECHNIQUES • Column chromatography • Thin layer chromatography • MPLC ( medium pressure liquid chromatography) • HPLC (High pressure liquid chromatography) STATIONARY PHASE • Silica-nonpolar • Bonded silica-C18, C-8, CN etc • Polyacrylamide • Polysaccharides; sephadex

34. SOME CLASSES OF NATURAL PRODUCTS • Alkaloids • Terpenes • Polyketides • Steroids

35. ALKALOIDS

36. WHAT IS AN ALKALOID ? • Naturally-occurring compounds that contain nitrogen • Many have heterocyclic rings as a part of their structure • They are found mostly in plants and also in certain animals • Many are physiologically active (often spectacularly) • Many are used by native peoples for religious or medicinal • purposes. • Many are basic (“alkaline”, due to an unshared pair on N) • Those nitrogen compounds that are found in all organisms • (i.e., amino acids, nucleic acids, etc.) are not considered • alkaloids. • Alkaloids are “secondary metabolites”, they are not • involved in primary metabolism.

37. Classification Schemes for Alkaloids • The heterocyclic ring systems found as a part of the compound’s structure • The plant or plant family where they originate Also alkaloids are classified as: 1- True alkaloids ( Characterized by heterocyclic ring with a nitrogen atom, and are derived from amino acids). 2- Proto alkaloids ( characterized by absence of the heterocyclic ring but also derived from amino acids) 3- Pseudo alkaloids ( characterized by a heterocyclic ring with a nitrogen atom, but are not derived from amino acids. ( Steroidal alkaloids)

38. In general the alkaloids are classified according to the chemical structure in two broad divisions:- 1- Non-heterocyclic or typical alkaloids or biological amines. 2- Heterocyclic or typical alkaloids , divided into 14 groups according to their structure, as : 1) Pyrrol or pyrrolidine 2) pyrrolizidine 3) Pyridine and piperidine 4) Tropane 5) Quinoline 6) Isoquinoline 7) Aporphine 8) Norlupinane 9) Indole 10) Indolizidine 11) Imidazole 12) purine 13) steroid 14) Terpenoid

39. HETEROCYCLIC RING SYSTEMS

40. HETEROCYCLIC RING SYSTEMS (cont)

41. Alkaloids with Exocyclic Nitrogen (Protoalkaloids) (Non-Heterocyclic Alkaloids) Phenyl alkylamine alkaloids *This group of alkaloids have the nitrogen atom located in an amino group and is not a member of a heterocyclic ring. i.e. alkaloids characterized by the absence of heterocyclic ring in their molecules. *Many are simple derivatives of b-Phenylethylamine and as such, are derived from the common amino acids Phenyl alanine or Tyrosine. *They are Sympathomimetic drugs (as they rise the blood pressure). *They includes the alkaloids of: 1) Ephedra alkaloids 2) Khat alkaloids 3) Capsicum alkaloids 4) Colchicum alkaloids

42. Prefixes • "Nor-" designates N-demethylation or N-demethoxylation, e.g. norpseudoephedrine and nornicotine. • "Apo-" designates dehydration e.g. apomorphine. • "Iso-, pseudo-, neo-, and epi-" indicate different types of isomers. • Suffixes: • "-dine" designates isomerism as quinidine and cinchonidine. • "-ine" indicates, in case of ergot alkaloids, a lower pharmacological activity e.g. ergotaminine is less potent than ergotamine.

43. Ephedrine Recently banned from organic weight-loss supplements! found in ephedra spp Extract of ephedra was used in the early american west as a cure for asthma (Mormon Tea). ephedrine Chinese medicine “Ma Huang” for hay fever. Sympathomimetic Amine “Organic” appetite suppressant. compare R=H amphetamine R= H Norepinephrine R = CH3 methamphetamine R = CH3 Epinephrine (adrenalin) neurotransmitter / hormone “speed” or crystal “meth”

44. UTAH Ephedra

46. Ephedrine biosynthesis

47. S-adenosylmethionine SAM S-Adenosylmethionine is the principal methylating agent in the secondary metabolism of plants.