Phytochemistry-I (Introduction) PDF

PHYTOCHEMISTRY- I PGC 504 Prof. Dr. Abeer M. El-Sayed 1  Pharmacognosy: The knowledge of drugs, which provide scientific description of drugs of natural origin used in medicine Drugs are of: -Natural origin: plants, animals, minerals -Synthetic orig...

PHYTOCHEMISTRY- I PGC 504 Prof. Dr. Abeer M. El-Sayed 1  Pharmacognosy: The knowledge of drugs, which provide scientific description of drugs of natural origin used in medicine Drugs are of: -Natural origin: plants, animals, minerals -Synthetic origin Phytochemistry is the study of phytochemicals. Those derived from plants, mainly large number of 2ry metabolites found in plants. Many of these are known to provide: protection against insect attacks, plant diseases and exhibit a number of protective or healing effects for human consumers.  Plant constituents (Organic substances formed & accumulated by plants) Primary metabolites are commonly present in all organisms and are essential for life (carbohydrates, proteins, fats, nucleic acid, vitamins) (* Rare fatty acids or sugars also considered as 2ry metabolites) Secondary metabolites are * Found in specific organisms or gp. of organisms * Pharmacologically active constituents  Plant constituents Factors influencing production of 2ry metabolites: 1- Gene composition (Heredity) → Qualitative & Quantitative changes 2- Stage of development (Ontogeny) → Quantitative changes 3- Environmental changes → Quantitative changes Different forms of Plant products supplied based 1- The purpose of use, 2- The nature of the active ingredients, 3- Some economic factors Different forms of Plant products 1- Fresh plant materials: In Trad. medicine, used as flavor & perfumery industries 2- Dried plant materials Used as Flavoring agents, spices & drugs. 3- Acellular products (unorganized drugs) e.g., gum exudates, resins, balsams, fixed & volatile oils 4- Galenical preparations For direct use e.g., plant extracts or tinctures 5- Processed extracts i.e., standardized extracts (with known concn. of the active principle) 6- Pure compounds most desirable for pharm. form. * Of known quality, safety and efficacy  Role of Natural Products in Modern Medicine Nat Prod have significant role in modern medicine due to: 1- Provide useful drugs: that cannot be commercially produced by synthesis e.g., Opium & Ergot alkaloids, * cardiac glyc. * Antibiotics 2- Supply semisynthetic comps: by modification→more effective or less toxic drugs e.g., morphine analogues based on SAR 3- Used as template: to produce synthetic analogues through drug design e.g., procaine & related local anesthetics 4- As starting materials for production of more potent drugs (chemically or by biotransformation from less or inactive one) e.g., -Baccatin III → taxol -Stigmasterol→ hydrocortisone & related steroids.  Classification of plant constituents (PC) Classification is based on: 1→ Biosynthetic origin 2→ Solubility properties 3→ Presence of key functional gps (widely used). OR 1 and 3 4→Physicochemical properties. 5→Pharmacological activities. Based on Key functional groups 1.Phenolic compounds e.g., flavonoids, tannins, quinones, anthocyanins,.. - Precursor → shikimic acid - Hydrophilic - with at least one aromatic ring (with 1 or more oxygenation site)  Classification of plant constituents (PC) 2.Terpenoids e.g., vol. oils, sterols, carotenoids - Precursor → isopentenyl pyrophosphate (isoprene) - Lipophilic - Sub classified acc. to No. of isopentene units (=isoprene, C = 5): Monoterpenes (C = 10) in V.O. Sesquiterpenes (C = 15) in V.O. Diterpenes (C = 20), in plant families Triterpenes (C = 30), in plant families Tetraterpenes (C = 40), carotenoids.  Classification of plant constituents (PC) 3- Nitrogen containing compounds Amino acids, peptides, alkaloids - Precursor → different amino acids - Basic in nature - +ve reaction to either ninhydrin or Dragendorff`s reagents 4- Organic acids, lipids and related compounds e.g., Simple organic acids (citric, malic, etc..) Fatty acids (as FA ester in the form of fixed oils, fats or waxes) Other compounds (alkanes, polyacetylenes) - Precursor → acetate - Of varying solubility 5- Water-soluble carbohydrates & their derivatives: mono- & oligosaccharides, water soluble glycosides 6- Macromolecules Of high M. wt., nucleic acids, proteins, polysacch. Classification according to chemistry & common physical properties 1. Volatile oils, Resins & Resin combinations 2. Carbohydrates 3. Bitter Principles = Bitters 4. Tannins 5. Alkaloids 6. Glycosides Based on Pharmacological activity Analgesics, emetics, diuretics, laxatives, cardiotonics etc…. 11 Nomenclature of plant constituents (Trivial) The root names derived from: 1- Name of discoverer, e.g., Pelletierine 2- Plant common name, e.g., Vinca (vincristine, vinblastine) Ergot (ergometrine, ergotamine) 3- Plant Latin name, e.g. (Podophyllotoxin, Visnagin, Digitoxin) 4- The pharmacological action, e.g., emetine Prefixes Suffixes (indicate compds. Derived from others,) (added to root name, to indicate, 1- hydroxy- , methoxy- , methyl- , 1- -in a “constituent of” dihydro- , homo-, for added 2- -oside, sugar deriv. (glycoside) substitutions 3- -genin, aglycone 2- Deoxy- , demethoxy- , demethyl- 4- -toxin,poisonous constituent , dehydro- , nor-, for removed 5- -one, -al, -ol substitutions Phytochemistry concerns with Plant constituents from Physical, chemical, structure & of 2r metabolites (Natural drugs) biosynthesis of phytochemicals Techniques commonly used in the field of phytochemistry are extraction, isolation using various chromatographic techniques (MPLC, HPLC, LC-MS) & structural elucidation (MS,1D and 2D NMR) of natural products.  Extraction, isolation, characterization & quantitative evaluation of plant constituents The Role of Phytochemist is to focus on the Isolation Identification, Chemical structures, Methods of evaluation Biosynthesis, Metabolism Distribution Biological function OF Phytochemicals Therefore, Isolation of pure Phytochemical is the most important step Extraction of the plant material There are medicinally desired and undesired plant constituents (PC) or phytochemicals. Therefore, extraction & fractionation of plants guided by biological activity rather than on chemistry of comps is best approach. Extraction could be defined as “the process, which involves withdrawal of the desired AC from the crude drugs by the use of selected solvent and procedure. Preparation of plant material for extraction ❑Plant material (PM) should be authenticated ❑PM should be free from adulterants or contaminants. ❑PM should be subjected to deactivation of enzymes. ❑PM could be extracted fresh or dry. ❑PM should be kept in dry weather till extraction Selection of: ►Mode of EXTRACTION ►SOLVENT Role of solvent of choice is to dissolve or extract Desired or Active PC (?) OR remove Inactive or Inert PC (?). ❑ Polar solvents extract or dissolve polar comps. e.g., sugars, alkaloids, salts. ❑ Non-polar solvents extract or dissolve non-polar comps. e.g., fats, waxes, oils. ❑ Factors affecting solubilization of PC Polarity of the solvent H-bonding Chemical nature of PC Dipole-dipole forces Less polar: Cyclohexane, hexane, CCl4 Benzene, CHCl3, EtOAc, acetone, EtOH, MeOH, H2O, acid. H2O Most polar. Notes  Solubilization of certain types of comps is enhanced by their ability to H-bonding with solvent molecules.  The affinity of the solute for the org phase may be increased by using mix of solvents.  Fractionation between different solvents leads to better isolation.  Knowing the chemical nature of PC may help in the choice of suitable solvent.  Methods for extraction Classical extraction : 1- Extraction with organic solvents: percolation, maceration. 2- Extraction using a Soxhlet apparatus; and 3- Extraction with water: infusion, decoction, and steam distillation. Specific old method is the Extraction with cold fat, called enfleurage, used mainly for the extraction of fragrances (V.O.) from flowers Shaking, stirring or homogenization could be applied to increase efficiency of extraction  Methods for extraction 1- Mechanical pressing (no solvent) It is used to extract fixed oils using a hydraulic press. The yield is affected by the moisture content of the seeds and temperature. 2-Maceration: PM to be extracted is immersed in suitable solvent for 3 days at room temp. with continuous agitation, then filter. 3–Percolation: It is maceration followed by a continuous slow flow of fresh solvent through PM (using percolator). 4-Digestion:Maceration with gentle heat to increase solvent efficiency and NOT affect PC. 5-Infusion: It is a maceration for a short period of time with either cold or boiling water. Used for readily water- soluble PC. 6-Decoction: It involves boiling PM with water for 15 min, filter. Used for thermostable water-sol PC.  7-Continuous hot extraction “Soxhlet extractor”  The PM is repeatedly extracted with solvent & the dissolved PC are concentrated in the extraction flask.  Used for thermostable PC  In case of thermolabile PC, a low- boiling point solvent is used 8- Ultrasound-assisted extraction (UAE) -High temp from ultrasonic energy increase solubility & diffusivity of PC in solvent. -The oxidative energy from radicals created during sonolysis, increases the extraction power. -Also, application of pressure could favor penetration and transport at inter-phase between the solvent & PM. Application: Extraction of plant materials, isolation of polysaccharides, volatile oils, fatty acids and their esters 9- Microwave-assisted extraction (MAE) Extraction with MAE is based on ❑ Heating of the solvent in contact with sample by means of microwave energy. ❑ Disruption of H-bonds by MW energy ❑ Heat generated by MW enhances solvent penetration into PM, allowing better dissolution of the target comps. Advantages: Reduced solvent volume High recoveries Shorter time Good reproducibility Minimal sample manipulation. Application: extraction of lipids, fixed oil, pigments, glycyrhizic acid (liquorice), cocaine (coca leaves)& alkamides (Echinacea). 10-Pressurized liquid extraction (PLE) PLE (= Accelerated solvent extraction, ASE) It is based on static extraction with superheated liquids under high pressure using organic solvent Advantages:  Higher solubility of solutes in solvent at higher temp  Higher diffusion rate due to high temp,  Disruption of the strong solute PM matrix interaction caused by Van der Waals forces, H-bonding and dipole-dipole attractions between solute molecules. Application: PLE is suited for the extraction of various types of comps from different materials because you can change type of solvent and pressure and adapted to the respective matrix. 11 - Supercritical fluid extraction (SFE): Based on that Gases such as CO2 and N2O become fluid when certain temp & pressure reached a critical point called supercritical phase Above a certain temp & pre, single sub do not condense or evaporate but exist as a fluid; i.e., both gas & liquid possess the same density and no division exists between two phases “the critical state”, Gas become supercritical fluid. Supercritical fluid is used to extract target subs From solid samples. https://youtu.be/oqP6r2RBNy4 Caffeine extraction from green coffee with supercritical CO2 - YouTube  Advantages of the use of CO2 Available & inexpensive Non-inflammable Non-toxic Has a low critical temp (tc31oC, Pc=74 bar) Easy to eliminate Chemically inert Advantages of SFE technique Extracted PC are of close composition to natural PC The temp. & pressure can be changed, to adjust selectivity. Extraction power can be modified by addition of a modifier (MeOH) to increase the polarity. The absence of hydrolysis, decomposition or rearrangements of the extracted PC. Alkaloids: Decaffeination of green coffee Caffeine extraction from green coffee with supercritical CO2 - YouTube Diterpenes: Extraction of Taxol from Taxus. Fixed and V. oils: oil of evening primrose 12- Subcritical water extraction (SWE) (Pressurized hot water extraction (PHWE)superheated water extraction) It is based on the use of water as extractant in a dynamic state, at temp 100 to 374°C but below its critical point (< 374°C) and pressure high enough to maintain water in liquid state. https://extractionmagazine.com/2020/12/09/subcritical-water-extraction-of- ashwagandha/ https://www.youtube.com/watch?v=TgAaI3FqElo Rise of temp leads to increase in diffusion rate and decrease in viscosity and surface tension. Advantages: Easy manipulation of dielectric constant () of water, which can be changed within a wide range just by changing the temp under moderate pressure.  Subcritical water extraction (SWE) E.g., at room temp and pre, H2O has a dielectric constant () ca. 80 (polar solvent). Subcritical water at 250oC and pr over 40 bar, it has  = 27, of similar polarity of EtOH. This means we can change property of water to be non-polar to extract even fixed or V. oils Application: ❑Extraction of fixed and V.Oils. ❑Subcritical water (pre 125 bar 175ºC) extract rapidly oxygenated fragrance comps from rosemary, leaving monoterpenes, waxes and lipids. 13 - Extraction by distillation Steam distillation (in details in V oils) 14- Spouted bed extraction Production of annatto powder (coloring matter) from the seeds of Bixa orellana present in the pigment layer of seed coat by action of hot air jet. Collected seed coat Seeds v Hot air jet 15- Solid Phase MicroExtraction SPME is an extraction technique used for: -Analysis of some V.O. containing drugs. - In GC & LC analysis of dilute samples in both liquid or gaseous phases. Adsorptive phase: silicone derivative fibers Injection port Liquid sample Volatile sample adsorption of the sample Adsorption and desorption of volatile sample Advantages of SPME : It can reduce solvent consumption, Reduce plant mat., more rapid and reproducible, Less opportunity for oxidation of vol. comps. 6399 16- Solid Phase Extraction (SPE) SPE is a physical extraction process involving use of solid phases, in pre-packed columns containing stationary phases (of same nature in column) as in 001988 HPLC e.g., Bond ElutTM, Sep-PakTM, …. The objective: to isolate or purify a sub or sample of interest from a more complex sample in a pure conc. state, e.g biological sample (urine, serum,..  Fractionation of crude extracts by liquid/liquid fractionation To facilitate further isolation of their individual compds. A- Solvent/solvent precipitation a)- Addition of less polar miscible solvent e.g.,-Preparation of Crude saponin mix using MeOH/acetone - Purification of gum using water/MeOH. b) Addition of a more polar miscible solvent, e.g., Purification of resin using Alc/H2O or acid/H2O  Fractionation of crude extracts by liquid/liquid fractionation B- Liquid/liquid extraction: (partition of solute moles. between two immiscible solvents) based on: their relative solubility or partition coefficient constant (K`)& polarity. In the form: B-1 Single contact (in a sep. funnel) B-2 Multiple contact (CCE, DCCC or RLCCC)  B-2 Multiple contact B2-1.Counter-current Extraction (CCE): Used for separation of PC with small differences in their partition coeff. (K`) using special apparatus called: Craig's counter-current apparatus“ Based on the same principle of liq/liq extraction (shaking) Separation occurs in multiple process of partition and separation step-wise process in hundreds of special tubes (Craig’s unit). In a countercurrent extraction, the solvent and feed enter from opposite ends of the system, and these two immiscible phases pass each other in opposing streams. Explain the Counter Current Extraction? | Solvent Extraction | Analytical Chemstry (youtube.com) https://youtu.be/OUMWDLulK64  B-2 Multiple contact B-2-2 Droplet Counter Current Chromatograph (DCCC) B-2-3 Rotatory Locular Counter Current Chromatograph (RLCCC) DCC Principal Advantages: -It can be easily applied to separate of saponins (no foaming due to shaking, cf. from CCE) -NO solid support used for highly polar PC, e.g alkaloids, tannins, iridoids, anthraquinones etc…. The Key step in phytochemical Investigation of a PM is the isolation and purification steps of PC Other methods can be applied to simplify or isolate PC See more ………… 1)- Sublimation: - Caffeinefrom tea - Balsamic acids (benzoic & cinnamic acids) 2)- Fractional distillation: Separation of different components of volatile oils 3)- Fractional liberation: (through pH gradient) - Separation of a mixture of basic compounds (alkaloids), and acidic comps (Phenolic acids and comps containing COOH). 4)- Fractional crystallization: acc. to the difference in solubility in certain solvent. 5)- Chromatography using different techniques (Column, prep. PC, TLC, HPLC, GC, & Specific techniques: ion exchange, gel filtration, etc….. Identification of plant constituents Check for homogeneity & purity: by repeated chromatography (TLC, PC, HPLC, mp) Determination of the class of compound: - Its response to color tests, derivatization to common derivatives - Its UV spectral analysis (eg. Flavonoids) data. Identification within the class: - Melting points (solids), boiling points (liquids) - Optical rotation “for optically active compounds” - Spectral characteristics: UV, IR, NMR & MS Confirmation of identity: - Direct comparison with an authentic sample (UV, IR, MP, D ) OR - Comparison of its spectral data with published one For new compounds → spectral data and degradation & Lab. synthesis  Quantitative determination of plant constituents Crude plant exts. shld. be standardized before use in therapy Determination of total active const. by (depending on the nature of the constituents) 1- Gravimetric methods (% recovery) 2- Titrimetric methods * “acid-base det. of alkaloids in aq. or non-aq. media” * “Redox determination of sugars and peroxides”. 3- Spectrophotometric methods e.g., colorimetric methods. 4- Biological methods Special methods for standardization of extracts contain toxic materials & failed to be accurately evaluated with other methods → cardiac glycosides. 5- Chromatographic techniques: ideal methods HPLC & GC. tn_teacher300-Teacher-Teachers-1_0307-2214-0705